U.S. patent application number 15/516015 was filed with the patent office on 2017-10-19 for antibodies against pathological forms of tdp-43 and uses thereof.
This patent application is currently assigned to Academia Sinica. The applicant listed for this patent is Academia Sinica. Invention is credited to Yun-Ru CHEN.
Application Number | 20170298124 15/516015 |
Document ID | / |
Family ID | 59254049 |
Filed Date | 2017-10-19 |
United States Patent
Application |
20170298124 |
Kind Code |
A1 |
CHEN; Yun-Ru |
October 19, 2017 |
ANTIBODIES AGAINST PATHOLOGICAL FORMS OF TDP-43 AND USES
THEREOF
Abstract
Disclosed herein are novel pathological form of TDP-43,
monoclonal antibodies against such pathological form of TDP-43, and
uses thereof. The novel pathological form of TDP-43 is
characterized in having a spherical particle size of about 2 to 400
nm in diameter.
Inventors: |
CHEN; Yun-Ru; (Taipei,
TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Academia Sinica |
Taipei |
|
TW |
|
|
Assignee: |
Academia Sinica
Taipei
TW
|
Family ID: |
59254049 |
Appl. No.: |
15/516015 |
Filed: |
September 15, 2015 |
PCT Filed: |
September 15, 2015 |
PCT NO: |
PCT/US2015/050114 |
371 Date: |
March 30, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62059192 |
Oct 3, 2014 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/6896 20130101;
C07K 2317/76 20130101; A61P 21/02 20180101; A61P 25/28 20180101;
C07K 16/18 20130101; A61P 25/14 20180101; A61P 25/00 20180101; G01N
2800/2821 20130101; A61P 25/16 20180101; A61P 43/00 20180101 |
International
Class: |
C07K 16/18 20060101
C07K016/18; G01N 33/68 20060101 G01N033/68 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 9, 2015 |
CN |
104107500 |
Claims
1. An antibody or a fragment thereof that specifically binds to a
transactivation responsive (TAR)-DNA-binding protein 43 kDa
(TDP-43) oligomer comprising, a heavy chain variable region
comprising amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and
SEQ ID NO: 3; and a light chain variable region comprising amino
acid sequences of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.
2. The antibody of claim 1, wherein the heavy chain variable region
has the amino acid sequence of SEQ ID NO: 4, and the light chain
variable region has the amino acid sequence of SEQ ID NO: 8.
3. The antibody of claim 1, wherein the TDP-43 oligomer has a
spherical particle size of about 2 to 400 nm in diameter.
4. The antibody of claim 3, wherein the TDP-43 oligomer has a
particle size that is about 40 to 60 nm in diameter.
5. A pharmaceutical composition for the prophylaxis or treatment of
a TDP-43 oligomer associated disease comprising an effective amount
of the antibody of claim 1; and a pharmaceutically acceptable
carrier.
6. The pharmaceutical composition of claim 5, wherein the TDP-43
oligomer associated disease is Alzheimer's disease, argyrophilic
grain disease, amyotrophic lateral sclerosis (ALS),
ALS-parkinsonism dementia complex of Guam, vascular dementia,
frontotemporal dementia, semantic dementia, dementia with Lewy
bodies, Huntington's disease, Spinocerebellar ataxia, inclusion
body myopathy, inclusion body myositis, hippocampal sclerosis, or
Parkinson's disease.
7. The pharmaceutical composition of claim 5, wherein the heavy
chain variable region has the amino acid sequence of SEQ ID NO: 4,
and the light chain variable region has the amino acid sequence of
SEQ ID NO: 8.
8. A method of diagnosing a TDP-43 oligomer associated disease from
a biological sample of a subject, comprising: determining the
amount of the TDP-43 oligomer in the biological sample by
contacting the biological sample with an effective amount of the
antibody of claim 1; and comparing the detected amount of the
TDP-43 oligomer in the biological sample with that of a control
sample obtained from a healthy subject; wherein a significantly
higher or lower amount of the detected TDP-43 oligomer in the
biological sample than that of the control sample indicates that
the subject suffers from the TDP-43 oligomer associated
disease.
9. The method of claim 8, wherein the TDP-43 oligomer associated
disease is Alzheimer's disease, argyrophilic grain disease,
amyotrophic lateral sclerosis (ALS), ALS-parkinsonism dementia
complex of Guam, vascular dementia, frontotemporal dementia,
semantic dementia, dementia with Lewy bodies, Huntington's disease,
Spinocerebellar ataxia, inclusion body myopathy, inclusion body
myositis, hippocampal sclerosis, or Parkinson's disease.
10. The method of claim 8, wherein the biological sample is a brain
biopsy sample, a cerebrospinal fluid sample, a whole blood sample,
a serum sample, a plasma sample, a urine sample, or a mucus
sample.
11. The method of claim 8, wherein the heavy chain variable region
has the amino acid sequence of SEQ ID NO: 4, and the light chain
variable region has the amino acid sequence of SEQ ID NO: 8.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0001] The present disclosure relates to novel pathological form of
transactivation responsive (TAR)-DNA-binding protein 43 kDa
(TDP-43), antibodies against such pathological form of TDP-43, and
uses thereof.
2. Description of Related Art
[0002] Neurodegenerative diseases have become an important health
issue in the modern society. According to the report of WHO, more
than 75% of elder population in the world will suffer some kinds of
neurodegenerative disease in the year of 2025. Frontotemporal lobar
degeneration (FTLD) is the second most common form of dementia in
the USA in the people age less than 65, and transactivation
responsive (TAR)-DNA-binding protein of 43 kDa (TDP-43) was
identified to be the major disease protein in the majority of
sporadic and familial FTLD cases, as well as amyotrophic lateral
sclerosis (ALS). In addition, TDP-43 was present in up to 57% of
patients in Alzheimer's disease. Currently, there is no cure or
treatment for these diseases, and significant efforts have been
made to identify molecules that may modulate the formation of
pathological forms of TDP-43.
[0003] In view of the foregoing, there exist in the related art, a
need for identifying molecules that may modulate the pathological
forms of TDP-43, such molecules will be potential drug candidates
for the manufacture of a medicament for the prophylaxis or
treatment of neurodegenerative diseases resulted from the formation
of pathological forms of TDP-43.
SUMMARY
[0004] This invention is based on the finding that transactivation
responsive (TAR)-DNA-binding protein 43 kDa (TDP-43) oligomer are
capable of cross-seeding Alzheimer's amyloid-.beta. (A.beta.) to
form amyloid oligomers, and are neurotoxic in vitro and in vivo.
Accordingly, a molecule capable of binding (e.g., an antibody) such
TDP-43 oligomer may suppresses the TDP-43 proteinopathy and thus
are useful for the manufacture of a medicament (i.e., a vaccine and
passive immunization) suitable for diagnosing, preventing or
treating a neurodegenerative disease resulted from deposit of
pathological form of TDP-43.
[0005] Accordingly, the present disclosure aims to provide an
antibody or a fragment thereof that specifically binds to TDP-43
oligomer. The antibody comprises a heavy chain variable region
comprising amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and
SEQ ID NO: 3; and a light chain variable region comprising amino
acid sequences of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.
[0006] The TDP-43 is characterized with a particle size of about 2
to 400 nm in diameter. Preferably, TDP-43 oligomer has a spherical
particle size of about 40 to 60 nm in diameter.
[0007] According to some preferred embodiments, the heavy chain
variable region of the present antibody has the amino acid sequence
of SEQ ID NO: 4, and the light chain variable region of the present
antibody has the amino acid sequence of SEQ ID NO: 8.
[0008] In one example, the present antibody is produced by the
hybridoma cell line deposited in the Bioresource and Collection
Center (BCRC) of the Food Industry Development and Research
Institute (FIDRI) in Taiwan (HsinChu, Taiwan, R.O.C.) as accession
number of BCRC960494.
[0009] In another example, the present antibody is produced by the
hybridoma cell line deposited with the BCRC as accession number of
BCRC960495.
[0010] In still another example, the present antibody is produced
by the hybridoma cell line deposited with the BCRC as accession
number of BCRC960496.
[0011] In a further example, the present antibody is produced by
the hybridoma cell line deposited with the BCRC as accession number
of BCRC960497.
[0012] In still a further example, the present antibody is produced
by the hybridoma cell line deposited with the BCRC as accession
number of BCRC960498.
[0013] It is therefore the second aspect of this disclosure to
provide a use of the antibody as described above for manufacturing
a medicament or a pharmaceutical composition for the prophylaxis or
treatment of a TDP-43 oligomer associated disease. The medicament
or the pharmaceutical composition comprises an effective amount of
the antibody described above; and a therapeutically acceptable
excipient.
[0014] According to preferred embodiments of the present
disclosure, the present antibody comprises a heavy chain variable
region comprising amino acid sequences of SEQ ID NO: 1, SEQ ID NO:
2 and SEQ ID NO: 3; and a light chain variable region comprising
amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO:
7. In one specific example, the heavy chain variable region has the
amino acid sequence of SEQ ID NO: 4, and the light chain variable
region has the amino acid sequence of SEQ ID NO: 8.
[0015] According to other preferred embodiments, the antibody may
be produced by any of the hybridoma cell lines deposited with the
BCRC as accession number of BCRC960494, BCRC960495, BCRC960496,
BCRC960497, or BCRC960498.
[0016] The antibody of this invention is present at a level of
about 0.1% to 99% by weight, based on the total weight of the
pharmaceutical composition. In some embodiments, the antibody of
this invention is present at a level of at least 1% by weight,
based on the total weight of the pharmaceutical composition. In
certain embodiments, the antibody of this invention is present at a
level of at least 5% by weight, based on the total weight of the
pharmaceutical composition. In still other embodiments, the
antibody of this invention is present at a level of at least 10% by
weight, based on the total weight of the pharmaceutical
composition. In still yet other embodiments, the antibody of this
invention is present at a level of at least 25% by weight, based on
the total weight of the pharmaceutical composition.
[0017] The TDP-43 oligomer associated disease treatable by the
medicament or the pharmaceutical composition of the present
disclosure may be any of, Alzheimer's disease, argyrophilic grain
disease, amyotrophic lateral sclerosis (ALS), ALS-parkinsonism
dementia complex of Guam, vascular dementia, frontotemporal
dementia, semantic dementia, dementia with Lewy bodies,
Huntington's disease, Spinocerebellar ataxia, inclusion body
myopathy, inclusion body myositis, hippocampal sclerosis, or
Parkinson's disease.
[0018] It is therefore a third aspect of the present disclosure to
provide a method for the prophylaxis or treatment of a TDP-43
oligomer associated disease in a subject. The method includes the
step of, administering to the subject a therapeutically effective
amount of the antibody of this invention, so as to inhibit or
suppress the TDP-43 proteinopathies.
[0019] According to some preferred embodiments, the present
antibody comprises a heavy chain variable region comprising amino
acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; and
a light chain variable region comprising amino acid sequences of
SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7. In one specific
example, the heavy chain variable region has the amino acid
sequence of SEQ ID NO: 4, and the light chain variable region has
the amino acid sequence of SEQ ID NO: 8.
[0020] According to other preferred embodiments, the antibody may
be produced by the hybridoma cell line deposited with the BCRC as
accession number of BCRC960494, BCRC960495, BCRC960496, BCRC960497,
or BCRC960498.
[0021] The TDP-43 oligomer associated disease that may be treated
by the method of the present disclosure is Alzheimer's disease
(AD), argyrophilic grain disease, amyotrophic lateral sclerosis
(ALS), ALS-parkinsonism dementia complex of Guam, vascular
dementia, frontotemporal dementia, semantic dementia, dementia with
Lewy bodies, Huntington's disease, Spinocerebellar ataxia,
inclusion body myopathy, inclusion body myositis, hippocampal
sclerosis, or Parkinson's disease (PD).
[0022] It is the fourth aspect of the present disclosure to provide
a method for the diagnosis of a TDP-43 oligomer associated disease
in a subject. The method includes steps of, obtaining a biological
sample from the subject; determining the amount of the TDP-43
oligomer in the biological sample by contacting the biological
sample with an effective amount of the antibody of this invention;
and comparing the detected amount of the TDP-43 oligomer in the
biological sample with that of a control sample obtained from a
healthy subject; wherein a significantly higher or lower amount of
the detected TDP-43 oligomer in the biological sample than that of
the control sample indicates that the subject suffers from the
neurodegenerative disease.
[0023] According to preferred embodiments of the present
disclosure, the biological sample is brain biopsy sample, a
cerebrospinal fluid sample, a whole blood sample, a serum sample, a
plasma sample, a urine sample, or a mucus sample.
[0024] According to some preferred embodiments, the present
antibody comprises a heavy chain variable region comprising amino
acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; and
a light chain variable region comprising amino acid sequences of
SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7. In one specific
example, the heavy chain variable region has the amino acid
sequence of SEQ ID NO: 4, and the light chain variable region has
the amino acid sequence of SEQ ID NO: 8.
[0025] According to other preferred embodiments, the antibody may
be produced by any of the hybridoma cell lines deposited with the
BCRC as accession number of BCRC960494, BCRC960495, BCRC960496,
BCRC960497, or BCRC960498.
[0026] It is therefore the fifth aspect of the present disclosure
to provide a kit for the detection of a pathological form of TDP-43
in a biological sample. The detecting result may be used as a
reference for determining whether a subject of the biological
sample suffers from a TDP-43 oligomer associated disease. The kit
includes, at least, a container, and reagents for detecting TDP-43
oligomer in the biological sample, wherein the reagents comprise
the anti-TDP-43 oligomer antibody of this invention and a legend
associated with the container and indicating how to use the
anti-TDP-43 oligomer antibody of this invention for detecting the
pathological form of TDP-43 (i.e., TDP-43 oligomer as described
herein) in the biological sample. The legend may be in a form of
pamphlet, tape, CD, VCD or DVD. The kit may further include a
negative control that indicates either absence of TDP-43 oligomer
or the normal level of the TDP-43 oligomer in a healthy
subject.
[0027] The details of one or more embodiments of the invention are
set forth in the accompanying description below. Other features and
advantages of the invention will be apparent from the detail
descriptions, and from claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] The present description will be better understood from the
following detailed description read in light of the accompanying
drawings, where:
[0029] FIGS. 1A-1G depict that full-length TDP-43 forms
amyloid-like oligomers. FIG. 1A: Analytical size-exclusion
chromatography (SEC) of full-length TDP-43 purified from E. coli
monitored by absorption at 280 nm (solid line) or human HEK293
cells quantified by slot blotting intensity (dashed line). The
retention times of the molecular weight standards are indicated.
FIG. 1B: Dot blotting of TDP-43 using anti-amyloid oligomer
antibody, A11. Freshly purified TDP-43 before (pre-load TDP-43) and
after SEC (the void volume, TDP-43 oligomers) and the buffers were
immunostained with A11. FIG. 1C: Freshly purified TDP-43 was dotted
in buffer or buffers containing 9 M urea, 7.2 M GdnHCI, or 2% SDS
with or without heating at 90.degree. C. for 1 h. Three replicates
were generated and probed separately by A11 antibody,
anti-N-terminal TDP-43 antibody (residues 1-260), and
anti-C-terminal TDP-43 antibody (residues 250-414). FIG. 1D: TEM
image of TDP-43 oligomers (scale bar, 500 nm) and zoomed-in images
of a single oligomer are shown in the upper left (scale bar, 50
nm). FIG. 1E: AFM image of TDP-43 oligomers (scale bar, 500 nm) and
zoomed-in images of a single oligomer are shown in the upper left
(scale bar, 50 nm). FIGS. 1F and 1G: DLS analysis of the oligomer
fraction of TDP-43 from SEC. Particle diameters are plotted against
scattered light intensity and particle number.
[0030] FIGS. 2A-2D depict the conformation, ThT fluorescence, and
DNA binding of TDP-43. FIG. 2A: Far-UV CD spectra of full-length
TDP-43 (solid line) and short-form TDP-43 (dashed line). The
spectra from 250 to 190 nm are shown. FIG. 2B: Bis-ANS fluorescence
spectra of full-length TDP-43 (solid line) and short-form TDP-43
(dashed line). The buffer signal is shown (dotted line). FIG. 2C:
ThT binding of TDP-43 in comparison to A.beta. fibrils. The ThT
fluorescence emission spectra of full-length TDP-43 (.box-solid.,
solid line), short-form TDP-43 ( , dashed line), and A.beta.
fibrils (.tangle-solidup., dotted line). Only A.beta. fibrils
showed ThT binding signal. FIG. 2D: TAR DNA binding of TDP-43
monitored by fluorescence titration. Full-length (squares) and
short-form (circles) of TDP-43 were titrated with single strand TAR
DNA-A (solid symbols) and -B sites (empty symbols). The emission
maxima of TDP-43 or TDP-43s at 350 nm were monitored while exciting
at 280 nm. The data shown were normalized to the starting point and
the lines shown are fit lines.
[0031] FIGS. 3A-3C depict the cross-seeding of TDP-43 to A.beta..
FIG. 3A: ThT assay of A.beta. fibrillization without and with
TDP-43 in concentrations ranging from 0.4 to 4%. Percentages of
TDP-43 seeded in molar ratio are indicated. FIG. 3B: Photo-induced
cross-linking (PICUP) assay of A.beta. without and with TDP-43 at
time 0. The percentages of TDP-43 seeded in molar ratio are
indicated. FIG. 3C: TEM images of end-point products of A.beta.
without and with 4% TDP-43 (scale bar, 100 nm).
[0032] FIGS. 4A-4E depict that TDP-43 oligomers induce neurite
degeneration and are neurotoxic in vitro and in vivo. Cytotoxicity
of TDP-43 to human BE(2)-C cells performed by MTT (FIG. 4A) and LDH
assays (FIG. 4B) (n=3, mean.+-.S.E.M.). FIG. 4C: Cytotoxicity of
TDP-43 to primary neuronal cultures using MTT assay. Cell viability
in MTT assays is normalized against the buffer control (n=3,
mean.+-.standard deviation). The statistics was analyzed by one
way-ANOVA with Tukey post-test for FIG. 4A and FIG. 4C and two
tailed, unpaired Student's t-test for FIG. 4B (*p<0.05,
**p<0.01, ***p<0.001). FIG. 4D: The immunocytochemistry of
primary neurons treated with control or TDP-43, 0.44 .mu.M. The
samples were subjected to MAP-2 immunostaining, GFAP
immunostaining, and DAPI staining. FIG. 4E: Intrahippocampal TDP-43
injection showed neuronal loss in the CA regions of mouse
hippocampus. Hippocampal injections (n=3 each group) of the buffer
control and TDP-43 were performed. Immunohistochemistry of the
hippocampal regions with neuronal specific marker NeuN and nuclear
specific dye DAPI are shown. The lesions are indicated by arrows
(scale bar, 50 .mu.m).
[0033] FIGS. 5A-5E depict that TDP-O antibody recognizes TDP-43
oligomers specifically. FIG. 5A: Freshly purified TDP-43 in native
buffer or buffers containing 9 M urea, 7.2 M GdnHCI, or 2% SDS with
or without heating at 90.degree. C. for 1 h was subjected to dot
blotting probed by the newly generated polyclonal antibody, TDP-O,
using TDP-43 oligomers as immunogen. The immunoreactivity of TDP-43
conformation by TDP-O was similar to that by A11 as shown in FIG.
1C. FIG. 5B: A.beta. oligomers were subjected to dot blotting
probed by A11 and TDP-O antibodies. A11, but not TDP-O, was able to
recognize A.beta. oligomers. FIG. 5C: TDP-43 with (solid line) and
without (dotted line) 3 fold concentration were subjected to SEC.
The elution volumes of TDP-43 oligomer (.star-solid.), TDP-43
monomer ( ), and the molecular weight standards are indicated. FIG.
5D: The 1 ml SEC fractions were collected and subjected to dot
blotting by TDP-O (upper blot) and N.sub.1-260 antibodies (lower
blot). FIG. 5E: The purified TDP-43 oligomers and monomers from SEC
were characterized by ELISA. The TDP-43 samples were coated
dose-dependently onto the ELISA plates. The TDP-43 oligomers were
probed by TDP-O (.box-solid.) and N.sub.1-260 (.quadrature.)
antibodies and TDP-43 monomers were probed by TDP-O ( ) and
N.sub.1-260 (.largecircle.) antibodies. TDP-O antibody possesses
significant higher specificity against TDP-43 oligomers.
[0034] FIGS. 6A-6B depict that TDP-43 oligomers are present and
increase with age in transgenic mouse model of FTLD-TDP. FIG. 6A:
Immunofluorescent staining of human TDP-43 and TDP-43 oligomers in
the brain slices of wild type and 6- and 12-month-old TDP-43
Tg.sup.+/+ transgenic mice. Cells with anti-TDP oligomer staining,
TDP-43 staining, and DAPI staining are shown (scale bar, 100
.mu.m). Blocked area was presented at higher magnification in the
last column (scale bar, 25 .mu.m). FIG. 6B: Quantified results
showed that the ratio of cells with deposition of TDP-O and TDP-43
in cytosol were age dependent. (n=3 per group, random 5 views were
calculated for each mouse, data represent as mean.+-.S.E.M. The
statistics was analyzed by one way-ANOVA with Turkey post-test
(*p<0.05, **p<0.01, ***p<0.001).
[0035] FIG. 7 depicts that TDP-43 oligomers are present in FTLD-TDP
patients. A total of three FTLD-TDP cases, three neurologically and
pathologically normal age-matched controls, and three Alzheimer's
disease cases without TDP-43 inclusions (as "neurodegenerative
disease controls") were examined. Representative images are shown.
(A), (C), and (E) Immunohistochemical staining of TDP-43 in the
hippocampal (C) and frontal cortical sections (A, E) of FTLD-TDP
patients by the TDP-O antibody. TDP-O identified densely stained,
ovoid or irregularly shaped but discreet cytoplasmic inclusions
(arrow) as well as comma-shaped profiles in the neuropil
representing dystrophic neurites (arrowhead). In some cortical
areas such as shown in (E), neuronal cytoplasm showed coarse
granular immunoreactivities. (B), (D), and (F) TDP-O did not stain
the brains of control subjects. TDP-O antibody, in contrast to
antibodies for monomeric TDP-43, did not stain nuclei demonstrating
specificity toward misfolded TDP-43. Scale bars in all panels are
20 .mu.m.
[0036] FIGS. 8A-8B depict the immunogold labeling of TDP-43
oligomers immunoprecipitated from the diseased hippocampus. The
hippocampus of a FTLD-TDP patient was extracted and
immunoprecipated by TDP-O antibody. The eluent was subjected to EM
with immunogold labeling with the N-term TDP-43 antibody (scale
bar, 50 nm). FIG. 8A: TDP-43 oligomers in FTLD-TDP (scale bar, 100
nm). FIG. 8B: zoom-in images of TDP-43 oligomers (scale bar, 50
nm).
[0037] FIG. 9 depicts that TDP-O mAbs exhibit higher specificities
toward TDP-43 oligomer. Various concentrations of TDP-O mAbs
(1-2.times.10.sup.-5 .mu.g/mL) respectively produced by TDP-O-3,
-5, -8, -9, and -10 hybridoma cells were used in ELISA assay to
detect the SDS denatured or non-denatured TDP-43.
[0038] FIGS. 10A-10B depict that TDP-O mAbs exhibit higher
specificities toward purified TDP-43 oligomer. The conditional
medium of TDP-O hybridoma cells were used to detect purified TDP-43
oligomers and monomers from size-exclusion chromatography by ELISA
assay. FIG. 10A: TDP-43 oligomers and monomers were separated and
collected by Superdex 200 10/300 GL column. FIG. 10B: Conditional
medium of TDP-O-3, -5, -8, -9, and -10 hybridoma cell lines were
used to detect the TDP-43 oligomers and monomers by ELISA
assay.
[0039] FIG. 11 depicts the consensus amino acid sequences of heavy
chain variable region (V.sub.H) and light chain variable region
(V.sub.L) of the 5 isolated TDP-O monoclonal antibodies. X
represents any amino acid residues, and CDR is the abbreviation of
the complementarity determining region.
[0040] FIG. 12 depicts that TDP-O monoclonal antibody rescues
TDP-43 oligomers-induced cytotoxicity. MTT assay was performed to
examine cell viability of BE(2)-C cells. Data are presented as
mean.+-.standard deviation. Statistical analysis was performed by
one-way ANOVAs, *p<0.05, **p<0.01, ***p<0.001. The result
showed the toxicity induced by TDP-43 oligomers was significantly
rescued by the treatment of TDP-O antibody.
DESCRIPTION
[0041] The detailed description provided below in connection with
the appended drawings is intended as a description of the present
examples and is not intended to represent the only forms in which
the present example may be constructed or utilized. The description
sets forth the functions of the example and the sequence of steps
for constructing and operating the example. However, the same or
equivalent functions and sequences may be accomplished by different
examples.
I. Definition
[0042] The term "TDP-43 proteinopathy" as used herein refers to
diseases particularly linked to transactivation responsive
(TAR)-DNA-binding protein of 43 kDa (TDP-43). TDP-43 is a disease
protein known to link with frontotemporal lobar degeneration with
ubiquitin-positive inclusion (FTLD-U), and amyotrophic lateral
sclerosis (ALS). TDP-43 proteinopathy thus includes, but is not
limited to, Alzheimer's disease (AD), argyrophilic grain disease,
amyotrophic lateral sclerosis (ALS), ALS-parkinsonism dementia
complex of Guam, vascular dementia, frontotemporal dementia,
semantic dementia, dementia with Lewy bodies, Huntington's disease,
Spinocerebellar ataxia, inclusion body myopathy, inclusion body
myositis, hippocampal sclerosis, or Parkinson's disease (PD).
[0043] The term "an effective amount" as used herein refers to an
amount effective, at dosages, and for periods of time necessary, to
achieve the desired therapeutically result with respect to the
treatment of a TDP-43 oligomer associated disease.
[0044] The phrase "pharmaceutically acceptable" refers to molecular
entities and compositions that are "generally regarded as safe",
e.g., that are physiologically tolerable and do not typically
produce an allergic or similar untoward reaction, such as gastric
upset, dizziness and the like, when administered to a human.
Preferably, as used herein, the term "pharmaceutically acceptable"
means approved by a regulatory agency of the Federal or a state
government or listed in the U.S. Pharmacopeia or other generally
recognized pharmacopeia for use in animals, and more particularly
in humans.
[0045] The term "administered", "administering" or "administration"
are used interchangeably herein to refer means either directly
administering a bi-specific antibody or a composition of the
present disclosure.
[0046] The term "subject" or "patient" refers to an animal
including the human species that is treatable with the compositions
and/or methods of the present disclosure. The term "subject" or
"patient" intended to refer to both the male and female gender
unless one gender is specifically indicated. Accordingly, the term
"subject" or "patient" comprises any mammal which may benefit from
treatment of cancer. Examples of a "subject" or "patient" include,
but are not limited to, a human, rat, mouse, guinea pig, monkey,
pig, goat, cow, horse, dog, cat, bird and fowl. In an exemplary
embodiment, the patient is a human.
[0047] The term "treat" or "treatment" as used herein are intended
to mean obtaining a desired pharmacological and/or physiologic
effect, e.g., detecting the presence of pathologic form of TDP-43,
preventing or rescuing organ atrophy, or inhibiting development of
dementia or muscular weakness and stiffness. The effect may be
prophylactic in terms of completely or partially preventing a
disease or symptom thereof and/or therapeutic in terms of a partial
or complete cure for a disease and/or adverse effect attributable
to the disease. "Treatment" as used herein includes preventative
(e.g., prophylactic), curative or palliative treatment of a disease
in a mammal, particularly human; and includes: (1) preventative
(e.g., prophylactic), curative or palliative treatment of a disease
or condition (e.g., a cancer or heart failure) from occurring in an
individual who may be pre-disposed to the disease but has not yet
been diagnosed as having it; (2) inhibiting a disease (e.g., by
arresting its development); or (3) relieving a disease (e.g.,
reducing symptoms associated with the disease).
[0048] The term "antibody" or "antibodies" is used in the broadest
sense and specifically covers monoclonal antibodies, polyclonal
antibodies, multispecific antibodies (e.g., bi-specific
antibodies), and antibody fragments so long as they exhibit the
desired biological activity, that is, to specifically bind to an
antigen when it preferentially recognizes its target antigen in a
complex mixture of proteins and/or other molecules. According to
one embodiment of the present application, the antibody of this
invention is a polyclonal antibody that specifically recognizes
TDP-43 oligomer.
[0049] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, and is not to be constructed as requiring production of
the antibody by any particular method. In contrast to polyclonal
antibodies which typically include different antibodies directed to
different epitopes, each monoclonal antibody is directed against a
single determinant (i.e., epitope) on the antigen. The monoclonal
antibodies of the present disclosure may be made by hybridoma
method or by recombinant DNA methods. The monoclonal antibodies
herein specifically include "chimeric" or "recombinant" antibodies,
in which a portion of the heavy and/or light chain is identical
with or homologous to corresponding sequences in antibodies derived
from a particular species or belonging to a antibody class or
subclass, while the remainder of the chain identical with or
homologous to corresponding sequences in antibodies derived from
another species or belonging to another antibody class or subclass,
as well as fragments of such antibodies, as long as they exhibit
the desired biological activity.
[0050] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies which contain minimal sequence derived from
non-human immunoglobulin. Humanized antibodies are human
immunoglobulins in which hypervarible region residues are replaced
by hypervarible region residues from a non-human species such as
mouse, rat, rabbit, or non-human primate having the desired
specificity or affinity. In some instances, Fv framework region
(FR) residues of the human immunoglobulin are replaced by
corresponding non-human residues. In general, the humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the FR regions are those of a human immunoglobulin sequence. The
humanized antibody may optionally comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin.
[0051] The singular forms "a", "and", and "the" are used herein to
include plural referents unless the context clearly dictates
otherwise.
II. Description of the Invention
[0052] Transactivation responsive (TAR)-DNA-binding protein of 43
kDa (TDP-43) has been identified as the disease protein in
ubiquitinated misfolded aggregates of all subtypes of sporadic
frontotemporal lobar degeneration with ubiquitin-positive inclusion
(FTLD-U), as well as amyotrophic lateral sclerosis (ALS). In the
present study, inventors unexpectedly discovered that full length
TDP-43 forms structurally stable spherical oligomers that
cross-seed Alzheimer's amyloid-.beta. (A.beta.) to form amyloid
oligomers, thus if an agent (e.g., an antibody) could bind such
TDP-43 oligomer, such agent may suppress or inhibit TDP-43
proteinopathy and/or TDP-43 effect to a subsequent event such as,
but not limited to, A.beta. oligomerization, and accordingly is
useful for the manufacture of a medicament (e.g., a vaccine or
passive immunization) suitable for preventing or treating a
neurodegenerative disease resulted from the deposit and/or
aggregation of pathological form of TDP-43.
[0053] Accordingly, it is the first aspect of the present
disclosure to provide an antibody, particularly, an antibody that
recognizes the TDP-43 oligomer identified in the examples of the
present invention, thereby inhibits the aggregation of a protein,
particularly, TDP-43, which aggregation is associated with a
disease. In general, the protein aggregation process proceeds in a
self-propagating manner, once initiated, an aggregation cascade
ensues that involves induced conformation change and/or
polymerization of further protein molecules, leading to the
formation of toxic product that is resistant to proteolysis. The
thus formed protein aggregation is thought to be the proximal cause
of neurodegeneration diseases, such as Alzheimer's disease (AD),
argyrophilic grain disease, amyotrophic lateral sclerosis (ALS),
ALS-parkinsonism dementia complex of Guam, vascular dementia,
frontotemporal dementia, semantic dementia, dementia with Lewy
bodies, Huntington's disease, Spinocerebellar ataxia, inclusion
body myopathy, inclusion body myositis, hippocampal sclerosis, or
Parkinson's disease (PD).
[0054] According to preferred embodiment of the present disclosure,
the TDP-43 oligomer is characterized in having a particle size of
about 2 to 400 nm in diameter. Preferably, the TDP-43 oligomer has
a particle size of about 20 to 400 nm in diameter, such as 20 to 30
nm, 30 to 40 nm, 40 to 50 nm, 50 to 60 nm, 60 to 70 nm, 70 to 80
nm, 80 to 90 nm, 90 to 100 nm, 100 to 120 nm, 120 to 140 nm, 140 to
160 nm, 160 to 180 nm, 180 to 200 nm, 200 to 220 nm, 220 to 240 nm,
240 to 260 nm, 260 to 280 nm, 280 to 300 nm, 300 to 320 nm, 320 to
340 nm, 340 to 360 nm, 360 to 380 nm, and 380 to 400 nm in
diameter. More preferably, the TDP-43 oligomer has a spherical
particle size of about 40 to 60 nm in diameter.
[0055] The antibodies of the present invention specifically bind
the TDP-43 oligomer described above, its epitopes, as well as
various conformations and epitopes thereof. In preferred
embodiments, antibodies disclosed herein preferentially bind
pathologic TDP-43, specifically, full-length TDP-43 oligomer
characterized in having a spherical particle size of about 2 to 400
nm in diameter.
[0056] To produce the desired monoclonal antibodies, animals such
as mice, rats or rabbits are first immunized with TDP-43 oligomer
at a suitable dose. Generally, adjuvant and the TDP-43 oligomer
solution are mixed together when immunizing the animals with TDP-43
oligomer. Examples of adjuvants useful for this invention include
Freund's complete adjuvant (FCA), Freund's incomplete adjuvant
(FIA), and aluminum hydroxide adjuvant. Immunization is generally
carried out mainly by intravenous, subcutaneous, intraperitoneal or
intramuscular injection of the antigen. The immunization interval
is not particularly limited. Immunization may be carried out at
intervals of several days to several weeks, preferably 2 to 3
weeks, for 1 to 10 times, preferably 2 to 5 times. Once antibody
titers reaches 2 or more in the absorbance level as the result of
immunization, the animals are left for about 1 month. Then,
re-immunization is carried out for at least once. Several days,
preferably 3 to 5 days, after the final immunization, splenic cells
and regional lymph nodes are removed. Blood samples are taken
regularly after immunization and subject to centrifugation to
separate sera. The resultant sera are then subject to measurement
of antibody titers by any suitable method, which includes, and is
not limited to, enzyme linked immunosorbent assay (ELISA), enzyme
immunoassay (EIA), or radio immunoassay (RIA). In one preferred
example, antibody titers are measured by ELISA. Then, final
immunization is given to those animals showing high antibody titers
to TDP-43 oligomer.
[0057] Antibody-producing cells are prepared from splenic cells and
regional lymph nodes or the like of the immunized animals. In the
preparation of antibody-producing cells, it is preferably to remove
tissue debris and erythrocytes as much as possible. Commercial
erythrocyte remover may be used to this purpose. Alternatively, a
buffer ammonium chloride and Tris may be prepared and used. The
thus prepared antibody-producing cells are immediately fused with
immortal cells such as myeloma cells to produce hybridoma cells,
which semi-eternally continue to proliferate while producing
antibodies. Commonly available cell strain derived from an animal
such as mouse may be used. A preferable cell strain to be used in
this invention should be those that fuse efficiently, support
stable high level production of antibody and are sensitive to HAT
selection medium, which contains hypoxanthine, thymidine and
aminopterin, and should survive there only when fused with
antibody-producing cells. Examples of myeloma cells include, but
are not limited to, mouse myeloma cell line (such as myeloma FO
cells) and human myeloma cell line (such as Karpas 707H).
[0058] Cell fusion is usually carried out by mixing splenic cells
or lymph node cells with a commercial available myeloma cells in
the presence of a cell-fusion promoter, such as PEG having an
average molecular weight from about 200 to 20,000 daltons or the
like. Alternatively, cell fusion may be carried out in a commercial
cell fusion device utilizing electric stimulation such as
electro-fusion. After the fusion, the resultant cells are then
diluted and cultured in HAT medium.
[0059] Hybridomas of interest are then selected from the fused
cells. The fused cells surviving cultured in HAT medium would form
colonies. The supernatant of each culture well is then collected
and examine for the presence or absence of antibody titers to
TDP-43 oligomer. As a method of confirmation, ELISA, EIA or RIA may
be used, in which TDP-43 oligomer or TDP-43 monomer is coated onto
the plates and used as a screening criteria. Once antibody-positive
wells are identified, cells are then cultured in a HT medium, which
does not contain aminopterin. After culturing for a while, antibody
titers in the culture supernatant are confirmed again. Cells that
are finally selected are then subject to cloning to obtain single
cells. Clones that exhibit high specificity to TDP-43 oligomer are
selected, and are proliferated to some extent to establish
hybridomas.
[0060] According to preferred embodiments of the present
disclosure, 5 hybridomas, TDP-O-3, TDP-O-5, TDP-O-8, TDP-O-9 and
TDP-O-10, were selected, and monoclonal antibodies may be isolated
or prepared by any known method. For example, antibodies may be
prepared from cultured supernatant obtained by culturing hybridomas
in a medium with low serum concentration. Alternatively, hybridomas
may be injected into abdominal cavities of animals and the
resultant abdominal dropsies are collected to prepare antibodies.
Antibodies may be purified or isolated by methods that employ
affinity column, gel filtration chromatography, ion exchange
chromatography or the like. Any of these known methods may be
appropriately selected or used in combination.
[0061] According to specific embodiments, the monoclonal antibodies
of the present disclosure are respectively produced by the
hybridoma cell line TDP-O-3, TDP-O-5, TDP-O-8, TDP-O-9 and
TDP-O-10, which are deposited in the Bioresource Collection and
Research Center (BCRC) of the Food Industry Development and
Research Institute (FIDRI) (HsinChu, Taiwan, R.O.C.) as accession
number of BCRC960494, BCRC960495, BCRC960496, BCRC960497, or
BCRC960498.
[0062] According to preferred embodiments, the monoclonal
antibodies disclosed herein comprise consensus sequences
respectively located at the heavy and light chain variable regions.
Accordingly, the monoclonal antibodies described herein
respectively comprise a heavy chain variable region comprising
amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO:
3; and a light chain variable region comprising amino acid
sequences of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.
Preferably, the humanized monoclonal antibody comprises a heavy
chain variable region of SEQ ID NO: 4, and a light chain variable
region of SEQ ID NO: 8. Among the amino acid sequences described in
the present disclosure, particularly SEQ ID NOs: 1 to 6, and 8, Xaa
represents any L- or D-form amino acid residues known in the art.
In one example, Xaa is an acidic amino acid residue (e.g.,
aspartate or glutamate). In another example, Xaa is a basic amino
acid residue (e.g., lysine, arginine, or histidine). In a further
example, Xaa is a nonpolar amino acid residue (e.g., alanine,
valine, leucine, isoleucine, proline, phenylalanine, methionine, or
tryptophan). In still another example, Xaa is an uncharged polar
amino acid residue (e.g., glycine, asparagine, glutamine, cysteine,
serine, threonine, or tyrosine).
[0063] Alternatively, anti-TDP-43 oligomer monoclonal antibodies
may be produced by DNA cloning or DNA synthesis. DNA encoding
anti-TDP-43 oligomer mAbs may be easily isolated and sequenced by
use of conventional procedures, such as using oligonucleotide
probes that are capable of binding specifically to genes encoding
the heavy and light chains of the monoclonal antibodies. The
hybridoma cells (e.g., TDP-O-3, TDP-O-5, TDP-O-8, TDP-O-9 or
TDP-O-10 hybridoma) serve as a preferred source of such DNA. Once
isolated, the DNA may be placed into expression vectors, which are
then transfected into host cells such as E. Coli cells, simian COS
cells or Chinese hamster ovary (CHO) cells or myeloma cells that do
not produce immunoglobulin proteins, to synthesize the desired
monoclonal antibodies in the recombinant host cells.
[0064] The monoclonal antibodies thus produced and the DNA encoding
such antibodies can then be used to produce chimeric antibodies
(e.g., bi-specific antibodies), humanized antibodies and/or
antibody fragments derived thereof.
[0065] The major concern of a non-human origin monoclonal antibody
is its immunogenicity to the recipient, in some cases, caused
dangerous allergic reactions. Most monoclonal antibodies are of
murine origin, and have been found to be immunogenic when injected
to human. To reduce the immunogenicity of anti-TDP-43 oligomer mAbs
of this invention, humanized antibodies are produced by attaching
variable domains in the heavy and light chains of murine
anti-TDP-43 oligomer Abs onto the constant regions of human
antibodies.
[0066] To create humanized anti-TDP-43 oligomer antibodies, the DNA
encoding such antibodies was isolated and sequenced, and then used
to create humanized constructs.
[0067] According to preferred embodiments of the present
disclosure, CDR (complementary determining region) grafting is
employed, in which the CDR regions in the VH and VL genes of a
human antibody are replaced with the appropriate CDR coding
segments (such as those DNA segments in anti-TDP-43 oligomer Abs
that code amino acid segments responsible for binding TDP-43
oligomer). The resulting antibodies therefore have variable regions
in which only the CDRs are from the original mouse antibodies,
while the framework regions in the VH and VL genes as well as the
constant region genes (i.e., CK or CH1-H--CH2-CH3) are those of
human IgG.
[0068] In preferred embodiments, the humanized anti-TDP-43 oligomer
mAb comprises a heavy chain variable domain and a light chain
variable domain. Once produced, the humanized anti-TDP-43 oligomer
mAbs may be purified according to standard procedures in the art,
including cross-flow filtration, affinity column chromatography,
gel filtration and the like. It should be understood that the
humanized antibodies shall perform in a manner identical or
substantially similar to that of murine anti-TDP-43 oligomer Abs.
Preferably, the humanized anti-TDP-43 oligomer Abs (either in the
form of Fab or full length IgG) shall be more advantages to use in
a human subject, as compared to the murine version. In some
embodiments, the humanized anti-TDP-43 oligomer Abs are used in the
production of bi-specific antibodies of the present disclosure.
[0069] Anti-TDP-43 oligomer antibodies of the present invention can
be characterized by use of any in vivo or in vitro models of TDP-43
proteinopathies. A skilled artisan readily understands that an
anti-TDP-43 oligomer antibody of the present invention can be
characterized in a mouse model, such as the animal model described
in Example 7. Alternatively, the anti-TDP-43 oligomer antibody of
the present invention can be characterized by human AD samples as
described in Example 8.
[0070] According to the in vitro data described in one example, the
anti-TDP-43 oligomer antibody of the present invention possesses an
inhibitory effect on the cell cytotoxicity induced by TDP-43
oligomers. According to the example, the concentration of the
present anti-TDP-43 oligomer antibody sufficient to inhibit the
TDP-43 oligomer-induced cytotoxicity is about 0.001-5.0 mg/ml; for
example, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008,
0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1,
0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0,
3.5, 4.0, 4.5, or 5.0 mg/ml. Preferably, the concentration is about
0.01-1.0 mg/ml. More preferably, the concentration is about
0.02-0.08 mg/ml.
[0071] A skilled artisan understands that an experimental model of
TDP-43 proteinopathy can be used in a preventive setting or in a
therapeutic setting. In a preventive setting, the dose of animals
starts prior to the onset of the TDP-43 proteinopathy or symptoms
thereof, and the Anti-TDP-43 oligomer antibody of the present
invention is evaluated for its ability to prevent, reduce or delay
the onset of TDP-43 proteinopathy or symptoms thereof. In a
therapeutic setting, the dose of animals starts after the onset of
the TDP-43 proteinopathy or symptoms thereof, and the Anti-TDP-43
oligomer antibody of the present invention is evaluated for its
ability to treat, reduce or alleviate the onset of TDP-43
proteinopathy or symptoms thereof. Symptoms of TDP-43 proteinopathy
include, but are not limited to, accumulation of pathological
TDP-43 deposits in the brain, spinal cord, cerebrospinal fluid or
serum of a test subject.
[0072] Accordingly, the disclosure provides a pharmaceutical
composition or a medicament for treating a neurodegenerative
disease associated with the aggregation of A.beta.. The composition
comprises an effective amount of the anti-TDP-43 oligomer antibody
of the present invention as described herein; and a
pharmaceutically acceptable excipient. The TDP-43 oligomer
associated disease treatable by the pharmaceutical composition or
the medicament of the present disclosure includes, but is not
limited to, Alzheimer's disease, argyrophilic grain disease,
amyotrophic lateral sclerosis (ALS), ALS-parkinsonism dementia
complex of Guam, vascular dementia, frontotemporal dementia,
semantic dementia, dementia with Lewy bodies, Huntington's disease,
Spinocerebellar ataxia, inclusion body myopathy, inclusion body
myositis, hippocampal sclerosis, or Parkinson's disease.
[0073] Generally, the anti-TDP-43 oligomer antibody of this
invention is present at a level of about 0.1% to 99% by weight,
based on the total weight of the pharmaceutical composition. In
some embodiments, the anti-TDP-43 oligomer antibody of this
invention is present at a level of at least 1% by weight, based on
the total weight of the pharmaceutical composition. In certain
embodiments, the anti-TDP-43 oligomer antibody is present at a
level of at least 5% by weight, based on the total weight of the
pharmaceutical composition. In still other embodiments, the
anti-TDP-43 oligomer antibody is present at a level of at least 10%
by weight, based on the total weight of the pharmaceutical
composition. In still yet other embodiments, the anti-TDP-43
oligomer antibody) is present at a level of at least 25% by weight,
based on the total weight of the pharmaceutical composition.
[0074] In some embodiments, the medicament of said pharmaceutical
composition of this invention further includes an agent that is
known to improve the symptoms of a neurodegenerative disease.
Examples of such agent include, and are not limited to, AChEI, an
A.beta. inhibitor, or a muscarinic receptor agonist, and the
like.
[0075] The medicament or said pharmaceutical composition is
prepared in accordance with acceptable pharmaceutical procedures,
such as described in Remington's Pharmaceutical Sciences, 17.sup.th
edition, ed. Alfonoso R. Gennaro, Mack Publishing Company, Easton,
Pa. (1985). Pharmaceutically acceptable excipients are those that
are compatible with other ingredients in the formulation and
biologically acceptable.
[0076] The anti-TDP-43 oligomer antibody of this invention may be
administered by any means known in the art, such as orally,
intraperitoneally, intracranially, intrathecally, intramuscularly,
intravenously, transdermally, rectally or by inhalation, alone or
in combination with conventional pharmaceutically acceptable
excipients. In one preferred embodiment, the anti-TDP-43 oligomer
antibody of this invention is administered intravenously to the
subject. In another preferred embodiment, the anti-TDP-43 oligomer
antibody of this invention is administered intrathecally to the
subject.
[0077] Applicable solid excipients may include one or more
substances that may also act as flavoring agents, lubricants,
solubilizers, suspending agents, fillers, glidants, compression
aids, binders or tablet-disintegrating agents or an encapsulating
material. In powders, the excipient is a finely divided solid that
is in admixture with the finely divided active ingredient. In
tablets, the active ingredient is mixed with an excipient having
the necessary compression properties in suitable proportions and
compacted in the shape and size desired. The powders and tablets
preferably contain up to 99% of the active ingredient. Suitable
solid excipient includes, for example, calcium phosphate, magnesium
stearate, talc, sugars, lactose, dextrin, starch, gelatin,
cellulose, methyl cellulose, sodium carboxymethyl cellulose,
polyvinylpyrrolidine and the like.
[0078] The anti-TDP-43 oligomer antibody of the present invention
may also be formulated into liquid pharmaceutical compositions,
which are sterile solutions or suspensions that can be administered
by, for example, intravenous, intramuscular, subcutaneous,
intrathecal, intraperitoneal, or intra-cerebella injection. Oral
administration may be either liquid or solid composition form.
[0079] The medicament or said pharmaceutical compositions of this
invention may also be formulated in a variety of dosage forms for
mucosal application, such as buccal and/or sublingual drug dosage
units for drug delivery through oral mucosal membranes. A wide
variety of biodegradable polymeric excipients may be used that are
pharmaceutically acceptable, provide both a suitable degree of
adhesion and the desired drug release profile, and are compatible
with the active agents to be administered and any other components
that may be present in the buccal and/or sublingual drug dosage
units. Generally, the polymeric excipient comprises hydrophilic
polymers that adhere to the wet surface of the oral mucosa.
Examples of polymeric excipients include, but are not limited to,
acrylic acid polymers and copolymers; hydrolyzed polyvinylalcohol;
polyethylene oxides; polyacrylates; vinyl polymers and copolymers;
polyvinylpyrrolidone; dextran; guar gum; pectins; starches; and
cellulosic polymers.
[0080] Accordingly, this invention also provides methods of
treating mammals, preferably humans, of a TDP-43 oligomer
associated disease, which comprises the administration of the
medicament or said pharmaceutical composition of this invention
that contains anti-TDP-43 oligomer antibody as described herein.
Such medicament or composition is administered to a mammal,
preferably human, by any route that may effectively transports the
active ingredient(s) of the composition to the appropriate or
desired site of action, such as oral, nasal, pulmonary,
transdermal, such as passive or iontophoretic delivery, or
parenteral, e.g., rectal, depot, subcutaneous, intravenous,
intrathecal, intramuscular, intranasal, intra-cerebella, ophthalmic
solution or an ointment. Further, the administration of the
compound of this invention with other active ingredients may be
concurrent or simultaneous.
[0081] In some embodiments, the effective dose administered to the
subject is from about 1 to 100 mg/Kg body weight of the subject,
such as about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mg/Kg body
weight of the subject, preferably about 50 to 70 mg/Kg body weight
of the subject, such as 50, 60 or 70 mg/Kg body weight of the
subject; most preferably about 50 mg/Kg body weight of the subject.
The dose can be administered in a single aliquot, or alternatively
in more than one aliquot.
[0082] According to optional embodiments of the present disclosure,
the method may further include the step of, administering to the
subject an acetylcholinesterase inhibitor (AChEI), an A.beta.
inhibitor, or a muscarinic receptor agonist, either simultaneously
or sequentially with the anti-TDP-43 oligomer antibody as described
above.
[0083] In some embodiments, the AChEI is any of alantamine,
cymserine, donepezil, ER 127528, galantamine, ganstigmine,
huperzine A, phenserine, phenethylnorcymserine, rivastigmine, RS
1259, SPH 1371, tacrine, thiacymserine, or zanapezil. In other
embodiments, the A.beta. inhibitor is any of bapineuzumab, PTB2,
scyllo-inositol, PPI 1019, RS 0406, SP 233, EGCG, Exberyl-1, or SEN
606. The muscarinic receptor agonist is oxotremorine or
xanomeline.
[0084] The anti-TDP-43 oligomer antibody of the present invention
may also be used as a tool for the detection or diagnosis of a
TDP-43 oligomer associated disease in a subject. Accordingly, this
invention provides a method for detecting or diagnosing a subject
having or suspected of having a TDP-43 oligomer associated disease.
The method includes steps of, obtaining a biological sample from
the subject; determining the amount of the TDP-43 oligomer in the
biological sample by contacting the biological sample with an
effective amount of the present antibody; and comparing the
detected amount of the TDP-43 oligomer in the biological sample
with that of a control sample obtained from a healthy subject;
wherein a significantly higher or lower amount of the detected
TDP-43 oligomer in the biological sample than that of the control
sample indicates that the subject suffers from the TDP-43 oligomer
associated disease.
[0085] The biological sample described herein includes, but is not
limited to, a brain biopsy sample, a cerebrospinal fluid sample, a
whole blood sample, a serum sample, a plasma sample, a urine
sample, a mucus sample and purified or filtered forms thereof.
[0086] Antibody binding may be detected by techniques known in the
art, such as radioimmunoassay, enzyme-linked immunosorbent assay
(ELISA), "sandwich" immunoassay, in situ immunoassays (e.g., using
colloidal gold, enzyme or radioisotope labels), western blot,
agglutination assay (e.g., gel agglutination assay,
hemagglutination assay and etc), complement fixation assay,
immunofluorescence assay, and immunoelectrophoresis assay and etc.
In one embodiment, antibody binding is detected by use of ELISA. In
some embodiments, autoantibodies are detected in bodily fluids,
including but are not limited to cerebrospinal fluid, whole blood,
serum, plasma, mucus, and purified or filtered forms thereof. In
one preferred example, antibodies were detected from a
cerebrospinal fluid sample. In other embodiments, antibodies are
detected from a brain biopsy sample.
[0087] To provide those skilled in the art tools to use the present
invention, the anti-TDP-43 oligomer antibody of the invention is
assembled into kits for the diagnosis, detection or confirmation of
a neurodegenerative disease. In preferred embodiments, the presence
of pathologic forms of TDP-43 reactive to the anti-TDP-43 oligomer
antibody of this invention is used to provide prognosis to a
subject. For example, the detection of a significant different
level of pathologic forms of TDP-43 reactive to the anti-TDP-43
oligomer antibody of this invention, as compared to controls
(derived from a healthy subject), in a biological sample is
indicative of occurrence of the TDP-43 oligomer associated disease.
The information provided is also used to direct the course of
treatment. For example, if a subject is found to have pathologic
forms of TDP-43, therapies for the treatment of the TDP-43 oligomer
associated disease, such as AD, ALS and PD, may be started at an
earlier time when they are more likely to be effective.
[0088] In one embodiment, the present invention provides a kit for
the diagnosis of a TDP-43 oligomer associated disease by use of the
anti-TDP-43 oligomer antibody of this invention. The components
included in the kits are: a container, reagents for detecting
TDP-43 oligomer in a biological sample, wherein the reagents
comprise the anti-TDP-43 oligomer antibody of this invention
prepared in accordance with the procedure described in one example
of this invention; and a legend associated with the container and
indicating how to use the anti-TDP-43 oligomer antibody of this
invention for detecting the pathological form of TDP-43 (or TDP-43
oligomer as described herein) in the biological sample. The legend
may be in a form of pamphlet, tape, CD, VCD or DVD. The kit may
further comprise a negative control that indicates the normal level
of the TDP-43 oligomer that forms a complex with the anti-TDP-43
oligomer antibody in a healthy subject.
[0089] The present invention will now be described more
specifically with reference to the following embodiments, which are
provided for the purpose of demonstration rather than
limitation.
EXAMPLES
[0090] Materials and Methods.
[0091] Materials.
[0092] The isolated human TDP-43 from HEK cells was a gift from
OriGene Technologies, Inc. (Rockville, Md., USA). Based on the
product description, TDP-43 was obtained from transiently
transfected human HEK 293 cells with a TrueORF clone, RC210639. The
recombinant TDP-43 possessed a C-terminal Myc-DDK tag. The
overexpressed recombinant TDP-43 protein was purified using an
anti-DDK affinity column. The short-form of TDP-43 (residues
101-285) is prepared in accordance with procedures described by Kuo
et al (Nucleic Acids Res (2009) 37, 1799-1808). Anti-N-terminal
residues 1-260 TDP-43 antibody (denoted as N.sub.1-260) for dot
blotting and anti-TDP-43 antibody for immunohistochemistry were
both purchased from Abcam (Cambridge, UK). Anti-C-terminal residues
350-414 TDP-43 antibody (denoted as C.sub.350-414) was from Novus
(Littleton, Colo., USA) and anti-amyloid oligomer antibody A11 was
from BioSource (Invitrogen, Carlsbad, Calif., USA). Anti-DDK
monoclonal antibody was from Origene Technologies, Inc. A.beta.
peptide was synthesized by peptide synthesis facility in the
Genomics Research Center, Academia Sinica. Other chemicals were
purchased either from Sigma Aldrich (St. Louis, Mo., USA) or
Amresco (Solon, Ohio, USA). All cell culturing reagents were
purchased from Gibco (Invitrogen) except for further
indication.
[0093] Purification of TDP-43.
[0094] The N-terminal His-Tag TDP-43 was cloned from the plasmid
pCMV-Taq2B containing the cDNA encoding full-length TDP-43. The
amplified product was double digested with XhoI/BamHI and subcloned
into pET14b vector (Novagen, Merck KGaA, Darmstadt, Germany) to
generate an N-terminal His-tag. The N-terminal His-tagged TDP-43
was transformed and overexpressed in E. coli strain Rosetta 2.
(Novagen, Merck KGaA, Darmstadt, Germany). The cells were harvested
and lysed by a microfluidizer on ice in 30 mM Tris-HCl buffer, pH
8, containing 500 mM NaCl, 10% glycerol, 1 mM DTT, 2% RNase A, 2%
DNase I, and protease inhibitor cocktail (Complete, EDTA-free,
Roche Applied Science, Mannheim, Germany). The lysate was
centrifuged at 27,000.times.g, 4.degree. C. The supernatant was
loaded onto Ni-NTA affinity column (GE healthcare Bio-Sciences AB,
Uppsala, Sweden) equilibrated in a buffer containing 30 mM Tris, pH
8, 500 mM NaCl, 1 mM DTT, 20 mM imidazole, and 10% glycerol.
Imidazole step gradients in the same running buffer were performed.
TDP-43 was eluted at approximately 200 mM imidazole. Purified
his-tagged TDP-43 protein was run on SDS-PAGE and identified by
Coomassie blue staining. The recombinant TDP-43 contained extra
N-terminal residues MGSSHHHHHHSSGLVPRGSHMLE. The calculated
molecular mass is 47,147 Da. The protein was further dialyzed as
indicated. Protein concentration was quantified after background
subtraction by absorption at 280 nm with the extinction coefficient
of 44,920 cm.sup.-1M.sup.-1 according to the equation described by
Nick Pace (Pace et al., (1995) Protein Sci 4, 2411-2423). For the
short-form of TDP-43, the extinction coefficient of 15,470
cm.sup.-1M.sup.-1 was used.
[0095] Size Exclusion Chromatography (SEC).
[0096] Superdex-200 10/300 GL analytical gel-filtration column (GE
healthcare Bio-Sciences AB, Uppsala, Sweden) was standardized by
molecular weight markers, ferritin (440 kDa), .beta.-amylase (200
kDa), bovine serum albumin (66 kDa), cytochrome C (12.4 kDa) in the
running buffer containing 30 mM Tris, pH 7.4, 150 mM NaCl. The flow
rate was 0.5 ml/min. A volume of 300 .mu.l of the recombinant
TDP-43 from E. coli filtered by 0.2 .mu.m filter membrane was
injected into the Superdex-200 column. One ml fractions were
collected automatically by a fraction collector. The preload sample
and the oligomer fraction were collected and subjected to dot
blotting probed by A11 (1:1000) with different exposure times due
to much diluted signal from the oligomer fraction. The oligomer
fraction was characterized with dynamic light scattering. A volume
of 100 .mu.l of TDP-43 obtained from HEK cells at 2 .mu.M were also
examined with the same procedure. The fractions were analyzed by
slot blotting.
[0097] Slot Blotting.
[0098] Fractions of the size-exclusion chromatography of TDP-43
isolated from HEK293 cells were examined by slot blotting because
of their low concentration. A 200 .mu.l aliquot of every 1 ml
fraction was loaded onto a Bio-Dot SF microfiltration apparatus
(Bio-Rad, Hercules, Calif., USA) equipped with an in-house vacuum
system. Anti-DDK monoclonal antibody (OriGene Technologies, Inc.,
Rockville, Md., USA) was used as primary antibody for the
detection. Intensity was quantified by Image J 1.42 (National
Institutes of Health, MD, USA).
[0099] Dot Blotting.
[0100] Purified TDP-43 was diluted 10 times into 10 mM Tris-HCl
buffer, pH 8, with or without denaturants. Each sample in different
conditions contains either no denaturant, 9 M urea, 7.2 M GdnHCI,
or 2% SDS. The final TDP-43 concentration was 0.4 .mu.M. The
samples were either incubated for 1 hr at the room temperature or
90.degree. C. The TDP-43 samples, 2 .mu.l, were dotted onto
nitrocellulose membranes and were subjected to dot blotting.
Briefly, after blocking and washing with Tris-buffered saline
containing 0.002% Tween 20 (TBST), the membranes were subjected to
anti-N-terminal residues 1-260 TDP-43 antibody (1:2,000),
Anti-C-terminal residues 350-414 TDP-43 antibody (1:2,000), and
anti-amyloid oligomer antibody, A11, (1:1,000) in 5% milk with TBST
followed by the corresponding horseradish peroxidase-conjugated
secondary antibodies anti-rabbit or anti-mouse IgG (1:5,000;
Millipore, Billerica, Mass., USA). The membranes were developed
with ECL chemiluminescence reagent (Millipore).
[0101] Transmission Electron Microscopy.
[0102] Freshly purified TDP-43 was dialyzed into a buffer
containing 10 mM Tris, pH 8, at 4.degree. C. overnight. The sample
was centrifuged at 17,000.times.g, 4.degree. C., for 30 min to
remove precipitates and the supernatant was quantified and
subjected to TEM imaging. The TDP-43 samples were placed on
glow-discharged, 400-mesh Formvar carbon-coated copper grids (EMS
Inc., Hatfield, Pa., USA) for 5 min, rinsed, and negatively stained
with 2% uranyl acetate. The samples were examined with Tecnai G2
Spirit TWIN TEM (FEI, Hillsboro, Oreg., USA) or Hitachi H-7000 TEM
(Hitachi Inc., Japan) with an accelerating voltage of 75 kV.
[0103] Atomic Force Microscopy.
[0104] Freshly purified TDP-43 was dialyzed into a buffer
containing 10 mM Tris, pH 8, at 4.degree. C. overnight. The sample
was centrifuged at 17,000.times.g, 4.degree. C., for 30 min to
remove precipitates and the supernatant was quantified and
subjected to AFM imaging. A volume of 10 .mu.L TDP-43 was dropped
onto freshly sliced mica (Ted Pella, Redding, Calif., U.S.A.) and
incubated for 5 min for sample adhesion. The sample was washed by 1
ml ddH.sub.2O and gently removed from the top of the tilted mica.
The sample was left to dry in the room temperature and subjected to
AFM imaging (Nanonics, Jerusalem, Israel) using tapping mode. AFM
tips, PPP-ZEILR (Nanosensors, Neuchatel, Switzerland) with a spring
constant of 1.6 N/m and the tip radius <10 nm were employed for
the experiment.
[0105] Dynamic Light Scattering.
[0106] The eluted oligomer fraction from SEC was used for dynamic
light scattering. The sample was in a buffer containing 30 mM Tris,
pH 7.4, and 150 mM NaCl. Data were obtained with a Zetasizer Nano
ZS dynamic light scattering instrument (Malvern Instruments,
Worcestershire, UK) equipped with 50 mW later fiber. Appropriate
parameters of viscosity and refractive index were set for each
solution and the temperature was kept at 25.degree. C.
[0107] Circular Dichroism.
[0108] Freshly purified TDP-43 was dialyzed into a buffer
containing 10 mM Tris, pH 8, at 4.degree. C. overnight. The sample
was centrifuged at 17,000.times.g, 4.degree. C., for 30 min to
remove precipitates and the supernatant was quantified and
subjected to CD measurement. Far-UV CD spectra were measured in a
circular quartz cell (Hellma, Forest Hills, N.Y., USA) by Jasco
J-815 spectropolarimeter (Jasco Inc., Easton, Md., USA) with 1 mm
path length at room temperature. The spectra were collected from
250 to 190 nm and corrected with buffer background.
[0109] Intrinsic and Bis-ANS Fluorescence Spectroscopy.
[0110] Freshly purified TDP-43 was dialyzed into a buffer
containing 10 mM Tris, pH 8, at 4.degree. C. overnight. The sample
was centrifuged at 17,000.times.g, 4.degree. C., for 30 min to
remove precipitates and the supernatant was quantified. The
intrinsic fluorescence of TDP-43, at 1.5 .mu.M, was collected from
305 to 400 nm while excitation at 280 or 295 nm. The Bis-ANS
spectra of TDP-43, TDP-43s, and buffer control were collected from
450 to 600 nm with excitation at 400 nm. The samples contain 0.8
.mu.M TDP-43 and 5 .mu.M Bis-ANS. All experiments were done at
25.degree. C. with a circulating water bath using FluoroMax-3
spectrofluorometer (Horiba Jobin Yvon, Kyoto, Japan).
[0111] Thioflavin T Binding.
[0112] Freshly purified TDP-43 was dialyzed into a buffer
containing 10 mM Tris, pH 8, at 4.degree. C. overnight. The sample
was centrifuged at 17,000.times.g, at 4.degree. C., for 30 min to
remove precipitates and the supernatant was quantified. The TDP-43
sample and A.beta.40 fibrils were used to detect thioflavin T
binding. The samples, 1 .mu.M, were mixed with equimolar thioflavin
T and excited at 442 nm, and the emission spectra from 455 to 505
nm were collected. A.beta.40 fibril stock, 25 .mu.M, was prepared
as previously described (Chen and Glabe, J. Biol. Chem. (2006) 281,
24414-24422). Briefly, synthetic A.beta. was dissolved in 6 M
GdnHCI and refolded to 10 mM phosphate buffer, pH 7.4. Then
A.beta., 25 .mu.M, was incubated at 25.degree. C. in quiescence for
more than 10 days. The mature fibrils were then diluted to 1 .mu.M
prior to the experiments. The spectra were subtracted from the
buffer background.
[0113] Congo Red Spectroscopy.
[0114] Absorbance of dialyzed TDP-43 and mature A.beta. fibrils,
0.5 .mu.M, was measured from 400 nm to 600 nm in the presence of f
10 .mu.M Congo Red by a UV/vis spectrophotometer DU800 (Beckman
Coulter, CA).
[0115] Dot Blotting by OC Antibody.
[0116] The dialyzed TDP-43 and A.beta. fibrils (2 .mu.l, 0.5 .mu.M)
were dotted onto nitrocellulose membranes and standard dot blot
procedure was performed. OC antibody (Millipore, 1:10,000) and
HRP-conjugated anti-rabbit IgG (1:10,000) were employed.
[0117] Fluorescence Titration for DNA Binding.
[0118] Fluorescence titration was employed to monitor protein
conformational changes upon DNA binding. Freshly purified and
dialyzed full-length TDP-43 and short-form of TDP-43 (residues
101-285), at 1.5 .mu.M, in 30 mM Tris-HCl, pH 8, were titrated with
single strand TAR DNAs including TAR DNA A-site (SEQ ID NO: 9) and
B-site (SEQ ID NO: 10). The intrinsic protein fluorescence was
excited at 280 nm to monitor the conformational change of TDP-43.
The emission spectra were collected by FluoroMax-3
spectrofluorometer (Horiba Jobin Yvon, NJ, USA). The emission
maxima at 350 nm for full-length or short-form TDP-43 were
collected, subtracted with the DNA controls, and corrected with the
dilution factor. Then, the data were normalized to the starting
intensity and further fitted to a single protein and ligand binding
equation (Chen et al., Protein Sci (2004) 13, 2196-2206):
P+LPL
r=(2Pt).sup.-1*[(Kd+Lt+Pt)-((Kd+Lt+Pt).sup.2-4PtLt).sup.1/2]
where .gamma. is the fraction of the observed signal changes
representing the bound protein fraction, P.sub.t is the total
TDP-43 concentration, L.sub.t is the total ligand concentration,
and Kd is the dissociation constant. The data and fits are plotted
against the ratio of DNA and TDP-43.
[0119] Oligomer Cross-Seeding and ThT Assay.
[0120] A.beta. was prepared following our previous protocol (Chen
et al., J. Biol. Chem. (2011) 286, 9646-9656; Ni et al., FASEB
(2011) 25, 1390-1401). A.beta.40 was dissolved in buffer A (10 mM
sodium phosphate, pH 7.4) containing 8 M GdnHCI, refolded, and
quantified using absorption at 280 nm (.epsilon.=1,280
cm.sup.-1M.sup.-1). The experimental samples containing 25 .mu.M
A.beta., 50 .mu.M ThT, and different concentrations of freshly
dialyzed TDP-43 ranging from 0 to 1 .mu.M (0-4%) were prepared in
10 mM Tris-HCl, pH 8, and 150 mM NaCl. The samples were then
incubated in a 96-well ELISA plate in quiescence, sealed with a
transparent film, and monitored by a microplate reader (SpectraMax
M5, Molecule Devices) at 25.degree. C. at various times. ThT
emission was measured at 485 nm while excitation was at 442 nm.
[0121] Photo-Induced Cross-Linking (PICUP).
[0122] The experiment was performed as described previously (Bitan
et al., J. Biol. Chem. (2001) 276, 35176-35184). Briefly, the
A.beta. stock was prepared by use of a buffer containing 8 M urea
and 10 mM sodium phosphate, pH 7.4, to facilitate running on
SDS-PAGE. A.beta. samples at 25 .mu.M were prepared in a buffer
containing 10 mM Tris-HCl, pH 8, and 150 mM NaCl with or without
TDP-43 at different concentrations as indicated. The samples were
immediately subjected to PICUP assay. A 90% A.beta. solution was
mixed with 5% each of 3 mM Ru(Bpy) and 20 mM APS. After mixing,
samples were exposed to a blue-light LED in a closed chamber with a
manual switch for 30 s. The cross-linking reaction was stopped by
adding the SDS-PAGE sample buffer, and the samples were run on 16%
Tris-tricine SDS-PAGE. All actions were performed without delay.
The gel was subjected to Western blotting with anti-A.beta.
antibody 6E10 (Chemicon Inc., Billerica, Mass.) recognizing A.beta.
residues 1-17 and anti-N-terminal TDP-43 1-260 antibody.
[0123] Cytotoxicity of TDP-43 Using Human Neuroblastoma.
[0124] To perform cytotoxicity of TDP-43, MTT
(3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) and
lactate dehydrogenase (LDH) (Promega, Madison, Wis., United States)
assays were first employed using human neuroblastoma BE(2)-C cells
(ATCC number CRL-2268). The cells were cultured in RPMI growth
medium (Gibco) with 10% fetal bovine serum (FBS; Biological
Industry) at 37.degree. C. under 5% CO.sub.2 and humidified
atmosphere. A total of 60,000 cells per well was seeded in
transparent 96-well ELISA plate (Corning, N.Y., USA) and incubated
overnight. The cells were then washed and replaced by 40 .mu.l of
serum-free RPMI media followed by addition of 10 .mu.l serially
diluted TDP-43 samples with the dialysis buffer. The TDP-43 samples
were freshly dialyzed and centrifuged. The supernatant was employed
and the dialysis buffer served as a buffer control. The cells were
further incubated for 24 hr, and MTT assay was performed following
the standard protocol. In brief, a volume of 7 .mu.l of 5 mg/ml MTT
was added in each well and cells were incubated for 3 hr. Then, the
media were discarded and DMSO was added to lyze the cells until the
purple formazan crystals were fully dissolved. Absorbance at 570
and 690 nm were measured by an ELISA plate reader (SpectraMax M5,
Molecule Devices, USA) and the signals at 570 nm were subtracted
from that of 690 nm. The data obtained from five replicas were
averaged and corrected with the sample background without cells.
The cell viability was shown after normalization of the data using
the cell treated with buffer as 100%. LDH assay was performed
following the manufacturer's protocol. Briefly, a number of 20,000
cells was seeded in the growth medium and incubated for 24 hr. The
samples were treated as described in MTT assay. LDH substrate was
mixed with the assay buffer and kept at room temperature. The cells
were cooled to room temperature for 30 min before substrate
addition. After 1 hr of dark incubation, the substrate fluorescence
was excited at 560 nm and emission at 590 nm was measured by the
ELISA plate reader. The data were obtained from three replicas,
averaged, and corrected by the sample background without cells. The
p-values of MTT and LDH assays were calculated using unpaired
Student's t-test.
[0125] Cytotoxicity of TDP-43 Using Mouse Primary Cortical
Neurons.
[0126] The pregnant C57BL/6JNarl mice were purchased from National
Laboratory Animal Center (NLAC, Taiwan) with the approval of the
Institutional Animal Care and Use Committee (IACUC) at National
Yang Ming University, Taipei, Taiwan. Primary cortical neuronal
cultures, were generated from embryonic day 19 mice and were seeded
in 96-well plates (2.times.10.sup.5 cells/well) with neurobasal
medium (21103049, GIBCO, USA) containing 2 .mu.M FdU. After growing
8 days in vitro, primary neuronal culture were treated with freshly
dialyzed soluble TDP-43 for 24 hr, and then incubated with 0.5
mg/ml MTT for 3 h. The dialysis buffer was served as buffer
control. MTT formazan crystals were then dissolved by equal volume
of lysis buffer containing 10% SDS and 20 mM HCl overnight. The
absorbance at wavelength 570 nm in each well was measured on an
ELISA plate reader (TECAN, Switzerland). The data were obtained
from three replicas, averaged, and corrected by the sample
background without cells. The p-values for MTT assay were
calculated using unpaired Student's t-test.
[0127] Immunocytochemistry.
[0128] Primary neuron cells were cultured on 24-well plates with 12
mm glass coverslips (3.times.10.sup.5 cells/well), and each well
was coated with poly-D-lysine. Cells were treated with TDP-43 (0.6
.mu.M) or buffer control for 24 hr at 37.degree. C. in a humidified
atmosphere with 5% CO2, and were fixed by 4% paraformaldehyde for
20 min at room temperature. Then, the cells were washed with PBS 10
min for 3 times, and blocked with blocking buffer (PBS containing
0.3% triton x-100 and 10% FBS) for 1 hr at room temperature. After
blocking, cells were first hybridized by the primary antibodies of
microtubule associated protein-2 (MAP-2) (MAB378, Millipore, USA)
and polyclonal rabbit anti-glial fibrillary acidic protein (GFAP)
(Z0334, DAKO, Glostrup, Denmark) for 2 hr at room temperature and
then hybridized by the fluorescent dye conjugated secondary
antibodies of Texas-Red-conjugated goat anti-mouse IgG (AbD
Serotec, UK) or FITC-conjugated goat anti-rabbit IgG (Millipore,
USA) for 1 hr at room temperature. Finally, cells were mounted by
mounting gel containing DAPI (VECTASHIELD.RTM.+DAPI, H-1200, Vector
Lab.) and observed by Olympus fluorescence microscope (BX-61,
Olympus).
[0129] Intrahippocampal Injection of TDP-43.
[0130] Two-month-old male C57/BL6J mice were applied to
intrahippocampal injection that purchased and bred in animal center
of National Cheng Kung University (NCKU) (Tainan, Taiwan) following
the guidelines of IACUC of NCKU. Animals were anesthetized with
isoflurane inhalation (1.2% in oxygen) via a nose tube. Each animal
received bilateral intrahippocamal injection of recombinant
full-length TDP-43. The stereotaxic coordinates were in relation to
bregma as follows: anteroposterior (AP), -2 mm; mediolateral (ML),
.+-.1 mm; dorsoventral (DV), -2 mm. The injection needle slowly
approached to the desired depth and 2 .mu.l TDP-43, 2.2 .mu.M, was
injected using a stainless-steel syringe needle (33-gauge)
connected to a microsyringe (Hamilton Company, NV, USA) at an
injection rate of 1 .mu.l/min. The needle was left in place for an
additional 5 min to limit the diffusion of the injected TDP-43.
[0131] Immunofluorescent Staining of the Mouse Brains.
[0132] Twelve-month-old male wild type mice, and 6- and
12-month-old TDP-43 Tg.sup.+/+ transgenic mice were adopt for
immunofluorescent staining. All mice were also cared in animal
center of NCKU following the guidelines of IACUC of NCKU. Two weeks
after TDP-43 injection, the adult mice were deeply anesthetized and
perfused transcardially with 4% paraformaldehyde (PFA)/0.01M
phosphate buffered saline (PBS), pH 7.4. The brain was removed and
then stored in 30% sucrose/4% PFA solution overnight.
10-.mu.m-thick sections were treated with a blocking solution
containing 0.2% Triton X-100 and 5% normal donkey serum in 0.01 M
PBS at room temperature for 1 h. For the experiments of
intrahippocampal injection, the sections were incubated in mouse
monoclonal anti-NeuN (1:300, Millipore, Temecula, Calif.) at
4.degree. C. overnight and Alexa Fluor 555-conjugated donkey
anti-mouse antibody (1:300, Chemicon, Temecula, Calif.) at room
temperature for 1 h. The sections were then counterstained with
DAPI and mounted with fluorescent mounting medium (DAKO, Glostrup,
Denmark). All sections were examined under an upright fluorescent
microscope (BX51, Olympus, Tokyo, Japan). For the colocalization
study in transgenic mice, the sections were incubated with
anti-TDP-43 antibody (1:1000, Abcam, ab104223), TDPO (1:1000, LTK
BioLaboratories, Taiwan), Alexa Fluor 488-conjugated goat
anti-rabbit antibodies and the Alex Fluor 555-conjugated donkey
anti-mouse antibodies (1:300, Invitrogen). The sections were then
incubated with DAPI and coverslipped with fluorescent mounting
medium (fluorescent mounting medium; Dako). All sections were
examined in a laser scanning confocal microscope (Nikon
TE2000EPFS-C1-Si).
[0133] TDP-43 Oligomer-Specific Polyclonal Antibody Production and
Characterization.
[0134] The purified full-length TDP-43 was dialyzed into 10 mM
Tris-HCl, pH 8.0, at 4.degree. C., and concentrated to
approximately 0.2 mg/ml to serve as immunogen. New Zealand white
rabbits were immunized with the immunogen following the standard
protocol for polyclonal antibody production (LTK BioLaboratories,
Taiwan). Briefly, the immunogen was injected 0.5 ml into the rabbit
at 2-week intervals. After 6 injections, the rabbit was sacrificed,
and the blood serum was obtained for usage. For characterization of
TDP-O by dot blotting and ELISA, full-length TDP-43 was subjected
to SEC (Superdex-200 10/300 GL, GE healthcare Bio-Sciences AB,
Uppsala, Sweden) in a buffer containing 30 mM Tris-HCl, pH 8.0, and
150 mM NaCl with a flow rate of 0.3 ml/min. One ml fractions were
collected and subjected to dot blotting probed by TDP-O and
N.sub.1-260 antibodies. The TDP-43 oligomer and monomer fractions
were further quantified by Micro BCA.TM. Protein Assay Kit (Thermo
Scientific, Rockford, Ill.) and coated onto ELISA plate with serial
dilution. ELISA following the standard protocol was performed.
Briefly, the coated samples were incubated overnight at 4.degree.
C., then blocked for 2 hr at room temperature with 10% skim milk in
TBST. The plates were washed and probed by TDP-O (1:12,500 in 3%
skim milk in TBST) or anti-N-terminal residues 1-260 TDP-43
antibody (1:1,000 dilution in 3% skim milk in TBST) for 2 hr at
room temperature. After washing, the plates were subjected to
anti-rabbit horseradish peroxidase-conjugated secondary antibodies
(1:1,000; Merck Millipore, Billerica, Mass., USA) for 2 hr at room
temperature, washed, then developed by 100 .mu.l
3,3,5,5-tetramethylbenzidine (TMB, Merck Millipore, Billerica,
Mass., USA). The reaction was stopped with addition of 100 .mu.l of
250 mM HCl and the absorbance was read at 450 nm by SpectraMax M5
(Molecular Device, Sunnyvale, Calif.).
[0135] Generation of .beta.5 Fibrils.
[0136] .beta.5 peptide within RRM2 domain of TDP-43 fragment was
chemically synthesized (MDBio, Inc., USA). The peptide was
solubilized by acetonitrile and diluted 10 times with PBS buffer
(pH 7.4) at 5 mg/ml. The supernatant was collected after
centrifugation at 17,000.times.g for 30 min and filtered through a
0.2 .mu.m filter membrane (Pall, USA). Then, the sample was
incubated in quiescence at room temperature for 7 days. .beta.5
fibrils were formed and the morphology was observed by TEM. The
sample was subjected to dot blotting probed by TDP-O antibody
following the aforementioned procedure.
[0137] TDP-O Staining of the Brain Tissues of FTLD-TDP
Patients.
[0138] Paraffin-embedded tissue sections were de-waxed with three
washes of Xylene. Tissues were then hydrated through an alcohol
gradient, followed by a five-min wash in 1.times.TBS. Antigen
recovery was performed using 10 mM sodium citrate (pH 6) by
microwaving for 15 min at full power (1,500 watts). Endogenous
peroxidase activity was blocked by incubating the tissue in 3%
hydrogen peroxide in 1.times.TBS for 20 min, followed by a five-min
wash in 1.times.TBS. Non-specific binding was blocked by incubating
the tissues in 10% normal goat serum (Invitrogen) for 1 hr at
ambient temperature. Primary antibody was diluted at 1:1000 in
antibody buffer (1.times.TBS supplemented with 5% bovine serum
albumin and 0.5% Tween-20) and applied to the tissues overnight at
4.degree. C. Slides were washed three times in wash buffer
(1.times.TBS/0.5% Tween-20). Biotinylated secondary antibodies were
applied at 1:500 dilution in antibody buffer for 1 hr at ambient
temperature. Tissue was washed three times in wash buffer, then
incubated with Vectastain ABC peroxidase reagent (Vector Labs,
PK-6100) for 30 minutes, followed by one wash in 1.times.TBS. The
tissues were then exposed to DAB peroxidase substrate reagent
(Vector Labs, SK-4100) for 2-10 min to achieve optimal staining.
Tissues were counterstained with Mayers Hematoxylin Solution (Sigma
Aldrich), and then dehydrated through an alcohol gradient followed
by a xylene wash. Tissue slides were mounted using Permount (Sigma
Aldrich). Post-mortem brain samples from three control, three
FTLD-TDP, and three AD patients were provided by the Alzheimer's
Disease Center at University of California Davis. The study was
approved by the Institutional Review Board. Informed consent to
share research tissue after death was obtained from all
patients.
[0139] IP and Immunolabeling of TDP-43 Oligomers in FTLD-TDP
Brain.
[0140] The frozen brain tissues of two control and one FTLD-TDP
patient were lysed at .about.0.05 g/ml with a lysis buffer
containing 50 mM Tris, pH 7.4, 150 mM NaCl, 0.5% Triton X-100, and
protease inhibitor cocktail (Calbiochem, Merck Millipore). The
lysed samples were homogenized on ice by a tissue grinder (Wheaton,
N.J., USA). After homogenization, the soluble fractions were
collected after centrifugation at 17,000.times.g for 10 min at
4.degree. C. and saved for IP analysis. Prior to tissue
fractioning, crossed-linked TDP-O antibodies on the IP beads were
prepared following the manufacturer's procedure. Briefly, a total
of 40 .mu.g of TDP-O antibody was incubated with 10 .mu.l each of
protein A and G Mag Sepharose.TM. Xtra (GE healthcare) in the
binding buffer (50 mM Tris pH 7.5, 150 mM NaCl) at room temperature
for 1 hr. After antibody binding on the beads, crosslinking was
performed by crosslink reagent (50 mM dimethyl pimelimidate
dihydrochloride and 200 mM triethanolamine, pH 8.9) at RT for 1 hr
and the reaction was stopped by the addition of 100 mM
ethanolamine, pH 8.9, at RT for 30 min. Next, the soluble fractions
of hippocampus were incubated with the TDP-O antibody-crosslinked
IP beads at room temperature for 1 hr. The unbound species were
removed by at least 6 washes with the binding buffer. The targeted
protein was eluted by Gentle Ag/Ab Elution Buffer (Thermo), pH 6.6,
and the eluent was saved for EM imaging and immunogold labeling.
For immunogold labeling, 10 .mu.l of eluent was placed on 400-mesh
Formvar carbon-coated copper grids (EMS Inc., Hatfield, Pa., USA)
for 5 min, washed by PBS, and then blocked by 1% BSA in PBS
solution at RT for 1 hr. After blocking, the grids were labeled by
N.sub.1-260 antibody (1:5,000, ab57105, Abcam) in 0.1% BSA
containing PBS solution at RT for 1 hr, and washed by high salt
tween (HST) buffer (50 mM Tris, pH 7.5, 500 mM NaCl, and 0.1%
Tween-20) and PBS. After washing, grids were incubated with 6 nm
gold-conjugated secondary anti-mouse IgG antibody (1:40, Jackson
ImmunoResearch) at RT for 1 hr. The unbound antibody was removed by
HST and PBS washes. Then, the grids were fixed by 1% glutaraldehyde
containing PBS at RT for 10 min and washed 6 times by ddH.sub.2O.
Finally, the grids were rinsed, negatively stained with 2% uranyl
acetate, and subjected to TEM imaging with FEI Tecnai G2 F20 S-TWIN
TEM.
[0141] Purification of Monoclonal TDP-O Antibodies.
[0142] The monoclonal TDP-O antibodies were purified by protein G
agarose kit (KPL) following the manufacturer's procedure. Briefly,
Protein G agarose (1.5 ml) supplied in 20% ethanol was poured into
a disposable column. The resin was washed with 10 ml wash buffer
(0.1 M sodium phosphate, pH 7.4, 0.15 M NaCl). Conditional medium
of TDP-O hybridoma cells (5 ml) was diluted with 5 ml binding
buffer. Then, the mixed sample solution was loaded into the column
and the column was inverted for mixing. Then, the column was washed
by 5 ml wash buffer for 4 times until OD 280 nm was close to 0. The
antibody was then eluted gently by adding 1 ml elution buffer (0.2
M glycine, pH 2.85) and collected in a collection tube that
contained 240 .mu.l 5.times. wash buffer. The antibody was
concentrated, and the buffer was exchanged to wash buffer by Amicon
Ultra-4 30 kDa cutoff (Millipore). The concentration of the
antibody was quantified by absorbance at 280 nm (absorbance of 1 mg
antibody=1.34).
[0143] Indirect ELISA.
[0144] ELISA 96-well plates (Nunc MaxiSorp) were coated with 20 ng
full-length TDP-43 oligomer protein dissolved in 200 .mu.l in TBS
(50 mM Tris, pH 7.4, 150 mM NaCl) at 4.degree. C. overnight. After
removing the coating solution, TBS buffer with and without 0.2% SDS
were added into the coated wells at room temperature for 1 hr.
TDP-43 oligomers treated with SDS will denature, whereas, TDP-43
oligomers treated without SDS will maintain the oligomer
conformation. After treatment, the buffer was removed and the wells
were blocked with 200 .mu.l 10% skim milk in TBST buffer (20 mM
Tris, pH 7.6, 137 mM NaCl, 0.001% Tween 20) for 2 hr at room
temperature. After washing three times with PBST (300 .mu.l/well),
100 .mu.l of diluted mTDP-O antibodies (from 0.00001 to 2 .mu.g/ml)
in TBST containing 5% skim milk was added and incubated for 1 hr at
room temperature. After washing 2 times with 300 .mu.l PBS, the
bound antibodies were detected by using the following 100 .mu.l HRP
conjugated anti-mouse antibody (1:1,000) diluted in 5% skim milk in
TBST. After incubation for 1 hr at room temperature and washing
twice by 300 .mu.l PBS, 3, 3', 5, 5'-tetramethylbenzidine (KPL
SureBlue) (100 .mu.l) was added to each well and the mixture was
incubated for 10 min at room temperature. The reaction was stopped
by adding 100 .mu.l 250 mM HCl to the mixture, and the optical
density at 450 nm was measured by SpectraMax M5 (Molecular
Devices).
[0145] Preparation of TDP-43 Oligomers and Monomers for Indirect
ELISA Assay.
[0146] TDP-43 protein (180 .mu.g/ml) was concentrated from 3 to 1
ml by using Amicon Ultra-4 30 kDa cutoff. Concentrated TDP-43
protein solution, 500 .mu.l, was loaded onto a Superdex 200 10/300
GL column and eluted with 30 mM Tris, pH 8.0, 50 mM NaCl at a flow
rate of 0.3 ml/min. Fractions (500 .mu.l per fraction) were
collected and the fractions containing TDP-43 oligomers or monomers
were pooled separately. The protein concentration was quantified by
micro BCA assay (Thermo). TDP-43 monomers or oligomers (300 ng)
were coated on 96-well ELISA plates (Nunc MaxiSorp). Conditional
medium of TDP-O hybridoma cell (100 .mu.l) (1:1,000) were used to
detect TDP-43 oligomers by indirect ELISA assay as mentioned
above.
[0147] Determination of Antibody Isotypes.
[0148] The isotyping of mTDP-O antibody was performed by an ELISA
mouse monoclonal antibody (mAb) isotyping Kit (Thermo) following
the manufacturer's procedure. In this assay, ELISA strip-well
plates with individual wells pre-coated with anti-mouse heavy-chain
capture antibody (anti-IgG.sub.1, IgG.sub.2a, IgG.sub.2b,
IgG.sub.3, IgA and IgM) or anti-mouse light-chain capture antibody
(kappa or lambda) were used. Briefly, the conditional medium of
TDP-O hybridoma cell lines were diluted in 50 fold in TBS and the
samples were added to each well of the 8-well strip. Then, the HRP
conjugated anti-mouse IgG+IgA+IgM (50 .mu.l) was added to each well
of the 8-well strip and the reaction was incubated for an hr at
room temperature. After 3 times washing, TMB substrate (75 .mu.l)
was added to each well and the plate was incubated at room
temperature in dark for 10 min. Next, the reaction was stopped by
adding 75 .mu.l of 0.18 M sulfuric acid. The absorbance of each
well was read by SpectraMax M5 (Molecular Devices) at 450 nm.
[0149] Cloning and Sequencing the Ig Variable (V) Genes.
[0150] Total RNA was prepared from 2.times.10.sup.6 hybridoma cells
using GeneJET RNA purification kit (Thermo). cDNA synthesis was
synthesized by using Maxima First Strand cDNA synthesis kit
(Thermo). Then, mouse Ig-Primer Sets (Novagen) and GoTaq.RTM. G2
Green Master Mix (Promega) were used to amplified Ig heavy and
kappa genes. The PCR products were cloned by TOPO.RTM. TA
Cloning.RTM. Kit (Invitrogen). Finally, DNA sequencing was
performed by Mission Biotech.
[0151] Cytotoxicity Assay.
[0152] MTT assay was employed to examine the inhibitory effect of
monoclonal TDP-O antibody on TDP-43 oligomers-induced cytotoxicity.
Human neuroblastoma BE(2)-C cells (ATCC #CRL-2268) were incubated
at 37.degree. C. under 5% CO.sub.2 and cultured in RPMI media with
10% fetal bovine serum. Cells were seeded in RPMI media in a
96-well plate with 40,000 cells per well and incubated for 24 hr.
Monoclonal TDP-O-3 (5 mg/ml) was prepared without further dilution,
or with 2- or 4-folds dilution with Dulbecco's PBS. The media were
discarded, and the cells were washed once by RPMI media without
FBS. RPMI media (40 .mu.l) were added to each well followed by the
addition of 20 .mu.l of 1.5 .mu.M TDP-43 oligomers and 1 .mu.l of
5, 2.5, or 1.25 mg/ml TDP-O antibody solution. The cells were
incubated for an additional 24 hr. Afterwards, 7 .mu.l of 5 mg/ml
MTT solution was added and incubated for 3 hr. The media were
discarded, and DMSO was used to lyse the formazan crystals.
Absorbance at 570 and 690 nm was measured by an ELISA microplate
reader SpectraMax M5 (Molecular Devices). The absorbance
differences between 570 and 690 nm were calculated, averaged (n=3),
and obtained by subtracting the background without cells. Each data
set was normalized using the buffer control. Statistical analysis
was performed by one-way ANOVAs.
Example 1 Full-Length TDP-43 Readily Forms Aggregates
[0153] In this example, the mechanism of TDP-43 pathology was
investigated. A recombinant full-length human TDP-43 in E. coli
(TDP-43 with N-terminal His-Tag, MW 47,145 Da) and a TDP-43 protein
purified from HEK293 cells were respectively obtained in accordance
with the procedures described in "Materials and Methods." The thus
obtained TDP-43 proteins were then subject to SEC analysis, and
results are depicted in FIG. 1A. As evidenced in FIG. 1A, more than
86% of TDP-43 was eluted in the void volume, and slot blotting
confirmed the majority of the recombinant full-length TDP-43
proteins from two different sources readily formed large
aggregates. Since TDP proteinopathies are characterized by
inclusion body (IB) formation and high-molecular-weight aggregates
were found in the recombinant full-length TDP-43, we speculated
that TDP-43 may form oligomers resembling the amyloid oligomers in
amyloidosis. Therefore, the conformation-dependent anti-amyloid
oligomer-specific antibody, A11, generated against A.beta. oligomer
mimics, was used to examine the TDP-43 oligomers, and we found both
TDP-43 samples, but not the corresponding buffers, were
immunoreactive with A11 (FIG. 1B). The oligomer fraction containing
diluted TDP-43 exhibited weaker intensity.
[0154] To confirm whether the recognition is
conformation-dependent, different denaturing methods were employed
to destruct TDP-43 oligomers before subjecting them to dot blotting
by A11 as well as anti-N-terminal and C-terminal TDP-43 antibodies
(FIG. 1C). The protein was incubated in various buffers, namely,
with or without 9 M urea, 7.2 M GdnHCI, or 2% SDS, and further
treated at 90.degree. C. for 1 h. For A11 detection, heating did
not significantly alter the detection signal in the native buffer.
However, the signals were weakened in the presence of high
concentrations of urea or GdnHCI and were nearly abolished by
additional heat treatment. The signals were not detected in the
presence of 2% SDS, regardless of heating. By contrast, the
anti-N-terminal antibody recognized TDP-43 consistently across all
conditions, confirming that equal amounts of proteins were dotted.
The recognition by the anti-C-terminal antibody was diminished or
completely lost when the samples were denatured in urea and heating
or when the samples were denatured in GdnHCI, with or without
heating. These findings indicate that the epitope recognized by the
anti-C-terminal TDP-43 antibody is altered in these denaturing
conditions.
[0155] Taken together, the results indicated that the recombinant
full-length TDP-43 readily forms high-molecular-weight species,
which share a common epitope with amyloid oligomers. These species
are highly stable under chemical denaturation but are
SDS-sensitive.
Example 2 Full-Length TDP-43 Forms Heterogeneous Spherical
Oligomers
[0156] In this example, the morphology of the aggregates was
observed by transmission electron microscopy (TEM) (FIG. 1D) and
atomic force microscopy (AFM) (FIG. 1E). TEM revealed heterogeneous
spherical species with several spheroid-shaped and ring-shaped
features, whereas, AFM revealed mostly spheroids but very few
ring-shaped structures (FIG. 1E, inset). The particle size
distribution was calculated based on TEM images. Majority of the
spherical particles measured around 40 to 60 nm in diameter (data
not shown). The ring-shaped oligomer examined by AFM was 6 nm in
height (data not shown). In addition, dynamic light scattering
(DLS) of the oligomer fraction eluted from the SEC also showed a
heterogeneous distribution, with the particle size ranging from 40
to 400 nm in diameter (FIG. 1F). The largest population of these
particles had a diameter of around 50 to 60 nm (FIG. 1G), which is
consistent with the results from the TEM studies. Combining the
imaging and size distribution findings, our results showed that
full-length TDP-43 formed heterogeneous particles with spherical
ultra-structures. The results are consistent with a previous
report, suggesting wild type TDP-43 and ALS-linked TDP-43 mutants
formed oligomers (Johnson et al., J. Biol. Chem (2009) 284,
20329-20339). In sum, we found that structurally and
immunologically, these TDP-43 species closely resembled spherical
amyloid oligomers that are widely considered neurotoxic in several
neurodegenerative diseases.
Example 3 TDP-43 Oligomers are Conformational and Functional
Distinct
[0157] To further examine the TDP-43 oligomers, the secondary
structure of TDP-43 oligomers were characterized by far-UV circular
dichroism (CD), and results are illustrated in FIG. 2A. Two double
minima were respectively observed near 210 and 222 nm, which likely
represent .alpha.-helical structures. The spectrum differs from
that of the short-form mouse TDP-43 (residues 101-265, denoted as
TDP-43s in plots), which comprises two RRMs with mostly
.beta.-strands in its crystal structure (Kuo et al., Nucleic Acids
Res (2009) 37, 1799-1808). The fluorescence spectra resulting from
the excitation of the intrinsic aromatic residues showed emission
maxima at approximately 340 nm, indicating the tyrosinyl and
tryptophanyl residues of TDP-43 were solvent-exposed (data not
shown). Furthermore, to detect the exposed hydrophobic protein
surfaces, an extrinsic fluorescence dye Bis-ANS that usually probes
partially unfolded intermediates upon protein folding was employed,
and results are depicted in FIG. 2B. As illustrated in FIG. 2B, the
full-length TDP-43 reacted with Bis-ANS to a greater extent than
the short-form TDP-43, which further suggest that full-length
TDP-43 oligomers possesses different hydrophobic-exposed surface
area than the short-form TDP-43.
[0158] In addition, the classic amyloid dye thioflavin T (ThT) was
also used to examine whether full-length TDP-43 binds to ThT, and
results are depicted in FIG. 2C. No fluorescence emission peak was
found for either full-length or short-form TDP-43 resulting from
ThT binding. By contrast, the A.beta. fibrils at the same protein
concentration, 1 .mu.M, showed strong fluorescence emission (FIG.
2C), which is consistent with the pathological examination result
using thioflavin. Similarly, TDP-43 oligomers binding with Congo
Red and its immunoreactivity with anti-fibrillar antibody OC all
came out negative (data not shown). These results and the CD data
suggest that TDP-43 oligomers are unlikely to adopt cross-.beta.
sheet structure. However, atomic level structural studies are
needed for further characterization.
[0159] Given that normal, functional TDP-43 binds to a specific
nucleic acid sequence, we then investigated whether TDP-43
oligomers would interfere with the DNA binding capability.
Fluorescence titration was adopted to monitor protein
conformational changes upon DNA binding, and results are
illustrated in FIG. 2D. Briefly, TDP-43 oligomers were titrated
with single-strand TAR DNA-A or -B sites and compared with the
short-form TDP-43 that has been reported to bind these ssDNAs in
submicromolar affinity (Kuo et al., Nucleic Acids Res (2009) 37,
1799-1808). As evidenced in FIG. 2D, both TAR DNA-A and -B sites
quenched the short-form TDP-43 fluorescence in low ssDNA
concentrations, whereas the level of quenching was significantly
reduced in full-length TDP-43. The data fitted well with a single
protein and ligand binding equation as described in the "Material
and Methods" section indicating a 1:1 stoichiometry of TDP-43 and
ssDNA. The dissociation constant, Kd, obtained from the fits are
7.05.+-.0.82, 6.27.+-.0.68, 0.20.+-.0.09, and 0.38.+-.0.10 .mu.M
for full-length TDP-43 with TAR-A, full-length TDP-43 with TAR-B,
short-form TDP-43 with TAR-A, and short-form TDP-43 with TAR-B,
respectively. The results indicated that ssDNA binding for the
short-form is more than 15 fold stronger than that for the full
length TDP-43, in which the TAR-A and -B sites have similar
affinities to TDP-43. The finding indicates that RRMs of
full-length TDP-43 oligomers either possess abnormal conformations
with reduced DNA binding capability or are hindered from DNA
binding because of masking of the binding regions. The emission
change that remained within the full-length TDP-43 could be
attributed to the existence of a small population of TDP-43
monomers in the sample. Taken together, these results demonstrated
that TDP-43 oligomers have different biophysical and biochemical
properties from those of the short-form TDP-43, indicating their
conformations are distinct.
Example 4 TDP-43 Oligomers Convert A.beta. to Amyloid Oligomers
[0160] In this example, whether TDP-43 may influence A.beta.
fibrillization pathway was investigated by cross-seeding
experiments.
[0161] First, A.beta.40 fibrillization was examined by ThT assay in
the absence and presence of TDP-43 oligomers, ranging from 0.4 to
4%, with results illustrated in FIG. 3A. It was found that TDP-43
potently inhibited A.beta. fibrillization in a dose-dependent
manner (FIG. 3A). The presence of 4% TDP-43 completely suppressed
A.beta. fibrillization during the entire experimental time of
approximately 180 hr. Photo-induced cross-linking (PICUP) was then
performed to examine the transient A.beta. species appeared in the
starting time point, and results indicated that A.beta. alone
formed primarily monomers, dimers, trimers, and tetramers after
cross-linking, whereas TDP-43 oligomer served to seed more
higher-molecular-weight A.beta. species (FIG. 3B). A.beta.
pentamers were observed dose-dependently upon TDP-43 addition.
Also, two larger assemblies, migrating at .about.55 kDa and
smearing from .about.105 to >210 kDa, were observed. In addition
to the SDS-irresistant TDP-43 monomers, a .about.55 kDa species and
some species causing a smear from .about.80 to >210 kDa were
found. Further analysis using TEM imaging showed that A.beta. did
not undergo fibril formation, but rather was transformed into
spherical oligomers with a diameter of <10 nm in the presence of
4% TDP-43 oligomers, whereas A.beta. alone still formed mature
amyloid fibrils as expected (FIG. 3C). The TDP-43 oligomer seeds
retained the diameter of >50 nm which was larger than those of
A.beta. oligomers (data not shown). These results supported that
TDP-43 oligomers can induce the A.beta. oligomer formation,
indicating again that TDP-43 shares common properties with
amyloids.
Example 5 TDP-43 Oligomers Induce Neurite Degeneration and
Toxicity
[0162] In this example, whether TDP-43 oligomers may cause
neurotoxicity and neurite degeneration was investigated.
[0163] Briefly, human neuroblastoma BE(2)-C cells were treated with
serially-diluted TDP-43 sample containing predominantly oligomers,
and the cytotoxic effect was examined by MTT and LDH assays, and
results are depicted in FIGS. 4A to 4C. MTT assay indicated
approximately 20% reduction in cell viability in the presence of
0.44 .mu.M TDP-43, as compared with that of the buffer controls
(FIG. 4A). The poor solubility of recombinant TDP-43 made toxicity
experiments with higher TDP-43 concentrations impossible. Similar
results were found in LDH assay, with 0.44 .mu.M TDP-43 induced a
significant level of cell death (FIG. 4B). TDP-43 produced
dose-dependent neurotoxicity in the primary cortical neurons of
mice, in which 0.6 .mu.M of TDP-43 caused approximately 20%
reduction in cell viability (FIG. 4C).
[0164] In addition, immunohistochemistry study from the cortical
neurons of mice revealed that 0.6 .mu.M TDP-43 induced shrinkage of
neurites and reduced neuron density and numbers (FIG. 4D). To
further test the neurotoxic effects of TDP-43 oligomers in vivo, 2
.mu.l of 2.2 .mu.M TDP-43 was injected to the hippocampal region of
mice and the survival of neuronal cells was examined by
immunofluorescence staining. Substantial amount of neuronal cells
was found lost in the CA1 layer in TDP-43-injected mice (FIG. 4E),
but not in the buffer-injected mice, as determined by the neuronal
marker NeuN immunoreactivity and DAPI staining. The result
indicated that injection of recombinant full-length TDP-43
oligomers in the hippocampus does induce toxicity.
Example 6 Production and Characterization of TDP-43
Oligomer-Specific Polyclonal Antibody
[0165] Since the anti-amyloid oligomer antibody (i.e., A11)
cross-reacted with different amyloids, to validate whether the
TDP-43 oligomers are present in the disease, a polyclonal antibody
were generated using the recombinant TDP-43 oligomers as immunogen
in rabbit in accordance with procedures described in "Materials and
Methods" section, and the thus obtained polyclonal antibody is
named TDP-O.
[0166] The specificity of TDP-O antibody against the full-length
TDP-43 oligomeric conformation was examined by dot blotting under
various denaturing conditions as described in "Materials and
Methods" section, and results are illustrated in FIG. 5A. TDP-O
with 1:125,000 dilution was capable of reacting with full-length
TDP-43 in the native buffer regardless of heating (90.degree. C., 1
h); however, the reactivity between the antibody and the TDP-43
oligomer disappeared when 2% SDS was present, with or without
heating. Further, the reactivity did not diminish when the
full-length TDP-43 was treated with either 7.2 M GdnHCI or 9 M urea
at room temperature for more than one hr, which indicated that the
protein was stable under high concentration of chemical
denaturants; however, the reactivity signal started to decrease
when additional heating was employed, indicating that TDP-O is a
conformational dependent antibody. In sum, the dot blotting result
from TDP-O was qualitatively identical to that of A11 (FIG. 5A vs
FIG. 1C).
[0167] To test whether TDP-O is TDP-43 specific rather than a
common amyloid oligomer antibody like A11, the antibody specificity
against A.beta. oligomers was examined using A11 and/or TDP-O. As
depicted in FIG. 5B, TDP-O failed to recognize A.beta. oligomers,
which demonstrates that it is specific toward TDP-43 oligomers.
ELISA was also performed to quantitatively characterize the
reactivity between TDP-O and TDP-43 oligomers and monomers; while
the TDP-43 antibody recognizing N-terminal residues 1-260
(N.sub.1-260) was employed for comparison purpose. To prepare the
oligomer and monomer fractions of the full-length TDP-43, the
full-length TDP-43 at approximately 1 .mu.M or three times more
concentrated TDP-43 samples was loaded into SEC, and 1 ml eluted
fractions were collected (FIG. 5C). The SEC fractions of the
concentrated TDP-43 from fractions 1 to 18 were then subjected to
dot blotting with TDP-O or N.sub.1-260 antibodies, and results are
illustrated in FIG. 5D. It was found that TDP-O reacted strongly
with fraction numbers 5 to 7, which corresponded to the TDP-43
oligomers eluted in the void volume. By contrast, N.sub.1-260
exhibited strong reactivity with fraction 6, and fractions 15 to
18; which indicates that this antibody may recognize both
oligomeric and monomeric form of TDP-43.
[0168] Further, the TDP-43 oligomer specificity with TDP-O was
quantified by ELISA, where the N.sub.1-260 antibody was used as
control. After protein quantification by micro-BCA assay, the
TDP-43 oligomer and monomer fractions were respectively coated onto
ELISA plate with serial dilutions. TDP-O and N.sub.1-260 were
applied and developed following standard ELISA protocol. Results
depicted in FIG. 5E indicated that TDP-O antibody exhibited strong
reactivity towards TDP-43 oligomers (EC50<0.5 .mu.g/ml) than to
TDP-43 monomer, whereas N.sub.1-260 antibody exhibited similar
reactivity toward ether TDP-43 oligomer or monomer (FIG. 5E).
Further, to insure TDP-O recognizes the oligomeric rather than
fibrillar form of TDP-43 as previously reported (Wang et al., J
Biol Chem (2013) 288, 9049-9057), the .beta.5 fibrils generated
from the 5.sup.th .beta.-strand within RRM2 domain of TDP-43 were
dotted (data not shown), and result clearly showed that TDP-O
recognizes only TDP-43 oligomers but not the .beta.5 fibrils. Taken
together, the polyclonal TDP-O antibody of this application may
specifically recognize TDP-43 oligomeric conformation without
cross-reacting with A.beta. species.
Example 7 TDP-43 Oligomers Exist in Transgenic Mice Brain
[0169] To examine the presence of TDP-43 oligomers in vivo,
immunohistochemical staining on the brain sections of wild type
mice and FTLD-TDP-43 transgenic mice at 6 and 12 month of age was
performed, and results are illustrated in FIGS. 6A and 6B.
FTLD-TDP-43 transgenic mice expressing full-length TDP-43 in the
forebrain has been reported to recapitulate FTLD-TDP-like pathology
(Tsai et al., J Exp Med (2010) 207, 1661-1673), for their
deficiency in the learning/memory capabilities and motor functions
as they age. In this example, the brain sections of FTLD-TDP-43
transgenic mice were immunofluorescently stained with anti-TDP-43
and TDP-O and counterstained with DAPI. As expected, in the wild
type mice, TDP-43 was found predominantly in the nucleus. By
contrast, TDP-43 was mainly found in the cytosol in the FTLD-TDP
transgenic mice, while TDP-O signals were also detected.
Significant amount of TDP-43 and TDP-O signals were found to
colocalize in the cells, and number of cells with double positive
signals increased with age. The appearance and colocalization of
the signals suggested that amyloid-like TDP-43 oligomers do exist
in the FTLD-TDP transgenic mice brain and the amount increases with
age.
Example 8 TDP-43 Oligomers are Present in Brains of FTLD-TDP
Patients
[0170] To evaluate the relevance of TDP-43 oligomers in human
diseases, the presence of TDP-43 oligomers in FTLD-TDP patients was
investigated using the subgroup of FTLD patients with TDP-43
immunoreactive inclusions. The TDP-O polyclonal antibodies were
used to immunostain hippocampal and frontal cortical sections of
three cases of pathologically confirmed FTLD-TDP, three cases of
age-matched non-dementic controls, and three cases of
pathologically confirmed AD without TDP-43 pathology as the
"disease controls". The TDP-O antibody identified various neuronal
cytoplasmic inclusions (FIG. 7, panels A and C) and dystrophic
neurites (FIG. 7, panel A) that appeared to be similar to the
TDP-43 inclusions in FTLD-TDP patients. In some areas, neuronal
cytoplasm was intensively stained in a granular pattern (FIG. 7,
panel E). By contrast, except the non-specific reactivities from
neuronal lipofuscin, TDP-O did not exhibit significant
immune-reactivity towards the control brains (FIG. 7, panels B, D,
and F), nor to AD brains lacking TDP-43 pathology (data not shown).
TDP-O polyclonal antibody failed to stain the nuclei in normal
tissue, which is consistent with our biochemical observation that
it does not react with TDP-43 monomer normally present in the
nuclei. In addition, to validate the morphology of TDP-43
recognized by TDP-O polyclonal antibody, we performed
immunoprecipitation (IP) by TDP-O with the control and diseased
human hippocampus. The Triton soluble fractions of hippocampus were
prepared and subjected to IP with cross-linked TDP-O polyclonal
antibody. The eluent was subjected to EM immunolabeled with
N-terminal TDP-43 antibody (N.sub.1-260) (FIGS. 8A and 8B). We
successfully observed rounded, spherical TDP-43 aggregates with a
diameter of approximately 50 nm in the diseased sample but not in
the control sample, and the species were recognized by N.sub.1-260
antibody indicating that they are not N-terminal truncated.
Overall, by using our TDP-O polyclonal antibody, we demonstrated
that TDP-43 oligomers exist in the brains of FTLD-TDP patients.
Example 9 Production and Characterization of TDP-43
Oligomer-Specific Monoclonal Antibody (mAb)
[0171] In this example, ELISA assay was performed to examine the
specificity of each mAbs produced from TDP-O-3, -5, -8, -9, and -10
hybridoma cells on TDP-43 oligomers with or without SDS
denaturation. Results are depicted in FIG. 9. Monoclonal antibodies
produced from any of the five hybridoma cell lines exhibited higher
binding activities toward non-denatured TDP-43 oligomers than the
denatured TDP-43 protein. The results indicated that these mAbs
have specificity toward TDP-43 oligomers and they are
conformational-dependent antibodies.
[0172] To confirm the binding specificity of the isolated mAbs,
TDP-43 oligomers and monomers were purified by gel filtration (FIG.
10A), and then respectively coated on plates for ELISA to confirm
the specificity of each mAbs. Results as depicted in FIG. 10B
confirmed that each of the mAbs produced from TDP-O-3, -5, -8, -9,
and -10 hybridoma cell lines may specifically recognize TDP-43
oligomers.
[0173] ELISA mouse mAb isotyping Kit (Thermo) was also used to
determine the isotypes of the TDP-O mAbs. Results are summarized in
Table 1.
TABLE-US-00001 TABLE 1 Isotypes of TDP-O mAbs OD 450 nm TDP-O-3
TDP-O-5 TDP-O-8 TDP-O-9 TDP-O-10 IgG.sub.1 0.2101 0.2261 0.1271
0.1717 0.2299 IgG.sub.2a 1.0523 1.2430 0.9270 1.0333 1.1781
IgG.sub.2b 0.0565 0.0520 0.0509 0.0564 0.0755 IgG.sub.3 0.0581
0.0539 0.0509 0.0556 0.0757 IgA 0.0563 0.0498 0.0533 0.0533 0.0680
IgM 0.0565 0.0502 0.0490 0.0519 0.0710 Kappa 2.1870 2.6605 2.2863
1.9313 2.3966 Lamda 0.0574 0.0603 0.0513 0.0572 0.0801
[0174] According to results presented in Table 1, the mAbs produced
by TDP-O-3, -5, -8, -9, and -10 hybridoma cell lines are more
reactive toward IgG.sub.2a and kappa light chain. Therefore, these
mAbs belongs to IgG.sub.2a and kappa light chain subclasses.
[0175] The 5 mAbs thus isolated were subjected to sequence
analysis, and consensus sequence were determined and depicted in
FIG. 11.
Example 10 TDP-43 Oligomer-Specific mAb Inhibits TDP-43
Oligomers-Induced Cytotoxicity
[0176] To examine whether TDP-O antibody can inhibit the cell
cytotoxicity induced by TDP-43 oligomers, human neuroblastoma
BE(2)-C cells were respectively treated by TDP-43 oligomers with or
without monoclonal TDP-O antibody (i.e., TDP-O-3) at the indicated
dosages as described in "Materials and Methods". The cell viability
was then measured by MTT assay. The result indicated that TDP-43
oligomers induced significant toxicity towards BE(2)-C cells, and
such cytotoxic phenomenon was successfully reversed by the
administration of TDP-O antibody (FIG. 12).
[0177] In conclusion, the present disclosure unexpectedly discovers
a pathological form of TDP-43, which cross-seeds A.beta. to form
amyloid oligomers and is associated with neurodegenerative
diseases. Accordingly, the present disclosure aims at providing an
antibody useful in suppressing the TDP-43 proteinopathy. Results
from the foregoing working examples confirm and support that the
present anti-TDP-43 antibodies specifically bind to the
pathological form of TDP-43 and suppress the TDP-43-induced
cytotoxicity; thus, they may act as a potential means to prevent
and/or treat the TDP-43-associated neurodegenerative diseases.
[0178] It will be understood that the above description of
embodiments is given by way of example only and that various
modifications may be made by those with ordinary skill in the art.
The above specification, examples and data provide a complete
description of the structure and use of exemplary embodiments of
the invention. Although various embodiments of the invention have
been described above with a certain degree of particularity, or
with reference to one or more individual embodiments, those with
ordinary skill in the art could make numerous alterations to the
disclosed embodiments without departing from the spirit or scope of
this invention.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 10 <210> SEQ ID NO 1 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: VH region-CDR1 <220>
FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: 3,5,6
<223> OTHER INFORMATION: Xaa is any of amino acid <400>
SEQUENCE: 1 Gly Tyr Xaa Phe Xaa Xaa Tyr Trp 1 5 <210> SEQ ID
NO 2 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: VH region-CDR2 <220> FEATURE: <221>
NAME/KEY: VARIANT <222> LOCATION: 4,6,7,8 <223> OTHER
INFORMATION: Xaa is any of amino acid <400> SEQUENCE: 2 Ile
Asn Pro Xaa Thr Xaa Xaa Xaa 1 5 <210> SEQ ID NO 3 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: VH
region-CDR3 <220> FEATURE: <221> NAME/KEY: VARIANT
<222> LOCATION: 1,7 <223> OTHER INFORMATION: Xaa is any
of amino acid <400> SEQUENCE: 3 Xaa Arg Gly Gly Lys Tyr Xaa
Gly Gly Ala Met Asp Tyr 1 5 10 <210> SEQ ID NO 4 <211>
LENGTH: 109 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: VH
region <220> FEATURE: <221> NAME/KEY: VARIANT
<222> LOCATION:
18,23,24,25,28,30,31,37,54,56,57,58,60,63,67,69,74,77,
81,84,95,97,103 <223> OTHER INFORMATION: Xaa is any of amino
acid <400> SEQUENCE: 4 Gln Val Gln Leu Gln Gln Ser Gly Ala
Glu Leu Ala Lys Pro Gly Ala 1 5 10 15 Ser Xaa Lys Met Ser Cys Xaa
Xaa Xaa Gly Tyr Xaa Phe Xaa Xaa Tyr 20 25 30 Trp Met His Trp Xaa
Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile
Asn Pro Xaa Thr Xaa Xaa Xaa Glu Xaa Asn Gln Xaa Phe 50 55 60 Lys
Asp Xaa Ala Xaa Leu Thr Ala Asp Xaa Ser Ser Xaa Thr Ala Tyr 65 70
75 80 Xaa Gln Leu Xaa Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Xaa
Cys 85 90 95 Xaa Arg Gly Gly Lys Tyr Xaa Gly Gly Ala Met Asp Tyr
100 105 <210> SEQ ID NO 5 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: VL region-CDR1 <220>
FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: 4
<223> OTHER INFORMATION: Xaa is any of amino acid <400>
SEQUENCE: 5 Ser Ser Val Xaa Tyr 1 5 <210> SEQ ID NO 6
<211> LENGTH: 3 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: VL region-CDR-2 <220> FEATURE: <221>
NAME/KEY: VARIANT <222> LOCATION: 1 <223> OTHER
INFORMATION: Xaa is any of amino acid <400> SEQUENCE: 6 Xaa
Thr Ser 1 <210> SEQ ID NO 7 <211> LENGTH: 9 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: VL region-CDR-3 <400>
SEQUENCE: 7 Gln Gln Arg Ser Ser Tyr Pro Leu Thr 1 5 <210> SEQ
ID NO 8 <211> LENGTH: 96 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: VL region <220> FEATURE: <221>
NAME/KEY: VARIANT <222> LOCATION:
6,9,10,11,18,21,26,30,35,36,48,49,74,80 <223> OTHER
INFORMATION: Xaa is any of amino acid <400> SEQUENCE: 8 Gln
Ile Val Leu Thr Xaa Ser Pro Xaa Xaa Xaa Ser Ala Ser Pro Gly 1 5 10
15 Glu Xaa Val Thr Xaa Thr Cys Ser Ala Xaa Ser Ser Val Xaa Tyr Met
20 25 30 His Trp Xaa Xaa Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp
Ile Xaa 35 40 45 Xaa Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg
Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Xaa
Ser Arg Met Glu Ala Xaa 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln
Gln Arg Ser Ser Tyr Pro Leu Thr 85 90 95 <210> SEQ ID NO 9
<211> LENGTH: 12 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: TAR DNA A-site <400> SEQUENCE: 9 ctttttgcct gt
12 <210> SEQ ID NO 10 <211> LENGTH: 12 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: TAR DNA B-site <400>
SEQUENCE: 10 tgggtctctc tg 12
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 10 <210>
SEQ ID NO 1 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: VH region-CDR1 <220> FEATURE: <221>
NAME/KEY: VARIANT <222> LOCATION: 3,5,6 <223> OTHER
INFORMATION: Xaa is any of amino acid <400> SEQUENCE: 1 Gly
Tyr Xaa Phe Xaa Xaa Tyr Trp 1 5 <210> SEQ ID NO 2 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: VH
region-CDR2 <220> FEATURE: <221> NAME/KEY: VARIANT
<222> LOCATION: 4,6,7,8 <223> OTHER INFORMATION: Xaa is
any of amino acid <400> SEQUENCE: 2 Ile Asn Pro Xaa Thr Xaa
Xaa Xaa 1 5 <210> SEQ ID NO 3 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: VH region-CDR3
<220> FEATURE: <221> NAME/KEY: VARIANT <222>
LOCATION: 1,7 <223> OTHER INFORMATION: Xaa is any of amino
acid <400> SEQUENCE: 3 Xaa Arg Gly Gly Lys Tyr Xaa Gly Gly
Ala Met Asp Tyr 1 5 10 <210> SEQ ID NO 4 <211> LENGTH:
109 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: VH region
<220> FEATURE: <221> NAME/KEY: VARIANT <222>
LOCATION: 18,23,24,25,28,30,31,37,54,56,57,58,60,63,67,69,74,77,
81,84,95,97,103 <223> OTHER INFORMATION: Xaa is any of amino
acid <400> SEQUENCE: 4 Gln Val Gln Leu Gln Gln Ser Gly Ala
Glu Leu Ala Lys Pro Gly Ala 1 5 10 15 Ser Xaa Lys Met Ser Cys Xaa
Xaa Xaa Gly Tyr Xaa Phe Xaa Xaa Tyr 20 25 30 Trp Met His Trp Xaa
Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile
Asn Pro Xaa Thr Xaa Xaa Xaa Glu Xaa Asn Gln Xaa Phe 50 55 60 Lys
Asp Xaa Ala Xaa Leu Thr Ala Asp Xaa Ser Ser Xaa Thr Ala Tyr 65 70
75 80 Xaa Gln Leu Xaa Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Xaa
Cys 85 90 95 Xaa Arg Gly Gly Lys Tyr Xaa Gly Gly Ala Met Asp Tyr
100 105 <210> SEQ ID NO 5 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: VL region-CDR1 <220>
FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: 4
<223> OTHER INFORMATION: Xaa is any of amino acid <400>
SEQUENCE: 5 Ser Ser Val Xaa Tyr 1 5 <210> SEQ ID NO 6
<211> LENGTH: 3 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: VL region-CDR-2 <220> FEATURE: <221>
NAME/KEY: VARIANT <222> LOCATION: 1 <223> OTHER
INFORMATION: Xaa is any of amino acid <400> SEQUENCE: 6 Xaa
Thr Ser 1 <210> SEQ ID NO 7 <211> LENGTH: 9 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: VL region-CDR-3 <400>
SEQUENCE: 7 Gln Gln Arg Ser Ser Tyr Pro Leu Thr 1 5 <210> SEQ
ID NO 8 <211> LENGTH: 96 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: VL region <220> FEATURE: <221>
NAME/KEY: VARIANT <222> LOCATION:
6,9,10,11,18,21,26,30,35,36,48,49,74,80 <223> OTHER
INFORMATION: Xaa is any of amino acid <400> SEQUENCE: 8 Gln
Ile Val Leu Thr Xaa Ser Pro Xaa Xaa Xaa Ser Ala Ser Pro Gly 1 5 10
15 Glu Xaa Val Thr Xaa Thr Cys Ser Ala Xaa Ser Ser Val Xaa Tyr Met
20 25 30 His Trp Xaa Xaa Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp
Ile Xaa 35 40 45 Xaa Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg
Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Xaa
Ser Arg Met Glu Ala Xaa 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln
Gln Arg Ser Ser Tyr Pro Leu Thr 85 90 95 <210> SEQ ID NO 9
<211> LENGTH: 12 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: TAR DNA A-site <400> SEQUENCE: 9 ctttttgcct gt
12 <210> SEQ ID NO 10 <211> LENGTH: 12 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: TAR DNA B-site <400>
SEQUENCE: 10 tgggtctctc tg 12
* * * * *