U.S. patent application number 12/729902 was filed with the patent office on 2011-06-09 for prevention and treatment of synucleinopathic disease.
This patent application is currently assigned to JANSSEN ALZHEIMER IMMUNOTHERAPY. Invention is credited to Eliezer Masliah, Dale B. Schenk.
Application Number | 20110135660 12/729902 |
Document ID | / |
Family ID | 32312587 |
Filed Date | 2011-06-09 |
United States Patent
Application |
20110135660 |
Kind Code |
A1 |
Schenk; Dale B. ; et
al. |
June 9, 2011 |
PREVENTION AND TREATMENT OF SYNUCLEINOPATHIC DISEASE
Abstract
The invention provides improved agents and methods for treatment
of diseases associated with synucleinopathic diseases, including
Lewy bodies of alpha-synuclein in the brain of a patient. Such
methods entail administering agents that induce a beneficial
immunogenic response against the Lewy body. The methods are
particularly useful for prophylactic and therapeutic treatment of
Parkinson's disease.
Inventors: |
Schenk; Dale B.;
(Burlingame, CA) ; Masliah; Eliezer; (San Diego,
CA) |
Assignee: |
JANSSEN ALZHEIMER
IMMUNOTHERAPY
LITTLE ISLAND
CA
THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
OAKLAND
|
Family ID: |
32312587 |
Appl. No.: |
12/729902 |
Filed: |
March 23, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10699517 |
Oct 31, 2003 |
7727957 |
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12729902 |
|
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60423012 |
Nov 1, 2002 |
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Current U.S.
Class: |
424/172.1 ;
424/184.1; 424/193.1 |
Current CPC
Class: |
A61P 37/04 20180101;
A61K 39/0007 20130101; A61P 25/28 20180101; A61P 43/00 20180101;
A61K 2039/55566 20130101; A61K 51/1078 20130101; A61K 38/1709
20130101; A61P 25/00 20180101; A61K 45/06 20130101; A61P 25/16
20180101 |
Class at
Publication: |
424/172.1 ;
424/184.1; 424/193.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 39/00 20060101 A61K039/00; A61K 39/385 20060101
A61K039/385; A61P 37/04 20060101 A61P037/04; A61P 25/00 20060101
A61P025/00 |
Claims
1-70. (canceled)
71. A method of therapeutically treating a patient suffering from a
Lewy body disease, the method comprising: administering to the
patient an effective regime of an agent and thereby therapeutically
treating the disease; wherein (i) the agent is A.beta. or an
immunogenic fragment thereof and the agent is linked to a carrier
that helps elicit an immune response to the agent or is
administered with an adjuvant that augments an immune response to
the agent, or (ii) the agent is an antibody to A.beta..
72. The method of claim 71, wherein the agent is A.beta. or an
immunogenic fragment thereof.
73. The method of claim 71, wherein the agent is an antibody to
A.beta..
74. The method of claim 71, wherein the agent is administered
peripherally.
75. The method of claim 71, wherein the effective regime comprises
administering multiple dosages over a period of at least six
months.
76. The method of claim 71, wherein the administering improves
motor characteristics of the patient.
77. The method of claim 71, wherein the patient is free of
Alzheimer's disease.
78. The method of claim 71, wherein the patient is free of
Alzheimer's disease and has no genetic risk factors thereof.
79. A method of therapeutically treating a patient suffering from a
Lewy body disease, comprising administering to the patient an
effective regime of a first agent and a second agent and thereby
therapeutically treating the disease; wherein (i) the first agent
is alpha synuclein or an immunogenic fragment thereof, and the
first agent is linked to a carrier that helps elicit an immune
response to the first agent or is administered with an adjuvant
that augments an immune response to the first agent, or (ii) the
first agent is an antibody to alpha synuclein, and wherein (i) the
second agent is A.beta. or an immunogenic fragment thereof and the
second agent is linked to a carrier that helps elicit an immune
response to the second agent or is administered with an adjuvant
that augments an immune response to the second agent, or (ii) the
second agent is an antibody to A.beta..
80. A method of reducing the risk, lessening the severity, or
delaying the outset of disease in a patient having a known genetic
risk of a Lewy body disease, the method comprising administering to
the patient an effective regime of an agent and thereby reducing
the risk, lessening the severity, or delaying the outset of the
disease; wherein (i) the agent is A.beta. or an immunogenic
fragment thereof, and the agent is linked to a carrier that helps
elicit an immune response to the agent or is administered with an
adjuvant that augments an immune response to the agent, or (ii) the
agent is an antibody to A.beta..
81. The method of claim 80, wherein the agent is A.beta. or an
immunogenic fragment thereof.
82. The method of claim 80, wherein the agent is an antibody to
A.beta..
83. A method of reducing the risk, lessening the severity, or
delaying the outset of disease in a patient having a known genetic
risk of a Lewy body disease, comprising administering to the
patient an effective regime of a first agent and a second agent
that induces an immunogenic response against A.beta. in the patient
and thereby reducing the risk, lessening the severity, or delaying
the outset of the disease; wherein (i) the first agent is alpha
synuclein or an immunogenic fragment thereof and the first agent is
linked to a carrier that helps elicit an immune response to the
first agent or is administered with an adjuvant that augments an
immune response to the first agent, or (ii) the first agent is an
antibody to alpha synuclein, and wherein (i) the second agent is
A.beta. or an immunogenic fragment thereof and the second agent is
linked to a carrier that helps elicit an immune response to the
second agent or is administered with an adjuvant that augments an
immune response to the second agent, or (ii) the second agent is an
antibody to A.beta..
84. The method of claim 83, wherein the agent is administered
peripherally.
85. The method of claim 83, wherein the effective regime comprises
administering multiple dosages over a period of at least six
months.
86. The method of claim 83, wherein the patient is free of
Alzheimer's disease.
87. The method of claim 83, wherein the patient is free of
Alzheimer's disease and has no genetic risk factors thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119(e) of U.S. Application No. 60/423,012, filed Nov. 1,
2002, which is incorporated by reference herein for all
purposes.
BACKGROUND OF THE INVENTION
[0002] Alpha-synuclein (alphaSN) brain pathology is a conspicuous
feature of several neurodegenerative diseases, including
Parkinson's disease (PD), dementia with Lewy bodies (DLB), the Lewy
body variant of Alzheimer's disease (LBVAD), multiple systems
atrophy (MSA), and neurodegeneration with brain iron accumulation
type-1 (NBIA-1). Common to all of these diseases, termed
synucleinopathies, are proteinaceous insoluble inclusions in the
neurons and the glia which are composed primarily of alphaSN.
[0003] Lewy bodies and Lewy neurites are intraneuronal inclusions
which are composed primarily of alphaSN. Lewy bodies and Lewy
neurites are the neuropathological hallmarks Parkinson's disease
(PD). PD and other synucleinopathic diseases have been collectively
referred to as Lewy body disease (LBD). 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). Other LBDs include diffuse Lewy body disease
(DLBD), Lewy body variant of Alzheimer's disease (LBVAD), combined
PD and Alzheimer's disease (AD), and multiple systems atrophy.
[0004] 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 are
neither curable nor preventable and understanding the causes and
pathogenesis of PD is critical towards developing new treatments
(Tanner et al., Epidemiology of Parkinson's disease and akinetic
syndromes, Curr. Opin. Neurol. (2000) 13:427-30). The cause for PD
is controversial and multiple factors have been proposed to play a
role, including various neurotoxins and genetic susceptibility
factors.
[0005] In recent years, new hope for understanding the pathogenesis
of PD has emerged. Specifically, several studies have shown that
the synaptic protein alpha-SN plays a central role in PD
pathogenesis since: (1) this protein accumulates in LBs
(Spillantini et al., Nature (1997) 388:839-40; Takeda et al., AM.
J. Pathol. (1998) 152:367-72; Wakabayashi et al., Neurosci. Lett.
(1997) 239:45-8), (2) mutations in the alpha-SN gene co-segregate
with rare familial forms of parkinsonism (Kruger et al., Nature
Gen. (1998) 18:106-8; Polymeropoulos M H, et al., Science (1997)
276:2045-7) and, (3) its overexpression in transgenic mice (Masliah
et al., Science (2000) 287.1265-9) and Drosophila (Feany et al.,
Nature (2000) 404:394-8) mimics several pathological aspects of PD.
Thus, the fact that accumulation of alpha-SN 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.
[0006] An alpha-SN fragment, previously determined to be a
constituent of AD amyloid plaques, was termed the non-amyloid-beta
(non-A.beta.) component of AD amyloid (NAC) (Iwai A., Biochim.
Biophys. Acta (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). Although the precise function of NAC is not known, it
may play a critical role in synaptic events, such as neural
plasticity during development, and learning and degeneration of
nerve terminals under pathological conditions in LBD, AD, and other
disorders (Hasimoto et al., Alpha-Synuclein in Lewy body disease
and Alzheimer's disease, Brain Pathol (1999) 9:707-20; Masliah, et
al., (2000).
[0007] AD, PD, and dementia with Lewy bodies (DLB) are the most
commonly found neurodegenerative disorders in the elderly. Although
their incidence continues to increase, creating a serious public
health problem, to date these disorders are neither curable nor
preventable. Recent epidemiological studies have demonstrated a
close clinical relationship 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, DLB and LBD 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 the link
between AD, PD and DLB.
[0008] The neuritic plaques that are the classic pathological
hallmark of AD contain beta-amyloid (A.beta.) peptide and non-beta
amyloid component (NAC) peptide. AP is derived from a larger
precursor protein termed amyloid precursor protein (APP). NAC is
derived from a larger precursor protein termed the non-beta amyloid
component of APP, now more commonly referred to as alpha-SN. NAC
comprises amino acid residues 60-87 or 61-95 of alpha-SN. Both
A.beta. 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.
[0009] Alpha-SN is part of a large family of proteins including
beta- and gamma-synuclein and synoretin. Alpha-SN 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-SN that enhance the aggregation of alpha-SN 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-SN to aggregate
are unknown.
[0010] Despite the fact that a number of mutations can be found in
APP and alpha-SN in the population, most cases of AD and PD are
sporadic. The most frequent sporadic forms of these diseases are
associated with an abnormal accumulation of A.beta. and alpha-SN,
respectively. However, the reasons for over accumulation of these
proteins is unknown. A.beta. is secreted from neurons and
accumulates in extracellular amyloid plaques. Additionally A.beta.
can be detected inside neurons. Alpha-SN accumulates in
intraneuronal inclusions called LBs. Although the two proteins are
typically found together in extracellular neuritic AD plaques, they
are also occasionally found together in intracellular
inclusions.
[0011] The mechanisms by which alpha-SN accumulation leads to
neurodegeneration and the characteristics symptoms of PD are
unclear. However, identifying the role of factors promoting and/or
blocking alpha-SN aggregation is critical for the understanding of
LBD pathogenesis and development of novel treatments for its
associated disorders. Research for identifying treatments has been
directed toward searching for compounds that reduce alpha-SN
aggregation (Hashimoto, et al.) or testing growth factors that will
promote the regeneration and/or survival of dopaminergic neurons,
which are the cells primarily affected (Djaldetti et al., New
therapies for Parkinson's disease, J. Neurol (2001) 248:357-62;
Kirik et al., Long-term rAAV-mediated gene transfer of GDNF in the
rat Parkinson's model: intrastriatal but not intranigral
transduction promotes functional regeneration in the lesioned
nigrostriatal system, J. Neurosci (2000) 20:4686-4700). Recent
studies in a transgenic mouse model of AD have shown that
antibodies against A.beta. 1-42 facilitate and stimulate the
removal of amyloid from the brain, improve AD-like pathology and
resulting in improve cognitive performance (Schenk et al.,
Immunization with amyloid-.beta. attenuates Alzheimer-disease-like
pathology in PDAPP mouse, Nature (1999) 408:173-177; Morgan et al.,
A-beta peptide vaccination prevents memory loss in an animal model
of Alzheimer's disease, Nature (2000) 408:982-985; Janus et al.,
A-beta peptide immunization reduces behavioral impairment and
plaques in a model of Alzheimer's disease, Nature (2000)
408:979-82). In contrast to the extracellular amyloid plaques found
in the brains of Alzheimer's patients, Lewy bodies are
intracellular, and antibodies do not typically enter the cell.
[0012] Surprisingly, given the intracellular nature of LBs in brain
tissue, the inventors have succeeded in reducing the number of
inclusions in transgenic mice immunized with synuclein. The present
invention is directed inter alia to treatment of PD and other
diseases associated with LBs by administration of synuclein,
fragments of synuclein, antigens that mimic synuclein or fragments
thereof, or antibodies to certain epitopes of synuclein to a
patient under conditions that generate a beneficial immune response
in the patient. The inventors have also surprisingly succeeded in
reducing the number of inclusions in transgenic mice immunized with
A.beta.. The present invention is directed inter alia to treatment
of PD and other diseases associated with LBs by administration of
A.beta., fragments of A.beta., antigens that mimic A.beta. or
fragments thereof, or antibodies to certain epitopes of A.beta. to
a patient under conditions that generate a beneficial immune
response in the patient. The invention thus fulfills a longstanding
need for therapeutic regimes for preventing or ameliorating the
neuropathology and, in some patients, the cognitive impairment
associated with PD and other diseases associated with LBs.
BRIEF SUMMARY OF THE INVENTION
[0013] In one aspect, the invention provides methods of preventing
or treating a disease characterized by Lewy bodies or alpha-SN
aggregation in the brain. Such methods entail, inducing an
immunogenic response against alpha-SN. Such induction may be
achieved by active administration of an immunogen or passive by
administration of an antibody or a derivative of an antibody to
synuclein. In some methods, the patient is asymptomatic. In some
methods, the patient has the disease and is asymptomatic. In some
methods the patient has a risk factor for the disease and is
asymptomatic. In some methods, the disease is Parkinson's disease.
In some methods, the disease is Parkinson's disease, and the
administering the agent improves motor characteristics of the
patient. In some methods, the disease is Parkinson's disease
administering the agent prevents deterioration of motor
characteristics of the patient. In some methods, the patient is
free of Alzheimer's disease.
[0014] For treatment of patients suffering from Lewy bodies or
alpha-SN aggregation in the brain, one treatment regime entails
administering a dose of alpha-SN or an active fragment thereof to
the patient to induce the immune response. In some methods the
alpha-SN or an active fragment thereof is administered in multiple
doses over a period of at least six months. The alpha-SN or an
active fragment thereof can be administered, for example,
peripherally, intraperitoneally, orally, subcutaneously,
intracranially, intramuscularly, topically, intranasally or
intravenously. In some methods, the alpha-SN or an active fragment
thereof is administered with an adjuvant that enhances the immune
response to the alpha-SN or an active fragment thereof. In some
methods, the immunogenic response comprises antibodies to alpha-SN
or an active fragment thereof.
[0015] In some methods, the agent is amino acids 35-65 of alpha-SN.
In some methods, the agent comprises amino acids 130-140 of
alpha-SN and has fewer than 40 amino acids total. In some methods,
the C-terminal amino acids of the agent are the C-terminal amino
acid of alpha-SN. In some of the above methods, the alpha-SN or
active fragment is linked to a carrier molecule to form a
conjugate. In some of the above methods, the alpha-SN or active
fragment is linked to a carrier at the N-terminus of the alpha-SN
or active fragment.
[0016] For treatment of patients suffering from Lewy bodies or
alpha-SN aggregation in the brain, one treatment regime entails
administering a dose of an antibody to alpha-SN or an active
fragment thereof to the patient to induce the immune response. The
antibody used may be human, humanized, chimeric, or polyclonal and
can be monoclonal or polyclonal. In some methods the isotype of the
antibody is a human IgG1. In some methods, the antibody is prepared
from a human immunized with alpha-SN peptide and the human can be
the patient to be treated with antibody. In some methods, the
antibody binds to the outer surface of neuronal cells having Lewy
bodies thereby dissipating the Lewy bodies. In some methods, the
antibody is internalized within neuronal cells having Lewy bodies
thereby dissipating the Lewy bodies.
[0017] In some methods, the antibody is administered with a
pharmaceutical carrier as a pharmaceutical composition. In some
methods, antibody is administered at a dosage of 0.0001 to 100
mg/kg, preferably, at least 1 mg/kg body weight antibody. In some
methods the antibody is administered in multiple doses over a
prolonged period, for example, at least six months. In some methods
antibodies may be administered as a sustained release composition.
The antibody can be administered, for example, peripherally,
intraperitoneally, orally, subcutaneously, intracranially,
intramuscularly, topically, intranasally or intravenously. In some
methods, the patient is monitored for level of administered
antibody in the blood of the patient.
[0018] In some methods, the antibody is administered by
administering a polynucleotide encoding at least one antibody chain
to the patient. The polynucleotide is expressed to produce the
antibody chain in the patient. Optionally, the polynucleotide
encodes heavy and light chains of the antibody and the
polynucleotide is expressed to produce the heavy and light chains
in the patient.
[0019] This invention further provides pharmaceutical compositions
comprising an antibody to alpha-SN and a pharmaceutically
acceptable carrier.
[0020] In another aspect, the invention provides methods of
preventing or treating a disease characterized by Lewy bodies or
alpha-SN aggregation in the brain comprising administering an agent
that induces an immunogenic response against alpha-SN, and further
comprising administering of a second agent that induces an
immunogenic response against A.beta. to the patient. In some
methods, the agent is A.beta. or an active fragment thereof. In
some methods, the agent is an antibody to A.beta..
[0021] In another aspect, the invention provides methods of
preventing or treating a disease characterized by Lewy bodies or
alpha-SN aggregation in the brain comprising administering an agent
that induces an immunogenic response against A.beta. to a patient.
In some methods, the agent is A.beta. or an active fragment
thereof. In some methods, the agent is an antibody to A.beta.. In
some methods the disease is Parkinson's disease. In some methods,
the patient is free of Alzheimer's disease and has no risk factors
thereof. In some methods, further comprise monitoring a sign or
symptom of Parkinson's disease in the patient. In some methods, the
disease is Parkinson's disease and administering the agent results
in improvement in a sign or symptom of Parkinson's disease.
[0022] This invention further provides pharmaceutical compositions
comprising an agent effective to induce an immunogenic response
against a component of a Lewy body in a patient, such as described
above, and a pharmaceutically acceptable adjuvant. In some
compounds, the agent is alpha-SN or an active fragment, for
example, NAC. In some compounds the agent is 6CHC-1 or an active
fragment. The invention also provides pharmaceutical compositions
comprising an antibody specific for a component of a Lewy body.
[0023] In another aspect, the invention provides for methods of
screening an antibody for activity in preventing or treating a
disease associated with Lewy bodies. Such methods entail,
contacting a neuronal cell expressing synuclein with the antibody.
Then one determines whether the contacting reduces synuclein
deposits in the cells compared with a control cells not contacted
with the antibody.
[0024] In another aspect, the invention provides for methods of
screening an antibody for activity in treating or preventing a Lewy
body disease in the brain of a patient. Such methods entail
contacting the antibody with a polypeptide comprising at least five
contiguous amino acids of alpha-SN. Then one determines whether the
antibody specifically binds to the polypeptide, specific binding
providing an indication that the antibody has activity in treating
the disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 shows the amino acid sequence of alpha-SN (SEQ ID: 1)
in alignment with two NAC amino acid sequences, SEQ ID NO: 2 and
SEQ ID NO: 3, respectively.
[0026] FIG. 2 shows immunohistostained brain sections from
nontransgenic mice (panels A, E, and I), alpha-SN transgenic mice
immunized with adjuvant alone (panels B, F, J), and alpha-SN
transgenic mice immunized with alpha-SN which developed low titers
(panels C, G, and K) and high titers (panels D, H, and I) of
antibodies to alpha-SN. Sections were subjected to staining with an
anti-alpha-synuclein antibody to detect synuclein levels (panels
A-D), an anti-IgG antibody to determine total IgG levels present in
the section (panels E-H), and for Glial Fibrillary Acidic Protein
(GFAP), a marker of astroglial cells.
[0027] FIG. 3 shows the effects of anti-mSYN polyclonal antibody on
synuclein aggregation in transfected GT1-7 cells as seen by light
microscopy.
[0028] FIG. 4 is a Western blot of synuclein levels in the
cytoplasm (C) and membranes (P) of GT1-7 .alpha.-syn cells treated
with preimmune sera and with 67-10 antibody at a concentration of
(1:50) for 48 hours prior to analysis.
[0029] FIG. 5 shows the results of studies of the effect of
A.beta.1-42 immunization amyloid deposition in the brains of
nontransgenic, SYN, APP and SYN/APP transgenic mice. The detectable
amyloid levels seen in APP and SYN/APP mice are reduced by
A.beta.1-42 immunization.
[0030] FIG. 6 shows the results of studies of the effect of
A.beta.1-42 immunization upon synuclein inclusion formation in the
brains of nontransgenic, SYN, APP and SYN/APP transgenic mice.
Synuclein inclusions detected in SYN and SYN/APP mice are reduced
by A.beta.1-42 immunization.
[0031] FIG. 7 shows direct and indirect mechanisms by which
antibodies block alpha-SN aggregation.
DEFINITIONS
[0032] The term "substantial identity" means that two peptide
sequences, when optimally aligned, such as by the programs GAP or
BESTFIT using default gap weights, share at least 65 percent
sequence identity, preferably at least 80 or 90 percent sequence
identity, more preferably at least 95 percent sequence identity or
more (e.g., 99 percent sequence identity or higher). Preferably,
residue positions which are not identical differ by conservative
amino acid substitutions.
[0033] 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.
[0034] 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, Wis.), or by visual
inspection (see generally Ausubel et al., supra). One 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 (NCBI)
website. Typically, default program parameters can be used to
perform the sequence comparison, although customized parameters can
also be used. 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 (see Henikoff & Henikoff, Proc. Natl.
Acad. Sci. USA 89, 10915 (1989)).
[0035] For purposes of classifying amino acids substitutions as
conservative or non-conservative, amino acids are grouped as
follows: Group I (hydrophobic sidechains): 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, gln, 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.
[0036] 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.
[0037] The phrase that a molecule "specifically binds" to a target
refers to a binding reaction which is determinative of the presence
of the molecule in the presence of a heterogeneous population of
other biologics. Thus, under designated immunoassay conditions, a
specified molecule binds preferentially to a particular target and
does not bind in a significant amount to other biologics present in
the sample. Specific binding of an antibody to a target under such
conditions requires the antibody be selected for its specificity to
the target. 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 immunoreactive 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. Specific binding between
two entities means an affinity of at least 10.sup.6, 10.sup.7,
10.sup.8, 10.sup.9 M.sup.-1, or 10.sup.10 M.sup.-1. Affinities
greater than 10.sup.8 M.sup.-1 are preferred.
[0038] 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 immunoglobulin 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. 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. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148,
1547-1553 (1992).
[0039] APP.sup.695, APP.sup.751, and APP.sup.770 refer,
respectively, to the 695, 751, and 770 amino acid residue long
polypeptides encoded by the human APP gene. See Kang et al., Nature
325, 773 (1987); Ponte et al., Nature 331, 525 (1988); and
Kitaguchi et al., Nature 331, 530 (1988). Amino acids within the
human amyloid precursor protein (APP) are assigned numbers
according to the sequence of the APP770 isoform. Terms such as
A.beta.39, A.beta.40, A.beta.41, A.beta.42 and A.beta.43 refer to
an A.beta. peptide containing amino acid residues 1-39, 1-40, 1-41,
1-42 and 1-43.
[0040] An "antigen" is an entity to which an antibody specifically
binds.
[0041] The term "epitope" or "antigenic determinant" refers to a
site on an antigen to which B and/or T cells respond. B-cell
epitopes can be formed both from contiguous amino acids or
noncontiguous amino acids juxtaposed by tertiary folding of a
protein. Epitopes formed from contiguous amino acids are typically
retained on exposure to denaturing solvents whereas epitopes formed
by tertiary folding are typically lost on treatment with denaturing
solvents. An epitope typically includes at least 3, and more
usually, at least 5 or 8-10 amino acids in a unique spatial
conformation. Methods of determining spatial conformation of
epitopes include, for example, x-ray crystallography and
2-dimensional nuclear magnetic resonance. See, e.g., Epitope
Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn
E. Morris, Ed. (1996). Antibodies that recognize the same epitope
can be identified in a simple immunoassay showing the ability of
one antibody to block the binding of another antibody to a target
antigen. T-cells recognize continuous epitopes of about nine amino
acids for CD8 cells or about 13-15 amino acids for CD4 cells. T
cells that recognize the epitope can be identified by in vitro
assays that measure antigen-dependent proliferation, as determined
by .sup.3H-thymidine incorporation by primed T cells in response to
an epitope (Burke et al., J. Inf. Dis. 170, 1110-19 (1994)), by
antigen-dependent killing (cytotoxic T lymphocyte assay, Tigges et
al., J. Immunol. 156, 3901-3910) or by cytokine secretion.
[0042] The term "immunological" or "immune" response is the
development of a beneficial humoral (antibody mediated) and/or a
cellular (mediated by antigen-specific T cells or their secretion
products) response directed against an amyloid peptide in a
recipient patient. Such a response can be an active response
induced by administration of immunogen or a passive response
induced by administration of antibody or primed T-cells. A cellular
immune response is elicited by the presentation of polypeptide
epitopes in association with Class I or Class II MHC molecules to
activate antigen-specific CD4.sup.+ T helper cells and/or CD8.sup.+
cytotoxic T cells. The response may also involve activation of
monocytes, macrophages, NK cells, basophils, dendritic cells,
astrocytes, microglia cells, eosinophils or other components of
innate immunity. The presence of a cell-mediated immunological
response can be determined by proliferation assays (CD4.sup.+ T
cells) or CTL (cytotoxic T lymphocyte) assays (see Burke, supra;
Tigges, supra). The relative contributions of humoral and cellular
responses to the protective or therapeutic effect of an immunogen
can be distinguished by separately isolating antibodies and T-cells
from an immunized syngeneic animal and measuring protective or
therapeutic effect in a second subject.
[0043] An "immunogenic agent" or "immunogen" is capable of inducing
an immunological response against itself on administration to a
mammal, optionally in conjunction with an adjuvant.
[0044] The term "all-D" refers to peptides having .gtoreq.75%,
.gtoreq.80%, .gtoreq.85%, .gtoreq.90%, .gtoreq.95%, and 100%
D-configuration amino acids.
[0045] The term "naked polynucleotide" refers to a polynucleotide
not complexed with colloidal materials. Naked polynucleotides are
sometimes cloned in a plasmid vector.
[0046] 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.
[0047] The term "patient" includes human and other mammalian
subjects that receive either prophylactic or therapeutic
treatment.
[0048] 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-SN. Numerous
types of competitive binding assays are known, for example: solid
phase direct or indirect radioimmunoassay (RIA), solid phase direct
or indirect enzyme immunoassay (EIA), sandwich competition assay
(see Stahli et al., Methods in Enzymology 9:242-253 (1983)); solid
phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol.
137:3614-3619 (1986)); solid phase direct labeled assay, solid
phase direct labeled sandwich assay (see Harlow and Lane,
Antibodies, A Laboratory Manual, Cold Spring Harbor Press (1988));
solid phase direct label RIA using I-125 label (see Morel et al.,
Molec. Immunol. 25(1):7-15 (1988)); solid phase direct
biotin-avidin EIA (Cheung et al., Virology 176:546-552 (1990)); and
direct labeled RIA (Moldenhauer et al., Scand. J. Immunol. 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%.
[0049] The term "symptom" or "clinical symptom" refers to a
subjective evidence of a disease, such as altered gait, as
perceived by the patient. A "sign" refers to objective evidence of
a disease as observed by a physician.
[0050] Compositions or methods "comprising" one or more recited
elements may include other elements not specifically recited. For
example, a composition that comprises alpha-SN peptide encompasses
both an isolated alpha-SN peptide and alpha-SN peptide as a
component of a larger polypeptide sequence.
DETAILED DESCRIPTION OF THE INVENTION
I. General
[0051] The invention provides methods of preventing or treating
several diseases and conditions characterized by presence of
deposits of alpha-SN peptide aggregated to an insoluble mass in the
brain of a patient, in the form of Lewy bodies. Such diseases are
collectively referred to as Lewy Body diseases (LBD) and include
Parkinson's disease (PD). Such diseases are characterized by
aggregates of alpha-SN that have a .beta.-pleated sheet structure
and stain with thioflavin-S and Congo-red (see Hasimoto, Ibid). The
invention provides methods of preventing or treating such diseases
using an agent that can generate an immunogenic response to
alpha-SN. The immunogenic response acts to prevent formation of, or
clear, synuclein deposits within cells in the brain. Although an
understanding of mechanism is not essential for practice of the
invention, the immunogenic response may induce clearing as a result
of antibodies to synuclein that are internalized within cells
and/or which interact with the membrane of such cells and thereby
interfere with aggregation of synuclein. In some methods, the
clearing response can be effected at least in part by Fc receptor
mediated phagocytosis. Immunization with synuclein can reduce
synuclein accumulation at synapses in the brain. Although an
understanding of mechanism is not essential for practice of the
invention, this result can be explained by antibodies to synuclein
being taken up by synaptic vesicles.
[0052] Optionally, agents generating an immunogenic response
against alpha-SN can be used in combination with agents that
generate an immunogenic response to A.beta.. The immunogenic
response is useful in clearing deposits of A.beta. in individuals
having such deposits (e.g., individuals having both Alzheimer's and
Parkinson's disease); however, the immunogenic response also has
activity in clearing synuclein deposits. Thus, the present
invention uses such agents alone, or in combination with agents
generating an immunogenic response to alpha-SN in individuals with
LBD but who are not suffering or at risk of developing Alzheimer's
disease.
II. Agents Generating an Immunogenic Response Against Alpha
Synuclein
[0053] An immunogenic response can be active, as when an immunogen
is administered to induce antibodies reactive with alpha-SN in a
patient, or passive, as when an antibody is administered that
itself binds to alpha-SN in a patient.
[0054] 1. Agents Inducing Active Immune Response
[0055] Therapeutic agents induce an immunogenic response
specifically directed to certain epitopes within the alpha-SN
peptide. Preferred agents are the alpha-SN peptide itself and
fragments thereof. Variants of such fragments, analogs and mimetics
of natural alpha-SN peptide that induce and/or cross-react with
antibodies to the preferred epitopes of alpha-SN peptide can also
be used.
[0056] Alpha synuclein was originally identified in human brains as
the precursor protein of the non-.beta.-amyloid component of (NAC)
of AD plaques. (Ueda et al., Proc. Natl. Acad. Sci. U.S.A. 90
(23):11282-11286 (1993). Alpha-SN, also termed the precursor of the
non-A.beta. component of AD amyloid (NACP), is a peptide of 140
amino acids. Alpha-SN has the amino acid sequence:
TABLE-US-00001 (SEQ ID NO: 1)
MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVV
HGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKD
QLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA (Ueda et al., Ibid.;
GenBank accession number: P37840).
[0057] The non-A.beta. component of AD amyloid (NAC) is derived
from alpha-SN. NAC, a highly hydrophobic domain within alpha
synuclein, is a peptide consisting of at least 28 amino acids
residues (residues 60-87) (SEQ ID NO: 3) and optionally 35 amino
acid residues (residues 61-95) (SEQ ID NO: 2). See FIG. 1. NAC
displays a tendency to form a beta-sheet structure (Iwai, et al.,
Biochemistry, 34:10139-10145). Jensen et al. have reported NAC has
the amino acid sequence:
TABLE-US-00002 (SEQ ID NO: 2) EQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFV
(Jensen et al., Biochem. J. 310 (Pt 1): 91-94(1995); GenBank
accession number S56746).
[0058] Ueda et al. have reported NAC has the acid sequence:
TABLE-US-00003 KEQVTNVGGAVVTGVTAVAQKTVEGAGS (SEQ ID NO: 3) (Ueda et
al., PNAS USA 90: 11282-11286 (1993).
[0059] Disaggregated alpha-SN or fragments thereof, including NAC,
means monomeric peptide units. Disaggregated alpha-SN or fragments
thereof are generally soluble, and are capable of self-aggregating
to form soluble oligomers. Oligomers of alpha-SN and fragments
thereof are usually soluble and exist predominantly as
alpha-helices. Monomeric alpha-SN may be prepared in vitro by
dissolving lyophilized peptide in neat DMSO with sonication. The
resulting solution is centrifuged to remove any insoluble
particulates. Aggregated alpha-SN or fragments thereof, including
NAC, means oligomers of alpha-SN or fragments thereof which have
associate into insoluble beta-sheet assemblies. Aggregated alpha-SN
or fragments thereof, including NAC, means also means fibrillar
polymers. Fibrils are usually insoluble. Some antibodies bind
either soluble alpha-SN or fragments thereof or aggregated alpha-SN
or fragments thereof. Some antibodies bind both soluble and
aggregated alpha-SN or fragments thereof.
[0060] Alpha-SN, the principal component of the Lewy bodies
characteristic of PD, and epitopic fragments thereof, such as, for
example, NAC, or fragments other than NAC, can be used to induce an
immunogenic response. Preferably such fragments comprise four or
more amino acids of alpha-SN or analog thereof. Some active
fragments include an epitope at or near the C-terminus of alpha-SN
(e.g., within amino acids 70-140, 100-140, 120-140, 130-140, or
135-140). In some active fragments, the C terminal residue of the
epitope is the C-terminal residue of alpha-SN. Other components of
Lewy bodies, for example, synphilin-1, Parkin, ubiquitin,
neurofilament, beta-crystallin, and epitopic fragments thereof can
also be used to induce an immunogenic response.
[0061] Unless otherwise indicated, reference to alpha-SN includes
the natural human amino acid sequences indicated above as well as
analogs including allelic, species and induced variants,
full-length forms and immunogenic fragments thereof. Analogs
typically differ from naturally occurring peptides at one, two or a
few positions, often by virtue of conservative substitutions.
Analogs typically 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 at
position 139 of alpha-SN 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, O-phosphoserine, N-acetylserine,
N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,
omega-N-methylarginine, .beta.-alanine, ornithine, norleucine,
norvaline, hydroxyproline, thyroxine, gamma-amino butyric acid,
homoserine, citrulline, and isoaspartic acid. Therapeutic agents
also include analogs of alpha-SN fragments. Some therapeutic agents
of the invention are all-D peptides, e.g., all-D alpha-SN or all-D
NAC, and of all-D peptide analogs. Fragments and analogs can be
screened for prophylactic or therapeutic efficacy in transgenic
animal models in comparison with untreated or placebo controls as
described below.
[0062] Alpha-SN, 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, Calif. Recombinant expression can be in
bacteria, such as E. coli, yeast, insect cells or mammalian cells.
Procedures for recombinant expression are described by Sambrook et
al., Molecular Cloning: A Laboratory Manual (C.S.H.P. Press, NY 2d
ed., 1989). Some forms of alpha-SN peptide are also available
commercially, for example, at BACHEM and American Peptide Company,
Inc.
[0063] Therapeutic agents also include longer polypeptides that
include, for example, an active fragment of alpha-SN peptide,
together with other amino acids. For example, preferred agents
include fusion proteins comprising a segment of alpha-SN fused to a
heterologous amino acid sequence that induces a helper T-cell
response against the heterologous amino acid sequence and thereby a
B-cell response against the alpha-SN segment. Such polypeptides can
be screened for prophylactic or therapeutic efficacy in animal
models in comparison with untreated or placebo controls as
described below. The alpha-SN peptide, analog, active fragment or
other polypeptide can be administered in associated or multimeric
form or in dissociated form therapeutic agents also include
multimers of monomeric immunogenic agents. The therapeutic agents
of the invention may include polylysine sequences.
[0064] In a further variation, an immunogenic peptide, such as a
fragment of alpha-SN, can be presented by a virus or bacteria as
part of an immunogenic composition. A nucleic acid encoding the
immunogenic peptide is incorporated into a genome or episome of the
virus or bacteria. Optionally, the nucleic acid is incorporated in
such a manner that the immunogenic peptide is expressed as a
secreted protein or as a fusion protein with an outer surface
protein of a virus or a transmembrane protein of bacteria so that
the peptide is displayed. Viruses or bacteria used in such methods
should be nonpathogenic or attenuated. Suitable viruses include
adenovirus, HSV, Venezuelan equine encephalitis virus and other
alpha viruses, vesicular stomatitis virus, and other rhabdo
viruses, vaccinia and fowl pox. Suitable bacteria include
Salmonella and Shigella. Fusion of an immunogenic peptide to HBsAg
of HBV is particularly suitable.
[0065] Therapeutic agents also include peptides and other compounds
that do not necessarily have a significant amino acid sequence
similarity with alpha-SN but nevertheless serve as mimetics of
alpha-SN and induce a similar immune response. For example, any
peptides and proteins forming beta-pleated sheets can be screened
for suitability. Anti-idiotypic antibodies against monoclonal
antibodies to alpha-SN or other Lewy body components can also be
used. Such anti-Id antibodies mimic the antigen and generate an
immune response to it (see Essential Immunology, Roit ed.,
Blackwell Scientific Publications, Palo Alto, Calif. 6th ed., p.
181). Agents other than alpha-SN should induce an immunogenic
response against one or more of the preferred segments of alpha-SN
listed above (e.g., NAC). Preferably, such agents induce an
immunogenic response that is specifically directed to one of these
segments without being directed to other segments of alpha-SN.
[0066] 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 polypeptides,
beta-turn mimetics, polysaccharides, phospholipids, 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
95/30642 (each of which is incorporated herein by reference for all
purposes). Peptide libraries can also be generated by phage display
methods. See, e.g., Devlin, W0 91/18980.
[0067] Combinatorial libraries and other compounds are initially
screened for suitability by determining their capacity to bind to
antibodies or lymphocytes (B or T) known to be specific for
alpha-SN or other Lewy body components. For example, initial
screens can be performed with any polyclonal sera or monoclonal
antibody to alpha-SN or a fragment thereof. Compounds can then be
screened for binding to a specific epitope within alpha-SN (e.g.,
an epitope within NAC). Compounds can be tested by the same
procedures described for mapping antibody epitope specificities.
Compounds identified by such screens are then further analyzed for
capacity to induce antibodies or reactive lymphocytes to alpha-SN
or fragments thereof. For example, multiple dilutions of sera can
be tested on microtiter plates that have been precoated with
alpha-SN or a fragment thereof and a standard ELISA can be
performed to test for reactive antibodies to alpha-SN or the
fragment. Compounds can then be tested for prophylactic and
therapeutic efficacy in transgenic animals predisposed to a disease
associated with the presence of Lewy body, as described in the
Examples. Such animals include, for example, transgenic mice over
expressing alpha-SN or mutant thereof (e.g., alanine to threonine
substitution at position 53) as described, e.g., in WO 98/59050,
Masliah, et al., Science 287: 1265-1269 (2000), and van der Putter,
et al., J. Neuroscience 20: 6025-6029 (2000), or such transgenic
mice that also over express APP or a mutant thereof. Particularly
preferred are such synuclein transgenic mice bearing a 717 mutation
of APP described by Games et al., Nature 373, 523 (1995) and mice
bearing a 670/671 Swedish mutation of APP such as described by
McConlogue et al., U.S. Pat. No. 5,612,486 and Hsiao et al.,
Science 274, 99 (1996); Staufenbiel et al., Proc. Natl. Acad. Sci.
USA 94, 13287-13292 (1997); Sturchler-Pierrat et al., Proc. Natl.
Acad. Sci. USA 94, 13287-13292 (1997); Borchelt et al., Neuron 19,
939-945 (1997)). Examples of such synuclein/APP transgenic animals
are provided in WO 01/60794. Additional animal models of PD include
6-hydroxydopamine, rotenone, and
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal models
(M. Flint Beal, Nature Reviews Neuroscience 2:325-334 (2001)). The
same screening approach can be used on other potential analogs of
alpha-SN and longer peptides including fragments of alpha-SN,
described above and other Lewy body components and analog or
fragments thereof.
[0068] 2. Agents for Passive Immune Response
[0069] Therapeutic agents of the invention also include antibodies
that specifically bind to alpha-SN or other components of Lewy
bodies. Antibodies immunoreactive for alpha-SN are known (see, for
example, Arima, et al., Brian Res. 808: 93-100 (1998); Crowther et
al., Neuroscience Lett. 292: 128-130 (2000); Spillantini, et al.
Nature 388: 839-840 (1997). Such antibodies can be monoclonal or
polyclonal. Some such antibodies bind specifically to insoluble
aggregates of alpha-SN without binding to the soluble monomeric
form. Some bind specifically to the soluble monomeric form without
binding to the insoluble aggregated form. Some bind to both
aggregated and soluble monomeric forms. Some such antibodies bind
to a naturally occurring short form of alpha-SN (e.g., NAC) without
binding to a naturally occurring full length alpha-SN. Some
antibodies bind to a long form without binding to a short form.
Some antibodies bind to alpha-SN without binding to other
components of LBs. Antibodies used in therapeutic methods usually
have an intact constant region or at least sufficient of the
constant region to interact with an Fc receptor. Human isotype IgG1
is preferred because of it having highest affinity of human
isotypes for the FcRI receptor on phagocytic cells. Bispecific Fab
fragments can also be used, in which one arm of the antibody has
specificity for alpha-SN, and the other for an Fc receptor. Some
antibodies bind to alpha-SN with a binding affinity greater than or
equal to about 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, or 10.sup.10
M.sup.-1.
[0070] Polyclonal sera typically contain mixed populations of
antibodies binding to several epitopes along the length of
alpha-SN. However, polyclonal sera can be specific to a particular
segment of alpha-SN, such as NAC. Monoclonal antibodies bind to a
specific epitope within alpha-SN that can be a conformational or
nonconformational epitope. Prophylactic and therapeutic efficacy of
antibodies can be tested using the transgenic animal model
procedures described in the Examples. Preferred monoclonal
antibodies bind to an epitope within NAC. In some methods, multiple
monoclonal antibodies having binding specificities to different
epitopes are used. Such antibodies can be administered sequentially
or simultaneously. Antibodies to Lewy body components other than
alpha-SN can also be used. For example, antibodies can be directed
to neurofilament, ubiquitin, or synphilin. Therapeutic agents also
include antibodies raised against analogs of alpha-SN and fragments
thereof. Some therapeutic agents of the invention are all-D
peptides, e.g., all-D alpha-SN or all-D NAC.
[0071] When an antibody is said to bind to an epitope within
specified residues, such as alpha-SN 1-5, for example, what is
meant is that the antibody specifically binds to a polypeptide
containing the specified residues (i.e., alpha-SN 1-5 in this an
example). Such an antibody does not necessarily contact every
residue within alpha-SN 1-5. Nor does every single amino acid
substitution or deletion with in alpha-SN1-5 necessarily
significantly affect binding affinity. Epitope specificity of an
antibody can be determined, for example, by forming a phage display
library in which different members display different subsequences
of alpha-SN. The phage display library is then selected for members
specifically binding to an antibody under test. A family of
sequences is isolated. Typically, such a family contains a common
core sequence, and varying lengths of flanking sequences in
different members. The shortest core sequence showing specific
binding to the antibody defines the epitope bound by the antibody.
Antibodies can also be tested for epitope specificity in a
competition assay with an antibody whose epitope specificity has
already been determined.
[0072] Some antibodies of the invention specifically binds to an
epitope within NAC. Some antibodies specifically binds to an
epitope within a 22-kilodalton glycosylated form of synuclein,
e.g., P22-synuclein (H. Shimura et al., Science 2001 Jul.
13:293(5528):224-5). Some antibodies binds to an epitope at or near
the C-terminus of alpha-SN (e.g., within amino acids 70-140,
100-140, 120-140, 130-140 or 135-140. Some antibodies bind to an
epitope in which the C-terminal residue of the epitope is the
C-terminal residue of alpha-SN. In some methods, the antibody
specifically binds to NAC without binding to full length
alpha-SN.
[0073] Monoclonal or polyclonal antibodies that specifically bind
to a preferred segment of alpha-SN without binding to other regions
of alpha-SN have a number of advantages relative to monoclonal
antibodies binding to other regions or polyclonal sera to intact
alpha-SN. First, for equal mass dosages, dosages of antibodies that
specifically bind to preferred segments contain a higher molar
dosage of antibodies effective in clearing amyloid plaques. Second,
antibodies specifically binding to preferred segments can induce a
clearing response against LBs without inducing a clearing response
against intact alpha-SN, thereby reducing the potential for side
effects.
[0074] i. General Characteristics of Immunoglobulins
[0075] The basic antibody structural unit is known to comprise a
tetramer of subunits. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal
portion of each chain includes a variable region of about 100 to
110 or more amino acids primarily responsible for antigen
recognition. The carboxy-terminal portion of each chain defines a
constant region primarily responsible for effector function.
[0076] Light chains are classified as either kappa or lambda. Heavy
chains are classified as gamma, mu, alpha, delta, or epsilon, and
define the antibody's isotype as IgG, IgM, IgA, IgD and IgE,
respectively. Within light and heavy chains, the variable and
constant regions are joined by a "J" region of about 12 or more
amino acids, with the heavy chain also including a "D" region of
about 10 more amino acids. (See generally, Fundamental Immunology,
Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989, Ch. 7 (incorporated
by reference in its entirety for all purposes).
[0077] The variable regions of each light/heavy chain pair form the
antibody binding site. Thus, an intact antibody has two binding
sites. Except in bifunctional or bispecific antibodies, the two
binding sites are the same. The chains all exhibit the same general
structure of relatively conserved framework regions (FR) joined by
three hypervariable regions, also called complementarity
determining regions or CDRs. The CDRs from the two chains of each
pair are aligned by the framework regions, enabling binding to a
specific epitope. From N-terminal to C-terminal, both light and
heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3
and FR4. The assignment of amino acids to each domain is in
accordance with the definitions of Kabat, Sequences of Proteins of
Immunological Interest (National Institutes of Health, Bethesda,
Md., 1987 and 1991); Chothia & Lesk, J. Mol. Biol. 196:901-917
(1987); or Chothia et al., Nature 342:878-883 (1989).
[0078] ii. Production of Nonhuman Antibodies
[0079] 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 IgG1 and IgG4. Human isotype IgG1 is
preferred. In some methods, the isotype of the antibody is human
IgG1. 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.
[0080] 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. Sci. USA 86:10029-10033 (1989), WO
90/07861, U.S. Pat. No. 5,693,762, U.S. Pat. No. 5,693,761, U.S.
Pat. No. 5,585,089, U.S. Pat. No. 5,530,101, and Winter, U.S. Pat.
No. 5,225,539 (each of which is incorporated by reference in its
entirety for all purposes). The constant region(s), if present, are
also substantially or entirely from a human immunoglobulin. The
human variable 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.
[0081] For example, when an amino acid differs between a murine
variable region framework residue and a selected human variable
region framework residue, the human framework amino acid should
usually be substituted by the equivalent framework amino acid from
the mouse antibody when it is reasonably expected that the amino
acid:
(1) noncovalently binds antigen directly, (2) is adjacent to a CDR
region, (3) otherwise interacts with a CDR region (e.g. is within
about 6 A of a CDR region), or (4) participates in the VL-VH
interface.
[0082] Other candidates for substitution are acceptor human
framework amino acids that are unusual for a human immunoglobulin
at that position. These amino acids can be substituted with amino
acids from the equivalent position of the mouse donor antibody or
from the equivalent positions of more typical human
immunoglobulins. Other candidates for substitution are acceptor
human framework amino acids that are unusual for a human
immunoglobulin at that position. The variable region frameworks of
humanized immunoglobulins usually show at least 85% sequence
identity to a human variable region framework sequence or consensus
of such sequences.
[0083] iv. Human Antibodies
[0084] Human antibodies against alpha-SN 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, such as one of
the mouse monoclonals described in Example XI. Human antibodies can
also be screened for a particular epitope specificity by using only
a fragment of alpha-SN as the immunogen, and/or by screening
antibodies against a collection of deletion mutants of alpha-SN.
Human antibodies preferably have isotype specificity human
IgG1.
(1) Trioma Methodology
[0085] The basic approach and an exemplary cell fusion partner,
SPAZ-4, for use in this approach have been described by Oestberg et
al., Hybridoma 2:361-367 (1983); Oestberg, U.S. Pat. No. 4,634,664;
and Engleman et al., U.S. Pat. No. 4,634,666 (each of which is
incorporated by reference in its entirety for all purposes). The
antibody-producing cell lines obtained by this method are called
triomas, because they are descended from three cells-two human and
one mouse. Initially, a mouse myeloma line is fused with a human
B-lymphocyte to obtain a non-antibody-producing xenogeneic hybrid
cell, such as the SPAZ-4 cell line described by Oestberg, supra.
The xenogeneic cell is then fused with an immunized human
B-lymphocyte to obtain an antibody-producing trioma cell line.
Triomas have been found to produce antibody more stably than
ordinary hybridomas made from human cells.
[0086] The immunized B-lymphocytes are obtained from the blood,
spleen, lymph nodes or bone marrow of a human donor. If antibodies
against a specific antigen or epitope are desired, it is preferable
to use that antigen or epitope thereof for immunization.
Immunization can be either in vivo or in vitro. For in vivo
immunization, B cells are typically isolated from a human immunized
with alpha-SN, a fragment thereof, larger polypeptide containing
alpha-SN or fragment, or an anti-idiotypic antibody to an antibody
to alpha-SN. In some methods, B cells are isolated from the same
patient who is ultimately to be administered antibody therapy. For
in vitro immunization, B-lymphocytes are typically exposed to
antigen for a period of 7-14 days in a media such as RPMI-1640 (see
Engleman, supra) supplemented with 10% human plasma.
[0087] The immunized B-lymphocytes are fused to a xenogeneic hybrid
cell such as SPAZ-4 by well known methods. For example, the cells
are treated with 40-50% polyethylene glycol of MW 1000-4000, at
about 37 degrees C., for about 5-10 min. Cells are separated from
the fusion mixture and propagated in media selective for the
desired hybrids (e.g., HAT or AH). Clones secreting antibodies
having the required binding specificity are identified by assaying
the trioma culture medium for the ability to bind to alpha-SN or a
fragment thereof. Triomas producing human antibodies having the
desired specificity are subcloned by the limiting dilution
technique and grown in vitro in culture medium. The trioma cell
lines obtained are then tested for the ability to bind alpha-SN or
a fragment thereof.
[0088] Although triomas are genetically stable they do not produce
antibodies at very high levels. Expression levels can be increased
by cloning antibody genes from the trioma into one or more
expression vectors, and transforming the vector into standard
mammalian, bacterial or yeast cell lines.
(2) Transgenic Non-Human Mammals
[0089] Human antibodies against alpha-SN can also be produced from
non-human transgenic mammals having transgenes encoding at least a
segment of the human immunoglobulin locus. Usually, the endogenous
immunoglobulin locus of such transgenic mammals is functionally
inactivated. Preferably, the segment of the human immunoglobulin
locus includes unrearranged sequences of heavy and light chain
components. Both inactivation of endogenous immunoglobulin genes
and introduction of exogenous immunoglobulin genes can be achieved
by targeted homologous recombination, or by introduction of YAC
chromosomes. The transgenic mammals resulting from this process are
capable of functionally rearranging the immunoglobulin component
sequences, and expressing a repertoire of antibodies of various
isotypes encoded by human immunoglobulin genes, without expressing
endogenous immunoglobulin genes. The production and properties of
mammals having these properties are described in detail by, e.g.,
Lonberg et al., WO93/1222, U.S. Pat. No. 5,877,397, U.S. Pat. No.
5,874,299, U.S. Pat. No. 5,814,318, U.S. Pat. No. 5,789,650, U.S.
Pat. No. 5,770,429, U.S. Pat. No. 5,661,016, U.S. Pat. No.
5,633,425, U.S. Pat. No. 5,625,126, U.S. Pat. No. 5,569,825, U.S.
Pat. No. 5,545,806, Nature 148, 1547-1553 (1994), Nature
Biotechnology 14, 826 (1996), Kucherlapati, WO 91/10741 (each of
which is incorporated by reference in its entirety for all
purposes). Transgenic mice are particularly suitable. Anti-alpha-SN
antibodies are obtained by immunizing a transgenic nonhuman mammal,
such as described by Lonberg or Kucherlapati, supra, with alpha-SN
or a fragment thereof. Monoclonal antibodies are prepared by, e.g.,
fusing B-cells from such mammals to suitable myeloma cell lines
using conventional Kohler-Milstein technology. Human polyclonal
antibodies can also be provided in the form of serum from humans
immunized with an immunogenic agent. Optionally, such polyclonal
antibodies can be concentrated by affinity purification using
alpha-SN or other amyloid peptide as an affinity reagent.
(3) Phage Display Methods
[0090] A further approach for obtaining human anti-alpha-SN
antibodies is to screen a DNA library from human B cells according
to the general protocol outlined by Huse et al., Science
246:1275-1281 (1989). As described for trioma methodology, such B
cells can be obtained from a human immunized with alpha-SN,
fragments, longer polypeptides containing alpha-SN or fragments or
anti-idiotypic antibodies. Optionally, such B cells are obtained
from a patient who is ultimately to receive antibody treatment.
Antibodies binding to alpha-SN or a fragment thereof are selected.
Sequences encoding such antibodies (or binding fragments) are then
cloned and amplified. The protocol described by Huse is rendered
more efficient in combination with phage-display technology. See,
e.g., Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047,
U.S. Pat. No. 5,877,218, U.S. Pat. No. 5,871,907, U.S. Pat. No.
5,858,657, U.S. Pat. No. 5,837,242, U.S. Pat. No. 5,733,743 and
U.S. Pat. No. 5,565,332 (each of which is incorporated by reference
in its entirety for all purposes). In these methods, libraries of
phage are produced in which members display different antibodies on
their outer surfaces. Antibodies are usually displayed as Fv or Fab
fragments. Phage displaying antibodies with a desired specificity
are selected by affinity enrichment to an alpha-SN peptide or
fragment thereof.
[0091] In a variation of the phage-display method, human antibodies
having the binding specificity of a selected murine antibody can be
produced. See Winter, WO 92/20791. In this method, either the heavy
or light chain variable region of the selected murine antibody is
used as a starting material. If, for example, a light chain
variable region is selected as the starting material, a phage
library is constructed in which members display the same light
chain variable region (i.e., the murine starting material) and a
different heavy chain variable region. The heavy chain variable
regions are obtained from a library of rearranged human heavy chain
variable regions. A phage showing strong specific binding for
alpha-SN (e.g., at least 10.sup.8 and preferably at least 10.sup.9
M.sup.-1) is selected. The human heavy chain variable region from
this phage then serves as a starting material for constructing a
further phage library. In this library, each phage displays the
same heavy chain variable region (i.e., the region identified from
the first display library) and a different light chain variable
region. The light chain variable regions are obtained from a
library of rearranged human variable light chain regions. Again,
phage showing strong specific binding for alpha-SN are selected.
These phage display the variable regions of completely human
anti-alpha-SN antibodies. These antibodies usually have the same or
similar epitope specificity as the murine starting material.
[0092] v. Selection of Constant Region
[0093] 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 IgG1 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 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.
[0094] vi. Expression of Recombinant Antibodies
[0095] Chimeric, humanized and human antibodies are typically
produced by recombinant expression. Recombinant polynucleotide
constructs typically include an expression control sequence
operably linked to the coding sequences of antibody chains,
including naturally associated or heterologous promoter regions.
Preferably, the expression control sequences are eukaryotic
promoter systems in vectors capable of transforming or transfecting
eukaryotic host cells. Once the vector has been incorporated into
the appropriate host, the host is maintained under conditions
suitable for high level expression of the nucleotide sequences, and
the collection and purification of the crossreacting
antibodies.
[0096] These expression vectors are typically replicable in the
host organisms either as episomes or as an integral part of the
host chromosomal DNA. Commonly, expression vectors contain
selection markers, e.g., ampicillin-resistance or
hygromycin-resistance, to permit detection of those cells
transformed with the desired DNA sequences.
[0097] E. coli is one prokaryotic host particularly useful for
cloning the DNA sequences of the present invention. Microbes, such
as yeast are also useful for expression. Saccharomyces is a
preferred yeast host, with suitable vectors having expression
control sequences, an origin of replication, termination sequences
and the like as desired. Typical promoters include
3-phosphoglycerate kinase and other glycolytic enzymes. Inducible
yeast promoters include, among others, promoters from alcohol
dehydrogenase, isocytochrome C, and enzymes responsible for maltose
and galactose utilization.
[0098] Mammalian cells are a preferred host for expressing
nucleotide segments encoding immunoglobulins or fragments thereof.
See Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). A
number of suitable host cell lines capable of secreting intact
heterologous proteins have been developed in the art, and include
CHO cell lines, various COS cell lines, HeLa cells, L cells, human
embryonic kidney cell, and myeloma cell lines. Preferably, the
cells are nonhuman. Expression vectors for these cells can include
expression control sequences, such as an origin of replication, a
promoter, an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)),
and necessary processing information sites, such as ribosome
binding sites, RNA splice sites, polyadenylation sites, and
transcriptional terminator sequences. Preferred expression control
sequences are promoters derived from endogenous genes,
cytomegalovirus, SV40, adenovirus, bovine papillomavirus, and the
like. See Co et al., J. Immunol. 148:1149 (1992).
[0099] Alternatively, antibody coding sequences can be incorporated
in transgenes for introduction into the genome of a transgenic
animal and subsequent expression in the milk of the transgenic
animal (see, e.g., U.S. Pat. No. 5,741,957, U.S. Pat. No.
5,304,489, U.S. Pat. No. 5,849,992). Suitable transgenes include
coding sequences for light and/or heavy chains in operable linkage
with a promoter and enhancer from a mammary gland specific gene,
such as casein or beta lactoglobulin.
[0100] The vectors containing the DNA segments of interest can be
transferred into the host cell by well-known methods, depending on
the type of cellular host. For example, calcium chloride
transfection is commonly utilized for prokaryotic cells, whereas
calcium phosphate treatment, electroporation, lipofection,
biolistics or viral-based transfection can be used for other
cellular hosts. Other methods used to transform mammalian cells
include the use of polybrene, protoplast fusion, liposomes,
electroporation, and microinjection (see generally, Sambrook et al.
supra). For production of transgenic animals, transgenes can be
microinjected into fertilized oocytes, or can be incorporated into
the genome of embryonic stem cells, and the nuclei of such cells
transferred into enucleated oocytes.
[0101] Once expressed, antibodies can be purified according to
standard procedures of the art, including HPLC purification, column
chromatography, gel electrophoresis and the like (see generally,
Scopes, Protein Purification (Springer-Verlag, NY, 1982)).
[0102] 3. Conjugates
[0103] Some agents for inducing an immune response contain the
appropriate epitope for inducing an immune response against LBs but
are too small to be immunogenic. In this situation, a peptide
immunogen can be linked to a suitable carrier molecule to form a
conjugate which helps elicit an immune response. Suitable carriers
include serum albumins, keyhole limpet hemocyanin, immunoglobulin
molecules, thyroglobulin, ovalbumin, tetanus toxoid, or a toxoid
from other pathogenic bacteria, such as diphtheria, E. coli,
cholera, or H. pylori, or an attenuated toxin derivative. T cell
epitopes are also suitable carrier molecules. Some conjugates can
be formed by linking agents of the invention to an
immunostimulatory polymer molecule (e.g., tripalmitoyl-S-glycerine
cysteine (Pam.sub.3Cys), mannan (a manose polymer), or glucan (a
beta 1.fwdarw.2 polymer)), cytokines (e.g., IL-1, IL-1 alpha and
beta peptides, IL-2, gamma-INF, IL-10, GM-CSF), and chemokines
(e.g., MIP1alpha and beta, and RANTES). Immunogenic agents can also
be linked to peptides that enhance transport across tissues, as
described in O'Mahony, WO 97/17613 and WO 97/17614. Immunogens may
be linked to the carries with or with out spacers amino acids
(e.g., gly-gly).
[0104] Some conjugates can be formed by linking agents of the
invention to at least one T cell epitope. Some T cell epitopes are
promiscuous while other T cell epitopes are universal. Promiscuous
T cell epitopes are capable of enhancing the induction of T cell
immunity in a wide variety of subjects displaying various HLA
types. In contrast to promiscuous T cell epitopes, universal T cell
epitopes are capable of enhancing the induction of T cell immunity
in a large percentage, e.g., at least 75%, of subjects displaying
various HLA molecules encoded by different HLA-DR alleles.
[0105] A large number of naturally occurring T-cell epitopes exist,
such as, tetanus toxoid (e.g., the P2 and P30 epitopes), Hepatitis
B surface antigen, pertussis, toxoid, measles virus F protein,
Chlamydia trachomitis major outer membrane protein, diphtheria
toxoid, Plasmodium falciparum circumsporozite T, Plasmodium
falciparum CS antigen, Schistosoma mansoni triose phosphate
isomersae, Escherichia coli TraT, and Influenza virus hemagluttinin
(HA). The immunogenic peptides of the invention can also be
conjugated to the T-cell epitopes described in Sinigaglia F. et
al., Nature, 336:778-780 (1988); Chicz R. M. et al., J. Exp. Med.,
178:27-47 (1993); Hammer J. et al., Cell 74:197-203 (1993); Falk K.
et al., Immunogenetics, 39:230-242 (1994); WO 98/23635; and,
Southwood S. et al. J. Immunology, 160:3363-3373 (1998) (each of
which is incorporated herein by reference for all purposes).
Further examples include:
[0106] Influenza Hemagluttinin: HA.sub.307-319 PKYVKQNTLKLAT (SEQ
ID NO: 4)
[0107] Malaria CS: T3 epitope EKKIAKMEKASSVFNV (SEQ ID NO: 5)
[0108] Hepatitis B surface antigen: HBsAg.sub.19-28 FFLLTRILTI (SEQ
ID NO: 6)
[0109] Heat Shock Protein 65: hsp65.sub.153-171 DQSIGDLIAEAMDKVGNEG
(SEQ ID NO: 7)
[0110] bacille Calmette-Guerin QVHFQPLPPAVVKL (SEQ ID NO: 8)
[0111] Tetanus toxoid: TT.sub.830-844 QYIKANSKFIGITEL (SEQ ID NO:
9)
[0112] Tetanus toxoid: TT.sub.947-967 FNNFTVSFWLRVPKVSASHLE (SEQ ID
NO: 10)
[0113] HIV gp120 T1: KQIINMWQEVGKAMYA (SEQ ID NO: 11)
[0114] Alternatively, the conjugates can be formed by linking
agents of the invention to at least one artificial T-cell epitope
capable of binding a large proportion of MHC Class II molecules.,
such as the pan DR epitope ("PADRE"). PADRE is described in U.S.
Pat. No. 5,736,142, WO 95/07707, and Alexander J et al., Immunity,
1:751-761 (1994) (each of which is incorporated herein by reference
for all purposes). A preferred PADRE peptide is AKXVAAWTLKAAA (SEQ
ID NO: 12), (common residues bolded) wherein X is preferably
cyclohexylalanine, tyrosine or phenylalanine, with
cyclohexylalanine being most preferred.
[0115] Immunogenic agents can be linked to carriers by chemical
crosslinking. Techniques for linking an immunogen to a carrier
include the formation of disulfide linkages using
N-succinimidyl-3-(2-pyridyl-thio) propionate (SPDP) and
succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC)
(if the peptide lacks a sulfhydryl group, this can be provided by
addition of a cysteine residue). These reagents create a disulfide
linkage between themselves and peptide cysteine resides on one
protein and an amide linkage through the epsilon-amino on a lysine,
or other free amino group in other amino acids. A variety of such
disulfide/amide-forming agents are described by Immun. Rev. 62, 185
(1982). Other bifunctional coupling agents form a thioether rather
than a disulfide linkage. Many of these thio-ether-forming agents
are commercially available and include reactive esters of
6-maleimidocaproic acid, 2-bromoacetic acid, and 2-iodoacetic acid,
4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid. The carboxyl
groups can be activated by combining them with succinimide or
1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt.
[0116] Immunogenicity can be improved through the addition of
spacer residues (e.g., Gly-Gly) between the T.sub.h epitope and the
peptide immunogen of the invention. In addition to physically
separating the T.sub.h epitope from the B cell epitope (i.e., the
peptide immunogen), the glycine residues can disrupt any artificial
secondary structures created by the joining of the T.sub.h epitope
with the peptide immunogen, and thereby eliminate interference
between the T and/or B cell responses. The conformational
separation between the helper epitope and the antibody eliciting
domain thus permits more efficient interactions between the
presented immunogen and the appropriate T.sub.h and B cells.
[0117] To enhance the induction of T cell immunity in a large
percentage of subjects displaying various HLA types to an agent of
the present invention, a mixture of conjugates with different
T.sub.h cell epitopes can be prepared. The mixture may contain a
mixture of at least two conjugates with different T.sub.h cell
epitopes, a mixture of at least three conjugates with different
T.sub.h cell epitopes, or a mixture of at least four conjugates
with different T.sub.h cell epitopes. The mixture may be
administered with an adjuvant.
[0118] Immunogenic peptides can also be expressed as fusion
proteins with carriers (i.e., heterologous peptides). The
immunogenic peptide can be linked at its amino terminus, its
carboxyl terminus, or both to a carrier. Optionally, multiple
repeats of the immunogenic peptide can be present in the fusion
protein. Optionally, an immunogenic peptide can be linked to
multiple copies of a heterologous peptide, for example, at both the
N and C termini of the peptide. Some carrier peptides serve to
induce a helper T-cell response against the carrier peptide. The
induced helper T-cells in turn induce a B-cell response against the
immunogenic peptide linked to the carrier peptide.
[0119] Some agents of the invention comprise a fusion protein in
which an N-terminal fragment of alpha-SN is linked at its
C-terminus to a carrier peptide. In such agents, the N-terminal
residue of the fragment of alpha-SN constitutes the N-terminal
residue of the fusion protein. Accordingly, such fusion proteins
are effective in inducing antibodies that bind to an epitope that
requires the N-terminal residue of alpha-SN to be in free form.
Some agents of the invention comprise a plurality of repeats of NAC
linked at the C-terminus to one or more copy of a carrier peptide.
Some fusion proteins comprise different segments of alpha-SN in
tandem.
[0120] In some fusion proteins, NAC is fused at its N-terminal end
to a heterologous carrier peptide. NAC can be used with C-terminal
fusions. Some fusion proteins comprise a heterologous peptide
linked to the N-terminus or C-terminus of NAC, which is in turn
linked to one or more additional NAC segments of alpha-SN in
tandem.
[0121] Some examples of fusion proteins suitable for use in the
invention are shown below. Some of these fusion proteins comprise
segments of alpha-SN linked to tetanus toxoid epitopes such as
described in U.S. Pat. No. 5,196,512, EP 378,881 and EP 427,347.
Some fusion proteins comprise segments of alpha-SN linked to at
least one PADRE Some heterologous peptides are promiscuous T-cell
epitopes, while other heterologous peptides are universal T-cell
epitopes. In some methods, the agent for administration is simply a
single fusion protein with an alpha-SN segment linked to a
heterologous segment in linear configuration. The therapeutic
agents of the invention may be represented using a formula. For
example, in some methods, the agent is multimer of fusion proteins
represented by the formula 2.sup.x, in which x is an integer from
1-5. Preferably x is 1, 2, or 3, with 2 being most preferred. When
x is two, such a multimer has four fusion proteins linked in a
preferred configuration referred to as MAP4 (see U.S. Pat. No.
5,229,490).
[0122] The MAP4 configuration is shown below, where branched
structures are produced by initiating peptide synthesis at both the
N terminal and side chain amines of lysine. Depending upon the
number of times lysine is incorporated into the sequence and
allowed to branch, the resulting structure will present multiple N
termini. In this example, four identical N termini have been
produced on the branched lysine-containing core. Such multiplicity
greatly enhances the responsiveness of cognate B cells.
##STR00001##
[0123] Z refers to the NAC peptide, a fragment of the NAC peptide,
or other active fragment of alpha-SN as described in section I. 2
above. Z may represent more than one active fragment, for
example:
Z=alpha-SN 60-72 (NAC region) peptide=NH2-KEQVTNVCGGAVVT-COOH (SEQ
ID NO: 13) Z=alpha-SN 73-84 (NAC region)
peptide=NH2-GVTAVAQKTVECG-COOH (SEQ ID NO: 14) Z=alpha-SN 102-112
peptide=NH2-C-amino-heptanoic acid-KNEEGAPCQEG-COOH (SEQ ID NO: 15)
alpha-SN 128-140 peptide
[0124] Other examples of fusion proteins include:
Z-Tetanus toxoid 830-844 in a MAP4 configuration:
TABLE-US-00004 Z-QYIKANSKFIGITEL (SEQ ID NO: 16)
Z-Tetanus toxoid 947-967 in a MAP4 configuration:
TABLE-US-00005 Z-FNNFTVSFWLRVPKVSASHLE (SEQ ID NO: 17)
Z-Tetanus toxoid.sub.830-844 in a MAP4 configuration:
TABLE-US-00006 Z-QYIKANSKFIGITEL (SEQ ID NO: 18)
[0125] Z-Tetanus toxoid.sub.830-844+Tetanus toxoid.sub.947-967 in a
linear configuration:
TABLE-US-00007 (SEQ ID NO: 19)
Z-QYIKANSKFIGITELFNNFTVSFWLRVPKVSASHLE
[0126] PADRE peptide (all in linear configurations), wherein X is
preferably cyclohexylalanine, tyrosine or phenylalanine, with
cyclohexylalanine being most preferred-Z:
TABLE-US-00008 AKXVAAWTLKAAA-Z (SEQ ID NO: 20)
3Z-PADRE peptide:
TABLE-US-00009 Z-Z-Z-AKXVAAWTLKAAA (SEQ ID NO: 21)
[0127] Further examples of fusion proteins include:
TABLE-US-00010 (SEQ ID NO: 22) AKXVAAWTLKAAA-Z-Z-Z-Z (SEQ ID NO:
23) Z-AKXVAAWTLKAAA (SEQ ID NO: 24) Z-ISQAVHAAHAEINEAGR (SEQ ID NO:
25) PKYVKQNTLKLAT-Z-Z-Z (SEQ ID NO: 26) Z-PKYVKQNTLKLAT-Z (SEQ ID
NO: 27) Z-Z-Z-PKYVKQNTLKLAT (SEQ ID NO: 28) Z-Z-PKYVKQNTLKLAT (SEQ
ID NO: 29) Z-PKYVKQNTLKLAT-EKKIAKMEKASSVFNV-QYIKANSKFIGITEL-
FNNFTVSFWLRVPKVSASHLE-Z-Z-Z-Z-QYIKANSKFIGITEL-FNN
FTVSFWLRVPKVSASHLE (SEQ ID NO: 30)
Z-QYIKANSKFIGITELCFNNFTVSFWLRVPKVSASHLE-Z-QYIKANS
KFIGITELCFNNFTVSFWLRVPKVSASHLE-Z
Z-QYIKANSKFIGITEL (SEQ ID NO: 31) on a 2 branched resin:
##STR00002##
[0128] The same or similar carrier proteins and methods of linkage
can be used for generating immunogens to be used in generation of
antibodies against alpha-SN for use in passive immunization. For
example, alpha-SN or a fragment linked to a carrier can be
administered to a laboratory animal in the production of monoclonal
antibodies to alpha-SN.
[0129] 4. Nucleic Acid Encoding Therapeutic Agents
[0130] Immune responses against Lewy bodies can also be induced by
administration of nucleic acids encoding segments of alpha-SN
peptide, and fragments thereof, other peptide immunogens, or
antibodies and their component chains used for passive
immunization. Such nucleic acids can be DNA or RNA. A nucleic acid
segment encoding an immunogen is typically linked to regulatory
elements, such as a promoter and enhancer that allow expression of
the DNA segment in the intended target cells of a patient. For
expression in blood cells, as is desirable for induction of an
immune response, promoter and enhancer elements from light or heavy
chain immunoglobulin genes or the CMV major intermediate early
promoter and enhancer are suitable to direct expression. The linked
regulatory elements and coding sequences are often cloned into a
vector. For administration of double-chain antibodies, the two
chains can be cloned in the same or separate vectors. The nucleic
acid encoding therapeutic agents of the invention may also encode
at least one T cell epitope. The disclosures herein which relates
to the use of adjuvants and the use of apply mutatis mutandis to
their use with the nucleic acid encoding therapeutic agents of the
present invention.
[0131] A number of viral vector systems are available including
retroviral systems (see, e.g., Lawrie and Tumin, Cur. Opin. Genet.
Develop. 3, 102-109 (1993)); adenoviral vectors (see, e.g., Bett et
al., J. Virol. 67, 5911 (1993)); adeno-associated virus vectors
(see, e.g., Zhou et al., J. Exp. Med. 179, 1867 (1994)), viral
vectors from the pox family including vaccinia virus and the avian
pox viruses, viral vectors from the alpha virus genus such as those
derived from Sindbis and Semliki Forest Viruses (see, e.g.,
Dubensky et al., J. Virol. 70, 508-519 (1996)), Venezuelan equine
encephalitis virus (see U.S. Pat. No. 5,643,576) and rhabdoviruses,
such as vesicular stomatitis virus (see WO 96/34625) and
papillomaviruses (Ohe et al., Human Gene Therapy 6, 325-333 (1995);
Woo et al., WO 94/12629 and Xiao & Brandsma, Nucleic Acids.
Res. 24, 2630-2622 (1996)).
[0132] DNA encoding an immunogen, or a vector containing the same,
can be packaged into liposomes. Suitable lipids and related analogs
are described by U.S. Pat. No. 5,208,036, U.S. Pat. No. 5,264,618,
U.S. Pat. No. 5,279,833, and U.S. Pat. No. 5,283,185. Vectors and
DNA encoding an immunogen can also be adsorbed to or associated
with particulate carriers, examples of which include polymethyl
methacrylate polymers and polylactides and
poly(lactide-co-glycolides), (see, e.g., McGee et al., J. Micro
Encap. 1996).
[0133] Gene therapy vectors or naked DNA can be delivered in vivo
by administration to an individual patient, typically by systemic
administration (e.g., intravenous, intraperitoneal, nasal, gastric,
intradermal, intramuscular, subdermal, or intracranial infusion) or
topical application (see e.g., U.S. Pat. No. 5,399,346). Such
vectors can further include facilitating agents such as bupivacine
(see e.g., U.S. Pat. No. 5,593,970). DNA can also be administered
using a gene gun. See Xiao & Brandsma, supra. The DNA encoding
an immunogen is precipitated onto the surface of microscopic metal
beads. The microprojectiles are accelerated with a shock wave or
expanding helium gas, and penetrate tissues to a depth of several
cell layers. For example, The Accel.TM. Gene Delivery Device
manufactured by Agacetus, Inc. Middleton, Wis. is suitable.
Alternatively, naked DNA can pass through skin into the blood
stream simply by spotting the DNA onto skin with chemical or
mechanical irritation (see WO 95/05853).
[0134] In a further variation, vectors encoding immunogens can be
delivered to cells ex vivo, such as cells explanted from an
individual patient (e.g., lymphocytes, bone marrow aspirates, and
tissue biopsy) or universal donor hematopoietic stem cells,
followed by reimplantation of the cells into a patient, usually
after selection for cells which have incorporated the vector.
III. Agents for Inducing Immunogenic Response Against A.beta.
[0135] A.beta., also known as .beta.-amyloid peptide, or A4 peptide
(see U.S. Pat. No. 4,666,829; Glenner & Wong, Biochem. Biophys.
Res. Commun. 120, 1131 (1984)), is a peptide of 39-43 amino acids,
which is the principal component of characteristic plaques of
Alzheimer's disease. A.beta. is generated by processing of a larger
protein APP by two enzymes, termed .beta. and .gamma. secretases
(see Hardy, TINS 20, 154 (1997)). Known mutations in APP associated
with Alzheimer's disease occur proximate to the site of .beta. or
.gamma. secretase, or within A.beta.. For example, position 717 is
proximate to the site of .gamma.-secretase cleavage of APP in its
processing to A.beta., and positions 670/671 are proximate to the
site of .beta.-secretase cleavage. It is believed that the
mutations cause AD by interacting with the cleavage reactions by
which A.beta. is formed so as to increase the amount of the 42/43
amino acid form of A.beta. generated.
[0136] A.beta. has the unusual property that it can fix and
activate both classical and alternate complement cascades. In
particular, it binds to C1q and ultimately to C3bi. This
association facilitates binding to macrophages leading to
activation of B cells. In addition, C3bi breaks down further and
then binds to CR2 on B cells in a T cell dependent manner leading
to a 10,000 increase in activation of these cells. This mechanism
causes A.beta. to generate an immune response in excess of that of
other antigens.
[0137] A.beta. has several natural occurring forms. The human forms
of A.beta. are referred to as A.beta.39, A.beta.40, A.beta.41,
A.beta.42 and A.beta.43. The sequences of these peptides and their
relationship to the APP precursor are illustrated by FIG. 1 of
Hardy et al., TINS 20, 155-158 (1997). For example, A.beta.42 has
the sequence:
TABLE-US-00011 (SEQ ID NO: 33)
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAT
[0138] A.beta.41, A.beta.40 and A.beta.39 differ from A.beta.42 by
the omission of Ala, Ala-Ile, and Ala-Ile-Val respectively from the
C-terminal end. A.beta.43 differs from A.beta.42 by the presence of
a Thr residue at the C-terminus.
[0139] Analogous agents to those described above for alpha-SN have
previously been described for A.beta. (see WO 98/25386 and WO
00/72880, both of which are incorporated herein for all purposes).
These agents include A.beta. and active fragments thereof,
conjugates of A.beta., and conjugates of A.beta. active fragments,
antibodies to A.beta. and active fragments thereof (e.g., mouse,
humanized, human, and chimeric antibodies), and nucleic acids
encoding antibody chains. Active fragments from the N-terminal half
of A.beta. are preferred. Preferred immunogenic fragments include
A.beta.1-5, 1-6, 1-7, 1-10, 3-7, 1-3; and 1-4. The designation
A.beta.1-5 for example, indicates a fragment including residues 1-5
of A.beta. and lacking other residues of A.beta.. Fragments
beginning at residues 1-3 of A.beta. and ending at residues 7-11 of
A.beta. are particularly preferred.
[0140] The disclosures herein which relates to agents inducing an
active immune response, agents for inducing a passive immune
response, conjugates, and nucleic acids encoding therapeutic agents
(see Sections II. 1, 2, 3, and 4, above) apply mutatis mutandis to
the use of A.beta. and fragments thereof. The disclosures herein
which relate to agents inducing an active immune response, agents
for inducing a passive immune response, conjugates, and nucleic
acids encoding therapeutic agents (see Sections II. 1, 2, 3, and 4,
above) apply mutatis mutandis to the use of A.beta. and fragments
thereof. The disclosures herein which relate to patients amendable
to treatment, and treatment regimes (see Sections IV and V, below)
apply mutatis mutandis to the use of A.beta. and fragments
thereof.
[0141] Disaggregated A.beta. or fragments thereof means monomeric
peptide units. Disaggregated A.beta. or fragments thereof are
generally soluble, and are capable of self-aggregating to form
soluble oligomers. Oligomers of A.beta. and fragments thereof are
usually soluble and exist predominantly as alpha-helices or random
coils. Aggregated A.beta. or fragments thereof, means oligomers of
alpha-SN or fragments thereof that have associate into insoluble
beta-sheet assemblies. Aggregated A.beta. or fragments thereof,
means also means fibrillar polymers. Fibrils are usually insoluble.
Some antibodies bind either soluble A.beta. or fragments thereof or
aggregated A.beta. or fragments thereof. Some antibodies bind both
soluble A.beta. or fragments thereof and aggregated A.beta. or
fragments thereof.
Some examples of conjugates include: AN90549 (A.beta.1-7-Tetanus
toxoid 830-844 in a MAP4 configuration):
TABLE-US-00012 DAEFRHD-QYIKANSKFIGITEL (SEQ ID NO: 34)
AN90550 (A.beta.1-7-Tetanus toxoid 947-967 in a MAP4
configuration):
TABLE-US-00013 DAEFRHD-FNNFTVSFWLRVPKVSASHLE (SEQ ID NO: 35)
AN90542 (A.beta.1-7-Tetanus toxoid 830-844+947-967 in a linear
configuration):
TABLE-US-00014 (SEQ ID NO: 36)
DAEFRHD-QYIKANSKFIGITELFNNFTVSFWLRVPKVSASHLE
AN90576: (A.beta.3-9)-Tetanus toxoid 830-844 in a MAP4
configuration):
TABLE-US-00015 EFRHDSG-QYIKANSKFIGITEL (SEQ ID NO: 37)
[0142] PADRE peptide (all in linear configurations), wherein X is
preferably cyclohexylalanine, tyrosine or phenylalanine, with
cyclohexylalanine being most preferred:
[0143] AN90562 (PADRE-A.beta.1-7):
TABLE-US-00016 AKXVAAWTLAAA-DAEFRHD (SEQ ID NO: 38)
[0144] AN90543 (3 PADRE-A.beta.1-7):
TABLE-US-00017 (SEQ ID NO: 39)
DAEFRHD-DAEFRHD-DAEFRHD-AKXVAAWTLKAAA
[0145] Other examples of fusion proteins (immunogenic epitope of
A.beta. bolded) include:
TABLE-US-00018 (SEQ ID NO: 40)
AKXVAAWTLKAAA-DAEFRHD-DAEFRHD-DAEFRHD (SEQ ID NO: 41)
DAEFRHD-AKXVAAWTLKAAA (SEQ ID NO: 42) DAEFRHD-ISQAVHAAHAEINEAGR
(SEQ ID NO: 43) FRHDSGY-ISQAVHAAHAEINEAGR (SEQ ID NO: 44)
EFRHDSG-ISQAVHAAHAEINEAGR (SEQ ID NO: 45)
PKYVKQNTLKLAT-DAEFRHD-DAEFRHD-DAEFRHD (SEQ ID NO: 46)
DAEFRHD-PKYVKQNTLKLAT-DAEFRHD (SEQ ID NO: 47)
DAEFRHD-DAEFRHD-DAEFRHD-PKYVKQNTLKLAT (SEQ ID NO: 48)
DAEFRHD-DAEFRHD-PKYVKQNTLKLAT (SEQ ID NO: 49)
DAEFRHD-PKYVKQNTLKLAT-EKKIAKMEKASSVFNV-QYIKANSKFI
GITEL-FNNFTVSFWLRVPKVSASHLE-DAEFRHD (SEQ ID NO: 50)
DAEFRHD-DAEFRHD-DAEFRHD-QYIKANSKFIGITELNNFTVSFWLR VPKVSASHLE (SEQ
ID NO: 51) DAEFRHD-QYIKANSKFIGITELCFNNFTVSFWLRVPKVSASHLE (SEQ ID
NO: 52) DAEFRHD-QYIKANSKFIGITELCFNNFTVSFWLRVPKVSASHLE-DAE FRHD (SEQ
ID NO: 53) DAEFRHD-QYIKANSKFIGITEL on a 2 branched resin.
##STR00003##
[0146] Preferred monoclonal antibodies bind to an epitope within
residues 1-10 of A.beta. (with the first N terminal residue of
natural A.beta. designated 1). Some preferred monoclonal antibodies
bind to an epitope within amino acids 1-5, and some to an epitope
within 5-10. Some preferred antibodies bind to epitopes within
amino acids 1-3, 1-4, 1-5, 1-6, 1-7 or 3-7. Some preferred
antibodies bind to an epitope starting at resides 1-3 and ending at
residues 7-11 of A.beta.. Other antibodies include those binding to
epitopes with residues 13-280 (e.g., monoclonal antibody 266).
Preferred antibodies have human IgG1 isotype.
IV. Screening Antibodies for Clearing Activity
[0147] The invention provides methods of screening an antibody for
activity in clearing a Lewy body or any other antigen, or
associated biological entity, for which clearing activity is
desired. To screen for activity against a Lewy body, a tissue
sample from a brain of a patient with PD or an animal model having
characteristic Parkinson's pathology is contacted with phagocytic
cells bearing an Fc receptor, such as microglial cells, and the
antibody under test in a medium in vitro. The phagocytic cells can
be a primary culture or a cell line, such as BV-2, C8-B4, or THP-1.
In some methods, the components are combined on a microscope slide
to facilitate microscopic monitoring. In some methods, multiple
reactions are performed in parallel in the wells of a microtiter
dish. In such a format, a separate miniature microscope slide can
be mounted in the separate wells, or a nonmicroscopic detection
format, such as ELISA detection of alpha-SN can be used.
Preferably, a series of measurements is made of the amount of Lewy
body in the in vitro reaction mixture, starting from a baseline
value before the reaction has proceeded, and one or more test
values during the reaction. The antigen can be detected by
staining, for example, with a fluorescently labeled antibody to
alpha-SN or other component of amyloid plaques. The antibody used
for staining may or may not be the same as the antibody being
tested for clearing activity. A reduction relative to baseline
during the reaction of the LBs indicates that the antibody under
test has clearing activity. Such antibodies are likely to be useful
in preventing or treating PD and other LBD.
[0148] Analogous methods can be used to screen antibodies for
activity in clearing other types of biological entities. The assay
can be used to detect clearing activity against virtually any kind
of biological entity. Typically, the biological entity has some
role in human or animal disease. The biological entity can be
provided as a tissue sample or in isolated form. If provided as a
tissue sample, the tissue sample is preferably unfixed to allow
ready access to components of the tissue sample and to avoid
perturbing the conformation of the components incidental to fixing.
Examples of tissue samples that can be tested in this assay include
cancerous tissue, precancerous tissue, tissue containing benign
growths such as warts or moles, tissue infected with pathogenic
microorganisms, tissue infiltrated with inflammatory cells, tissue
bearing pathological matrices between cells (e.g., fibrinous
pericarditis), tissue bearing aberrant antigens, and scar tissue.
Examples of isolated biological entities that can be used include
alpha-SN, viral antigens or viruses, proteoglycans, antigens of
other pathogenic microorganisms, tumor antigens, and adhesion
molecules. Such antigens can be obtained from natural sources,
recombinant expression or chemical synthesis, among other means.
The tissue sample or isolated biological entity is contacted with
phagocytic cells bearing Fc receptors, such as monocytes or
microglial cells, and an antibody to be tested in a medium. The
antibody can be directed to the biological entity under test or to
an antigen associated with the entity. In the latter situation, the
object is to test whether the biological entity is vicariously
phagocytosed with the antigen. Usually, although not necessarily,
the antibody and biological entity (sometimes with an associated
antigen) are contacted with each other before adding the phagocytic
cells. The concentration of the biological entity and/or the
associated antigen, if present, remaining in the medium is then
monitored. A reduction in the amount or concentration of antigen or
the associated biological entity in the medium indicates the
antibody has a clearing response against the antigen and/or
associated biological entity in conjunction with the phagocytic
cells.
[0149] Antibodies or other agents can also be screened for activity
in clearing Lewy bodies using the in vitro assay described in
Example II. Neuronal cells transfected with an expression vector
expressing synuclein form synuclein inclusions that can be
visualized microscopically. The activity of an antibody or other
agent in clearing such inclusions can be determined comparing
appearance or level of synuclein in transfected cells treated with
agent with appearance or level of synuclein in control cells not
treated with the agent. A reduction in size or intensity of
synuclein inclusions or a reduction in level of synuclein signals
activity in clearing synuclein. The activity can be monitored
either by visualizing synuclein inclusions microscopically or by
running cell extracts on a gel and visualizing a synuclein band. As
noted in Example 1, section 2, the change in level of synuclein is
most marked if the extracts are fractionated into cytosolic and
membrane fractions, and the membrane fraction is analyzed.
V. Patients Amenable to Treatment
[0150] Patients amenable to treatment include individuals at risk
of a synucleinopathic disease but not showing symptoms, as well as
patients presently showing symptoms. Patients amenable to treatment
also include individuals at risk of disease of a LBD but not
showing symptoms, as well as patients presently showing symptoms.
Such diseases include Parkinson's disease (including idiopathic
Parkinson's disease), DLB, DLBD, LBVAD, pure autonomic failure,
Lewy body dysphagia, incidental LBD, inherited LBD (e.g., mutations
of the alpha-SN gene, PARK3 and PARK4) and multiple system atrophy
(e.g., olivopontocerebellar atrophy, striatonigral degeneration and
Shy-Drager syndrome). 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
synuclein or Parkin, UCHLI, and CYP2D6 genes; particularly
mutations at position 53 of the 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.
[0151] In some methods, is free of clinical symptoms, signs and/or
risk factors of any amyloidogenic disease and suffers from at least
one synucleinopathic disease. In some methods, the patient is free
of clinical symptoms, signs and/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 A.beta. peptide. In some methods, the patient is free of
clinical symptoms, signs and/or risk factors of Alzheimer's
disease. In some methods, the patient is free of clinical symptoms,
signs and/or risk factors of Alzheimer's disease, cognitive
impairment, mild cognitive impairment and Down's syndrome. 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 disease and a disease characterized
synuclein accumulation. In some methods, the patient has concurrent
Alzheimer's and Parkinson's disease.
[0152] In asymptomatic patients, treatment can begin at any age
(e.g., 10, 20, or 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 the therapeutic agent (e.g., alpha-SN
peptide or A.beta., or both) over time. If the response falls, a
booster dosage is indicated.
[0153] Optionally, presence of absence of symptoms, signs or risk
factors of a disease is determined before beginning treatment.
Vi. Treatment Regimes
[0154] In general treatment regimes involve administering an agent
effective to induce an immunogenic response to alpha-SN and/or an
agent effective to induce an immunogenic response to A.beta. to a
patient. 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 prophylactically-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.
[0155] In some methods, administration of an agent results in
reduction of intracellular levels of aggregated synuclein. In some
methods, administration of an agent results in improvement in a
clinical symptom of a LBD, such as motor function in the case of
Parkinson's disease. In some methods, reduction in intracellular
levels of aggregated synuclein or improvement in a clinical symptom
of disease is monitored at intervals after administration of an
agent.
[0156] 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. The amount of
immunogen depends on whether adjuvant is also administered, with
higher dosages being required in the absence of adjuvant. The
amount of an immunogen for administration sometimes varies from
1-500 .mu.g per patient and more usually from 5-500 .mu.g per
injection for human administration. Occasionally, a higher dose of
1-2 mg per injection is used. Typically about 10, 20, 50 or 100
.mu.g is used for each human injection. The mass of immunogen also
depends on the mass ratio of immunogenic epitope within the
immunogen to the mass of immunogen as a whole. Typically, 10.sup.-3
to 10.sup.-5 micromoles of immunogenic epitope are used for
microgram of immunogen. 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 .mu.g/patient and usually greater than 10
.mu.g/patient if adjuvant is also administered, and greater than 10
.mu.g/patient and usually greater than 100 .mu.g/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.
[0157] For passive immunization with an antibody, 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 mgs or 700 mgs or within the range of 70-700
mgs, 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 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-SN in the patient. In some methods, dosage is adjusted to
achieve a plasma antibody concentration of 1-1000 ug/ml and in some
methods 25-300 ug/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.
[0158] Doses for nucleic acids encoding immunogens range from about
10 ng to 1 g, 100 ng to 100 mg, 1 .mu.g to 10 mg, or 30-300 .mu.g
DNA per patient. Doses for infectious viral vectors vary from
10-100, or more, virions per dose.
[0159] Agents for inducing an immune response 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 are 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 Medipad.TM. device.
[0160] As noted above, agents inducing an immunogenic response
against alpha-SN and A.beta. respectively can be administered in
combination. The agents can be combined in a single preparation or
kit for simultaneous, sequential or separate use. The agents can
occupy separate vials in the preparation or kit or can be combined
in a single vial. These agents of the invention can optionally be
administered in combination with other agents that are at least
partly effective in treatment of LBD. In the case of Parkinson's
Disease and Down's syndrome, in which LBs occur in the brain,
agents of the invention can also be administered in conjunction
with other agents that increase passage of the agents of the
invention across the blood-brain barrier.
[0161] Immunogenic agents of the invention, such as peptides, are
sometimes administered in combination with an adjuvant. A variety
of adjuvants can be used in combination with a peptide, such as
alpha-SN, 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 (MPL.TM.) (see GB 2220211 (RIBI ImmunoChem Research Inc.,
Hamilton, Mont., now part of Corixa). Stimulon.TM. 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); U.S. Pat. No.
5,057,540), (Aquila BioPharmaceuticals, Framingham, Mass.). 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 al., N. Engl. J. Med. 336,
86-91 (1997)), pluronic polymers, and killed mycobacteria. Another
adjuvant is CpG (WO 98/40100). Alternatively, alpha-SN or A.beta.
can be coupled to an adjuvant. However, such coupling should not
substantially change the conformation of alpha-SN 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.
[0162] 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-isoglutamine (nor-MDP),
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'dipalmitoyl-sn-
-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE),
N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxy
propylamide (DTP-DPP) Theramide.TM.), or other bacterial cell wall
components. Oil-in-water emulsions include (a) MF59 (WO 90/14837),
containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally
containing various amounts of MTP-PE) formulated into submicron
particles using a microfluidizer such as Model 110Y microfluidizer
(Microfluidics, Newton Mass.), (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) Ribi.TM. adjuvant
system (RAS), (Ribi ImmunoChem, Hamilton, Mont.) containing 2%
squalene, 0.2% Tween 80, and one or more bacterial cell wall
components from the group consisting of monophosphoryllipid A
(MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS),
preferably MPL+CWS (Detox.TM.).
[0163] Another class of preferred adjuvants is saponin adjuvants,
such as Stimulon.TM. (QS-21, Aquila, Framingham, Mass.) or
particles generated therefrom such as ISCOMs (immunostimulating
complexes) and ISCOMATRIX. 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
IL-15), macrophage colony stimulating factor (M-CSF),
granulocyte-macrophage colony stimulating factor (GM-CSF), and
tumor necrosis 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 immuno-modulators or adjuvants (see
U.S. Pat. No. 4,855,283). Heat shock proteins, e.g., HSP70 and
HSP90, may also be used as adjuvants.
[0164] An adjuvant can be administered with an immunogen as a
single composition, or can be administered before, concurrent with
or after administration of the immunogen. Immunogen and adjuvant
can be packaged and supplied in the same vial or can be packaged in
separate vials and mixed before use. Immunogen and adjuvant are
typically packaged with a label indicating the intended therapeutic
application. If immunogen 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, MPL and QS-21 are preferred.
Optionally, two or more different adjuvants can be used
simultaneously. Preferred combinations include alum with MPL, alum
with QS-21, MPL with QS-21, MPL or RC-529 with GM-CSF, and alum,
QS-21 and MPL together. Also, Incomplete Freund's adjuvant can be
used (Chang et al., Advanced Drug Delivery Reviews 32, 173-186
(1998)), optionally in combination with any of alum, QS-21, and MPL
and all combinations thereof.
[0165] 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 (15th ed., Mack
Publishing Company, Easton, Pa., 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.
[0166] 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 copolymers, and
lipid aggregates (such as oil droplets or liposomes). Additionally,
these carriers can function as immunostimulating agents (i.e.,
adjuvants).
[0167] 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 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 NaCl,
adjusted to pH 6.0 with HCl. Compositions for parenteral
administration are typically substantially sterile, substantially
isotonic and manufactured under GMP conditions of the FDA or
similar body. For example, compositions containing biologics are
typically sterilized by filter sterilization. Compositions can be
formulated for single dose administration.
[0168] 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 Delivery 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.
[0169] Additional formulations suitable for other modes of
administration include oral, intranasal, and pulmonary
formulations, suppositories, and transdermal applications.
[0170] 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%.
[0171] 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 et al., 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.
[0172] Alternatively, transdermal delivery can be achieved using a
skin path or using transferosomes (Paul et al., Eur. J. Immunol.
25, 3521-24 (1995); Cevc et al., Biochem. Biophys. Acta 1368,
201-15 (1998)).
VII. Methods of Monitoring and Methods of Diagnosis
[0173] The invention provides methods of detecting an immune
response against alpha-SN peptide and/or A.beta. peptide in a
patient suffering from or susceptible to a LBD. The methods are
particularly useful for monitoring a course of treatment being
administered to a patient. The methods can be used to monitor both
therapeutic treatment on symptomatic patients and prophylactic
treatment on asymptomatic patients. The methods are useful for
monitoring both active immunization (e.g., antibody produced in
response to administration of immunogen) and passive immunization
(e.g., measuring level of administered antibody).
[0174] 1. Active Immunization
[0175] Some methods entail determining a baseline value of an
immune response in a patient before administering a dosage of
agent, and comparing this with a value for the immune response
after treatment. A significant increase (i.e., greater than the
typical margin of experimental error in repeat measurements of the
same sample, expressed as one standard deviation from the mean of
such measurements) in value of the immune response signals a
positive treatment outcome (i.e., that administration of the agent
has achieved or augmented an immune response). If the value for
immune response does not change significantly, or decreases, a
negative treatment outcome is indicated. In general, patients
undergoing an initial course of treatment with an immunogenic agent
are expected to show an increase in immune response with successive
dosages, which eventually reaches a plateau. Administration of
agent is generally continued while the immune response is
increasing. Attainment of the plateau is an indicator that the
administered of treatment can be discontinued or reduced in dosage
or frequency.
[0176] In other methods, a control value (i.e., a mean and standard
deviation) of immune response is determined for a control
population. Typically the individuals in the control population
have not received prior treatment. Measured values of immune
response in a patient after administering a therapeutic agent are
then compared with the control value. A significant increase
relative to the control value (e.g., greater than one standard
deviation from the mean) signals a positive treatment outcome. A
lack of significant increase or a decrease signals a negative
treatment outcome. Administration of agent is generally continued
while the immune response is increasing relative to the control
value. As before, attainment of a plateau relative to control
values in an indicator that the administration of treatment can be
discontinued or reduced in dosage or frequency.
[0177] In other methods, a control value of immune response (e.g.,
a mean and standard deviation) is determined from a control
population of individuals who have undergone treatment with a
therapeutic agent and whose immune responses have reached a plateau
in response to treatment. Measured values of immune response in a
patient are compared with the control value. If the measured level
in a patient is not significantly different (e.g., more than one
standard deviation) from the control value, treatment can be
discontinued. If the level in a patient is significantly below the
control value, continued administration of agent is warranted. If
the level in the patient persists below the control value, then a
change in treatment regime, for example, use of a different
adjuvant may be indicated.
[0178] In other methods, a patient who is not presently receiving
treatment but has undergone a previous course of treatment is
monitored for immune response to determine whether a resumption of
treatment is required. The measured value of immune response in the
patient can be compared with a value of immune response previously
achieved in the patient after a previous course of treatment. A
significant decrease relative to the previous measurement (i.e.,
greater than a typical margin of error in repeat measurements of
the same sample) is an indication that treatment can be resumed.
Alternatively, the value measured in a patient can be compared with
a control value (mean plus standard deviation) determined in a
population of patients after undergoing a course of treatment.
Alternatively, the measured value in a patient can be compared with
a control value in populations of prophylactically treated patients
who remain free of symptoms of disease, or populations of
therapeutically treated patients who show amelioration of disease
characteristics. In all of these cases, a significant decrease
relative to the control level (i.e., more than a standard
deviation) is an indicator that treatment should be resumed in a
patient.
[0179] The tissue sample for analysis is typically blood, plasma,
serum, mucous or cerebrospinal fluid from the patient. The sample
is analyzed for indication of an immune response to any form of
alpha-SN, typically NAC, or A.beta.. The immune response can be
determined from the presence of, e.g., antibodies or T-cells that
specifically bind to alpha-SN or AB. ELISA methods of detecting
antibodies specific to alpha-SN are described in the Examples
section. Methods of detecting reactive T-cells have been described
above (see Definitions). In some methods, the immune response is
determined using a clearing assay, such as described in Section III
above. In such methods, a tissue or blood sample from a patient
being tested is contacted with LBs (e.g., from a synuclein/hAPP
transgenic mouse) and phagocytic cells bearing Fc receptors.
Subsequent clearing of the LBs is then monitored. The existence and
extent of clearing response provides an indication of the existence
and level of antibodies effective to clear alpha-SN in the tissue
sample of the patient under test.
[0180] 2. Passive Immunization
[0181] In general, the procedures for monitoring passive
immunization are similar to those for monitoring active
immunization described above. However, the antibody profile
following passive immunization typically shows an immediate peak in
antibody concentration followed by an exponential decay. Without a
further dosage, the decay approaches pretreatment levels within a
period of days to months depending on the half-life of the antibody
administered. For example the half-life of some human antibodies is
of the order of 20 days.
[0182] In some methods, a baseline measurement of antibody to
alpha-SN in the patient is made before administration, a second
measurement is made soon thereafter to determine the peak antibody
level, and one or more further measurements are made at intervals
to monitor decay of antibody levels. When the level of antibody has
declined to baseline or a predetermined percentage of the peak less
baseline (e.g., 50%, 25% or 10%), administration of a further
dosage of antibody is administered. In some methods, peak or
subsequent measured levels less background are compared with
reference levels previously determined to constitute a beneficial
prophylactic or therapeutic treatment regime in other patients. If
the measured antibody level is significantly less than a reference
level (e.g., less than the mean minus one standard deviation of the
reference value in population of patients benefiting from
treatment) administration of an additional dosage of antibody is
indicated.
[0183] 3. Diagnostic Kits
[0184] The invention further provides diagnostic kits for
performing the diagnostic methods described above. Typically, such
kits contain an agent that specifically binds to antibodies to
alpha-SN. The kit can also include a label. For detection of
antibodies to alpha-SN, the label is typically in the form of
labeled anti-idiotypic antibodies. For detection of antibodies, the
agent can be supplied prebound to a solid phase, such as to the
wells of a microtiter dish. Kits also typically contain labeling
providing directions for use of the kit. The labeling may also
include a chart or other correspondence regime correlating levels
of measured label with levels of antibodies to alpha-SN. The term
labeling refers to any written or recorded material that is
attached to, or otherwise accompanies a kit at any time during its
manufacture, transport, sale or use. For example, the term labeling
encompasses advertising leaflets and brochures, packaging
materials, instructions, audio or video cassettes, computer discs,
as well as writing imprinted directly on kits.
[0185] The invention also provides diagnostic kits for performing
in vivo imaging. Such kits typically contain an antibody binding to
an epitope of alpha-SN, preferably within NAC. Preferably, the
antibody is labeled or a secondary labeling reagent is included in
the kit. Preferably, the kit is labeled with instructions for
performing an in vivo imaging assay.
VIII. In Vivo Imaging
[0186] The invention provides methods of in vivo imaging LBs in a
patient. Such methods are useful to diagnose or confirm diagnosis
of PD, or other disease associated with the presence of LBs in the
brain, 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, e.g.
PD. 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 Parkinson's disease.
[0187] The methods work by administering a reagent, such as
antibody that binds to alpha-SN in the patient and then detecting
the agent after it has bound. Preferred antibodies bind to alpha-SN
deposits in a patient without binding to full length NACP
polypeptide. Antibodies binding to an epitope of alpha-SN within
NAC are particularly preferred. If desired, the 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. In general,
antibodies binding to epitopes N-terminal of alpha-SN do not show
as strong signal as antibodies binding to epitopes C-terminal,
presumably because the N-terminal epitopes are inaccessible in LBs
(Spillantini et al PNAS, 1998). Accordingly, such antibodies are
less preferred.
[0188] 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-SN 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.
[0189] 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.
EXAMPLES
I. Immunization of Human Alpha-Synuclein Transgenic Mice with Human
Alpha-Synuclein Results in the Production of High Titer
Anti-Alpha-Synuclein Antibodies that Cross the Blood-Brain
Barrier
[0190] Full-length recombinant human alpha-SN was resuspended at a
concentration of 1 mg/ml in 1.times. phosphate buffered saline
(PBS). For each injection, 50 .mu.l of alpha-SN was used; giving a
final concentration of 50 .mu.g per injection to which 150 .mu.l of
1.times.PBS was added. Complete Freund's adjuvant (CFA) was then
added 1:1 to either alpha-SN or PBS alone (control), vortexed and
sonicated to completely resuspend the emulsion. For the initial
injections, eight D line human alpha-SN transgenic (tg) single
transgenic 4-7 months old mice (Masliah, et al. Science
287:1265-1269 (2000) received injections of human alpha-SN in CFA
and, as control, four D line human alpha-SN tg mice received
injections of PBS in CFA. Mice received a total of 6 injections.
Three injections were performed at two weeks intervals and then 3
injections at one month intervals. Animals were sacrificed using
NIH Guidelines for the humane treatment of animals 5 months after
initiation of the experiment. After blood samples were collected
for determination of antibody titers, brains were immersion-fixed
for 4 days in 4% paraformaldehyde in PBS. Levels of antibodies
against human alpha-SN by ELISA are shown in Table 1. The treated
mice are divided into two groups by titer. The first group
developed a moderate titer of 2-8,000. The second group developed a
high titer of 12000-30000. No titer was found in control mice.
Neuropathological analysis showed that mice producing high titers
had a marked decrease in the size of synuclein incusions. Mice
producing moderate titers showed a smaller decrease. FIG. 2 (panels
a-d) show synuclein inclusions in (a) a nontransgenic mouse, (b) a
transgenic mouse treated with CFA only, (c) a transgenic mouse
immunized with alpha synuclein and CFA that developed a moderate
titer and (d) a transgenic mouse immunized with alpha synuclein and
CFA that developed a higher titer. Samples were visualized by
immunostaining with an anti-human alpha-SN antibody. FIG. 2 shows
synuclein inclusions in panel (b) but not panel (a). In panel (c),
treated mouse, moderate titers, the inclusions are somewhat reduced
in intensity. In panel (d) the inclusions are markedly reduced in
intensity. Panels (e)-(h) show levels of anti-IgG in the brains
same four mice as panels (a) to (d) respectively. It can be seen
that IgG is present in panels (g) and to a greater extent in panel
(h). The data shows that peripherally administered antibodies to
alpha-SN cross the blood brain harrier and reach the brain. Panels
(i) to (l) showing staining for GAP, a marker of astroglial cells,
again for the same four mice as in the first two rows of the
figure. It can be seen that panels (k) and (l) show moderately
increased staining compared with (i) and (j). These data show that
clearing of synuclein deposits is accompanied by a mild astroglial
and microglial reaction.
TABLE-US-00019 TABLE 1 Syn (+) Group Genotype n= Age at Sac
Treatment/Length Titers inclusions/mm2 I Syn Tg 4 10-13 mo a-syn +
CFA 2,000-8,000 15-29 50 ug/inj for 3 mo sac'd 3 mo later II Syn Tg
4 10-13 mo a-syn + CFA 12,000-30,000 10-22 50 ug/inj for 3 mo sac'd
3 mo later III Syn Tg 4 10-13 mo PBS + CFA for 0 18-29 3 mo sac'd 3
mo later
II. In Vitro Screen for Antibodies Clearing Synuclein
Inclusions
[0191] GT1-7 neuronal cell (Hsue et al. Am. J. Pathol. 157:401-410
(2000)) were transfected with a pCR3.1-T expression vector
(Invitrogen, Carlsbad, Calif.) expressing murine alpha-SN and
compared with cells transfected with expression vector alone (FIG.
3, panels B and A respectively). Cells transfected with vector
alone (panel A) have a fibroblastic appearance while cells
transfected with alpha-SN are rounded, with inclusion bodies at the
cell surface visible via both light and confocal scanning
microscopy. Transfected cells were then treated with rabbit
preimmune serum (panel C) or 67-10, an affinity purified rabbit
polyclonal antibody against a murine alpha-SN C terminal residues
131-140 (Iwai, et al., Neuron 14:467 (1995) (panel D). It can be
seen that the inclusion bodies stain less strongly in panel D than
in panel C indicating that the antibody against alpha synuclein was
effective in clearing or preventing the development of inclusions.
FIG. 4 shows a gel analysis of particulate and cytosolic fractions
of GT1-7 transfected cells treated with the rabbit preimmune serum
and 67-10 polyclonal antibody. It can be seen that the synuclein
levels in the cytosolic fraction is largely unchanged by treatment
with preimmune serum or antibody to alpha-SN. However, the alpha-SN
band disappears in the membrane fraction of GT1-7 cells treated
with antibody to alpha-SN. These data indicates that the alpha
synuclein antibody activity results in the clearance of synuclein
associated with the cellular membrane.
[0192] Transfected GT1-7 cells can be used to screen antibodies for
activity in clearing synuclein incusions with detection either by
immunohistochemical analysis, light microscopy as in FIG. 3 or by
gel analysis as in FIG. 4.
III. Prophylactic and Therapeutic Efficacy of Immunization with
Alpha-Synuclein
[0193] i. Immunization of Human Alpha-Synuclein tg Mice
[0194] For this study, heterozygous human alpha-SN transgenic (tg)
mice (Line D) (Masliah et al., Am. J. Pathol (1996) 148:201-10) and
nontransgenic (nontg) controls are used. Experimental animals are
divided into 3 groups. For group I, the preventive effects of early
immunization by immunizing mice for 8 months beginning at 2 months
of age are tested. For group II, young adult mice are vaccinated
for 8 months beginning at the age of 6 months to determine whether
immunization can reduce disease progression once moderate pathology
had been established. For group III, older mice are immunized for 4
months beginning at the age of 12 months to determine whether
immunization can reduce the severity of symptoms once robust
pathology has been established. For all groups, mice are immunized
with either recombinant human alpha-SN plus CFA or CFA alone, and
for each experiment 20 tg and 10 nontg mice are used. Of them, 10
tg mice are immunized with human alpha-SN+CFA and other 10 tg with
CFA alone. Similarly, 5 nontg mice are immunized with human
alpha-SN+CFA and the other 5 with CFA alone. Briefly, the
immunization protocol consists of an initial injection with
purified recombinant human alpha-SN (2 mg/ml) in CFA, followed by a
reinjection 1 month later with human alpha-SN in combination with
IFA. Mice are then re-injected with this mixture once a month. In a
small subset of human alpha-SN tg (n=3/each; 6-months-old) and
nontg (n=3/each; 6-month-old) mice, additional experiments
consisting of immunization with murine (m) alpha-SN, human beta
synuclein or mutant (A53T) human alpha-SN are performed.
[0195] Levels of alpha-SN antibody are determined using 96-well
microtiter plates coated with 0.4 .mu.g per well of purified
full-length alpha-SN by overnight incubation at 4.degree. C. in
sodium carbonate buffer, pH 9.6. Wells are washed 4.times. with 200
.mu.l each PBS containing 0.1% Tween and blocked for 1 hour in
PBS-1% BSA at 37.degree. C. Serum samples are serially diluted
"in-well", 1:3, starting in row A, ranging from a 1:150 to
1:328,050 dilution. For control experiments, a sample of mouse
monoclonal antibody is run against alpha-SN, no protein, and
buffer-only blanks. The samples are incubated overnight at
4.degree. C. followed by a 2-hour incubation with goat anti-mouse
IgG alkaline phosphatase-conjugated antibody (1:7500, Promega,
Madison, Wis.). Atto-phos.RTM. alkaline phophatase fluorescent
substrate is then added for 30 minutes at room temperature. The
plate is read at an excitation wavelength of 450 nm and an emission
wavelength of 550 nm. Results are plotted on a semi-log graph with
relative fluorescence units on the ordinate and serum dilution on
the abscissa. Antibody titer is defined as the dilution at which
there was a 50% reduction from maximal antibody binding.
[0196] For each group, at the end of the treatment, mice undergo
motor assessment in the rotarod, as described (Masliah, et al.
(2000)). After analysis, mice are euthanized and brains are removed
for detailed neurochemical and neuropathological analysis as
described below. Briefly, the right hemibrain is frozen and
homogenized for determinations of aggregated and non-aggregated
human alpha-SN immunoreactivity by Western blot (Masliah, et al.
(2000)). The left hemibrain is fixed in 4% paraformaldehyde,
serially sectioned in the vibratome for immunocytochemistry and
ultrastructural analysis.
[0197] ii. Immunocytochemical and Neuropathological Analysis.
[0198] In order to determine if immunization decreases, human
alpha-SN aggregation sections are immunostained with a rabbit
polyclonal antibody against human alpha-SN (1:500). After an
overnight incubation at 4.degree. C., sections are incubated with
biotinylated anti-rabbit secondary antibody followed by Avidin
D-Horseradish peroxidase (HRP) complex (1:200, ABC Elite, Vector).
Sections are also immunostained with biotinylated anti-rabbit,
mouse or human secondary alone. The experiments with the anti-mouse
secondary determine whether the antibodies against human alpha-SN
cross into the brain. The reaction is visualized with 0.1%
3,3,-diaminobenzidine tetrahydrochloride (DAB) in 50 mM Tris-HCl
(pH 7.4) with 0.001% H.sub.2O.sub.2 and sections are then mounted
on slided under Entellan. Levels of immunoreactivity are
semiquantitatively assessed by optical densitometry using the
Quantimet 570C. These sections are also studied by image analysis
to determine the numbers of alpha-SN immunoractive inclusions and
this reliable measure of alpha-SN aggregation acts as a valuable
index of the anti-aggregation effects of vaccination (Masliah, et
al. (2000)).
[0199] 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-immunoreactive
terminals displaying pixel intensity within a linear range are
included. A scale is set to determine the pixel to .mu.m ratio.
Then, this information is used to calculate the % area of the
neuropil covered by TH-immunoractive terminals. These same sections
are also utilized to evaluate the numbers of TH neurons in the
SN.
[0200] To assess the patterns of immune response to immunization,
immunocytochemical and ultrastructural analysis with antibodies
against human GFAP, MCH class II, Mac 1, TNF-alpha, IL1beta and IL6
are performed in the brain sections of nontg and alpha-SN tg mice
immunized with recombinant human alpha-SN and control
immunogens.
[0201] iii. Behavioral Analysis.
[0202] For locomotor activity mice are analyzed for 2 days in the
rotarod (San Diego) Instruments, San Diego, Calif.), as previously
described (Masliah, et al. (2000)). On the first day mice are
trained for 5 trials: the first one at 10 rpm, the second at 20 rpm
and the third to fifth at 40 rpm. On the second day, mice are
tested for 7 trials at 40 rpm 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.
IV. Immunization with Alpha-Synuclein Fragments
[0203] Human alpha-SN transgenic mice 10-13 months of age are
immunized with 9 different regions of alpha-SN to determine which
epitopes convey the efficacious response. The 9 different
immunogens and one control are injected i.p. as described above.
The immunogens include four human alpha-SN peptide conjugates, all
coupled to sheep anti-mouse IgG via a cystine link. Alpha-SN and
PBS are used as positive and negative controls, respectively.
Titers are monitored as above and mice are euthanized at the end of
3-12 months of injections. Histochemistry, alpha-SN levels, and
toxicology analysis is determined post mortem.
[0204] i. Preparation of Immunogens
[0205] Preparation of coupled alpha-SN peptides: H alpha-SN peptide
conjugates are prepared by coupling through an artificial cysteine
added to the alpha-SN peptide using the crosslinking reagent
sulfo-EMCS. The alpha-SN peptide derivatives are synthesized with
the following final amino acid sequences. In each case, the
location of the inserted cysteine residue is indicated by
underlining.
[0206] alpha-synuclein 60-72 (NAC region) peptide:
TABLE-US-00020 NH2-KEQVTNVCGGAVVT-COOH (SEQ ID NO: 54)
[0207] alpha-synuclein 73-84 (NAC region) peptide:
TABLE-US-00021 NH2-GVTAVAQKTVECG-COOH (SEQ ID NO: 55)
[0208] alpha-synuclein 102-112 peptide:
TABLE-US-00022 (SEQ ID NO: 56) NH2-C-amino-heptanoic
acid-KNEEGAPCQEG-COOH
[0209] alpha-synuclein 128-140 peptide:
TABLE-US-00023 Ac-NH-PSEEGYQDYEPECA-COOH (SEQ ID NO: 57)
[0210] To prepare for the coupling reaction, ten mg of sheep
anti-mouse IgG (Jackson ImmunoResearch Laboratories) is dialyzed
overnight against 10 mM sodium borate buffer, pH 8.5. The dialyzed
antibody is then concentrated to a volume of 2 mL using an Amicon
Centriprep tube. Ten mg sulfo-EMCS
[0211] [N (.epsilon.-maleimidocuproyloxy) succinimide] (Molecular
Sciences Co.) is dissolved in one mL deionized water. A 40-fold
molar excess of sulfo-EMCS is added drop wise with stirring to the
sheep anti-mouse IgG and then the solution is stirred for an
additional ten min. The activated sheep anti-mouse IgG is purified
and buffer exchanged by passage over a 10 mL gel filtration column
(Pierce Presto Column, obtained from Pierce Chemicals) equilibrated
with 0.1 M NaPO4, 5 mM EDTA, pH 6.5. Antibody containing fractions,
identified by absorbance at 280 nm, are pooled and diluted to a
concentration of approximately 1 mg/mL, using 1.4 mg per OD as the
extinction coefficient. A 40-fold molar excess of alpha-SN peptide
is dissolved in 20 mL of 10 mM NaPO4, pH 8.0, with the exception of
the alpha-SN peptide for which 10 mg is first dissolved in 0.5 mL
of DMSO and then diluted to 20 mL with the 10 mM NaPO4 buffer. The
peptide solutions are each added to 10 mL of activated sheep
anti-mouse IgG and rocked at room temperature for 4 hr. The
resulting conjugates are concentrated to a final volume of less
than 10 mL using an Amicon Centriprep tube and then dialyzed
against PBS to buffer exchange the buffer and remove free peptide.
The conjugates are passed through 0.22 .mu.m-pore size filters for
sterilization and then aliquoted into fractions of 1 mg and stored
frozen at -20.degree. C. The concentrations of the conjugates are
determined using the BCA protein assay (Pierce Chemicals) with
horse IgG for the standard curve. Conjugation is documented by the
molecular weight increase of the conjugated peptides relative to
that of the activated sheep anti-mouse IgG.
V. Passive Immunization with Antibodies to Alpha-Synuclein
[0212] Human alpha-SN mice each are injected with 0.5 mg in PBS of
anti-alpha-SN monoclonals as shown below. All antibody preparations
are purified to have low endotoxin levels. Monoclonals can be
prepared against a fragment by injecting the fragment or longer
form of alpha-SN into a mouse, preparing hybridomas and screening
the hybridomas for antibody that specifically binds to a desired
fragment of alpha-SN without binding to other nonoverlapping
fragments of alpha-SN.
[0213] Mice are injected ip as needed over a 4 month period to
maintain a circulating antibody concentration measured by ELISA
titer of greater than 1:1000 defined by ELISA to alpha-SN or other
immunogen. Titers are monitored as above and mice are euthanized at
the end of 6 months of injections. Histochemistry, alpha-SN levels
and toxicology are performed post mortem.
VI. A.beta. Immunization of Syn/APP Transgenic Mice
[0214] This experiment compares the effects of A.beta. immunization
on three types of transgenic mice: transgenic mice with an alpha
synuclein transgene (SYN), APP mice with an APP transgene (Games et
al.) and double transgenic SYN/APP mice produced by crossing the
single transgenic. The double transgenic mice are described in
Masliah et al., PNAS USA 98:12245-12250 (2001). These mice
represent a model of individuals having both Alzheimer's and
Parkinson's disease. Table 2 shows the different groups, the age of
the mice used in the study, the treatment procedure and the titer
of antibodies to A.beta.. It can be seen that a significant titer
was generated in all three types of mice. FIG. 5 shows the % area
covered by amyloid plaques of A.beta. in the brain determined by
examination of brain sections from treated subjects by microscopy.
Substantial deposits accumulate in the APP and SYN/APP mice but not
in the SYN mice or nontransgenic controls. The deposits are greater
in the SYN/APP double transgenic mice. Immunization with
A.beta.1-42 reduces the deposits in both APP and SYN/APP mice. FIG.
6 shows synuclein deposits in the various groups of mice as
detected by confocal laser scanning and light microscopy. Synuclein
deposits accumulate in the SYN and SYN/APP mice treated with CFA
only. However, in the same types of mice treated with A.beta.1-42
and CFA there is a marked reduction in the level of synuclein
deposit. These data indicate that treatment with A.beta. is
effective not only in clearing A.beta. deposits but also in
clearing deposits of synuclein. Therefore, treatment with A.beta.
or antibodies thereto is useful in treating not only Alzheimer's
disease but combined Alzheimer's and Parkinson's disease, and
Parkinson's disease in patients free of Alzheimer's disease. The
titer of antiA.beta. antibodies in SYN/APP mice correlated with
decreased formation of synuclein inclusions (r=-0.71,
p<0.01).
TABLE-US-00024 TABLE 2 Group n= Age Treatment/Length Ab Titers SYN
4 12-20 mo Ab inj. 50 ug/inj 10,000-58,000 for 6 mo SYN 2 12-20 mo
Sal inj. for 6 mo 0 APP 2 12-20 mo Ab inj. 50 ug/inj 25,000 for 6
mo APP 2 12-20 mo Sal inj. for 6 mo 0 SYN/APP 4 12-20 mo Ab inj. 50
ug/inj 1,000-50,000 for 6 mo SYN/APP 2 12-20 mo Sal inj. for 6 mo 0
Sequence CWU 1
1
571140PRTHomo sapiens 1Met Asp Val Phe Met Lys Gly Leu Ser Lys Ala
Lys Glu Gly Val Val1 5 10 15Ala Ala Ala Glu Lys Thr Lys Gln Gly Val
Ala Glu Ala Ala Gly Lys 20 25 30Thr Lys Glu Gly Val Leu Tyr Val Gly
Ser Lys Thr Lys Glu Gly Val 35 40 45Val His Gly Val Ala Thr Val Ala
Glu Lys Thr Lys Glu Gln Val Thr 50 55 60Asn Val Gly Gly Ala Val Val
Thr Gly Val Thr Ala Val Ala Gln Lys65 70 75 80Thr Val Glu Gly Ala
Gly Ser Ile Ala Ala Ala Thr Gly Phe Val Lys 85 90 95Lys Asp Gln Leu
Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu Gly Ile 100 105 110Leu Glu
Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro 115 120
125Ser Glu Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala 130 135
140235PRTHomo sapiens 2Glu Gln Val Thr Asn Val Gly Gly Ala Val Val
Thr Gly Val Thr Ala1 5 10 15Val Ala Gln Lys Thr Val Glu Gly Ala Gly
Ser Ile Ala Ala Ala Thr 20 25 30Gly Phe Val 35328PRTHomo sapiens
3Lys Glu Gln Val Thr Asn Val Gly Gly Ala Val Val Thr Gly Val Thr1 5
10 15Ala Val Ala Gln Lys Thr Val Glu Gly Ala Gly Ser 20
25413PRTInfluenza virus 4Pro Lys Tyr Val Lys Gln Asn Thr Leu Lys
Leu Ala Thr1 5 10516PRTPlasmodium sp. 5Glu Lys Lys Ile Ala Lys Met
Glu Lys Ala Ser Ser Val Phe Asn Val1 5 10 15610PRTHepatitis B virus
6Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile1 5 10719PRTHomo sapiens
7Asp Gln Ser Ile Gly Asp Leu Ile Ala Glu Ala Met Asp Lys Val Gly1 5
10 15Asn Glu Gly814PRTMycobacterium bovis 8Gln Val His Phe Gln Pro
Leu Pro Pro Ala Val Val Lys Leu1 5 10915PRTClostridium tetani 9Gln
Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu1 5 10
151021PRTClostridium tetani 10Phe Asn Asn Phe Thr Val Ser Phe Trp
Leu Arg Val Pro Lys Val Ser1 5 10 15Ala Ser His Leu Glu
201116PRTHuman immunodeficiency virus 11Lys Gln Ile Ile Asn Met Trp
Gln Glu Val Gly Lys Ala Met Tyr Ala1 5 10 151213PRTArtificial
SequenceFusion protein 12Ala Lys Xaa Val Ala Ala Trp Thr Leu Lys
Ala Ala Ala1 5 101314PRTArtificial SequenceFusion protein 13Lys Glu
Gln Val Thr Asn Val Cys Gly Gly Ala Val Val Thr1 5
101413PRTArtificial SequenceFusion protein 14Gly Val Thr Ala Val
Ala Gln Lys Thr Val Glu Cys Gly1 5 101512PRTArtificial
SequenceFusion protein 15Xaa Lys Asn Glu Glu Gly Ala Pro Cys Gln
Glu Gly1 5 101616PRTArtificial SequenceFusion protein 16Xaa Gln Tyr
Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu1 5 10
151722PRTArtificial SequenceFusion protein 17Xaa Phe Asn Asn Phe
Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val1 5 10 15Ser Ala Ser His
Leu Glu 201816PRTArtificial SequenceFusion protein 18Xaa Gln Tyr
Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu1 5 10
151937PRTArtificial SequenceFusion protein 19Xaa Gln Tyr Ile Lys
Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu1 5 10 15Phe Asn Asn Phe
Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser 20 25 30Ala Ser His
Leu Glu 352014PRTArtificial SequenceFusion protein 20Ala Lys Xaa
Val Ala Ala Trp Thr Leu Lys Ala Ala Ala Xaa1 5 102116PRTArtificial
SequenceFusion protein 21Xaa Xaa Xaa Ala Lys Xaa Val Ala Ala Trp
Thr Leu Lys Ala Ala Ala1 5 10 152217PRTArtificial SequenceFusion
protein 22Ala Lys Xaa Val Ala Ala Trp Thr Leu Lys Ala Ala Ala Xaa
Xaa Xaa1 5 10 15Xaa2314PRTArtificial SequenceFusion protein 23Xaa
Ala Lys Xaa Val Ala Ala Trp Thr Leu Lys Ala Ala Ala1 5
102418PRTArtificial SequenceFusion protein 24Xaa Ile Ser Gln Ala
Val His Ala Ala His Ala Glu Ile Asn Glu Ala1 5 10 15Gly
Arg2516PRTArtificial SequenceFusion protein 25Pro Lys Tyr Val Lys
Gln Asn Thr Leu Lys Leu Ala Thr Xaa Xaa Xaa1 5 10
152615PRTArtificial SequenceFusion protein 26Xaa Pro Lys Tyr Val
Lys Gln Asn Thr Leu Lys Leu Ala Thr Xaa1 5 10 152716PRTArtificial
SequenceFusion protein 27Xaa Xaa Xaa Pro Lys Tyr Val Lys Gln Asn
Thr Leu Lys Leu Ala Thr1 5 10 152815PRTArtificial SequenceFusion
protein 28Xaa Xaa Pro Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala
Thr1 5 10 1529106PRTArtificial SequenceFusion protein 29Xaa Pro Lys
Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Glu Lys1 5 10 15Lys Ile
Ala Lys Met Glu Lys Ala Ser Ser Val Phe Asn Val Gln Tyr 20 25 30Ile
Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Phe Asn Asn 35 40
45Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser Ala Ser His
50 55 60Leu Glu Xaa Xaa Xaa Xaa Gln Tyr Ile Lys Ala Asn Ser Lys Phe
Ile65 70 75 80Gly Ile Thr Glu Leu Phe Asn Asn Phe Thr Val Ser Phe
Trp Leu Arg 85 90 95Val Pro Lys Val Ser Ala Ser His Leu Glu 100
1053077PRTArtificial SequenceFusion protein 30Xaa Gln Tyr Ile Lys
Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu1 5 10 15Cys Phe Asn Asn
Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val 20 25 30Ser Ala Ser
His Leu Glu Xaa Gln Tyr Ile Lys Ala Asn Ser Lys Phe 35 40 45Ile Gly
Ile Thr Glu Leu Cys Phe Asn Asn Phe Thr Val Ser Phe Trp 50 55 60Leu
Arg Val Pro Lys Val Ser Ala Ser His Leu Glu Xaa65 70
753116PRTArtificial SequenceFusion protein 31Xaa Gln Tyr Ile Lys
Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu1 5 10
153226PRTArtificial SequenceFusion protein 32Glu Gln Val Thr Asn
Val Gly Gly Ala Ile Ser Gln Ala Val His Ala1 5 10 15Ala His Ala Glu
Ile Asn Glu Ala Gly Arg 20 253343PRTHomo sapiens 33Asp Ala Glu Phe
Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys1 5 10 15Leu Val Phe
Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30Gly Leu
Met Val Gly Gly Val Val Ile Ala Thr 35 403422PRTArtificial
SequenceConjugate 34Asp Ala Glu Phe Arg His Asp Gln Tyr Ile Lys Ala
Asn Ser Lys Phe1 5 10 15Ile Gly Ile Thr Glu Leu 203528PRTArtificial
SequenceConjugate 35Asp Ala Glu Phe Arg His Asp Phe Asn Asn Phe Thr
Val Ser Phe Trp1 5 10 15Leu Arg Val Pro Lys Val Ser Ala Ser His Leu
Glu 20 253643PRTArtificial SequenceConjugate 36Asp Ala Glu Phe Arg
His Asp Gln Tyr Ile Lys Ala Asn Ser Lys Phe1 5 10 15Ile Gly Ile Thr
Glu Leu Phe Asn Asn Phe Thr Val Ser Phe Trp Leu 20 25 30Arg Val Pro
Lys Val Ser Ala Ser His Leu Glu 35 403722PRTArtificial
SequenceConjugate 37Glu Phe Arg His Asp Ser Gly Gln Tyr Ile Lys Ala
Asn Ser Lys Phe1 5 10 15Ile Gly Ile Thr Glu Leu 203820PRTArtificial
SequenceConjugate 38Ala Lys Xaa Val Ala Ala Trp Thr Leu Lys Ala Ala
Ala Asp Ala Glu1 5 10 15Phe Arg His Asp 203934PRTArtificial
SequenceConjugate 39Asp Ala Glu Phe Arg His Asp Asp Ala Glu Phe Arg
His Asp Asp Ala1 5 10 15Glu Phe Arg His Asp Ala Lys Xaa Val Ala Ala
Trp Thr Leu Lys Ala 20 25 30Ala Ala4034PRTArtificial SequenceFusion
protein 40Ala Lys Xaa Val Ala Ala Trp Thr Leu Lys Ala Ala Ala Asp
Ala Glu1 5 10 15Phe Arg His Asp Asp Ala Glu Phe Arg His Asp Asp Ala
Glu Phe Arg 20 25 30His Asp4120PRTArtificial SequenceFusion protein
41Asp Ala Glu Phe Arg His Asp Ala Lys Xaa Val Ala Ala Trp Thr Leu1
5 10 15Lys Ala Ala Ala 204224PRTArtificial SequenceFusion protein
42Asp Ala Glu Phe Arg His Asp Ile Ser Gln Ala Val His Ala Ala His1
5 10 15Ala Glu Ile Asn Glu Ala Gly Arg 204324PRTArtificial
SequenceFusion protein 43Phe Arg His Asp Ser Gly Tyr Ile Ser Gln
Ala Val His Ala Ala His1 5 10 15Ala Glu Ile Asn Glu Ala Gly Arg
204424PRTArtificial SequenceFusion protein 44Glu Phe Arg His Asp
Ser Gly Ile Ser Gln Ala Val His Ala Ala His1 5 10 15Ala Glu Ile Asn
Glu Ala Gly Arg 204534PRTArtificial SequenceFusion protein 45Pro
Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Asp Ala Glu1 5 10
15Phe Arg His Asp Asp Ala Glu Phe Arg His Asp Asp Ala Glu Phe Arg
20 25 30His Asp4627PRTArtificial SequenceFusion protein 46Asp Ala
Glu Phe Arg His Asp Pro Lys Tyr Val Lys Gln Asn Thr Leu1 5 10 15Lys
Leu Ala Thr Asp Ala Glu Phe Arg His Asp 20 254734PRTArtificial
SequenceFusion protein 47Asp Ala Glu Phe Arg His Asp Asp Ala Glu
Phe Arg His Asp Asp Ala1 5 10 15Glu Phe Arg His Asp Pro Lys Tyr Val
Lys Gln Asn Thr Leu Lys Leu 20 25 30Ala Thr4827PRTArtificial
SequenceFusion protein 48Asp Ala Glu Phe Arg His Asp Asp Ala Glu
Phe Arg His Asp Pro Lys1 5 10 15Tyr Val Lys Gln Asn Thr Leu Lys Leu
Ala Thr 20 254979PRTArtificial SequenceFusion protein 49Asp Ala Glu
Phe Arg His Asp Pro Lys Tyr Val Lys Gln Asn Thr Leu1 5 10 15Lys Leu
Ala Thr Glu Lys Lys Ile Ala Lys Met Glu Lys Ala Ser Ser 20 25 30Val
Phe Asn Val Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile 35 40
45Thr Glu Leu Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro
50 55 60Lys Val Ser Ala Ser His Leu Glu Asp Ala Glu Phe Arg His
Asp65 70 755056PRTArtificial SequenceFusion protein 50Asp Ala Glu
Phe Arg His Asp Asp Ala Glu Phe Arg His Asp Asp Ala1 5 10 15Glu Phe
Arg His Asp Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly 20 25 30Ile
Thr Glu Leu Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro 35 40
45Lys Val Ser Ala Ser His Leu Glu 50 555144PRTArtificial
SequenceFusion protein 51Asp Ala Glu Phe Arg His Asp Gln Tyr Ile
Lys Ala Asn Ser Lys Phe1 5 10 15Ile Gly Ile Thr Glu Leu Cys Phe Asn
Asn Phe Thr Val Ser Phe Trp 20 25 30Leu Arg Val Pro Lys Val Ser Ala
Ser His Leu Glu 35 405251PRTArtificial SequenceFusion protein 52Asp
Ala Glu Phe Arg His Asp Gln Tyr Ile Lys Ala Asn Ser Lys Phe1 5 10
15Ile Gly Ile Thr Glu Leu Cys Phe Asn Asn Phe Thr Val Ser Phe Trp
20 25 30Leu Arg Val Pro Lys Val Ser Ala Ser His Leu Glu Asp Ala Glu
Phe 35 40 45Arg His Asp 505322PRTArtificial SequenceFusion protein
53Asp Ala Glu Phe Arg His Asp Gln Tyr Ile Lys Ala Asn Ser Lys Phe1
5 10 15Ile Gly Ile Thr Glu Leu 205414PRTHomo sapiens 54Lys Glu Gln
Val Thr Asn Val Cys Gly Gly Ala Val Val Thr1 5 105513PRTHomo
sapiens 55Gly Val Thr Ala Val Ala Gln Lys Thr Val Glu Cys Gly1 5
105612PRTHomo sapiensMISC_FEATURE(1)..(1)X = amino-heptanoic acid
56Xaa Lys Asn Glu Glu Gly Ala Pro Cys Gln Glu Gly1 5 105714PRTHomo
sapiensMOD_RES(1)..(1)ACETYLATION X= Aecylated proline 57Xaa Ser
Glu Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Cys Ala1 5 10
* * * * *