Note: Descriptions are shown in the official language in which they were submitted.
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TRUNCATED FRAGMENTS OF
ALPHA-SYNUCLEIN IN LEWY BODY DISEASE
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority from U.S. 60/471,929, filed May
19, 2003.
BACKGROUND
[0002] Lewy body diseases (LBDs) are characterized by degeneration of the
dopaminergic
system, motor alterations, cognitive impairment, and formation of Lewy bodies
(LBs).
(McKeith el al., Clinical and pathological diagnosis of dementia with Lewy
bodies (DLB):
Report of the CDLB International Workshop, Neurology (1996) 47:1113-24). LBDs
include
Parkinson's disease, Diffuse Lewy body disease (DLBD), Lewy body variant of
Alzheimer's
disease (LBV), and combined PD and Alzheimer's disease (AD) and the syndromes
identified
as multiple system atrophy (MSA). Dementia with Lewy bodies (DLB) is a term
coined to
reconcile differences in the terminology of LBDs. Disorders with LBs continue
to be a
common cause for movement disorders and cognitive deterioration in the aging
population
(Galasko et aL, Clinical-neuropathological correlations in Alzheimer's disease
and related
dementias. Arch. NeuroL (1994) 51:888-95). Although their incidence continues
to increase
creating a serious public health problem, to date these disorders lack
approved treatments
(Tanner et al., Epidemiology of Parkinson 's disease and akinetic syndromes,
Curr. Opin.
Neural. (2000) 13:427-30). The cause for LBD's is controversial and multiple
factors have been
proposed to play a role, including various neurotoxins and genetic
susceptibility factors.
[0003] AD, PD, and DLBD are the most commonly found neurodegenerative
disorders in the
elderly. Recent epidemiological studies have demonstrated a close clinical
relationship
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between AD and PD, as about 30% of Alzheimer's patients also have PD. Compared
to the
rest of the aging population, patients with AD are thus more likely to develop
concomitant
PD. Furthermore, PD patients that become demented usually have developed
classical AD.
Although each neurodegenerative disease appears to have a predilection for
specific brain =
regions and cell populations, resulting in distinct pathological features, PD,
AD, and DLBD
also share common pathological hallmarks. Patients with familial AD, Down
syndrome, or
sporadic AD develop LBs on the amygdala, which are the classical
neuropathological
hallmarks of PD. Additionally, each disease is associated with the
degeneration of neurons,
interneuronal synaptic connections and eventually cell death, the depletion of
neurotransmitters, and abnormal accumulation of misfolded proteins, the
precursors of which
participate in normal central nervous system function. Biochemical studies
have confirmed a
link between AD, PD and DLB.
[0004] In recent years, new hope for understanding the pathogenesis of LBD has
emerged.
Specifically, several studies have shown that the synaptic protein alpha-
synuclein plays a
central role in PD pathogenesis since: (1) this protein accumulates in LBs
(Spillantini et at.,
Nature (1997) 388:839-40; Takeda et al., J. PathoL (1998) 152;367-72;
Wakabayashi et at.,
Neurosci. Lett. (1997) 239:45-8), (2) mutations in the alpha-synuclein gene co-
segregate with
rare familial forms of parlcinsonism (Kruger et al., Nature Gen. (1998) 18:106-
8;
Polymeropoulos, et al., Science (1997) 276:2045-7) and, (3) its overexpression
in transgenic
mice (Masliah et at., Science (2000) 287:1265-9) and Drosophila (Feany at aL,
Nature
(2000) 404:394-8) mimics several pathological aspects of PD. Thus, the fact
that
accumulation of alpha-synuclein in the brain is associated with similar
morphological and
neurological alterations in species as diverse as humans, mice, and flies
suggests that this
molecule contributes to the development of PD.
[0005] The neuritic plaques that are the classic pathological hallmark of AD
consist
essentially of amyloid beta (AP) peptide, an amino acid proteolytic product of
the amyloid
precursor protein (APP), and NAC, a 35 amino acid proteolytic fragment of
alpha-synuelein.
Both AP and NAC were first identified in amyloid plaques as proteolytic
fragments of their =
respective full-length proteins, for which the full-length cDNAs were
identified and cloned.
(Iwai A., Biochinz. Biophys. Ada (2000) 1502:95-109); Masliah et al., AM J.
Pathol (1996)
148:201-10; Ueda et al., Proc. Natl. Acad. Sci. USA (1993) 90;11282-6).
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[0006] Alpha-synuclein is part of a large family of proteins including beta-
and gamma-
synuclein and synoretin. Alpha-synuclein is expressed in the normal state
associated with
synapses and is believed to play a role in neural plasticity, learning and
memory. Mutations
in human (h) alpha-synuclein that enhance the aggregation of alpha-synuclein
have been
identified (Ala30Pro and Ala53Thr) and are associated with rare forms of
autosomal
dominant forms of PD. The mechanism by which these mutations increase the
propensity of
alpha-synuclein to aggregate are unknown.
SUMMARY OF THE CLAIMED INVENTION
[0007] The invention provides methods of screening for an agent having a
pharmacological
activity useful for treating a Lewy Body Disease (LBD). The method involves
contacting the
agent with a fragment of alpha-synuclein, wherein the fragment is
characterized by presence
of at least 100 contiguous amino acids of intact (i.e., full-length) alpha-
synuclein and a
deletion of 1-25 contiguous amino acids from the C-terminus of intact alpha-
synuclein; and
determining the rate or extent of aggregation of the fragment of alpha-
synuclein, wherein a
reduction in the rate or extent of aggregation relative to a control lacking
the agent indicates
the agent has the pharmacological activity.
[0008] Optionally, the fragment has a C-terminus at a residue within residues
115 and 125
of intact alpha-synuclein with residues numbered according to SEQ ID NO: 1.
Preferred
fragments include alpha-synuclein SN1-115, SNI-116, SN1-117, SN1-118, SN1-119,
SN1-
120, SN1-121, SN1-122, SN1-123, SN1-124, and SN1-125. SN1-115, SN1-119, SN1-
122,
SN1-133 and SN1-135 are particularly preferred. Optionally, the fragment of
alpha-
synuclein is 1-X, wherein X is 130-139. Optionally, the fragment of alpha-
synuclein bears a
mutation associated with a hereditary LBD, such as an A53T mutation.
Optionally, the
method involves an additional step of conducting a trial in a human having a
LBD or an
animal model of LBD to determine whether the agent treats or inhibits a
symptom of the
LBD.
[0009] The invention further provides methods of screening for an agent having
a
pharmacological activity useful for treating a Lewy Body Disease (LBD). The
methods
. . involve contacting the agent with phosphorylated alpha-synuclein or a
phosphorylated
fragment thereof, wherein the fragment is characterized by presence of at
least 100
contiguous amino acids of intact alpha-synuclein and a deletion of 1-11
contiguous amino
acids from the C-terminus of intact alpha-synuclein; and determining the rate
or extent of
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aggregation of the alpha synuclein or fragment of alpha-synuclein, wherein a
reduction in the
rate or extent of aggregation relative to a control lacking the agent
indicates the agent has the
pharmacological activity. Optionally, the agent is contacted with intact alpha
synuclein or
SNI -133 or SN1-135 numbered according to SEQ ID NO:l. Optionally, intact
alpha
synuclein or the fragment of alpha-synuclein bears a mutation associated with
a hereditary
LBD. Optionally, the mutation is an A531 mutation. Optionally, the method
further
comprises conducting a trial in a human having a LBD or an animal model of LBD
to
determine whether the agent treats or inhibits a symptom of the LBD.
100101 The invention further provides methods of screening an agent for a
pharmacological
activity useful in treating a LBD (e.g., Parkinson's disease or DLBD). These
methods
comprise contacting a cell expressing alpha-synuclein and processing the alpha-
synuclein
into a fragment with an agent. The fragment is characterized by presence of at
least 100
contiguous amino acids of intact alpha-synuclein and a deletion of 1-25
contiguous amino
acids from the C-terminus of intact alpha-synuclein. One then determines a
level of the
fragment in the cell relative to a baseline level in the same cell type in the
absence of the
agent, a reduction in the level of the fragment relative to the baseline
indicating the agent has
the pharmacological activity useful in treating a LBD. Optionally, the
fragment of alpha-
synuclein has a C-terminus at a residue between 115 and 125 of intact alpha-
synuclein.
Preferred fragments are SN1-115, SN1-116, SN1-117, SN1-118, SN1-119, SN1-120,
SN1-
121, SN1-122, SN1-123, SN1-124, and SN1-125 of alpha synuclein. SN1-115, SN1-
119,
SN1-122, SN1-133 and SN1-135 are particularly preferred. Optionally, the
fragment of
alpha-synuclein is 1-X, wherein X is 130-139. Optionally, the fragment of
alpha-synuclein
bears a mutation associated with a hereditary LBD, such as an A53T mutation.
The cell can
be a human cell, a neuronal cell, a doparninergic cell or a nondopaminergic
cell. Optionally,
the cell is a PC12 or Sy5Y cell. Optionally, the method involves a step of
conducting a trial
in a human having a LBD or an animal model of LBD to detemiine whether the
agent treats
or inhibits a symptom of the LBD.
[0011] The invention further provides methods of screening for an agent having
a
pharmacological activity useful for treating a LBD (e.g., Parkinson's disease
or DLBD). The
methods involve contacting a transgenic animal expressing a fragment of alpha-
synuclein,
wherein the fragment is characterized by presence of at least 100 contiguous
amino acids of
intact alpha-synuclein and a deletion of 1-25 contiguous amino acids from the
C-terminus of
intact alpha-synuclein; and determining a level of aggregated forms of the
fragment in the
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brain of the transgenic animal relative to a baseline level of aggregated
forms of the fragment
in a comparable transgenic animal in the absence of the agent, a reduction in
the level of the
aggregated forms fragment relative to the baseline indicating the agent has a
pharmacological
activity useful in treating a LBD. Optionally, the fragment of alpha-synuclein
has a C-
terminus at a residue between 115 and 125 of intact alpha-synuclein. Preferred
fragments
include SN1-115, SN1-116, SN1-117, SN1-118, SN1-119, SN1-120, SN1-121, SN1-
122,
SN1-123, SN1-124, and SN1-125 of alpha synuclein. SN1-115, SN1-119, SN1-122,
SN133
and SN135 are particularly preferred. Optionally, the fragment of alpha-
synuclein is 1-X,
wherein X is 119-139. Optionally, the fragment of alpha-synuclein bears a
mutation
associated with a hereditary LBD, such as an A531 mutation. Optionally, the
transgenic
animal is a rodent. The transgenic animal can also be a Drosophila.
Optionally, the method
involves conducting a trial in a human having a LBD or an animal model of LBD
to
determine whether the agent treats or inhibits a symptom of the LBD.
[0012] The invention further provides methods of screening an agent for a
pharmacological
activity useful for treating a LBD (e.g., Parkinson's disease or DLBD). The
methods involve
contacting a transgenic animal expressing alpha-synuclein and processing the
alpha-synuclein
into a fragment with an agent, wherein the fragment is characterized by
presence of at least
100 contiguous amino acids of intact alpha-synuclein and a deletion of 1-25
contiguous
amino acids from the C-terminus of intact alpha-synuclein; and determining a
level of the
fragment in a neuronal cell relative to a baseline level in the absence of the
agent, a reduction
in the level of the fragments relative to the baseline indicating the agent
has the
pharmacological activity useful for treating the LBD. Optionally, the fragment
of alpha-
synuclein has a C-terminus at a residue between 115 and 125 of intact alpha-
synuclein.
Preferred fragments include SN1-115, SN1-116, SN1-117, SN1-118, SN1-119, SN1-
120,
SN1-121, SNI-122, SN1-123, SN1-124, and SN1-125 of alpha synuclein. SN1-115,
SN1-
119, SN1-122, 5N133 and SN135 are particularly preferred. Optionally, the
fragment of
alpha-synuclein is 1-X, wherein X is 130-139. Optionally, the fragment of
alpha-synuclein
bears a mutation associated with a hereditary LBD, such as an A53T mutation.
Optionally,
the transgenic animal is a rodent, mouse or Drosophila. Optionally, the method
involves a
step of conducting a trial in a human having a LBD or an animal model of LBD
to determine
whether the agent treats or inhibits a symptom of the LBD.
[00131 The invention further provides a transgenic animal having a genome
comprising a
txansgene comprising a promoter operably linked to a nucleic acid segment
encoding a
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fragment of alpha-synuclein wherein the fragment is characterized by presence
of at least 100
contiguous amino acids of intact alpha-synuclein and a deletion of 1-25
contiguous amino
acids from the C-terminus of intact alpha-synuclein; wherein expression of the
fragment in
the transgenic animal disposes the animal to develop at least one
characteristic of a LBD.
Optionally, the fragment of alpha-synuclein is selected from the group
consisting of SN1-
115, SN1-116, SN1-117, SN1-118, SN1-119, SN1-120, SN1-121, SN1-122, SN1-123,
SN1-
124, and SN1-125. SNI-115, SN1-119, SN1-122, SN133 and SN135 are particularly
preferred. Optionally, the fragment of alpha-synuclein is 1-X, wherein X is
130-139.
Optionally, the promoter is a PDGF promoter. Optionally, at least one
characteristic is an
impairment of motor function. Optionally, at least one characteristic of the
transgenic animal
is an impairment of cognitive function. Optionally, the transgenic animal is a
rodent, mouse
or Drosophila.
[0014] The invention further provides methods of detecting presence or
susceptibility to an
LBD in a patient The methods involve detecting a level of a fragment of alpha-
synuclein in
cerebrospinal fluid, wherein the fragment is characterized by presence of at
least 100
contiguous amino acids of intact alpha-synuclein and a deletion of 1-25
contiguous amino
acids from the C-terminus of intact alpha-synuclein. A change in level,
usually an increase,
relative to the baseline level in undiseased individuals indicating presence
or susceptibility to
LBD.
[0015] The invention further provides an antibody that specifically binds to a
fragment of
alpha-synuclein, wherein the fragment is characterized by presence of at least
100 contiguous
amino acids of intact alpha-synuclein and a deletion of 1-25 contiguous amino
acids from the
C-terminus of intact alpha-synuclein; without specifically binding to full-
length alpha
synuclein. Preferred fragments include SN1-115, SN1-116, SN1-117, SN1-118, SN1-
119,
SN1-120, SNI-121, SN1-122, SN1-123, SN1-124, and SN1-125 of alpha synuclein.
SNI -
115, SN1-119, SN1-122, SN133 and SN135 are particularly preferred. Optionally,
the
fragment is 1-X, wherein X is 130-139. Optionally, the antibody is a human,
humanized,
chimeric antibody. Optionally, the antibody is monoclonal. Optionally, the
antibody has
human isotype IgGl.
[0016j The invention further provides methods of diagnosing presence or
susceptibility to
LBD. The methods involve administering to a patient an antibody that
specifically binds to a
fragment of alpha-synuclein having a free C-terminus at residues 115-135
without
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specifically binding to full length synuclein; and determining a level of
binding of the
antibody in the patients, wherein a higher level of binding relative to a base
line level in
undiseased individuals indicates presence or susceptibility to the LBD.
Preferably the
antibody specifically binds to the free C-terminus of a fragment seledted from
the group
consisting of SN1-115, SN1-119, SN1-122, SN133 and S11135.
[0017] The invention further provides methods of effecting treatment or
prophylaxis of a
LBD, comprising administering to a patient suffering from or at risk of a LBD,
an effective
regime of a fragment of alpha-synuclein, wherein the fragment is characterized
by presence
of at least 100 contiguous amino acids of intact alpha-synuclein and a
deletion of 1-25
contiguous amino acids from the C-terminus of intact alpha-synuclein, and
thereby effecting
treatment or prophylaxis of the LBD. Optionally, the fragment of alpha-
synuclein is SN1-
115, SN1-116, SN1-117, SN1-118, SN1-119, SN1-120, SIN11-121, SN1-122, SNI-123,
SN1-
124, and SN1-125 of alpha synuclein. SN1-115, SN1-11 9, SN1-122, SN133 and
SN135 are
particularly preferred. Optionally, the fragment is 1-X, wherein X is 130-139.
Optionally,
the method further comprises administering an adjuvant that augments an immune
response
comprising antibodies to the fragment. Optionally, the fragment is linked to a
carrier forming
a fusion protein, wherein the carrier augments an immune response comprising
antibodies to
the fragment.
[0018] The invention further provides methods of effecting treatment or
prophylaxis of a
LBD. The method involves administering to a patient suffering from or at risk
of a LBD an
effective regime of an antibody that specifically binds to a fragment of alpha-
synuclein,
wherein the fragment is selected from the group consisting of SN1-115, SN1-
116, SN1-117,
SN1-118, SN1-119, SN1-120, SN1-121, SN1-122, SN1-1 23, SN1-124, and SN1-125
and 1-
X, wherein X is 130-139, without binding to intact alpha-synuclein, whereby
the antibody
effects prophylaxis or treatment of the disease. SN1-115, SN1-119, SN1-122,
SN133 and
SN135 are particularly preferred.
[0019] The invention further provides methods of effecting treatment or
prophylaxis of a
. ,
LBD. The methods comprise administering to a patient suffering from or at risk
of a LBD,
an effective regime of phosphorylated alpha synuclein or a phosphorylated
fragment of alpha-
synuclein, wherein the fragment is characterized by presence of at least 100
contiguous
amino acids of intact alpha-synuclein and a deletion of 1-10 contiguous amino
acids from the
C-terminus of intact alpha-synuclein; and thereby effecting treatment or
prophylaxis of the
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LBD. Optionally, the fragment is SNI-133 or SNI-135. Optionally, the method
further
comprises administering an adjuvant that augments an immune response
comprising
antibodies to the fragment. Optionally, the fragment is linked to a carrier
fonning a fusion
protein, wherein the carrier augments an immune response comprising antibodies
to the =
fragment.
[00201 The invention further provides a method of isolating a protease that
cleaves intact
alpha-synuclein to form a fragment, wherein the fragment is characterized by
presence of at
least 100 contiguous amino acids of intact alpha-synuclein and a deletion of 1-
25 contiguous
amino acids from the C-terminus of intact alpha-synuclein. SNI -115, SN1-119,
SN1-122,
SN133 and SN135 are particularly preferred. The method involves identifying an
inhibitor of
the protease; contacting the inhibitor with a cellular or tissue extract
containing the protease,
whereby the protease binds to the inhibitor; and releasing the protease from
the inhibitor.
Optionally, the inhibitor is a peptide of alpha-synuclein comprising a
contiguous segment of
at least 5 residues and up to 20 residues of intact alpha-synuclein between
positions 111 and
130. Alternatively the peptide is an inhibitor comprising least 5 residues of
intact alpha-
synuclein between positions 129 and 139. Optionally, the peptide comprises a
contiguous
segment of at least 5 residues between positions 118 and 122. Optionally, the
peptide
comprises a contiguous segment of at least four residues between positions 114
and 117.
Optionally, at least one of the residues is a transition state analog.
[00211 The invention further provides a monoclonal antibody that specifically
binds to an
epitope within residues 109-120 of alpha synuclein. Optionally, the monoclonal
antibody is
chimeric, humanized or human.
[00221 The invention further provides a monoclonal antibody that specifically
binds to an
epitope within residues 115-123 of alpha synuclein.
100231 The invention further provides a monoclonal antibody that specifically
binds to a
discontinuous epitope within residues 43-51 and 58-65 of alpha synuclein.
Optionally, the
antibody is chimeric, humanized or human.
10024] The invention further provides an end-specific monoclonal antibody that
specifically binds to isolated full-length alpha-synuclein having a free C-
terminus without
specifically binding to a fusion protein comprising alpha synuclein having a C-
tenninus
linked to a second polypeptide. Optionally, the antibody is chimeric,
humanized or human.
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[0025] The invention further provides methods of detecting presence or
susceptibility to
a Lewy body disease in a patient. The methods involves determining a level of
alpha-synuclein
phosphorylated at position 129 or phosphorylated or nitrated at position 125
of alpha-synuclein
in a sample from a brain of the patient, an elevated level relative to the
mean level in a
population of undiseased individuals indicating the patient has or is
susceptible to a Lewy body
disease. The invention provides other methods of detecting presence or
susceptibility to a Lewy
body disease in a patient by determining a level of ubiquitinated alpha
synuclein in a sample
from a brain of the patient, an elevated level relative to the mean level in a
population of
undiseased individuals indicating the patient has or is susceptible to a Lewy
body disease.
Optionally, levels of both phosphorylated alpha-synuclein and ubiquitinated
alpha-synuclein
can be detected.
[0025.1] In one aspect, the invention provides an in vitro method of
screening for an
agent having a pharmacological activity useful for treating Dementia with Lewy
Bodies
comprising: contacting the agent with a phosphorylated fragment of alpha-
synuclein, wherein
the fragment is SN1-135, wherein SN1-135 is a peptide of residues 1-135 of SEQ
ID NO:1;
determining the rate or extent of aggregation of the fragment of alpha-
synuclein, wherein a
reduction in the rate or extent of aggregation relative to a control lacking
the agent indicates the
agent has the pharmacological activity.
[0025.2] In another aspect, the invention provides an in vitro method of
screening for an
agent having a pharmacological activity useful for treating Dementia with Lewy
Bodies
comprising: contacting the agent with a fragment of alpha-synuclein, which is
SN1-135,
wherein SN1-135 is a peptide of residues 1-135 of SEQ ID NO:1; determining the
rate or
extent of aggregation of the alpha-synuclein or fragment of alpha-synuclein,
wherein a
reduction in the rate or extent of aggregation relative to a control lacking
the agent indicates the
agent has the pharmacological activity.
[0025.3] In another aspect, the invention provides an in vitro method of
screening an
agent for a pharmacological activity useful in treating Dementia with Lewy
Bodies comprising:
contacting a cell, which cell expresses alpha-synuclein and processes the
alpha-synuclein into a
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fragment, with an agent, wherein the fragment is SN I -135 wherein SN1-135 is
a peptide of
residues 1-135 of SEQ ID NO:1; determining a level of the fragment in the cell
relative to a
baseline level in the same cell type in the absence of the agent, a reduction
in the level of the
fragment relative to the baseline indicating the agent has the pharmacological
activity useful in
treating a LBD.
[0025.4] In another aspect, the invention provides a method of screening
for an agent
having a pharmacological activity useful for treating Dementia with Lewy
Bodies, comprising:
contacting a transgenic animal having a transgene expressing a fragment of
alpha-synuclein
with the agent, wherein the fragment is SN1-135 wherein SN1-135 is a peptide
of residues 1-
135 of SEQ ID NO:1; determining a level of aggregated forms of the fragment in
the brain of
the transgenic animal relative to a baseline level of aggregated forms of the
fragment in a
comparable transgenic animal in the absence of the agent, a reduction in the
level of the
aggregated forms fragment relative to the baseline indicating the agent has a
pharmacological
activity useful in treating a LBD.
[0025.5] In another aspect, the invention provides a method of screening an
agent for a
pharmacological activity useful for treating Dementia with Lewy Bodies,
comprising:
contacting a transgenic animal having a transgene expressing alpha-synuclein
and processing
the alpha-synuclein into a fragment with an agent, wherein the fragment is SN1-
135 wherein
SN1-135 is a peptide of residues 1-135 of SEQ ID NO:1; determining a level of
the fragment in
a neuronal cell relative to a baseline level in the absence of the agent, a
reduction in the level of
the fragments relative to the baseline indicating the agent has the
pharmacological activity
useful for treating the LBD.
[0025.6] In another aspect, the invention provides a method of detecting
presence or
susceptibility to Dementia with Lewy Bodies in a patient, comprising:
detecting a level of a
fragment of alpha-synuclein in body fluid, wherein the fragment is SN1-135,
wherein SN1-135
is a peptide of residues 1-135 of SEQ ID NO:1; a level greater than a baseline
level in
undiseased individuals indicating presence or susceptibility to LBD.
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[0025.7] In another aspect, the invention provides an antibody that
specifically binds to a
fragment of alpha-synuclein, wherein the fragment is SN1-135 or SN136-140
numbered
according to SEQ ID NO:1; without specifically binding to full-length alpha
synuclein.
[0025.8] In another aspect, the invention provides use of an antibody that
specifically
binds to SN1-135, wherein SN1-135 is a peptide of residues 1-135 of SEQ ID
NO:1 without
specifically binding to full length alpha-synuclein, for diagnosing presence
of or susceptibility
to Dementia with Lewy Bodies in a patient.
[0025.9] In another aspect, the invention provides a method of purifying a
protease that
cleaves intact alpha-synuclein to form a fragment, wherein the fragment is SN1-
135 wherein
SN1-135 is a peptide of residues 1-135 of SEQ ID NO:1; comprising: identifying
an inhibitor
of the protease; contacting the inhibitor with a cellular or tissue extract
containing the protease,
whereby the protease binds to the inhibitor; and releasing the protease from
the inhibitor,
wherein the inhibitor is a peptide of alpha-synuclein comprising a contiguous
segment of at
least 5 residues of intact alpha-synuclein between positions 129-139, and
wherein at least one
of the residues is a transition state analog.
[0025.10] In another aspect, the invention provides an isolated fragment of
alpha synuclein
wherein the fragment is SN1-135 or a peptide of less than 20 amino acids
including the C-
terminus of SN1-135, wherein SN1-135 is residues 1-135 of SEQ ID NO:1.
[0025.11] In another aspect, the invention provides a method of producing
an antibody that
specifically binds to a fragment of alpha-synuclein, wherein the fragment is
SN1-135,
comprising immunizing a non-human animal with a peptide of less than 20 amino
acids
including the C-terminus of SN1-135 to induce the antibody, wherein SN1-135 is
a peptide of
residues 1-135 of SEQ ID NO:1.
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BRIEF DESCRIPTION OF THE FIGURES
[0026] Figs. IA and B show a Western blot of various extracts from the cortex
and
hippocampus of a transgenic mouse (B) and a matched control (A) with a
polyclonal antibody
that binds to an epitope within SN115-122.
[0027] Fig. 2 shows a Western blot with the same antibody as Figs. IA and B to
compare the
level of the truncated form of alpha-synuclein in TritonTm-XI extractions of
the cortex and
hippocampus mice of 3 months and 12 months in age.
[0028] Figs. 3A and B shows a Western blot with a different antibody termed
12C1 (a
monoclonal binding to epitope at amino acids 43-51 and 58-65) of a Triton
extracts from the
brain of a transgenic mouse three months old (B) compared with an aged matched
control (A).
[0029] Fig. 4 shows a further Western blot using the same antibody as Fig. 3
on a Triton
extract from the brain of transgenic mice of three and twelve months of age.
[0030] Figs. 5 A, B, C, D, E show Western blots with four different antibodies
(B, C, D, E) and
an epitope map (A) of the binding sites (SEQ ID NOS:5, 6, 7, and 8) of the
antibodies to
various extracts from the brains of transgenic mice.
[0031] Figs. 6 A, B, C shows Iris extracts of the brain of a patient with Lewy
body disease
probed with three different antibodies (A, B, C), subject to 2-D gel
electrophoresis and
9c
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WO 2006/045037 PCT/US2005/037875
subjected to Western blotting. All 2D gels in this document are shown vvith
acidic proteins
on the left, more basic proteins on the right.
[0032] Fig. 7A, B, C, D shows additional blots of Tris extracts of the brain
of a patient with
Lewy body disease with four antibodies (A, B, C, D) of additional
specificities.
[0033] Fig. 8 summarizes the sites of cleavage relative to the epitopes (SEQ
ID NO:9)
bound by antibodies used in the Western blotting.
[00341 Figs. 9A, B compares the Iris soluble proteins (A) with proteins
extracted from
Lewy bodies (B) by 2D electrophoresis and Western blotting.
[00351 Figs. 10A, B, C, D show the immunoblots of proteins from Lewy bodies
reprobed
with various C-terminal antibodies.
[0036] Figs. 11A, B show Western blots of various extracts of an undiseased
and Contursi
patient probed with an antibody recognizing either total alpha synuclein (A)
or specific for
phospho-129 alpha synuclein (B).
100371 Fig. 12 Extracted ion chromatogram of C-terminal peptide of SN1-122.
[0038] Fig. 13: Extracted ion chromatogram of C-terminal peptide of SN1-119.
[0039] Figs. 14 A, B, C, D, E and F: 2D immunoblot with antibody recognizing
total
synuclein. Dashes mark positions of four rows of C-truncated synuclein
species. Figs. 14A,
B, C, E, and F show different preparations from different patients and Fig.
14D is a control.
[0040] Figs. 15 A, B: 2D immunoblots comparing ELADW101 (B), which is end
specific
for SN1-119 with an antibody to total alpha synuclein (A). Asterisks indicate
spots which
react with both antibodies. These spots are identified as SN1-119.
[0041] Figs. 16A and B respectively show labeling of Lewy bodies and neuritis
with the
SNI-119 end-specific polyclonal antibody ELADW-101.
[0042] Figs. 17A and B are controls from a normal individual stained with
ELADW-101.
[0043] Figs. 18A and B are brain sections from a DLBD patient stained with SN1-
119 end-
specific monoclonal antibody 12C6.
DEFINITIONS
[0044] The term "agent" is used to describe a compound that has or may have a
pharmacological activity. Agents include compounds that are known drags,
compounds for
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PCT/US2005/037875
which pharmacological activity has been identified but which are undergoing
further
therapeutic evaluation, and compounds that are members of collections and
libraries that are
to be screened for a pharmacological activity.
[0045] A "pharmacological" activity means that an agent exhibits an activity
in a screening
system that indicates that the agent is or may be useful in the prophylaxis or
treatment of a
disease. The screening system can be in vitro, cellular, animal or human.
Agents can be
described as having pharmacological activity notwithstanding that further
testing may be
required to establish actual prophylactic or therapeutic utility in treatment
of a disease.
[0046] In the context of molecular weight determinations based on gel
electrophoresis, the
term "about" indicates the standard deviation of molecular weight expected due
to
experimental error in repetitions of the method under the same conditions. The
molecular
weight determination of 12 kDa for certain fragments of alpha-synuclein
applies to
determinations using a tricine buffer.
[0047) The phrases "specifically binds" refers to a binding reaction which is
determinative
of the presence of the protein in the presence of a heterogeneous population
of proteins and
other biologics. Thus, under designated conditions, a specified ligand binds
preferentially to
a particular protein and does not bind in a significant amount to other
proteins present in the
sample. A molecule such as antibody that specifically binds to a protein often
has an
association constant of at least 106M4 or 107M-1, preferably 108M-1 to 109M-1,
and more
preferably, about 1010 M-1 to 1011 M-1 or higher. A variety of immunoassay
formats may be
used to select antibodies specifically immunoreactive with a particular
protein. For example,
solid-phase ELISA immunoassays are routinely used to select monoclonal
antibodies
specifically Mununoreactive with a protein. See, e.g., Harlow and Lane (1988)
Antibodies, A
Laboratory Manual, Cold Spring Harbor Publications, New York, for a
description of
immunoassay formats and conditions that can be used to determine specific
immunoreactivity.
[0048] For sequence comparison, typically one sequence acts as a reference
sequence, to
which test sequences are compared. When using a sequence comparison algorithm,
test and
reference sequences are input into a computer, subsequence coordinates are
designated, if
necessary, and sequence algorithm program parameters are designated. The
sequence
comparison algorithm then calculates the percent sequence identity for the
test sequence(s)
relative to the reference sequence, based on the designated program
parameters.
11
CA 02912912 2015-11-20
[0049] Optimal alignment of sequences for comparison can be conducted, e.g.,
by the local
homology algorithm of Smith & Waterman, Adv. AppL Math. 2:482 (1981), by the
homology
alignment algorithm of Needleman & Wunsch, J MoL Biol. 48:443 (1970), by the
search for
similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA
85:2444(1988), by
computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA
in the Wisconsin Genetics Software Package, Genetics Computer Group, 575
Science Dr.,
Madison, WI), or by visual inspection (see generally Ausubel et al., supra).
[0050] Another example of algorithm that is suitable for determining percent
sequence
identity and sequence similarity is the BLAST algorithm, which is described in
Altschul et
aL, J. Mol. Biol. 215:403-410(1990). Software for performing BLAST analyses is
publicly
available through the National Center for Biotechnology Information.
This algorithm involves first identifying high scoring
sequence pairs (HSPs) by identifying short words of length W in the query
sequence, which
either match or satisfy some positive-valued threshold score T when aligned
with a word of
the same length in a database sequence. T is referred to as the neighborhood
word score
threshold (Altschul et aL, supra.). These initial neighborhood word hits act
as seeds for
initiating searches to find longer HSPs containing them. The word hits are
then extended in
both directions along each sequence for as far as the cumulative alignment
score can be
increased. Cumulative scores are calculated using, for nucleotide sequences,
the parameters
M (reward score for a pair of matching residues; always >0) and N (penalty
score for
mismatching residues; always <0). For amino acid sequences, a scoring matrix
is used to
calculate the cumulative score. Extension of the word hits in each direction
are halted when:
the cumulative alignment score falls off by the quantity X from its maximum
achieved value;
the cumulative score goes to zero or below, due to the accumulation of one or
more negative-
scoring residue alignments; or the end of either sequence is reached. For
identifying whether
a nucleic acid or polypeptide is within the scope of the invention, the
default parameters of
the BLAST programs are suitable. The BLASTN program (for nucleotide sequences)
uses as
defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a
comparison of
both strands. For amino acid sequences, the BLASTP program uses as defaults a
word length
(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. The
TBLATN
program (using protein sequence for nucleotide sequence) uses as defaults a
word length (W)
of 3, an expectation (E) of 10, and a BLOSUM 62 scoring matrix. (see Henikoff
Henikoff,
Proc. Natl. Acad. Sc!. USA 89:10915 (1989)).
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[00511 In addition to calculating percent sequence identity, the BLAST
algorithm also
performs a statistical analysis of the similarity between two sequences (see,
e.g., Karlin &
Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of
similarity
provided by the BLAST algorithm is the smallest sum probability (P(N)), which
provides an
indication of the probability by which a match between two nucleotide or amino
acid
. ,
sequences would occur by chance. For example, a nucleic acid is considered
similar to a
reference sequence if the smallest sum probability in a comparison of the test
nucleic acid to
the reference nucleic acid is less than about 0.1, more preferably less than
about 0.01, and
most preferably less than about 0.001.
[0052] For purposes of classifying amino acids substitutions as conservative
or non-
conservative, amino acids are grouped as follows: Group I (hydrophobic side
chains):
norleucine, met, ala, val, leu, ile; Group II (neutral hydrophilic side
chains): cys, ser, thr;
Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn,
gin, his, lys, arg;
Group V (residues influencing chain orientation): gly, pro; and Group VI
(aromatic side
chains): trp, tyr, phe. Conservative substitutions involve substitutions
between amino acids
in the same class. Non-conservative substitutions constitute exchanging a
member of one of
these classes for a member of another.
(0053] Therapeutic agents of the invention are typically substantially pure
from undesired
contaminant. This means that an agent is typically at least about 50% w/w
(weight/weight)
purity, as well as being substantially free from interfering proteins and
contaminants.
Sometimes the agents are at least about 80% w/w and, more preferably at least
90 or about
95% w/w purity. However, using conventional protein purification techniques,
homogeneous
peptides of at least 99% w/w can be obtained.
[0054] The term "antibody" or "immunoglobulin" is used to include intact
antibodies and
binding fragments thereof. Typically, fragments compete with the intact
antibody from
which they were derived for specific binding to an antigen fragment including
separate heavy
chains, light chains Fab, Fab' F(ab')2, Fabc, and Fv. Fragments are produced
by recombinant
,
DNA techniques, or by enzymatic or chemical separation of intact
immunoglobulins. The
term "antibody" also includes one or more inununoglobulin chains that are
chemically
conjugated to, or expressed as, fusion proteins with other proteins. The term
"antibody" also
includes bispecific antibody. A bispecific or bifunctional antibody is an
artificial hybrid
antibody having two different heavy/light chain pairs and two different
binding sites.
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Bispecific antibodies can be produced by a variety of methods including fusion
of
hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann,
Clin. Exp.
Immunot 79:315-321 (1990); Kostelny etal., J. hninunoL 148, 1547-1553 (1992).
[0055] The term "adjuvant" refers to a compound that when administered in
conjunction
with an antigen augments the immune response to the antigen, but when
administered alone
does not generate an immune response to the antigen. Adjuvants can augment an
immune
response by several mechanisms including lymphocyte recruitment, stimulation
of B and/or T
cells, and stimulation of macrophages.
100561 The term "patient" includes human and other mammalian subjects that
receive either
prophylactic or therapeutic treatment.
[0057] Competition between antibodies is determined by an assay in which the
immunoglobulin under test inhibits specific binding of a reference antibody to
a common
antigen, such as alpha-synuclein. Numerous types of competitive binding assays
are known,
for example: solid phase direct or indirect radioimmunoassay (RT.A), solid
phase direct or
indirect enzyme immunoassay (ETA), sandwich competition assay (see Stahli et
al., Methods
in Enzymology 9:242-253 (1983)); solid phase direct biotin-avidin ETA (see
Kirkland etal., J.
ImmunoL 137:3614-3619 (1986)); solid phase direct labeled assay, solid phase
direct labeled
sandwich assay (see Harlow and Lane, Antibodies, A Laboratoq Manual, Cold
Spring
Harbor Press (1988)); solid phase direct label R1A using 1-125 label (see
Morel et aL, Molec.
immund. 25(1):7-15 (1988)); solid phase direct biotin-avidin EIA (Cheung
etal., Virology
176:546-552 (1990)); and direct labeled RIA (Moldenhauer etal., Scand. J.
Imnzunot 32:77-
82 (1990)). Typically, such an assay involves the use of purified antigen
bound to a solid
surface or cells bearing either of these, an unlabelled test immunoglobulin
and a labeled
reference immunoglobulin. Competitive inhibition is measured by determining
the amount of
label bound to the solid surface or cells in the presence of the test
immunoglobulin. Usually
the test immunoglobulin is present in excess. Antibodies identified by
competition assay
(competing antibodies) include antibodies binding to the same epitope as the
reference
antibody and antibodies binding to an adjacent epitope sufficiently proximal
to the epitope
bound by the reference antibody for steric hindrance to occur. Usually, when a
competing
antibody is present in excess, it will inhibit specific binding of a reference
antibody to a
common antigen by at least 50 or 75%.
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[0058] Epitope co-ordinates are approximate (2 amino acids). Not every amino
acid
within an epitope is necessarily required for binding.
[0059] Compositions or methods "comprising" one or more recited elements may
include
other elements not specifically recited. For example, a composition that
comprises alpha-
synuclein peptide encompasses both an isolated alpha-synuclein peptide and
alpha-synuclein
peptide as a component of a larger polypeptide sequence.
[0060] Unless otherwise apparent from the context, each embodiment, element,
step or
feature of the invention can be used in combination with any other.
DETAILED DESCRIPTION OF THE INVENTION
I. General
[0061] The invention is premised in part on the identification of novel
fragments of alpha-
synuclein in patients with Lewy Body Disease (LBD) and transgenic animal
models thereof.
These diseases are characterized by aggregations of alpha-synuclein. The
fragments have a
truncated C-terminus relative to full-length alpha-synuclein. Some fragments
are
characterized by a molecular weight of about 12 kDa (corresponding to SN1-
119), 12.5 kDa
(corresponding to SN1-122), 13.5 kDa (which fragment is found only in patients
with LBD)
and 15 kDa (probably corresponding to SN1-133 or SN1-135) as determined by SDS
gel
electrophoresis in tricine buffer and a truncation of at least ten contiguous
amino acids from
the C-terminus of natural alpha-synuclein. The site of cleavage preferably
occurs after
residue 115 and before residue 136 of natural human alpha-synuclein.
Particularly preferred
sites of cleavage are between residues 115 and 116, 119 and 120, between
residues 122 and
123, between residues 132 and 133 and between residues 135 and 136. The
identification of
these novel fragments of alpha-synuclein has a number of application in for
example, drug
discovery, diagnostics, therapeutics, and transgenic animals.
[0062] The invention is further premised in part on the result that
phosphorylation
synuclein partitions more to the particulate (Lewy body enriched fraction)
relative to the
soluble cytosolic fraction in patients with synucleinopathic disease relative
to controls.
Phosphorylation occurs at position 129 of alpha synuclein. Although an
understanding of
mechanism is not required for practice of the invention, it is proposed that
phosphorylation of
alpha synuclein drives subsequent processing to truncated forms (i.e.,
cleavages between
residues between residues 132 and 133 and between residues 135 and 136) and
aggregation
of alpha synuclein. The invention further shows that small amounts of alpha
synuclein in
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insoluble fractions from patients with synucleinopathic disease are
ubiquitinated at lysine
residues 6, 10, 12, 21, 23, 32 and 34. Ubiquitination is known to have a role
in degradation
o f proteins (see, e.g., Cierchanover, EMBO L 17, 7151-7160 (1998)).
Ubiquitination also
renders alpha synuclein prone to aggregation and changes its intracellular
path from
proteosomes to lysosomes. Thus, ubiquitination can both increase degradation
of alpha
synuclein and promote its aggregation. Modulation of ubiquitination can
therefore be useful
in treating synucleinopathic disease.
[0063] The invention provides several methods for screening agents for
activity useful in
treating LBDs. Some methods identify agents that inhibit the cleavage reaction
that generates
the novel fragments of the invention. Other method identify agents that
inhibit aggregation
of the products of the cleavage reaction. Such inhibitors are useful for
treatment of LBD's.
Inhibitors of the cleavage reaction are also useful for affinity purification
of the protease
responsible for the cleavage reaction.
[0064] The invention also provides transgenic animal models and cells
expressing
fragments of alpha-synuclein as described above. The transgenic animal models
and cells are
disposed to develop characteristics of Lewy body disease, including Lewy
bodies containing
aggregations of the fragments. The animal models and cells can be used in the
screening
methods described above.
[0065] The invention further provides end-specific antibodies that
specifically bind to
fragments of alpha-synuclein without specifically binding to intact alpha-
synuclein per se.
These antibodies are useful for in vivo imaging of alpha-synuclein
aggregations and also in
methods of treatment. The novel alpha-synuclein fragments can also be used in
methods of
treatment, optionally, in combination with an adjuvant.
Alpha-synuclein fragments
[0066] Human alpha-synuclein is a peptide of 140 amino acids having the
following amino
acid sequence:
MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH
GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL
GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA
(SEQ ID NO:1)
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(Ueda et al., Proc. Natl. Acad. Sci. USA (1993) 90:11282-6).; GenBank
accession number:
P37840). The protein has three recognized domains, a KTKE repeat domain
covering amino
acids 1-61, a NAC (Non-amyloid component) domain running from about amino
acids 60-95,
and a C-terminal acidic domain running from about amino acid 98 to 140.
[0067] Some novel fragments of the invention have C-terminal truncations of at
least ten
contiguous amino acids, preferably at least 15 contiguous amino acids, and
optionally at up to
20, 22, 23 or 25 amino acids. The fragments include all or substantially all
(i.e., at least 100
contiguous residues from alpha-synuclein other than the deletion). Some
fragments also have
relatively short truncations at the N-terminus of up to 20 amino acids, such
as deletions of
residues 1-4, 1-6, 1-10 and 1-12. Some fragments have N-terminal deletions of
residues 1-
23, 1-38 or 1-45. Preferred fragments are SN1-115, SNI-116, 1-117, SN1-118,
SN1-119,
SN1-120, SN1-121, SN1-122, SN1-123, SN1-124, SN1-125, SN1-126, SN1-127, SNI-
128,
SN 1-129 and SNI -130. Particularly preferred fragments are SN1-115, SNI -119,
SNI-120,
SN1-121, SN1-122, SN1-123, SN 1-124 and SN 1-125. Especially preferred
fragments are
SN1-115, SN1-119, SN1-122 SN1-133 and SN1-135. The cleavage reaction
preferably
occurs at a peptide bond between amino acid residues 115 and 116, or 118 and
136, e.g.,
particularly between residue 119 and 120 or residues 122 and 123 or residues
133-134 or
residues 135-136.
[0068] The C-terminal fragments resulting from cleavage are also included in
the invention
and can be used in the methods described below. These fragments include SN116-
140,
SN117-140, SN118-140, SN119-140, SN119-140, SN120-140, SN121-140, SN122-140,
SN123-140, SN124-140, SN125-140, SN! 26-140, SN1-127-140, SN128-140, SN129-
140,
SN 130-140 and SN131-140. Preferred fragments are SN116-140, SN120-140, SN123-
140,
SN 134-140 and 5N136-140.
[0069] Other fragments of the invention include N-terminal fragments of alpha-
synuclein
of about 6 to 7 kDa (as determined by SDS electrophoresis) or 50-80 amino
acids. Other
fragments of the invention include N-terminal fragments of alpha-synuclein
that are free of 1-
amino acids from the C-terminus of intact alpha-synuclein, i.e., SN 1-X,
wherein X is 130-
139. Some fragments are characterized by specific binding to antibodies
ELADW43 (free
N-terminus) and 5C12 (111-118) and lack of specific binding to 8A5 (free C-
terminus),
LB509 (115-123) and ELADW47 (115-122). Some fragments are characterized by
specific
binding to ELADW43 (intact N-terminus) and 5C12 (111-118), LB509 (115-123) and
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ELADW47 (115-122) and lack of specific binding to 8A5 (free C-terminus). Some
fragments are characterized by specific binding to ELADW43 (free N-terminus)
and 5C12
(111-11.8), LB509 (115-122) and ELADW47 (118-123) and 8A5 (free C-terminus)
and lack
of specific binding to ELADW43 (free N-terminus).
{0070] Some fragments or full-length alpha synuclein are phosphorylated at
position 125 or
129 or nitrated at the tyrosine residue occupying position 125 of alpha
synuclein. Fragments
retaining amino acid serine 125 or full-length alpha synuclein can also be
phosphorylated at
this position. Detection of enhanced phosphorylation or nitration at position
125 or
phosphorylation at position 129 in a patient relative to the mean in a
population of undiseased
individuals is an indication of a Lewy body disease. Detection can be
performed using an
antibody specific for alpha-synuclein phosphorylated or nitrated at position
125. A level is
considered enhanced if greater than the mean plus one standard deviation in a
population of
undiseased individuals.
[0071] The invention also provides isolated peptides of up to five or ten
contiguous
residues of alpha synuclein containing at least one of the above-mentioned
ubiquitination
sites. These peptides can be used to compete with sites in the full-length
alpha-synuclein for
ubiquitination or as immunogens to generate antibodies that block
ubiquitination of full-
length alpha-synuclein.
[0072] The fragments of the invention are distinct from the non-A.13 component
of
Alzheimer's disease amyloid (NAC) previously reported. This fragment
consisting of at least
28 amino acids residues (residues 60-87) and optionally 35 amino acid residues
(residues 61-
95). See Iwai, et al, Biochemist/3), 34:10139-10145); Jensen et al., Biochem.
.1. 310 (Pt 1):
91-94 (1995); GenBank accession number S56746.
[0073] Unless otherwise apparent from the context, reference to alpha-
synuclein or its
fragments includes the natural human amino acid sequence indicated above, or
fragments
thereof, as well as analogs including allelic, species and induced variants
(e.g., E83Q, A90V,
A76T). Amino acids of analogs are assigned the same numbers as corresponding
amino
acids in the natural human sequence when the analog and human sequence are
maximally
aligned. Analogs typically differ from naturally occurring peptides at one,
two or a few
positions, often by virtue of conservative substitutions. Some natural allelic
variants are
genetically associated with hereditary LBD. The term "allelic variant" is used
to refer to
variations between genes of different individuals in the same species and
corresponding
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PCT/US2005/037875
variations in proteins encoded by the genes. Allelic variants include E46K,
A3OP and A531
(the first letter indicates the amino acid in SEQ ID NO:1, the number is the
codon position in
SEQ ID NO:1, and the second letter is the amino acid in the allelic variant).
Analogs can
include any combination of allelic variants. The A53T variation is associated
with enhanced
levels of phosphorylation at position 129 of alpha synuclein in an individual
having the
mutation relative to the norm of phosphorylation in undiseased individuals who
lack the
mutation. Analogs exhibit at least 80 or 90% sequence identity with natural
peptides. Some
analogs also include unnatural amino acids or modifications of N or C terminal
amino acids
at one, two or a few positions. For example, the natural glutamic acid residue
can be replaced
with iso-aspartic acid. Examples of unnatural amino acids are D, alpha, alpha-
disubstituted
amino acids, N-alkyl amino acids, lactic acid, 4-hydroxyproline, gamma-
carboxyglutamate,
epsilon-N,N,N-trimethyllysine, epsilon-N-acetyllysine, 0-phosphoserine, N-
acetyl serine, N-
formylmethionine, 3-methylhistidine, 5-hydroxylysine, omega-N-methylarginine,
fl-alanine,
ornithine, norleucine, norvaline, hydroxproline, thyroxine, gamma-amino
butyric acid,
homoserine, citrulline, and isoaspartic acid. Analogs typically specifically
bind to a
polyclonal antibody population generated against natural human alpha-
synuclein, and each
end of an analog of a specific fragment of a natural human alpha synuclein
also specifically
bind to a monoclonal antibody that is end specific for the respective end of
the natural
fragment. The invention also provides D-peptides, in which D-amino acids can
be
substituted for corresponding natural L-amino acids of alpha-synuclein at most
or all
positions. A fragment designated in the form SNx-y means a fragment of alpha
synuclein
that begins at amino acid X and ends at amino acid Y, and contains each amino
acid between
X and Y. Such a fragment can (but need not) be linked to a heterologous
polypeptide but not
to other amino acids of human alpha synuclein such that the fragment begins
before X or
ends after Y. Residues in a fragment are numbered according to SEQ B) NO:1
when the
fragment is maximally aligned with SEQ ID NO:1 as described above using
default
parameters.
[0074] Alpha-synuclein, its fragments, and analogs can be synthesized by solid
phase
peptide synthesis or recombinant expression, or can be obtained from natural
sources.
Automatic peptide synthesizers are commercially available from numerous
suppliers, such as
Applied Biosystems, Foster City, California. Recombinant expression can be in
bacteria,
such as E. cofi, yeast, insect cells or mammalian cells. Procedures for
recombinant
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expression are described by Sambrook et al., Molecular Cloning: A Laboratmy
Manual
(C.S.H.P. Press, NY 2d ed., 1989).
III. Lewy Body Diseases
[0075] Lewy Body Disease (LBD) is characterized by degeneration of the
dopaminergic
system, motor alterations, cognitive impairment, and formation of Lewy bodies
(LBs).
(McKeith et al., Clinical and pathological diagnosis of dementia with Lewy
bodies (DLB):
Report of the CDLB International Workshop, Neurology (1996) 47:1113-24). Lewy
Bodies
are spherical protein deposits found in nerve cells. Their presence in the
brain disrupts the
brain's normal function interrupting the action of chemical messengers
including
acetylcholine and dopamine. Lewy Body diseases include Parkinson's disease
(including
idiopathic Parkinson's disease(PD)), Diffuse Lewy Body Disease (DLBD) also
known as
Dementia with Lewy Bodies (DLB), Combined Alzheimer's and Parkinson disease
and
multiple system atrophy (MSA). DLBD shares symptoms of both Alzheimer's and
Parkinson's disease. DLBD differs from Parkinson's disease mainly in the
location of Lewy
Bodies. In DLBD Lewy Bodies form mainly in the cortex. In Parkinson's disease,
they form
mainly in the substantia nigra. Other Lewy Body diseases include Pure
Autonomic Failure,
Lewy body dysphagia, Incidental LBD, Inherited LBD (e.g., mutations of the
alpha-synuclein
gene, PARK3 and PA1.K4) and Multiple System Atrophy (e.g.,
Olivopontocerebellar
Atrophy, Striatonigral Degeneration and Shy-Drager Syndrome).
IV. Transgenic Animals and Cells
[0076] The invention provides transgenic animals having a genome comprising a
transgene
comprising a nucleic acid segment encoding a C-terminal truncated form of
alpha-synuclein
as described above. Preferred truncated forms are SN1-115, SN1-119, SN1-122,
SN1-133
and SN1-135. The transgene is preferably present in all or substantially of
the somatic and
germline cells of the transgenic animal. The nucleic acid segment encoding the
C-terminal
truncated form of alpha-synuclein is operably linked to one or more regulatory
segments that
allow the truncated form of alpha-synuclein to be expressed in neuronal cells
of the animal.
Promoters such as the rat neuron specific enolase promoter, human beta-actin
gene promoter,
human platelet derived growth factor B (PDGF-B) chain gene promoter, rat
sodium channel
gene promoter, mouse myelin basic protein gene promoter, human copper-zinc
superoxide
dismutase gene promoter, and mammalian POU-domain regulatory gene promoter can
be
used. The PDGF promoter is particularly suitable. Optionally, an inducible
promoter is used.
The mouse metallothionine promoter, which can be regulated by addition of
heavy metals
CA 02 912 912 2015-11-20
14/0 2006/045037 PCT/US2005/037875
such as zinc to the mouse's water or diet, is suitable. Such transgenic
animals can be
produced by the same general approaches described by (Masliah et al., Am. J.
Pa/ho!. (1996)
148:201-10 and Feany et al., Nature (2000) 404:394-8)) for transgenic animals
with full-
- length alpha-syrtuclein or US 5,811,633 (for transgenic animals with a
mutant form of APP).
Optionally, transgenic animals bearing a transgene expressing a truncated
alpha-synuclein
protein can be crossed with other transgenic models of neurogenic disease,
such as models of
Alzheimer's disease. For example, transgenic animals bearing a transgene
expressing a
truncated alpha-synuclein protein can be crossed with transgenic animals
bearing a transgene
expressed APP with a FAD mutation as described by e.g., Games etal., Nature
373, 523
(1995) McConlogue etal., US 5,612,486, Hsiao etal., Science 274, 99(1996);
Staufenbiel et
al., Proc. Natl. Acad. Sci. USA 94, 13287-13292 (1997); Sturchler-Pierrat
etal., Proc. Natl.
Acad. ScL USA 94, 13287-13292 (1997); Borchelt etal., Neuron 19, 939-945
(1997)). The
procedure for performing such a cross is described by e.g., Masliah etal.,
PNAS USA
98:12245-12250 (2001), which reports a cross between transgenic mice
expressing a full
length alpha-synuclein with PDAPP mice as described by Games et al Transgenic
animals of
the invention are preferably rodents, such as mice or rats, or insects, such
as Drosophila.
Transgenic animals can be produced by introduction of a transgene at the
gerrnline stage in
which case all or substantially all (except for rare loss through somatic
mutation) of the cells
of the transgenic animal include the transgene integrated into the genome.
Transgenes can be
introduced by microinjection, nuclear transfer or viral infection into cells
or animals.
Lentiviruses are particularly suitable for the latter. Alternatively,
transgenes can be
introduced by viral infection into the brain of the animal. Such transgenes
are not part of the
germline of recipient animals but can be targeted to regions of the brain
responsible for
disease (e.g., the substantia nigra). Such animal models incorporate an alpha
synuclein into
the genome of brain cells and are disposed to develop at least one
characteristic of
synucleinopathic disease. Lentiviruses provide a suitable vehicle for so
introducing an alpha
synuelein transgene into the brain (see Brain Pathology 13, 364-372 (2003);
Bjorklund,
Trends Neurosci. 26, 386-92 (2003), Lotharius et al., J. Biol. Chem. 277,
38884-94 (2002),
Zhou et al., Brain Research 866, 33-43 (2000)) .
[0077] The expression of truncated forms of alpha-synuclein in animal models
gives rise to
animals disposed to develop at least one characteristic of a Lewy Body
disease. Such
characteristics include increased levels of intracellular deposits of alpha-
spuclein, increased
formation of Lewy bodies, and impaired cognitive and motor functions relative
to normal
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nontransgenic animals of the same species. Such transgenic animals are useful
for screening
agents for pharmacological activity in treating Lewy Body disease.
100781 The invention also provides cells transformed with truncated alpha -
synuclein which
form inclusion bodies containing aggregated truncated alpha-synuclein. The
transformed
cells are preferably neuronal cells, such as GT1-7 neuronal cells (Hsue et al.
Am. J. Patho. 1.
157:401-410 (2000)), PC12 cells or SY5Y neuroblastoma cells. PEAK cells can
also be
used. The cells are preferably human cells. A vector comprising a segment
encoding a
truncated form of alpha-synuclein operably linked to one or more regulatory
sequences that
ensure expression of the truncated expression is transfected into the cells.
Transfected celts
can be used to screen agents for activity in clearing alpha-synuclein
inclusions.
V. Screening Methods
100791 The invention provide several screening methods to identify agents
having a
pharmacological activity useful in treating a LBD. The methods include screens
that can be
performed in vitro, in cells or transgenic animals, and which test a variety
of parameters as an
indication of activity. Agents determined to have an activity in these screens
can be retested
in secondary screens of animal models of LED or in clinical trials to
determine activity
against behavioral or other symptoms of these diseases.
1. In vitro
100801 In vitro assays are performed to test the capacity of an agent to
inhibit aggregatiom
of truncated forms of alpha-synuclein, particularly SN1-115, SN1-119, SN1-122,
SN1-133
and SN1-135. The basis format for analyzing in vitro aggregation of alpha-
synuclein, albeit
in the context of full-length alpha-synuclein, is described by (Wood, J. Biol.
Chem. 274,
19509-19512 (1999)). Truncated fragments can be phosphorylated for performing
the assay.
The assay can also be performed with full-length phosphorylated alpha
synuclein.
Phosphorylation is preferably at position 129. Synuclein can be phosphorylated
in vitro using
a serine lcinase. In the present methods, the assay is performed in the
presence of an agent
being tested. The rate or extent of aggregation of alpha-synuclein in the
presence of an agent
is determined and compared with the rate or extent of aggregation of alpha-
synuclein in a
contemporaneous or historical control in which the agent was omitted. A
reduction in the
rate or extent of aggregation in the presence of the agent relative to the
control indicates that
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WO 2006/045037 PCT/US2005/037875
the agent has activity in inhibiting aggregation of truncated forms of alpha-
synuclein. This
activity is potentially useful in treating or preventing Lewy Body diseases.
2. Cellular Assays
[00811 Some cellular assays are performed on cells transfected with nucleic
acids encoding
truncated forms of alpha-synuclein as described above (particularly SN I -115,
SNI -119, SN I -
122, SN1-133 and SN1-135), optionally with a hereditary variation, such as
Ala30Pro or
Ala53Thr. Cells can also bear mutations in other genes associated with
Parkinson's disease,
such as leucine rich repeat kinase PARK8. Such cells are contacted with an
agent under test,
and the rate of extent of aggregation of the truncated alpha-synuclein is
measured. The rate
of extent of aggregation of alpha-synuclein is then compared to that of
similarly transfected
control cells in the absence of the agent. Aggregation can be monitored by
immunohistochemical analysis, light microscopy, sedimentation, or by gel
analysis. Gel
analysis can detect formation of dimmers, trimers or higher oligomers as well
as inability of
synuclein to enter gels due to a high level of oligomerization. A reduction in
the rate or
extent of aggregation in the presence of the test agent relative to the
control indicates the
agent has activity has a pharmacological activity in inhibiting aggregation of
truncated forms
of alpha-synuclein. This activity is potentially useful in treating or
preventing Lewy Body
diseases.
[0082] Other cellular assays are performed on cells transfected with nucleic
acids encoding
full-length alpha-synuclein, optionally with a hereditary variation, such as
Ala30Pro or
Ala53Thr. Cells can also have mutations in other genes associated with
Parkinson's disease
such as leucine rich repeat kinase, PARK& Similar assays can also be performed
on cells
naturally expressing alpha synuclein. Such cells are contacted with an agent
under test and
the rate or extent of formation of truncated forms of alpha-synuclein
(particularly SN1-115,
SN1-119, SN1-122, SN1-133 and SN 1-135) and/or phosphorylated or nitrated
forms of
synuclein is/are measured. The presence of these forms can be detected by
Western blotting
using one or more antibodies to alpha-synuclein. End specific antibodies
(i.e., antibodies that
bind to a truncated form without binding to full length aIpha-synuclein) are
particularly
useful for this analysis. Collections of antibodies having different epitope
specificities can
also be used. For example, presence of truncated forms of alpha-synuclein can
be shown by
presence of bands when blotted with antibodies recognizing an epitope N-
terminal of an
amino acid segment defined approximately by amino acids 115-125 or 118-135
(particularly
23
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PCT/US2005/037875
SN1-115, SN1-119, SN1-122, SN1-133, and SN1-135) of intact alpha-synuclein,
and, and
lack of bands when blotted with an antibody recognizing an epitope C-terminal
of this region.
The rate or extent of formation of truncated forms of alpha-synuclein and/or
phosphorylated
or nitrated forms in the presence of agent is compared with that of comparable
control cells
in the absence of agent. A reduction in the rate or extent of formation of
truncated forms of
alpha-synuclein in the presence of the test agent relative to the control
indicates that the agent
has a pharmacological activity that inhibits processing of alpha-synuclein to
its truncated
forms. This activity is useful for treating or preventing LBD.
3. Transgenic Animal Assays
[0083] Transgenic animals have a transgene expressing a truncated form of
alpha-synuclein
as described above (particularly SN1-115, SN I -119, SN1-122, SN1-133 or SN1-
135),
optionally with a hereditary variation, such as Ala30Pro or Ala53Thr.
Transgenic animals
can also bear mutations in other genes associated with Parkinson's disease
such as leucine
rich repeat lcinase, PARK8. Such an animal is contacted with an agent under
test, and the rate
of extent of aggregation of the truncated form of alpha-synuclein is measured
compared with
that in a contemporaneous or historical control. The control is usually a
similar transgenic
animal of the same species that has not been exposed to the agent. Aggregation
of alpha-
synuclein in a transgenic animal can be monitored by Western blotting or
immunohistochemistry as described in the examples. Alternatively or
additional, activity of
the agent in such transgenic animals can be determined from behavioral
characteristics such
as motor or cognitive characteristics, as described in the Examples. In such
assays,
pharmacological activity of the agent is shown by improved motor or cognitive
characteristics (i.e., decrease impairment of such characteristics) relative
to a comparable
control transgenic animal not exposed to the agent.
[0084] Other assays are performed on transgenic animals having a transgene
expressing a
full-length form of alpha-synuclein, optionally with a hereditary variation,
such as Ala30Pro
or Ala53, or mutations in other genes associated with Parkinson's disease such
as leucine rich
repeat lcinase, PARK8. Similar assays can be performed on nontransgenic
animals
expressing endogenous alpha synuclein. Such animals are contacted with an
agent under test,
and the rate or extent of appearance of truncated forms of alpha-synuclein
(particularly SN1-
115, SN1-119, SN1-122, SN1-133 or SN1-135) is detected, optionally with a
hereditary
variation, such as Ala30Pro or Ala53Th. Such forms can be detected using
Western blotting
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WO 2006/045037 PCT/US2005/037875
or immunohistochemical analysis using appropriate anti-alpha-synuclein
antibodies (as
described for the cellular assays). The rate of extent of appearance of
truncated forms of
alpha-synuclein and/or phosphorylated or nitrated forms is compared with the
rate or extent
of appearance of such forms in a contemporaneous or historical control
constituting a
comparable transgenic animal that has not been exposed to the agent. A
reduction in the rate
or extent of appearance of the truncated forms of alpha-synuclein in the
animal exposed to
the test agent relative to the control indicates that agent has activity in
inhibiting processing
of full-length alpha-synuclein to truncated forms.
4. Agents to be Screened
[0085] Agents to be screened include antibodies to alpha-synuclein, peptides
of alpha-
synuclein, drugs known or suspected to have activity in treating a LBD,
natural products, and
combinatorial libraries. Preferred peptides of alpha-synuclein are relatively
short peptides of
30, 25, 20 10, 5 or fewer amino acid including amino acids 114-117, 117-126,
118-125, 117-
120, 120-124, 130-136, 132-138, 131-135, 132-134, 133-137, 134-136 of alpha-
synuclein.
Optionally, an amino acid immediately on the N-terminal side of the cleavage
site that
generates C-terminal truncated forms of alpha-synuclein is replaced with a
transition state
analog amino acid that forms a nonhydrolizable bond between the two amino
acids flanking
the cleavage site, e.g., between residues 115-116, 119-120, 122-123, 133-134
and 135-136 of
alpha synuclein. Examples of analogs are transition state analogs are statine,
hydroxyethelene, hydroxyethelamine, AHPPA, ACHPA, and derivatives thereof. One
or
more amino acids of a natural alpha-synuclein sequence can also be substituted
with other
natural amino acids.
[00861 Natural products to be screened can also be obtained from the National
Cancer
Institute's Natural Product Repository, Bethesda, MD. Random libraries of
peptides or other
compounds can also be screened for suitability. Combinatorial libraries can be
produced for
many types of compounds that can be synthesized in a step-by-step fashion.
Such
compounds include polyp eptides, beta-turn mimetics, polysaccharides, phospho
lipids,
hormones, prostaglandins, steroids, aromatic compounds, heterocyclic
compounds,
benzodiazepines, oligomeric N-substituted glycines and oligocarbamates. Large
combinatorial libraries of the compounds can be constructed by the encoded
synthetic
libraries (ESL) method described in Affymax, WO 95/12608, Affymax, WO
93/06121,
Columbia University, WO 94/08051, Pharmacopeia, WO 95/35503 and Scripps, WO
CA 02912912 2015-11-20
95/30642. Peptide libraries can also be generated by phage display methods.
See, e.g., Devlin,
WO 91/18980. Combinatorial libraries and other compounds can initially be
screened for
suitability by determining their capacity to bind to alpha-synuclein.
VI. Toxicity Assays
[00871 Analogous strategies to those described in the screening assays can be
used to
determine whether existing drugs, foods, environmental toxins, and other
compounds exert
toxic effects via promotion of alpha-synuclein processing, phosphorylation or
aggregation.
Such assays are performed in the same manner as the screening assays. Toxic
activity is
indicated by the opposite result to pharmacological activity in the screening
assays.
VII. Isolation of Protease
[0088] Processing of full-length alpha-synuclein to the truncated forms of the
invention is
effected by a protease. The protease can be purified using an inhibitor
identified by the
screening methods discussed above. A preferred inhibitor is a peptide of alpha-
synttclein of e.g.,
up to 20 contiguous amino acids from SEQ ID NO: 1 including residues 114-117,
111-126,
113-126, 113-119, 117-121 or 120-125, or 130-136, 132-138, 131-135, 133-134,
133-137, or
135-136, in which a residue N-terminal to the cleavage site (e.g., between
residues 115-116,
119-120, 122-123, 133-134 and 135-136) has been replaced by a transition state
analog. Such
an inhibitor is used as an affinity purification reagent to purify the
protease from extracts of
brain cells. Such cells can be obtained from cadaver of a normal individual or
one who has
suffered from a LBD disease. Levels of protease may be elevated in the latter.
The protease can
be assayed by presenting it with an alpha-synuclein substrate and monitoring
formation of
cleavage products. End-specific antibodies described below are useful for
detection of cleavage
products. The substrate can be, for example, the natural human form of alpha-
synuclein
described above, a fragment thereof containing residues flanking both sides of
the cleavage site,
or a mutant form thereof in which the mutation is associated with a hereditary
form of LBD.
Optionally, the C-terminus of the substrate can be immobilized to the solid
phase, and the N-
terminus to a label. Cleavage of the substrate releases the label to the
liquid phase. The liquid
phase can readily be separated from the solid phase, and the amount of label
quantified as a
measure of proteolytic activity.
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VIII. End-specific antibodies
[0089] The invention provides end-specific antibodies. Such antibodies
specifically bind to
a truncated form of alpha-synuclein (at the C-terminus), preferably a form
selected of the
group consisting of SN1-115, SN1-116, SN1-117, SN1-118, SN1-119, 1-120, 1-121,
1-122,
1-123, 1-124, 1-125, 1-126 without specifically binding to full-length alpha-
synuclein.
Preferred antibodies are end-specific for SN1-115, SN1-119, SN1-122, SN1-133
and SNI-
135. Such antibodies are useful for in vivo imaging of alpha-synuclein
deposits, as
therapeutic agents (see below), and for detecting cleavage products resulting
from proteolytic
cleavage of alpha-synuclein in the screening methods described above. End-
specific
antibodies are also provided to corresponding C-terminal fragments, e.g., 116-
140, 117-140,
118-140, 119-140, 120-140, 121-140, 122-140, 123-140, 124-140, 125-140, 126-
140, 134-
140 and 136-140. Preferred fragments are 116-140, 120-140, 123-140, 134-140
and 136-140.
The end-specific antibodies recognize the N-terminus of these fragments such
that they
specifically bind to the fragment without specifically binding to full-length
alpha synuclein.
[0090] Preferred end specific antibodies are ELADW-101 (polyclonal) and 12C6
(monoclonal) specific for the C-terminus of SN1-119, and ELADW-105
(polyclonal) and
7G8 (monoclonal) specific for the C-terminus of SN1-122. The monoclonals are
mouse
monoclonals expressed by hybridomas produced by conventional methods.
[0091] Such antibodies can be generated by immunizing a laboratory animal with
alpha-
synuclein or a fragment thereof to induce antibodies, and screening the
resulting antibodies to
identify those having the desired binding specificity. Optionally,
immunization can be
performed with relatively short peptides of less than 20 amino acids, usually
7 or 8 amino
acids that include the C-terminus of the truncated fragments of the invention
(e.g., SN 99-
118, SN106-115, SN 110-119, SN-113-122, SN126-133, SN128-135 . Optionally,
such
short peptides are linked to a carrier that helps elicit an immune response.
For example,
the peptide CGGDMPVD (SEQ 1D NO:10) which corresponds to amino acids SN 115-
119
with a COG linker is useful for generating antibodies such as ELADW-101 and
12C6, and
the peptide
CGGVDPDN (SEQ II) NO:10) which corresponds to amino acids 118-122 with a CGG
linker is useful for generating antibodies ELADW-105 and 7G8.
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CA 02912912 2015-11-20
[0092] Optionally, specific binding to a labeled or immobilized truncated
fragment can be
performed in competition with unlabelled full-length alpha-synuclein.
Optionally, large
libraries of antibodies can be screened simultaneously using the phage display
technique.
100931 The production of non-human monoclonal antibodies, e.g., murine, guinea
pig, primate,
rabbit or rat, can be performed as described by Harlow & Lane, Antibodies, A
Laboratory
Manual (CSHP NY, 1988). Complete Freund's adjuvant followed by incomplete
adjuvant is
preferred for immunization of laboratory animals. Rabbits or guinea pigs are
typically used for
making polyclonal antibodies. Mice are typically used for making monoclonal
antibodies.
Binding can be assessed, for example, by Western blot or ELISA. The smallest
fragment to
show specific binding to the antibody defines the epitope of the antibody.
Alternatively, epitope
specificity can be determined by a competition assay is which a test and
referente antibody
compete for binding to alpha-synuclein. If the test and reference antibodies
compete, then they
bind to the same epitope or epitopes sufficiently proximal that binding of one
antibody
interferes with binding of the other.
[0094] Chimeric and humanized antibodies have the same or similar "binding
specificity and
affinity as a mouse or other nonhuman antibody that provides the starting
material for
construction of a chimeric or humanized antibody. Chimeric antibodies are
antibodies whose
light and heavy chain genes have been constructed, typically by genetic
engineering, from
immunoglobulin gene segments belonging to different species. For example, the
variable (V)
segments of the genes from a mouse monoclonal antibody may be joined to human
constant (C)
segments, such as IgGI and IgG4. Human isotype IgGI is preferred. In some
methods, the
isotype of the antibody is human IgGI. IgM antibodies can also be used in some
methods. A
typical chimeric antibody is thus a hybrid protein consisting of the V or
antigen-binding
domain from a mouse antibody and the C or effector domain from a human
antibody.
[0095] Humanized antibodies have variable region framework residues
substantially from a
human antibody (termed an acceptor antibody) and complementarity determining
regions
substantially from a mouse-antibody, (referred to as the donor
immunoglobulin). See, Queen et
al., Proc, Natl. Acad. Sc!. USA 86:10029-10033 (1989), WO 90/07861, US
5,693,762, US
5,693,761, US 5,585,089, US 5,530,101, and Winter, US 5,225,539. The constant
region(s), if
present, are also substantially or entirely from a human immunoglobulin. The
human variable
CA 02912912 2015-11-20
domains are usually chosen from human antibodies whose framework sequences
exhibit a high
degree of sequence identity with the murine variable region domains from which
the CDRs
were derived. The heavy and light chain variable region framework residues can
be derived
from the same or different human antibody sequences. The human antibody
sequences can be
the sequences of naturally occurring human antibodies or can be consensus
sequences of
several human antibodies. See Carter et al, WO 92/22653. Certain amino acids
from the human
variable region framework residues are selected for substitution based on
their possible
influence on CDR conformation and/or binding to antigen. Investigation of such
possible
influences is by modeling, examination of the characteristics of the amino
acids at particular
locations, or empirical observation of the effects of substitution or
mutagenesis of particular
amino acids.
[0096] Human antibodies against alpha-synuclein are provided by a variety of
techniques
described below. Some human antibodies are selected by competitive binding
experiments, or
otherwise, to have the same epitope specificity as a particular mouse
antibody. Techniques for
producing human antibodies include the trioma methodology of Oestberg et al,
Hybridoma
2:361-367 (1983); Oestberg, US Patent No. 4,634,664; and Engleman et al, US
Patent
4,634,666, use of non-human transgenic mammals having transgenes encoding at
least a
segment of the human immunoglobulin locus as described by, e.g., Lonberg et
al, WO 93/1222,
US 5,877,397, US 5,874,299, US 5,814,318, US 5,789,650, US 5,770,429, US
5,661,016, US
5,633,425, US 5,625,126, US 5,569,825, US 5,545,806, Nature 148, 1547-1553
(1994), Nature
Biotechnology 14, 826 (1996), Kucherlapati, WO 91/10741 and phage display
methods see, e.g.,
Dower et al,WO 91/17271 and McCafferty eta!, WO 92/01047, US 5,877,218, US
5,871,907,
US 5,858,657, US 5,837,242, US 5,733,743 and US 5,565,332.
100971 The heavy and light chain variable regions of chimeric, humanized, or
human
antibodies can be linked to at least a portion of a human constant region. The
choice of constant
region depends, in part, whether antibody-dependent complement and/or cellular
mediated
toxicity is desired. For example, isotopes IgGI and IgG3 have complement
activity and isotypes
IgG2 and IgG4 do not. Choice of isotype can also affect passage of antibody
into the brain.
Human isotype IgG1 is preferred. Light chain constant regions can be lambda or
kappa.
Antibodies can be expressed as tetramers containing two light and two heavy
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chains, as separate heavy chains, light chains, as Fab, Fab' F(ab')2, and Fv,
or as single chain
antibodies in which heavy and light chain variable domains are linked through
a spacer.
100981 In another embodiment, monoclonal antibodies specifically binding to an
epitope
within residues 109-120, or 115-122, of alpha synuclein, or a discontinuous
epitope within
residues 43-51 and 58-65, or end-specific to the C-terminus of alpha-synuclein
are also
provided, including humanized, chimeric and human forms thereof. An end-
specific
antibody to the C-terminus of alpha-synuclein can be recognized by capacity to
specifically
bind to alpha-synuclein as a free protein without specifically binding to
alpha synuclein as a
component of a fusion protein when the C-terminus of alpha-synuclein is linked
to a second
peptide. These antibodies can be screened for therapeutic activity, and if
positive results are
obtained, can be used in therapeutic methods. The antibodies can also be used
in detecting
fragments of alpha-synuclein as described above.
IX. Diagnostics
100991 The invention provides methods of in vivo imaging LBs in a patient.
Such methods
are useful to diagnose or confirm diagnosis of a Lewy Body disease of PD or
susceptibility
thereto. For example, the methods can be used on a patient presenting with
symptoms of
dementia. If the patient has LBs, then the patient is likely suffering from a
Lewy Body
disease. The methods can also be used on asymptomatic patients. Presence of
abnormal
deposits of amyloid indicates susceptibility to future symptomatic disease.
The methods are
also useful for monitoring disease progression and/or response to treatment in
patients who
have been previously diagnosed with a Lewy Body disease.
101001 The methods work by administering an end-specific antibody as described
above
that binds to alpha-synuclein in the patient and then detecting the antibody
after it has bound.
If desired, a clearing response can be avoided by using antibody fragments
lacking a full
length constant region, such as Fabs. In some methods, the same antibody can
serve as both a
treatment and diagnostic reagent.
10101] Diagnostic imaging can also be performed using an antibody specific for
phosphorylated alpha synuclein, such as the 11A5 (phosphor-specific) or 5C12
(binds to
phosphorylated and nonphosphorylated forms of alpha synuclein) monoclonal
described in
copending application 10/984,192. Presence of phosphorylated alpha synuclein
associated
with deposits of alpha synuclein is an indication of synucleinopathic disease
or susceptibility
thereto. Presence of ubiquitinated alpha-synuclein is also a marker of
disease. Such can be
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detected using a two step assay in which alpha-synuclein is precipitated with
a first antibody
to alpha-synuclein, and the amount of ubiquitinated alpha-synuclein is
detected using an
antibody to ubiquitin.
101021 Diagnostic reagents can be administered by intravenous injection into
the body of
the patient, or directly into the brain by intracranial injection or by
drilling a hole through the
skull. The dosage of reagent should be within the same ranges as for treatment
methods.
Typically, the reagent is labeled, although in some methods, the primary
reagent with affinity
for alpha-synuclein is unlabelled and a secondary labeling agent is used to
bind to the
primary reagent. The choice of label depends on the means of detection. For
example, a
fluorescent label is suitable for optical detection. Use of paramagnetic
labels is suitable for
tomographic detection without surgical intervention. Radioactive labels can
also be detected
using PET or SPECT.
[0103] Diagnosis is performed by comparing the number, size and/or intensity
of labeled
loci to corresponding base line values. The base line values can represent the
mean levels in
a population of undiseased individuals. Base line values can also represent
previous levels
determined in the same patient. For example, base line values can be
determined in a patient
before beginning treatment, and measured values thereafter compared with the
base line
values. A decrease in values relative to base line signals a positive response
to treatment.
[01041 End-specific antibodies are also useful to determine whether truncated
forms of
alpha-synuclein are present in cerebrospinal fluid or other body tissues or
fluids. Presence of
such forms at significantly changed, usually greater, levels (i.e., greater or
less than mean
plus one standard deviation) in a patient relative to the normal level in a
population of
undiseased individuals is indicative of presence or susceptibility to a LBD.
X. Methods of Treatment
[0105] The invention provides several methods of preventing or treating Lewy
Body
disease in patients suffering from or at risk of such disease. Therapeutic
agents include the
truncated forms of alpha-synuclein described above, particularly SN1-1 15, SN-
1-119, SN1-
'' 122, SN1-133 and SN1-135 and fragments thereof effective to induce
antibodies, end-
specific antibodies as described above, and inhibitors of aggregation of
truncated fragments
of alpha-synuclein or proteolytic processing of alpha-synuclein as described
above.
Optionally, the fragments are phosphorylated particularly at position 129.
Other agents
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include full-length phosphorylated alpha synuclein, preferably at position
129, agents that
inhibit phosphorylation of alpha synuclein, or which promote removal of
phosphorylated alpha
synuclein by ubiquitination or otherwise, or which promote or inhibit
ubiquitination. General
approaches for administering agents to patients suffering or at risk of LBD
are described in
copending application USSN 60/423,012 filed November 1, 2002, and WO
2000/072876 filed
June 1,2000, and WO 2004/041067, filed October 31, 2003.
101061 Patients amenable to treatment include individuals at risk of disease
of a LBD but not
showing symptoms, as well as patients presently showing symptoms. Therefore,
the present
methods can be administered prophylactically to individuals who have a known
genetic risk of
a LBD. Such individuals include those having relatives who have experienced
this disease, and
those whose risk is determined by analysis of genetic or biochemical markers.
Genetic markers
of risk toward PD include mutations in the alpha-synuclein or Parkin, UCHLI,
and CYP2D6
genes; particularly mutations at positions 30 and 53 of the alpha-synuclein
gene. Individuals
presently suffering from Parkinson's disease can be recognized from its
clinical manifestations
including resting tremor, muscular rigidity, bradykinesia and postural
instability.
101071 In some methods, the patient is free of clinical symptoms or risk
factors any
amyloidogenic disease other than one characterized by Lewy bodies. In some
methods, the
patient is free of clinical symptoms or risk factors of any disease
characterized by extracellular
amyloid deposits. In some methods, the patient is free of diseases
characterized by amyloid
deposits of All peptide. In some methods, the patient is free of clinical
symptoms and risk
factors of Alzheimer's disease. In some methods, the patient has concurrent
Alzheimer's disease
and a disease characterized by Lewy bodies. In some methods, the patient has
concurrent
Alzheimer's and Parkinson's disease.
[0108] In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,
30). Usually,
however, it is not necessary to begin treatment until a patient reaches 40,
50, 60 or 70.
Treatment typically entails multiple dosages over a period of time. Treatment
can be monitored
by assaying antibody, or activated T-cell or B-cell responses to a therapeutic
agent (e.g., a
truncated form of alpha-synuclein peptide) over time. If the response falls, a
booster dosage is
indicated.
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[0109] In prophylactic applications, pharmaceutical compositions or
medicaments are
administered to a patient susceptible to, or otherwise at risk of a LBD in
regime comprising
an amount and frequency of administration of the composition or medicament
sufficient to
eliminate or reduce the risk, lessen the severity, or delay the outset of the
disease, including
physiological, biochemical, histologic and/or behavioral symptoms of the
disease, its
complications and intermediate pathological phenotypes presenting during
development of
the disease. In therapeutic applications, compositions or medicates are
administered to a
patient suspected of, or already suffering from such a disease in a regime
comprising an
amount and frequency of administration of the composition sufficient to cure,
or at least
partially arrest, the symptoms of the disease (physiological, biochemical,
histologic and/or
behavioral), including its complications and intermediate pathological
phenotypes in
development of the disease. An amount adequate to accomplish therapeutic or
prophylactic
treatment is defined as a therapeutically- or prophylactically-effective dose.
A combination
of amount and dosage frequency adequate to accomplish therapeutic or
prophylactic
treatment is defined as a therapeutically or prophylatically-effective regime.
In both
prophylactic and therapeutic regimes, agents are usually administered in
several dosages until
a sufficient immune response has been achieved. Typically, the immune response
is
monitored and repeated dosages are given if the immune response starts to
wane.
101101 In some methods, administration of an agent results in reduction of
intracellular
levels of aggregated alpha-synuclein. In some methods, administration of the
agent results in
a reduction in levels of C-terminal truncated forms of alpha-synculein. In
some methods,
administration of an agent results in improvement in a clinical symptom of a
LBD, such as
motor or cognitive function in the case of Parkinson's disease. In some
methods, reduction in
intracellular levels of aggregated alpha-synuclein or improvement in a
clinical symptom of
disease is monitored at intervals after administration of an agent.
[0111] Effective doses of the compositions of the present invention, for the
treatment of the
above described conditions vary depending upon many different factors,
including means of
administration, target site, physiological state of the patient, whether the
patient is human or
an animal, other medications administered, and whether treatment is
prophylactic or
therapeutic. Usually, the patient is a human but nonhuman mammals including
transgenic
mammals can also be treated. Treatment dosages need to be titrated to optimize
safety and
efficacy.
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[0112] In some methods, the agent is a truncated fragment of alpha-synuclein
or a fragment
thereof capable of inducing antibodies to alpha-synuclein. The amount of such
a fragment
depends on whether adjuvant is also administered, with higher dosages being
required in the
absence of adjuvant. The amount of a fragment for administration sometimes
varies from 1-
500 jig per patient and more usually from 5-500 jig per injection for human
administration.
Occasionally, a higher dose of 1-2 mg per injection is used. Typically about
10, 20, 50 or
100 jig is used for each human injection. The mass of fragment also depends on
the mass
ratio of immunogenic epitope within the fragment to the mass of fragment as a
whole.
Typically, le to 10-5 micromoles of immunogenic epitope are used for microgram
of
fragment. The timing of injections can vary significantly from once a day, to
once a year, to
once a decade. On any given day that a dosage of immunogen is given, the
dosage is greater
than 1 jig/patient and usually greater than 10 lig/ patient if adjuvant is
also administered, and
greater than 10 jig/patient and usually greater than 100 jig/patient in the
absence of adjuvant.
A typical regimen consists of an immunization followed by booster injections
at time
intervals, such as 6 week intervals. Another regimen consists of an
immunization followed
by booster injections 1, 2 and 12 months later. Another regimen entails an
injection every
two months for life. Alternatively, booster injections can be on an irregular
basis as indicated
by monitoring of immune response.
101131 Truncated fragments of alpha-symiclein can also be administered in the
form of
nucleic acids encoding the fragments operably linked to one or more regulatory
elements to
ensure expression of the truncated fragment of alpha-synuclein. Doses for
nucleic acids
encoding immunogens range from about 10 ng to 1 g, 100 ng to 100 mg, 1 jig to
10 mg, or
30-300 jig DNA per patient. Doses for infectious viral vectors vary from 10-
100, or more,
virions per dose.
10114] Some methods involve passive immunization with an end-specific
antibody. In
such methods, the dosage ranges from about 0.0001 to 100 mg/kg, and more
usually 0.01 to 5
mg/kg, of the host body weight. For example dosages can be 1 mg/kg body weight
or 10
mg/kg body weight or within the range of 1-10 mg/kg or, in other words, 70 mg
or 700 mg or
within the range of 70-700 mg, respectively, for a 70 kg patient. An exemplary
treatment
regime entails administration once per every two weeks or once a month or once
every 3 to 6
months. In some methods, two or more monoclonal antibodies with different
binding
specificities are administered simultaneously, in which case the dosage of
each antibody
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administered falls within the ranges indicated. Antibody is usually
administered on multiple
occasions. Intervals between single dosages can be weekly, monthly or yearly.
Intervals can
also be irregular as indicated by measuring blood levels of antibody to alpha-
synuclein in the
patient. In some methods, dosage is adjusted to achieve a plasma antibody
concentration of
1-10004g/ml and in some methods 25-300 p.g/ml. Alternatively, antibody can be
administered as a sustained release formulation, in which case less frequent
administration is
required. Dosage and frequency vary depending on the half-life of the antibody
in the
patient. In general, human antibodies show the longest half life, followed by
humanized
antibodies, chimeric antibodies, and nonhuman antibodies. The dosage and
frequency of
administration can vary depending on whether the treatment is prophylactic or
therapeutic. In
prophylactic applications, a relatively low dosage is administered at
relatively infrequent
intervals over a long period of time. Some patients continue to receive
treatment for the rest
of their lives. In therapeutic applications, a relatively high dosage at
relatively short intervals
is sometimes required until progression of the disease is reduced or
terminated, and
preferably until the patient shows partial or complete amelioration of
symptoms of disease.
Thereafter, the patent can be administered a prophylactic regime.
[01151 Therapeutic agents can be administered by parenteral, topical,
intravenous, oral,
subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal,
intranasal or
intramuscular means for prophylactic and/or therapeutic treatment. The most
typical route of
administration of an immunogenic agent is subcutaneous although other routes
can be equally
effective. The next most common route is intramuscular injection. This type of
injection is
most typically performed in the arm or leg muscles. In some methods, agents
are injected
directly into a particular tissue where deposits have accumulated, for example
intracranial
injection. Intramuscular injection or intravenous infusion is preferred for
administration of
antibody. In some methods, particular therapeutic antibodies are injected
directly into the
cranium. In some methods, antibodies are administered as a sustained release
composition or
device, such as a MedipadT" device. Small molecules that act by inhibiting
protease
processing of alpha-synuclein can be administered intravenously if the small
molecules pass
through the blood brain barrier sufficiently for therapeutic or prophylactic
efficacy or directly
into the cranium otherwise.
[01161 Agents of the invention can optionally be administered in combination
with other
agents that are at least partly effective in treatment of LBD. Agents of the
invention can also
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be administered in conjunction with other agents that increase passage of the
agents of the
invention across the blood-brain barrier.
[01171 Immunogenic agents are sometimes administered in combination with an
adjuvant.
A variety of adjuvants can be used in combination with a peptide, such as
alpha-synuclein, to
elicit an immune response. Preferred adjuvants augment the intrinsic response
to an
immunogen without causing conformational changes in the immunogen that affect
the
qualitative form of the response. Preferred adjuvants include aluminum
hydroxide and
aluminum phosphate, 3 De-O-acylated monophosphoryl lipid A (M-PL) (see GB
2220211
(R.D3I InununoChem Research Inc., Hamilton, Montana, now part of Corixa).
StimulonTm
QS-21 is a triterpene glycoside or saponin isolated from the bark of the
Quillaja Saponaria
Molina tree found in South America (see Kensil et al., in Vaccine Design: The
Subunit and
Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); US Patent
No.
5,057,540), (Aquila BioPharmaceuticals, Framingham, MA). Other adjuvants are
oil in water
emulsions (such as squalene or peanut oil), optionally in combination with
immune
stimulants, such as monophosphoryl lipid A (see Stoute et at., N. Engl. .1.
Med. 336, 86-91
(1997)), pluronic polymers, and killed mycobacteria. Another adjuvant is CpG
(WO
98/40100). Alternatively, alpha-synuclein can be coupled to an adjuvant.
However, such
coupling should not substantially change the conformation of alpha-synuclein
so as to affect
the nature of the immune response thereto. Adjuvants can be administered as a
component of
a therapeutic composition with an active agent or can be administered
separately, before,
concurrently with, or after administration of the therapeutic agent.
[01181 A preferred class of adjuvants is aluminum salts (alum), such as alum
hydroxide,
alum phosphate, alum sulfate. Such adjuvants can be used with or without other
specific
immunostimulating agents such as MPL or 3-DMP, QS-21, polymeric or monomeric
amino
acids such as polyglutamic acid or polylysine. Another class of adjuvants is
oil-in-water
emulsion formulations. Such adjuvants can be used with or without other
specific
immunostimulating agents such as muramyl peptides (e.g., N-acetylmuramyl-L-
threonyl-D-
isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutaraine (nor-
MDP), N-
acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(r-2'dipalmitoyl-sn-glycero-
3-
hydroxyphosphoryloxy)-ethylamine (MTP-PE), N-acetylglucsaminyl-N-acetylmuramyl-
L-
Al-D-isoglu-L-Ala-dipalmitoxy propylamide (DTP-DPP) theramideTM), or other
bacterial
cell wall components. Oil-in-water emulsions include (a) MF59 (WO 90/14837),
containing
5% Squalene, 0.5% Tween 80TM, and 0.5% Span 85 (optionally containing various
amounts of
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MTP-PE) formulated into submicron particles using a rnicrofluidizer such as
Model 110Y
microfluidizer (Microfiuidics, Newton MA), (b) SAF, containing 10% Squalene,
0.4%
Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP, either microfluidized
into a
submicron emulsion or vortexed to generate a larger particle size emulsion,
and (c) RibiTM
adjuvant system (RAS), (Ribi ImmunoChem, Hamilton, MT) containing 2% squalene,
0.2%
Tween 80, and one or more bacterial cell wall components from the group
consisting of
monophosphoryllipid A (IVIPL), trehalose dimycolate (TDM), and cell wall
skeleton (CWS),
preferably MPL + CWS (DetoxTm).
[0119] Another class of preferred adjuvants is sapoain adjuvants, such as
Stimulonrm (QS-
21, Aquila, Framingham, MA) or particles generated there from such as ISCOMs
(immunostimulating complexes) and ISCOMATRLX. Other adjuvants include RC-529,
GM-
CSF and Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant
(IFA).
Other adjuvants include cytokines, such as interleukins (e.g., IL-1, IL-2, IL-
4, IL-6, IL-12,
IL13, and 11-15), macrophage colony stimulating factor (M-CSF), granulocyte-
macrophage
colony stimulating factor (GM-CSF), and tumor necro sis factor (TNF). Another
class of
adjuvants is glycolipid analogues including N-glycosylamides, N-glycosylureas
and N-
glycosylcarbamates, each of which is substituted in the sugar residue by an
amino acid, as
irnmuno-modulators or adjuvants (see US Pat. No. 4,8 55,283). Heat shock
proteins, e.g.,
HSP70 and HSP90, may also be used as adjuvants.
[0120] An adjuvant can be administered with an alpha-synuclein fragment as a
single
composition, or can be administered before, concurrent with or after
administration of the
alpha-synuclein fragment. The alpha-synuclein fragment and adjuvant can be
packaged and
supplied in the same vial or can be packaged in separate vials and mixed
before use. The
alpha-synuclein fragment and adjuvant are typically packaged with a label
indicating the
intended therapeutic application. If the alpha-Synuclein fragment and adjuvant
are packaged
separately, the packaging typically includes instructions for mixing before
use. The choice of
an adjuvant and/or carrier depends on the stability of the immunogenic
formulation
containing the adjuvant, the route of administration, the dosing schedule, the
efficacy of the
adjuvant for the species being vaccinated, and, in humans, a pharmaceutically
acceptable
adjuvant is one that has been approved or is approvable for human
administration by
pertinent regulatory bodies. For example, Complete Freund's adjuvant is not
suitable for
human administration. Alum, MR, and QS-21 are preferred. Optionally, two or
more
different adjuvants can be used simultaneously. Preferred combinations include
alum with
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MPL, alum with QS-21, MPL with QS-21, MPL or RC-529 with GNI-CSF, and alum, QS-
21
and MPL together. Also, Incomplete Fretmd's adjuvant can be used (Chang et
al., Advanced
Drug Delivety Reviews 32, 173-186 (1998)), optionally in combination with any
of alum,
QS-21, and MPL and all combinations thereof.
[0121] Agents of the invention are often administered as pharmaceutical
compositions
comprising an active therapeutic agent, i.e., and a variety of other
pharmaceutically
acceptable components. See Remington's Pharmaceutical Science ([5th ed., Mack
Publishing
Company, Easton, Pennsylvania, 1980). The preferred form depends on the
intended mode
of administration and therapeutic application. The compositions can also
include, depending
on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or
diluents, which
are defined as vehicles commonly used to formulate pharmaceutical compositions
for animal
or human administration. The diluent is selected so as not to affect the
biological activity of
the combination. Examples of such diluents are distilled water, physiological
phosphate-
buffered saline, Ringer's solutions, dextrose solution, and Hank's solution.
In addition, the
pharmaceutical composition or formulation may also include other carriers,
adjuvants, or
nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
[01221 Pharmaceutical compositions can also include large, slowly metabolized
macromolecules such as proteins, polysaccharides such as chitosan, polylactic
acids,
polyglycolic acids and copolymers (such as latex functionalized Sepharose(TM),
agarose,
cellulose, and the like), polymeric amino acids, amino acid copolyira ers, and
lipid aggregates
(such as oil droplets or liposomes). Additionally, these carriers can function
as
immunostimulating agents adjuvants).
[0123] For parenteral administration, agents of the invention can be
administered as
injectable dosages of a solution or suspension of the substance in a
physiologically acceptable
diluent with a pharmaceutical carrier that can be a sterile liquid such. as
water oils, saline,
glycerol, or ethanol. Additionally, auxiliary substances, such as wetting or
emulsifying
agents, surfactants, pH buffering substances and the like can be present in
compositions.
Other components of pharmaceutical compositions are those of petroleum,
animal, vegetable,
or synthetic origin, for example, peanut oil, soybean oil, and mineral oil. In
general, glycols
such as propylene glycol or polyethylene glycol are preferred liquid carriers,
particularly for
injectable solutions. Antibodies can be administered in the form of a depot
injection or
implant preparation which can be formulated in such a manner as to permit a
sustained
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release of the active ingredient. An exemplary composition comprises
monoclonal antibody
at 5 mg/mL, formulated in aqueous buffer consisting of 50 mM L-histidine, 150
mM NaC1,
adjusted to pH 6.0 with HCI. Compositions for parenteral administration are
typically
substantially sterile, substantially isotonic and manufactured under GMP
conditions of the
FDA or similar body.
[0124] Typically, compositions are prepared as injectables, either as liquid
solutions or
suspensions; solid forms suitable for solution in, or suspension in, liquid
vehicles prior to
injection can also be prepared. The preparation also can be emulsified or
encapsulated in
liposomes or micro particles such as polylactide, polyglycolide, or copolymer
for enhanced
adjuvant effect, as discussed above (see Langer, Science 249, 1527 (1990) and
Hanes,
Advanced Drug Delivety Reviews 28, 97-119 (1997). The agents of this invention
can be
administered in the form of a depot injection or implant preparation which can
be formulated
in such a manner as to permit a sustained or pulsatile release of the active
ingredient.
[0125] Additional formulations suitable for other modes of administration
include oral,
intranasal, and pulmonary formulations, suppositories, and transdermal
applications. For
suppositories, binders and carriers include, for example, polyalkylene glycols
or triglycerides;
such suppositories can be formed from mixtures containing the active
ingredient in the range
of 0.5% to 10%, preferably 1%-2%. Oral formulations include excipients, such
as
pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine,
cellulose, and magnesium carbonate. These compositions take the form of
solutions,
suspensions, tablets, pills, capsules, sustained release formulations or
powders and contain
10%-95% of active ingredient, preferably 25%-70%.
[0126] Topical application can result in transdermal or intradermal delivery.
Topical
administration can be facilitated by co-administration of the agent with
cholera toxin or
detoxified derivatives or subunits thereof or other similar bacterial toxins
(See Glenn etal.,
Nature 391, 851 (1998)). Co-administration can be achieved by using the
components as a
mixture or as linked molecules obtained by chemical crosslinking or expression
as a fusion
protein. Alternatively, transdermal delivery can be achieved using a skin path
or using
transferosomes (Paul eta),, Eur. J. Itnmunol. 25, 3521-24 (1995); Cevc et al.,
Biocheni.
Biophys. Acta 1368, 201-15 (1998)).
Examples
1. Detecting truncated forms of alpha-synuclein in a transgenic animal
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[0127] Transgenic mice having a nucleic acid encoding intact alpha-synuclein
operably
linked to a PDFG promoter were analyzed at 6 weeks, 3 months and 12 months
old. The
animals were euthanized and the cortex and hippocampus tissue from four mice
(2 male/2
female) was pooled. The tissue was homogenized in TBS (250 mM NaC1), and spun
at
150,000 x g for 15 minutes. The pellet was then extracted with 1% Triton-X 100
for 30 mm
at 4 degrees and spun as before. The resulting pellet was then extracted with
1% SDS for 30
min at 25 degrees and spun as before. Finally, the pellet was extracted with 8
M Urea/ 1%
SDS. This procedure resulted in four extracts which will be referred to as
Tris, Triton, SDS,
and Urea extracts in the description that follows.
[0128] Figs. IA and B show a Western blot of extracts from a transgenic mouse
and a
matched control with antibody ELADW-47. This antibody is a polyclonal that
binds to an
epitope within SN115-122 (but does not necessarily require each amino acid for
some
binding to occur). The antibody preferentially binds the human form of alpha-
synuclein but
also binds the mouse form to a lesser extent. Figs. lA and B shows an alpha-
synuclein band
at 14 kDa for both the control mouse and the transgenic mouse. The band is
stronger for the
transgenic mouse than the control. For the different extracts, the band is
most intense in the
Triton extract. This extract solubilizes membrane bound alpha-synuclein and
possibly Lewy
body-like inclusions. The Tris and particularly the Triton extractions of the
transgenic mouse
(but not the control) show a band at about 12 lcDa in a tricine buffer. This
is a truncated form
of alpha-synuclein. The molecular weight of the band corresponds to a length
of about 115-
120 amino acids.
[0129] Fig. 2 shows a Western blot with the same antibody as Figs. IA and B to
compare
the level of the truncated form of alpha-synuclein in mice of 3 months and 12
months in age.
The Figure shows that the truncated form appears more strongly in the 3 month
old mice.
Again, the truncated band does not appear in the control mice. The more
intense appearance
of the truncated form of alpha-synuclein early in development of the
transgenic mice
indicates that the truncated form of alpha-synuclein has a role early in the
pathogenesis of
Lewy Body disease.
[0130] Figs. 3A and B show a Western blot with a different antibody termed
12C1 (binds
epitope at amino acids 43-51 and 58-65, monoclonal, IgG1 k). This antibody
binds equally to
mouse and human forms of alpha-synuclein at an epitope including amino acids
43-51 and
58-65. Fig. 3 shows the truncated band of 12 IcDa in the Triton extract of the
transgenic
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mice. The same band appears much more faintly in the Triton extract of the
control mice.
Thus, processing of aIpha-synuclein to a truncated form occurs in both normal
mice and
transgenic mice, but more strongly in the latter. The greater extent of
processing in the
.= transgenic mice may be due to processing of the human alpha-
synuclein directly, or may be
due to the presence of human alpha-synuclein driving mouse alpha-synuclein
down a path
that is used to a lesser extent in nontransgenic mice.
[0131] Fig. 4 shows a further Western blot using the same antibody as Fig. 3.
This gel
shows two additional bands of molecular weights approximately 6 or 7 kDa. The
7 kDa band
appears more strongly in the transgenic mice than control mice. The 6 kDa band
appears
only in the transgenic mouse, and then only in the 3 mo sample. The 6 or 7 kDa
bands are
indicative of shorter N-terminal fragments of alpha-synuclein of length about
50-80 amino
acids.
[01321 Figs. 5A, B, C, D, E shows Western blots with four different antibodies
and epitope
maps of the binding sites of the antibodies. ELADW-44 is a polyclonal that
binds only to the
human form of alpha-synuclein (i.e., not the mouse form) It binds to at
epitope at amino
acids 103-105. ELADW-47 is a polyclonal that binds preferentially to the human
form but
also binds the mouse form. It binds to an epitope at amino acids 115-122.
ELADW-48 is a
polyclonal that binds the human and mouse forms equally. It binds to an
epitope between
amino acids 131 and 140. 8A5 is a monoclonal that binds to the human and mouse
forms
equally. It binds to the C-terminus of alpha-synuclein. Figs. 5A-E shows that
of these four
antibodies, only ELADW-47 generated a 12 kDa band indicative of a truncated
form of
alpha-spuclein. The result that ELADW48 did not give rise to this band is of
assistance in
mapping the site of cleavage. Because ELADW-47 did bind and ELADW-48 did not,
the site
of cleavage is bordered by the N-terminal end of the ELADW-47 epitope and the
C-terminal
amino acid of the ELADW48 epitope. Further, because some amino acids from the
ELADW-47 epitope must be present to allow binding and some of the ELADW-48
epitope
must be absent to prevent binding, the cleavage site is further confined to a
region
approximately within amino acids 118-135. When this data is considered with
the size of the
truncated fragment (about 115-120 amino acids) then the probable site of
cleavage is around
amino acids 118-121. The lack of binding by the C-terminal antibody 8A5 is
consistent with
this cleavage site. The lack of binding by the antibody ELADW-44, however,
requires
further comment. The lack of cleavage can be explained if a truncated form of
human alpha-
synuclein resulting from cleavage adapts a different conformation to intact
alpha-synuclein
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preventing binding of ELADW-44. Alternatively, the truncated form of alpha-
synuclein
present in transgenic mice to a greater extent than in normal mice represents
a form of mouse
alpha-synuelein. In this case, the greater amount of the truncated form in the
transgenic
mouse would be due to the presence of the human alpha-synuclein driving more
of the mouse
alpha-synuclein down a processing path that leads to truncated alpha-synuclein
relative to the
situation in a control mouse.
2. Detecting Truncated Forms of Alpha-Synuclein in the Brain of a Patient
with DLBD
[0133] This example compares alpha-synuclein species in LBs to those in the
remaining
soluble and particulate protein fractions of a DLBD brain. LBs and soluble
protein were
prepared from the cortex of a single DLBD patient (see Jensen et al., J. Biol.
Chem. 275
21500-21507 (2000)). Tissue was homogenized in Tris/sucrose (0.32 mM)/EDTA
(5mM)
and protease inhibitors buffer. The homogenate was spun at 1000 x g. The
supernatant was
subjected to a further spin at 150,000 x g. The supernatant from this spin was
used to prepare
a Tris soluble fraction of proteins. The pellet from the 1000 x g was
resuspended and used to
prepare a Lewy body fraction. Lewy bodies were purified by
hrununoprecipitation on
magnetic beads bearing anti-synuclein antibodies. The precipitate was then
extracted with 7
M Urea/2 M Thiourea/4% CHAPS. The unextracted material was reextracted with
Urea/Thiourea/CHAPS. The extracts from this step and the previous extraction
were then
pooled and analyzed by 2D PAGE and immunoblot. The unextracted residue was
subject to
further extraction with 90% formic acid. This extract was stored diluted to 9%
formic acid.
The extract was then analyzed by SDS PAGE and RP-HPLC. Little or no synuclein
was
found in this last extract, and what was present resembled the material
extracted by
Urea/Thiourea/CHAPS, indicating that the Urea/Thiourea/CHAPS gave a
comprehensive
extraction.
[0134] Synuclein species were resolved on 2-D gels and detected on Western
blots.
All 2D Western blots are shown with more acidic proteins on the left, more
basic on the right.
Multiple alpha-synuclein species, including phosphorylated and truncated
species, were
present in both LBs and the soluble brain fraction. The predominant
truncations were in the
C-terminal region of alpha-synuclein at approximately amino acids 118-125. An
additional
larger fragment cleaved close to the C-terminus was also observed. No beta or
gamma-
synuelein was detected in the LBs despite being found in the soluble protein
fraction. The
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CA 02912912 2015-11-20
WO 2006/045037 PCT/US2005/037875
alpha-synuclein in the LB preparation differed from that in the soluble
fraction in that it had
additional C-terminal cleavages, and that overall the truncated alpha-
synuclein species were
enriched in the LBs relative to the soluble protein fraction. In addition,
multiple alpha-
synuclein species species of higher molecular mass, approximately 25-35 kDa,
were detected only in
the LB preparation. These include ubiquitinated species, as identified by us
and by others
(Tofaris et al. J. Biol. Chem. 278(45): 44405-44411, 2003). The C-terminally
truncated
fragments are of the same size as those observed in the transgenic mouse model
of Example I
indicating a role in disease pathogenesis.
[0135] Figs. 6A, B, C shows Tris extracts probed with different antibodies,
subject to 2-D
gel electrophoresis and subjected to Western blotting. The dark spots present
toward the left
of the charts represent full-length alpha-synuclein. The most notable feature
is four spots in
the Syn-1 chart that are absent in the 8A5 chart. These four spots represent
truncated forms
of alpha-synuclein that are unable to bind the 8A5 antibody because of the
lack of a C-
terminal amino acid. These truncations correspond approximately to forms of SN
between 1-
118 and 1-125. Several additional spots are seen underneath and adjacent to
the full length
alpha-synuclein spots. The spots underneath the full length spots probably
represent minor
truncations from the C-terminus (i.e., synuclein 1-X, wherein X is 130-139) ,
since they react
with antibodies to phosphorylated S129 but not with 8A5. The spot adjacent the
full-length
spots but to the right represent a minor deletion from the N-terminus (due to
lack of this spot
in the blot with ELADW43).
[0136] Figs. 7A, B, C, D shows blots with additional antibodies. The four
spots are present
with 5C12 (111-118). Two of the spots are present with ELADW47 (115-122) and
the spots
are absent with LB509 (115-123). The spots may differ from each other both in
molecular
weight and in the presence or absence of posttranslational modification, such
as nitration or
phosphorylation. The shift of these fragments towards a basic pH, relative to
the full length
synuclein, is consistent with removal of a portion of the acidic C-terminal
sequence. These
results fix the sites of cleavage to within about amino acids 120-125 of alpha-
synuclein. Also
notable are several spots running slight below (lower molecular weight) or to
the left (lower
pH) than the unmodified synuclein spots. These likely may represent forms of
synuclein that
have undergone a small extent of N-terminal truncation and/or different
posttranslational
modification relative to the main spots. Note that some spots visualized by
BLADW43 and
8A5 in the blots of soluble protein are beta synuclein, particularly the
prominent spot to the
left and slightly above the full length alpha.
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WO 2006/045037 PCT/US2005/037875
[0137] Fig. 8 summarizes the sites of cleavage relative to the epitopes bound
by antibodies
used in the Western blotting.
[0138] Figs. 9A, B compare the Tris soluble proteins with proteins extracted
from Levvvy
bodies by 2D electrophoresis and Western blotting. The Tris blot on the Iefl
shows four spots
at lower molecular weight representing truncated forms of alpha-synuclein
(probably in the 1-
120 to 1-125 amino acid range). These are of relatively low intensity compared
to the spots
representative of full length alpha-synuclein. The blot of proteins from Lewy
bodies shows
more spots representative of truncated forms of alpha synuclein in the 1-120
to 1-125 range.
However, these arc of greater intensity relative to the spots representative
of full length alpha
synuclein. Also, apparent are two spots migrating faster than full-length
alpha synuclein but
slower than the collection of spots at the bottom of the blot. These spots
probably represent
truncations in the range 1-X wherein X is 130-139 amino acids. As above, these
spots react
with antibodies to phosphorylated S129 but not with 8A5.
[0139] Figs. 10A, B, C, D show the inununoblots of proteins from Lewy bodies
reprobed
with various C-terminal antibodies. All spots appear with Syn-1 (91-96) and
5C12 (111 -
118). With ELADW47, the spot running at the fastest speed and most basic
position in the
Syn-1 and 5C12 blots is missing. In the LB509 blot, the 12 kD spots
corresponding to those
in the Tris soluble samples are missing or faint, although the row of spots
just above them
("tier 3") still react. The absence or reduced intensity of certain spots in
the ELADW47 and
LB509 blots indicates that these spots represent truncated forms of alpha-
synuclein and are
consistent with cleavage occurring approximately between amino acids 120 and
125.
3. Detecting Aggregated Alpha-Synuclein in a Transgenic Animal
[0140] Transgenic animals are euthanized and brains are removed for
neurochemical and
neuropathological analysis Briefly, the right hemibrain is frozen and
homogenized for
determinations of aggregated and non-aggregated human alpha-synuclein
immunoreactivity
by Western blot (Masliah et aL, Science (2000) 287:1265). The left hemibrain
is fixed in 4%
paraformaldehyde, serially sectioned in the vibratome for immunocytochemistry
and
ultrastructural analysis.
[0141] Brain sections are immunostained with a rabbit polyclonal antibody
against human
alpha-synuclein (1:500). After an overnight incubation at 4 C, sections are
incubated with
biotinylated anti-rabbit secondary antibody followed by Avidin D-Horseradish
peroxidase
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CA 02912912 2015-11-20
WO 2006/045037 PCT/US2005/037875
(HR.?) complex (1 :200, ABC Elite, Vector). The reaction is visualized with
0.1% 3,3,-
diaminobenzidine tetrahydrochloride (DAB) in 50mM Tris-HC1 (pH 7.4) with
0.001% H202
and sections are then mounted on slides under Entellan. Levels of
itnmunoreactivity are semi
quantitatively assessed by optical densitometry using the Quantimet 570C.
These sections
are also studied by image analysis to determine the numbers of alpha-synuclein
immunoreactive inclusions and this reliable measure of alpha-synuclein
aggregation acts as a
valuable index of the anti-aggregation effects (Masliah et al. Science (2000)
287:1265).
10142] Analysis of patterns of neurodegeneration is achieved by analyzing
synaptic and
dendritic densities in the hippocampus, frontal cortex, temporal cortex and
basal ganglia
utilizing vibratome sections double-immunolabeled for synaptophysin and
microtubule-
associated protein 2 (MAP2) and visualized with LSCM. Additional analysis of
neurodegeneration is achieved by determining tyrosine hydroxylase (TH)
immunoreactivity
in the caudoputamen and substantia nigra (SN) as previously described
(Masliah, et al.
(2000)). Sections will be imaged with the LSCM and each individual image is
interactively
thresholded such that the TH-inununoreactive terminals displaying pixel
intensity within a
linear range are included. A scale is set to determine the pixel to Am ratio.
Then, this
information is used to calculate the % area of the neuropil covered by TH-
immunoreactive
terminals. These same sections are also utilized to evaluate the numbers of TH
neurons in the
SN.
4. Analysis of Alpha-Synuclein in LBD patients
[0143] To determine which species of a-synuclein are enriched in or unique to
disease tissue,
we have examined brain samples from patients with multiple system atrophy
(MSA) and a
familial Parkinson's disease mutation (A53T; Contursi kindred). Particulate
fractions of
MSA and Contursi brain were prepared by homogenizing brain tissue in 50 mM
Tris, 140
triM NaCl and 1%Triton respectively Age matched, control patients ("normals")
were
prepared identically to the disease brain. Samples were analyzed on western
blots of 1-D gels
and by ELLSA as described below, and also on 2-D gels. Part of the particulate
fraction was
analyzed. The rest was spun. The supernatant was also analyzed. The pellet was
extracted
in 7 M urea. The supernatant from this extraction was analyzed. The pellet was
further
extracted in 7 M urea/1% SDS. The supernatant was analyzed. Western blots
using an
antibody to detect total alpha-synuclein or to specifically alpha-synuclein
phosphorylated at
position 129 are shown in Figs. 11 A and B.
CA 02912912 2015-11-20
. .
[0144] The synuclein fractionated differently from the particulate fraction of
the Contursi
brain versus control brain. Most of the synuclein in the particulate fraction
of the normal
brain was soluble after homogenization in tris buffered sucrose but almost all
of the synuclein
in the Contursi brain required urea plus SDS for solubilization suggesting a
massive amount
of Lewy bodies in this patient. The synuclein in the Contursi patient was
strikingly different
from that in the control patient in the amount of ser 129 phosphorylation.
Only a small
amount of phosphorylated cc-synuclein was detected in the control patient
(left tracks),
whereas the Contursi patient (right tracks) had an extremely large amount of
phospho-
synuclein by comparison on western blots. Thus, the insolubility of synuclein
in the Contursi
brain was associated with a large increase in synuclein phosphorylation at ser
129.
The a-synuclein in the Contursi patient also differed from that in normal
brain in the
distribution of C-terminal truncations. C-terminally truncated a-synuclein
were observed in
both control and Contursi particulate brain fraction However, all detectable
truncations were
highly insoluble (urea/SDS extract) in the Contursi patient, whereas those in
the control brain
were soluble (tris buffered sucrose extract). The enrichment of the C-
terminally truncated
synuclein in a LB-enriched fraction of a Contursi patient is in agreement with
our finding of
C-terminally truncated synuclein enrichment in DLBD LBs. The MSA brain was
also
enriched in phospho (ser 129)-a-synuclein revealed C-terminal truncation and
an abundance
of phosphorylation and other acidic modifications also seen in LBs. High
levels of phospho
(ser 129) were also seen in the brain of a DLBD patient relative to an
undiseased control.
5. Identification of Truncated Svnuclein Species from DLBD Brain by LC-MS/MS
[0145] To generate an enriched pool of a-synuclein, a Urea/Thiourea/CHAPS
solubilized
particulate fraction of DLBD brain was first purified by anion exchange
chromatography.
The resulting fractions were analyzed by Western Blot and separated into crude
pools
enriched in truncated, full length, or phospho-synuclein. The truncated pool
was further
purified by affinity chromatography (5Cl2 antibody conjugated to SepharoseTm).
Next,
individual fractions were individually concentrated and separated by capillary
HPLC.
[0146] The three major peaks from one fraction (designated C6) were
subsequently digested
with trypsin and analyzed by LC-MS/MS to determine the identity and
composition of the
protein present in the sample. Peak 1 was analyzed and a-synuclein was
identified with
sequence coverage spanning amino acids 1-97. Since the sequence coverage ends
at the C-
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CA 02912912 2015-11-20
WO 21)06/045037 PCT/US2005/037875
terminus with a lysine residue, which is a trypsin cleavage site, additional
downstream
peptides may be present.
[0147] Peak 2 from fraction C6 was analyzed and found to contain a-synuclein
sequence
from positions 11-97. Again, since tryptic fragments on both the N and C
termini are absent;
the exact composition of the species is not able to be determined. Also, the
majority of the
protein present in this fraction is not synuclein, but a form of myelin basic
protein that co-
purified with this synuclein pool.
[0148] Analysis of Peak 3 identified two different synuclein
species, both beginning
at amino acid 1, with one terminating at position 1191) and the other at
position 122N. Both
of these truncated forms were identified using a database search designed to
detect truncated
C-terminal tryptic peptides. The database search results listing the truncated
C-terminal
peptide fragments are shown below (Table 1) along with their respective
extracted ion
chromatograms (Figures 12 and 13) which illustrate the intensity of the
precursor
(unfragmented) ion signals for these peptides when compared to baseline level.
Table 1: Database search results for Fraction-C6 Peak 3
Reference
Scan(s) Sequence MH+ Charge XCorr
#2 alpha_synuclein(C122)
2690 - 2694 DQLGKNEEGAPQEGILEDMPVDPDN. 2710.22 2
4.53
(SEQ. ID NO: 2)
2344 -2679 KDQLGKNEEGAPQEGILEDMPVDPDN. 2838.31 3 2.38
(SEQ. ID NO: 3)
#3 alpha_synuclein(C119)
2675 2512.19 2
1.94
(SEQ. ID NO: 4) KDOLGKNEEGAPQEGILEDMPVD.
[0149] The search identified the 122N truncation twice, finding two different
tryptic
sequences for the 122N species, with one peptide being the result of a missed
tryptic cleavage
of the N-terminal lysine residue. Also, the Xcorr, or cross correlation score,
for the first
listed sequence of the 122N variant is very high. Three additional truncated
forms SN1-115,
SN1-133 and SN1-35 have also been found in DLBD brains. Truncations were
identified by
matching MS/MS peptide fragmentation spectra from the truncated C-terminal a-
synuelein
peptides against a theoretical spectrum generated by TurboSequest Mass
spectral analysis
. , software (licensed from ThermoElectron, Inc). Both forms were
phosphorylated at Ser129.
The greater the Xcorr value, the higher the confidence in the match, which
lends more
support to the data.
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WO 2006/045037 PCT/US2005/037875
[0150] Figs. 14 A-F show inununoblots of 2D gels of extracts of Lewy body
preparations
from DLB patients P48 and P52 and MSA patient P2 (strictly speaking, tlie MSA
preparation
contains glial cortical inclusions) prepared as above compared with a control.
The probe is
antibody to total alpha synuclein. The identifying numbers (A/1, U/1, etc _)
refer to different
Lewy body preparations, done on different days and using different cortical
regions. Different
preparations from diseased patients are shown in Figs. 14 A, B, C, E and F.
The control is
shown in Fig. 14D. No synuclein modifications were consistently present or
absent in the
MSA preparation compared to those from DLB, so the two pathologies will be
discussed
together. Most preparations have the same groups of adducts migrating above
full-length
synuclein monomer although the relative amount of each group varies. The C-
truncated
synuclein between 16 and 12 kD can be grouped into four tiers, ranging from
full-length in
the first tier to the 12 Id3 fragment in the fourth. The relative amounts of
the different
truncations vary. P52U/2 and P28U/1 have none of the DLB-specific tier 3
detectable. P48
has very little C-truncated material; its tier 3 spots are barely visible
(although present), and
the tier 4 is not much more intense, if at all, than the soluble proteins.
Fig. 15B shows the
results of reprobing a 2D immunoblot of the P52U/2 Lewy body preparation with
ELADW101. A previous probe of the same blot with the monoclonal Syri-1 is
shown to the
left for comparison (Fig. 15A). Three asterisks mark spots that overlay each
other on the two
probes. The P28U/1 blot was also reprobed with identical results. The
polyclonal antibody
ELADW101, directed against termination at Asp119, specifically reacts with the
3 most
prominent spots in the 1210 fourth row of truncated species The multiple spots
suggest
different forms of SN1-119 differing by charge modifications, e.g.
phosphorylation, of the
remaining sequence.
[0151] Free-floating, cortical brain sections from a normal and DLBD patient
were probed
with the neo-epitope specific antibody ELADW-101. The results are shown in
Figs. 16A and
B (diseased brain) and Figs. 17A and B (controls). The boxed regions labeled A
and B are
shown magnified in the right panels. A typical LB and LN are labeled with
arrows. Only
faint synaptic staining is seen in the normal brain. The same experiment was
performed with
neo-epitope specific antibody 12C6. The results are shown in Figs. 18A and B.
The boxed
regions labeled A and B are shown magnified in the right panels. Typical LB
and LNs are
labeled with arrows. In panel B, an arrow indicates granular cytoplasmic
staining by 12C6.
This staining is not seen in normal brain, These results show that the
truncated alpha
synuclein fragment SN1-119 is enriched in patients with Lewy Body disease.
48
CA 02912912 2015-11-20
-
5. Behavioral Analysis in a Transgenic Animal
[0152] For locomotor activity, mice are analyzed for 2 days in the rotarod
(San Diego)
Instruments, San Diego, CA), as previously described (Masliah, et al. (2000)).
On the first day
mice are trained for 5 trials: the first one at lrpm, the second at 20rpm and
the third to fifth at
40rpm. On the second day, mice are tested for 7 trials at 40rpm each. Mice are
placed
individually on the cylinder and the speed of rotation is increased from 0 to
40 rpm over a
period of 240 sec. The length of time mice remain on the rod (fall Latency) is
recorded and
used as a measure of motor function.
[01531 Mice are tested for cognitive ability in the Morris Water maze (Morris,
Learn Motivat.
12;239-260 (1981)). In this procedure, the animal is placed in a circular pool
filled with water,
with an escape platform submerged just below the surface of the water. A
visible marker is
placed on the platform so that the animal can find it by navigating toward, a
proximal visual
cue. Alternatively, a more complex form of the test in which there are no
formal cues to mark
the platform's location will be given to the animals. In this form, the animal
must learn the
platform's location relative to distal visual cues. The length of time the
animal remains in the
water is inversely related to its cognitive ability.
6. Analysis of Aggregated Alpha-Svnuclein Fragments in a Cell Line
101541 GT1-7 neuronal cells (Hsue etal. Am. J. Pathol. 157:401-410 (2000)) are
transfected with a pCR3.1-T expression vector (Invitrogen, Carlsbad, CA)
expressing a
truncated fragment of alpha-synuclein as described above murine alpha-
synuclein and
compared with cells transfected with expression vector alone. Cells
transfected with vector
alone have a fibroblastic appearance while cells transfected with alpha-
synuclein are rounded,
with inclusion bodies at the cell surface visible via both light and confocal
scanning
microscopy. Transfected GT1-7 cells can be used to screen agents for activity
in clearing
synuclein inclusions.
101551 The above examples are illustrative only. Other variants will be
readily apparent to
those of ordinary skill in the art. The scope of the invention is encompassed
by the claims of
any patent(s) issuing herefrom.
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CA 02912912 2015-11-20
SEQUENCE LISTING IN ELECTRONIC FORM
In accordance with section 111(1) of the Patent Rules, this
description contains a sequence listing in electronic form in
ASCII text format (file: 92180-5d1seq19-11-15v1.txt).
A copy of the sequence listing in electronic form is available
from the Canadian Intellectual Property Office.
The sequences in the sequence listing in electronic form are
reproduced in the following table.
SEQUENCE TABLE
<110> Elan Pharmaceuticals, LLC.
<120> TRUNCATED FRAGMENTS OF ALPHA-SYNUCLEIN IN LEWY BODY DISEASE
<130> 92180-5D1
<150> 2,584,512
<151> 2005-10-19
<150> US 10/969,335
<151> 2004-10-19
<150> US 11/194,115
<151> 2005-07-29
<160> 11
<170> Patenrin version 3.3
<210> 1
<211> 140
<212> PRT
<213> Homo sapiens
<400> 1
Met Asp Val Phe Met Lys Gly Leu Ser Lys Ala Lys Glu Gly Val Val
1 5 10 15
Ala Ala Ala Glu Lys Thr Lys Gln Gly Val Ala Glu Ala Ala Gly Lys
20 25 30
Thr Lys Glu Gly Val Leu Tyr Val Gly Ser Lys Thr Lys Glu Gly Val
35 40 45
Val His Gly Val Ala Thr Val Ala Glu Lys Thr Lys Glu Gln Val Thr
50 55 60
Asn Val Gly Gly Ala Val Val Thr Gly Val Thr Ala Val Ala Gin Lys
65 70 75 80
Thr Val Glu Gly Ala Gly Ser Ile Ala Ala Ala Thr Gly Phe Val Lys
85 90 95
Lys Asp Gln Leu Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu Gly Ile
100 105 110
CA 02912912 2015-11-20
Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro
115 120 125
Ser Glu Glu Gly Tyr Gin Asp Tyr Glu Pro Glu Ala
130 135 140
<210> 2
<211> 25
<212> PRI
<213> Homo sapiens
<400> 2
Asp Gin Leu Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu Gly Ile Leu
1 5 10 15
Glu Asp Met Pro Val Asp Pro Asp Asn
20 25
<210> 3
<211> 26
<212> PRT
<213> Homo sapiens
<400> 3
Lys Asp Gin Leu Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu Gly Ile
1 5 10 15
Leu Glu Asp Met Pro Val Asp Pro Asp Asn
20 25
<210> 4
<211> 23
<212> PRT
<213> Homo sapiens
<400> 4
Lys Asp Gln Leu Gly Lys Asn Glu Glu Gly Ala Pro Gin Glu Gly Ile
1 5 10 15
Leu Glu Asp Met Pro Val Asp
<210> 5
<211> 8
<212> PRT
<213> Homo sapiens
<400> 5
Lys Asn Glu Glu Gly Ala Pre Gln
1 5
<210> 6
<211> 31
<212> PRT
<213> Homo sapiens
<400> 6
Glu Gly Ile Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr
1 5 10 15
51
CA 02912912 2015-11-20
Glu Met Pro Ser Glu Glu Gly Tyr Gin Asp Tyr Glu Pro Glu Ala
20 25 30
<210> 7
<211> 8
<212> PRT
<213> Mus musculus
<400> 7
Lys Gly Glu Glu Gly Val Pro Gin
1 5
<210> 8
<211> 31
<212> PRT
<213> Mus musculus
<400> 8
Glu Gly Ile Leu Glu Asp Met Pro Val Asp Pro Gly Ser Glu Ala Tyr
1 5 10 15
Glu Met Pro Ser Glu Glu Gly Tyr Gin Asp Tyr Glu Pro Glu Ala
20 25 30
<210> 9
<211> 11
<212> PRT
<213> Artificial
<220>
<223> Epitope bound by antibody as depicted in Figure 8
<400> 9
Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr
1 5 10
<210> 10
<211> 8
<212> PRT
<213> Artificial
<220>
<223> Immunogenic peptide
<400> 10
Cys Gly Gly Asp Met Pro Val Asp
1 5
<210> 11
<211> 8
<212> PRT
<213> Artificial
<220>
<223> Immunogenic peptide
<400> 11
Cys Gly Gly Val Asp Pro Asp Asn
1 5
52
