U.S. patent application number 11/842085 was filed with the patent office on 2008-04-24 for prevention and treatment of amyloidogenic disease.
This patent application is currently assigned to Elan Pharma International Limited. Invention is credited to Dale B. Schenk, Nicki Vasquez, Theodore Yednock.
Application Number | 20080096818 11/842085 |
Document ID | / |
Family ID | 26748211 |
Filed Date | 2008-04-24 |
United States Patent
Application |
20080096818 |
Kind Code |
A1 |
Schenk; Dale B. ; et
al. |
April 24, 2008 |
PREVENTION AND TREATMENT OF AMYLOIDOGENIC DISEASE
Abstract
The invention provides improved agents and methods for treatment
of diseases associated with amyloid deposits of A.beta. in the
brain of a patient. Such methods entail administering agents that
induce a beneficial immunogenic response against the amyloid
deposit. The methods are useful for prophylactic and therapeutic
treatment of Alzheimer's disease. Preferred agents including
N-terminal fragments of A.beta. and antibodies binding to the
same.
Inventors: |
Schenk; Dale B.;
(Burlingame, CA) ; Vasquez; Nicki; (San Francisco,
CA) ; Yednock; Theodore; (Forest Knolls, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Elan Pharma International
Limited
Athlone
IE
|
Family ID: |
26748211 |
Appl. No.: |
11/842085 |
Filed: |
August 20, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11245916 |
Oct 7, 2005 |
|
|
|
11842085 |
Aug 20, 2007 |
|
|
|
10933559 |
Sep 2, 2004 |
6972127 |
|
|
11245916 |
Oct 7, 2005 |
|
|
|
09724102 |
Nov 28, 2000 |
6787139 |
|
|
10933559 |
Sep 2, 2004 |
|
|
|
09201430 |
Nov 30, 1998 |
6787523 |
|
|
09724102 |
Nov 28, 2000 |
|
|
|
09322289 |
May 28, 1999 |
|
|
|
11842085 |
Aug 20, 2007 |
|
|
|
09201430 |
Nov 30, 1998 |
6787523 |
|
|
09322289 |
May 28, 1999 |
|
|
|
09723765 |
Nov 28, 2000 |
|
|
|
11842085 |
Aug 20, 2007 |
|
|
|
09580019 |
May 26, 2000 |
|
|
|
09723765 |
Nov 28, 2000 |
|
|
|
60080970 |
Apr 7, 1998 |
|
|
|
60080970 |
Apr 7, 1998 |
|
|
|
60067740 |
Dec 2, 1997 |
|
|
|
Current U.S.
Class: |
514/17.8 |
Current CPC
Class: |
A61K 31/00 20130101;
A61K 2039/53 20130101; C07K 2319/00 20130101; A61K 9/7023 20130101;
A61P 43/00 20180101; A61P 25/28 20180101; A61K 38/1709 20130101;
A61K 2039/55555 20130101; A61K 38/193 20130101; A61K 2039/605
20130101; A61K 2039/55505 20130101; A61K 2039/55572 20130101; A61K
9/2031 20130101; A61K 9/2009 20130101; A61K 9/0019 20130101; A61K
9/4866 20130101; C07K 14/4711 20130101; A61K 9/2054 20130101; A61K
2039/55566 20130101; A61K 39/0007 20130101; C07K 2317/24 20130101;
A61K 2300/00 20130101; A61K 2039/55577 20130101; A61P 37/00
20180101; C07K 16/18 20130101; A61K 38/193 20130101; A61K 2039/6037
20130101; A61P 25/00 20180101; A61K 31/739 20130101; A61K 39/00
20130101; A61K 47/646 20170801; A61K 2039/505 20130101; C07K
2317/567 20130101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 38/00 20060101
A61K038/00; A61P 25/28 20060101 A61P025/28 |
Claims
1. A method of preventing or treating a disease associated with
amyloid deposits of A.beta. in the brain of a patient, comprising
administering to the patient an effective dosage of a polypeptide
comprising an N-terminal segment of A.beta., the segment beginning
at residue 1-3 of A.beta. and ending at residues 7-11 of
A.beta..
2-29. (canceled)
33. A pharmaceutical composition comprising a polypeptide as
defined in claim 1 and an adjuvant.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation in part of U.S. Ser. No.
09/322,289, filed May 28, 1999, which is incorporated by reference
in its entirety for all purposes.
TECHNICAL FIELD
[0002] The invention resides in the technical fields of immunology
and medicine.
BACKGROUND OF THE INVENTION
[0003] Alzheimer's disease (AD) is a progressive disease resulting
in senile dementia. See generally Selkoe, TINS 16, 403-409 (1993);
Hardy et al., WO 92/13069; Selkoe, J. Neuropathol. Exp. Neurol. 53,
438-447 (1994); Duff et al., Nature 373, 476-477 (1995); Games et
al., Nature 373, 523 (1995). Broadly speaking, the disease falls
into two categories: late onset, which occurs in old age (65+years)
and early onset, which develops well before the senile period,
i.e., between 35 and 60 years. In both types of disease, the
pathology is the same but the abnormalities tend to be more severe
and widespread in cases beginning at an earlier age. The disease is
characterized by at least two types of lesions in the brain, senile
plaques and neurofibrillary tangles. Senile plaques are areas of
disorganized neuropil up to 150 .mu.m across with extracellular
amyloid deposits at the center visible by microscopic analysis of
sections of brain tissue. Neurofibrillary tangles are intracellular
deposits of microtubule associated tau protein consisting of two
filaments twisted about each other in pairs.
[0004] The principal constituent of the plaques is a peptide termed
A.beta. or .beta.-amyloid peptide. A.beta. peptide is an internal
fragment of 39-43 amino acids of a precursor protein termed amyloid
precursor protein (APP). Several mutations within the APP protein
have been correlated with the presence of Alzheimer's disease. See,
e.g., Goate et al., Nature 349, 704) (1991) (valine.sup.717 to
isoleucine); Chartier Harlan et al. Nature 353, 844 (1991))
(valine.sup.717 to glycine); Murrell et al., Science 254, 97 (1991)
(valine.sup.717 to phenylalanine); Mullan et al., Nature Genet. 1,
345 (1992) (a double mutation changing
lysine.sup.595-methionine.sup.596 to
asparagine.sup.595-leucine.sup.596). Such mutations are thought to
cause Alzheimer's disease by increased or altered processing of APP
to A.beta., particularly processing of APP to increased amounts of
the long form of A.beta. (i.e., A.beta.1-42 and A.beta.1-43).
Mutations in other genes, such as the presenilin genes, PS1 and
PS2, are thought indirectly to affect processing of APP to generate
increased amounts of long form A.beta. (see Hardy, TINS 20, 154
(1997)). These observations indicate that A.beta., and particularly
its long form, is a causative element in Alzheimer's disease.
[0005] McMichael, EP 526,511, proposes administration of
homeopathic dosages (less than or equal to 10.sup.-2 mg/day) of
A.beta. to patients with preestablished AD. In a typical human with
about 5 liters of plasma, even the upper limit of this dosage would
be expected to generate a concentration of no more than 2 pg/ml.
The normal concentration of A.beta. in human plasma is typically in
the range of 50-200 pg/ml (Seubert et al., Nature 359, 325-327
(1992)). Because EP 526,511 's proposed dosage would barely alter
the level of endogenous circulating A.beta. and because EP 526,511
does not recommend use of an adjuvant, as an immunostimulant, it
seems implausible that any therapeutic benefit would result.
[0006] By contrast, the present invention is directed inter alia to
treatment of Alzheimer's and other amyloidogenic diseases by
administration of fragments of A.beta., or antibody to certain
epitopes within 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 Alzheimer's disease.
[0007] This application is related to Townsend and Townsend and
Crew Attorney Docket 015270-004750PC, filed May 26, 2000,
PCT/US98/25386, filed Nov. 30, 1998, U.S. Ser. No. 60/067,740,
filed Dec. 2, 1997, U.S. Ser. No. 60/080,970, filed Apr. 7, 1998,
and U.S. Ser. No. 09/201,430, filed Nov. 30, 1998, each of which is
incorporated by reference in its entirety for all purposes.
SUMMARY OF THE CLAIMED INVENTION
[0008] In one aspect, the invention provides methods of preventing
or treating a disease associated with amyloid deposits of A.beta.
in the brain of a patient. Such diseases include Alzheimer's
disease, Down's syndrome and cognitive impairment. The latter can
occur with or without other characteristics of an amyloidogenic
disease. Some methods of the invention entail administering an
effective dosage of an antibody that specifically binds to a
component of an amyloid deposit to the patient. Such methods are
particularly useful for preventing or treating Alzheimer's disease
in human patients. Some methods entail administering an effective
dosage of an antibody that binds to A.beta.. Some methods entail
administering an effective dosage of an antibody that specifically
binds to an epitope within residues 1-10 of A.beta.. In some
methods, the antibody specifically binds to an epitope within
residues 1-6 of A.beta.. In some methods, the antibody specifically
binds to an epitope within residues 1-5 of A.beta.. In some
methods, the antibody specifically binds to an epitope within
residues 1-7 of A.beta.. In some methods, the antibody specifically
binds to an epitope within residues 3-7 of A.beta.. In some
methods, the antibody specifically binds to an epitope within
residues 1-3 of A.beta.. In some methods, the antibody specifically
binds to an epitope within residues 1-4 of A.beta.. In some
methods, the antibody binds to an epitope comprising a free
N-terminal residue of A.beta.. In some methods, the antibody binds
to an epitope within residues of 1-10 of A.beta. wherein residue 1
and/or residue 7 of A.beta. is aspartic acid. In some methods, the
antibody specifically binds to A.beta. peptide without binding to
full-length amyloid precursor protein (APP). In some methods, the
isotype of the antibody is human IgG1.
[0009] In some methods, the antibody binds to an amyloid deposit in
the patient and induces a clearing response against the amyloid
deposit. For example, such a clearing response can be effected by
Fc receptor mediated phagocytosis.
[0010] The methods can be used on both asymptomatic patients and
those currently showing symptoms of disease. The antibody used in
such methods can be a human, humanized, chimeric or nonhuman
antibody and can be monoclonal or polyclonal. In some methods, the
antibody is prepared from a human immunized with A.beta. peptide,
which human can be the patient to be treated with antibody.
[0011] 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 dosages over a
prolonged period, for example, of at least six months. In some
methods, the antibody is administered as a sustained release
composition. The antibody can be administered, for example,
intraperitoneally, orally, subcutaneously, intracranially,
intramuscularly, topically, intranasally or intravenously.
[0012] 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. The polynucleotide
is expressed to produce the heavy and light chains in the patient.
In some methods, the patient is monitored for level of administered
antibody in the blood of the patient.
[0013] In another aspect, the invention provides methods of
preventing or treating a disease associated with amyloid deposits
of A.beta. in the brain of patient. For example, the methods can be
used to treat Alzheimer's disease or Down's syndrome or cognitive
impairment. Such methods entail administering fragments of A.beta.
or analogs thereof eliciting an immunogenic response against
certain epitopes within A.beta.. Some methods entail administering
to a patient an effective dosage of a polypeptide comprising an
N-terminal segment of at least residues 1-5 of A.beta., the first
residue of A.beta. being the N-terminal residue of the polypeptide,
wherein the polypeptide is free of a C-terminal segment of A.beta..
Some methods entail administering to a patient an effective dosage
of a polypeptide comprising an N-terminal segment of A.beta., the
segment beginning at residue 1-3 of A.beta. and ending at residues
7-11 of A.beta.. Some methods entail administering to a patient an
effective dosage of an agent that induces an immunogenic response
against an N-terminal segment of A.beta., the segment beginning at
residue 1-3 of A.beta. and ending at residues 7-11 of A.beta.
without inducing an immunogenic response against an epitope within
residues 12-43 of A.beta.43.
[0014] In some of the above methods, the N-terminal segment of
A.beta. is linked at its C-terminus to a heterologous polypeptide.
In some of the above methods, the N-terminal segment of A.beta. is
linked at its N-terminus to a heterologous polypeptide. In some of
the above methods, the N-terminal segment of A.beta. is linked at
its N and C termini to first and second heterologous polypeptides.
In some of the above methods, the N-terminal segment of A.beta. is
linked at its N terminus to a heterologous polypeptide, and at its
C-terminus to at least one additional copy of the N-terminal
segment. In some of the above methods, the heterologous polypeptide
and thereby a B-cell response against the N-terminal segment. In
some of the above methods, the polypeptide further comprises at
least one additional copy of the N-terminal segment. In some of the
above methods, the polypeptide comprises from N-terminus to
C-terminus, the N-terminal segment of A.beta., a plurality of
additional copies of the N-terminal segment, and the heterologous
amino acid segment. In some of the above methods, the N-terminal
segment consists of A.beta.1-7. In some of the above methods, the
N-terminal segment consists of A.beta.3-7.
[0015] In some methods, the fragment is free of at least the 5
C-terminal amino acids in A.beta.43. In some methods, the fragment
comprises up to 10 contiguous amino acids from A.beta.. Fragments
are typically administered at greater than 10 micrograms per dose
per patient.
[0016] In some methods, the fragment is administered with an
adjuvant that enhances the immune response to the A.beta. peptide.
The adjuvant and fragment can be administered in either order or
together as a composition. The adjuvant can be, for example,
aluminum hydroxide, aluminum phosphate, MPLC, QS-21 (Stimulon.TM.)
or incomplete Freund's adjuvant.
[0017] The invention further provides pharmaceutical compositions
comprising fragments of A.beta. or other agents eliciting
immunogenic response to the same epitopes of A.beta., such as
described above, and an adjuvant. The invention also provides
pharmaceutical compositions comprising any of the antibodies
described above and a pharmaceutically acceptable carrier.
[0018] In another aspect, the invention provides methods of
screening an antibody for activity in treating a disease associated
with deposits of A.beta. in the brain of a patient (e.g.,
Alzheimer's disease). Such methods entail contacting the antibody
with a polypeptide comprising at least five contiguous amino acids
of an N-terminal segment of A.beta. beginning at a residue between
1 and 3 of A.beta., the polypeptide being free of a C-terminal
segment of A.beta.. One then determines whether the antibody
specifically binds to the polypeptide, specific binding providing
an indication that the antibody has activity in treating the
disease.
[0019] In another aspect, the invention provides methods of
screening an antibody for activity in clearing an
antigen-associated biological entity. Such methods entail combining
the antigen-associated biological entity and the antibody and
phagocytic cells bearing Fc receptors in a medium. The amount of
the antigen-associated biological entity remaining in the medium is
then monitored. A reduction in the amount of the antigen-associated
biological entity indicates the antibody has clearing activity
against the antigen-associated biological entity. The antigen can
be provided as a tissue sample or in isolated form. For example,
the antigen can be provided as a tissue sample from the brain of an
Alzheimer's disease patient or a mammal animal having Alzheimer's
pathology. Other tissue samples against which antibodies can be
tested for clearing activity include cancerous tissue samples,
virally infected tissue samples, tissue samples comprising
inflammatory cells, nonmalignant abnormal cell growths, or tissue
samples comprising an abnormal extracellular matrix.
[0020] In another aspect, the invention provides methods of
detecting an amyloid deposit in a patient. Such methods entail
administering to the patient an antibody that specifically binds to
an epitope within amino acids 1-10 of A.beta., and detecting
presence of the antibody in the brain of the patient. In some
methods, the antibody binds to an epitope within residues 4-10 of
A.beta.. In some methods, the antibody is labelled with a
paramagnetic label and detected by nuclear magnetic resonance
tomography.
[0021] The invention further provides diagnostic kits suitable for
use in the above methods. Such a kit comprises an antibody that
specifically binds to an epitope with residues 1-10 of A.beta..
Some kits bear a label describing use of the antibody for in vivo
diagnosis or monitoring of Alzheimer's disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1: Antibody titer after injection of transgenic mice
with A.beta.1-42.
[0023] FIG. 2: Amyloid burden in the hippocampus. The percentage of
the area of the hippocampal region occupied by amyloid plaques,
defined by reactivity with the A.beta.-specific monoclonal antibody
3D6, was determined by computer-assisted quantitative image
analysis of immunoreacted brain sections. The values for individual
mice are shown sorted by treatment group. The horizontal line for
each grouping indicates the median value of the distribution.
[0024] FIG. 3: Neuritic dystrophy in the hippocampus. The
percentage of the area of the hippocampal region occupied by
dystrophic neurites, defined by their reactivity with the human
APP-specific monoclonal 8E5, was determined by quantitative
computer-assisted image analysis of immunoreacted brain sections.
The values for individual mice are shown for the AN1792-treated
group and the PBS-treated control group. The horizontal line for
each grouping indicates the median value of the distribution.
[0025] FIG. 4: Astrocytosis in the retrosplenial cortex. The
percentage of the area of the cortical region occupied by glial
fibrillary acidic protein (GFAP)-positive astrocytes was determined
by quantitative computer-assisted image analysis of immunoreacted
brain sections. The values for individual mice are shown sorted by
treatment group and median group values are indicated by horizontal
lines.
[0026] FIG. 5: Geometric mean antibody titers to A.beta.1-42
following immunization with a range of eight doses of AN1792
containing 0.14, 0.4, 1.2, 3.7, 11, 33, 100, or 300 .mu.g.
[0027] FIG. 6: Kinetics of antibody response to AN1792
immunization. Titers are expressed as geometric means of values for
the 6 animals in each group.
[0028] FIG. 7: Quantitative image analysis of the cortical amyloid
burden in PBS- and AN1792-treated mice.
[0029] FIG. 8: Quantitative image analysis of the neuritic plaque
burden in PBS- and AN1792-treated mice.
[0030] FIG. 9: Quantitative image analysis of the percent of the
retrosplenial cortex occupied by astrocytosis in PBS- and
AN1792-treated mice.
[0031] FIG. 10: Lymphocyte Proliferation Assay on spleen cells from
AN1792-treated (FIG. 10A) or PBS-treated (FIG. 10B).
[0032] FIG. 11: Total A.beta. levels in the cortex. A scatterplot
of individual A.beta. profiles in mice immunized with A.beta. or
APP derivatives combined with Freund' adjuvant.
[0033] FIG. 12: Amyloid burden in the cortex was determined by
quantitative image analysis of immunoreacted brain sections for
mice immunized with the A.beta. peptide conjugates A.beta.1-5,
A.beta.1-12, and A.beta.13-28; the full length A.beta. aggregates
AN1792 (A.beta.1-42) and AN1528 (A.beta.1-40) and the PBS-treated
control group.
[0034] FIG. 13: Geometric mean titers of A.beta.-specific antibody
for groups of mice immunized with A.beta. or APP derivatives
combined with Freund's adjuvant.
[0035] FIG. 14: Geometric mean titers of A.beta.-specific antibody
for groups of guinea pigs immunized with AN1792, or a palmitoylated
derivative thereof, combined with various adjuvants.
[0036] FIGS. 15A-E: A.beta. levels in the cortex of 12-month old
PDAPP mice treated with AN1792 or AN1528 in combination with
different adjuvants. The A.beta. level for individual mice in each
treatment group, and the median, mean, and p values for each
treatment group are shown.
[0037] FIG. 15A: The values for mice in the PBS-treated control
group and the untreated control group.
[0038] FIG. 15B: The values for mice in the AN1528/alum and
AN1528/MPL-treatment groups.
[0039] FIG. 15C: The values for mice in the AN1528/QS21 and
AN1792/Freund's adjuvant treatment groups.
[0040] FIG. 15D: The values for mice in the AN1792/Thimerosol and
AN1792/alum treatment groups.
[0041] FIG. 15E: The values for mice in the AN1792/MPL and
AN1792/QS21 treatment groups.
[0042] FIG. 16: Mean titer of mice treated with polyclonal antibody
to A.beta..
[0043] FIG. 17: Mean titer of mice treated with monoclonal antibody
10D5 to A.beta..
[0044] FIG. 18: Mean titer of mice treated with monoclonal antibody
2F12 to A.beta..
[0045] FIG. 19: Epitope Map: Restricted N-terminal Response. Day
175 serum from cynomolgus monkeys was tested by ELISA against a
series of 10-mer overlapping peptides covering the complete AN1792
sequence. Animal number F10920M shows a representative N-terminal
restricted response to the peptide DAEFRHDSGY which covers amino
acids 1-10 of the AN1792 peptide which was used as immunizing
antigen.
[0046] FIG. 20: Epitope Map: Non-restricted N-terminal response.
Day 175 serum from cynomolgus monkeys was tested by ELISA against a
series of 10-mer overlapping peptides covering the complete AN1792
sequence. Animal number F10975F shows a representative
non-restricted N-terminal response. Reactivity is seen against the
two peptides N-terminal and one peptide C-terminal to the peptide
DAEFRHDSGY which covers amino acids 1-10 of the AN1792 peptide.
DEFINITIONS
[0047] 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.
[0048] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are input into a computer, subsequence coordinates are designated,
if necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percent sequence identity for the test sequence(s) relative to the
reference sequence, based on the designated program parameters.
[0049] Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, 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.
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 wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62
scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.
USA 89, 10915 (1989)).
[0050] For purposes of classifying amino acids substitutions as
conservative or nonconservative, 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.
[0051] 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.
[0052] 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.
[0053] 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').sub.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).
[0054] 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.
[0055] An "antigen" is an entity to which an antibody specifically
binds.
[0056] 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.
[0057] 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.
[0058] 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.
[0059] The term "naked polynucleotide" refers to a polynucleotide
not complexed with colloidal materials. Naked polynucleotides are
sometimes cloned in a plasmid vector.
[0060] 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.
[0061] The term "patient" includes human and other mammalian
subjects that receive either prophylactic or therapeutic
treatment.
[0062] Disaggregated or monomeric A.beta. means soluble, monomeric
peptide units of A.beta.. One method to prepare monomeric AD is to
dissolve lyophilized peptide in neat DMSO with sonication. The
resulting solution is centrifuged to remove any insoluble
particulates. Aggregated A.beta. is a mixture of oligomers in which
the monomeric units are held together by noncovalent bonds.
[0063] 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 A.beta.. 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 1-125 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 labelled 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%.
[0064] Compositions or methods "comprising" one or more recited
elements may include other elements not specifically recited. For
example, a composition that comprises A.beta. peptide encompasses
both an isolated A.beta. peptide and A.beta. peptide as a component
of a larger polypeptide sequence.
DETAILED DESCRIPTION
I. General
[0065] Several amyloidogenic diseases and conditions are
characterized by presence of deposits of A.beta. peptide aggregated
to an insoluble mass in the brain of a patient. Such diseases
include Alzheimer's disease, Down's syndrome and cognitive
impairment. The latter is a symptom of Alzheimer's disease and
Down's syndrome but can also without other characteristics of
either of these diseases. For example, mild cognitive impairment or
age-associated memory loss occurs in some patient who have not yet
developed, or may never develop full Alzheimer's disease. Mild
cognitive impairment can be defined by score on the Mini-Mental
State Exam in accordance with convention. Such diseases are
characterized by aggregates of A.beta. that have a .beta.-pleated
sheet structure and stain with Congo Red dye. The basic approach of
preventing or treating Alzheimer's disease or other amyloidogenic
diseases by generating an immunogenic response to a component of
the amyloid deposit in a patient is described in WO 99/27944
(incorporated by reference). The present application reiterates and
confirms the efficacy of the basic approach. The present
application is, however, principally directed to improved reagents
and methods. These improvements are premised, in part, on the
present inventors having localized the preferred epitopes within
A.beta. against which an immunogenic response should be directed.
The identification of preferred epitopes within A.beta. results in
agents and methods having increased efficacy, reduced potential for
side effects, and/or greater ease of manufacture, formulation and
administration.
II. Therapeutic Agents
[0066] An immunogenic response can be active, as when an immunogen
is administered to induce antibodies reactive with A.beta. in a
patient, or passive, as when an antibody is administered that
itself binds to A.beta. in a patient.
[0067] 1. Agents Inducing Active Immune Response
[0068] Therapeutic agents induce an immunogenic response
specifically directed to certain epitopes within A.beta. peptides.
Preferred agents are the A.beta. peptide itself and segments
thereof. Variants of such segments, analogs and mimetics of natural
A.beta. peptide that induce and/or crossreact with antibodies to
the preferred epitopes of A.beta. peptide can also be used.
[0069] 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.
[0070] A.beta. has the unusual property that it can fix and
activate both classical and alternate complement cascades. In
particular, it binds to Clq 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.
[0071] 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, AD42
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:
[0072]
H.sub.2N-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-G-
ln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-
-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala-OH (SEQ ID NO:42).
[0073] A.beta.41, A.beta.40 and A.beta.39 differ from A42 by the
omission of Ala, Ala-Ile, and Ala-Ile-Val respectively from the
C-terminal end. A043 differs from A.beta.42 by the presence of a
threonine residue at the C-terminus.
[0074] Immunogenic fragments of A.beta. are advantageous relative
to the intact molecule in the present methods for several reasons.
First, because only certain epitopes within A.beta. induce a useful
immunogenic response for treatment of Alzheimer's disease, an equal
dosage of mass of a fragment containing such epitopes provides a
greater molar concentration of the useful immunogenic epitopes than
a dosage of intact A.beta.. Second, certain immunogenic fragments
of A.beta. generate an immunogenic response against amyloid
deposits without generating a significant immunogenic response
against APP protein from which A.beta. derives. Third, fragments of
A.beta. are simpler to manufacture than intact A.beta. due to their
shorter size. Fourth, fragments of A.beta. do not aggregate in the
same manner as intact A.beta., simplifying preparation of
pharmaceutical compositions and administration thereof.
[0075] Some immunogenic fragments of A.beta. have a sequence of at
least 2, 3, 5, 6, 10 or 20 contiguous amino acids from a natural
peptide. Some immunogenic fragments have no more than 10, 9, 8, 7,
5 or 3 contiguous residues from A.beta.. 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. The
fragment A.beta.1-12 can also be used but is less preferred. In
some methods, the fragment is an N-terminal fragment other than
A.beta.1-10. Other less preferred fragments include A.beta.13-28,
17-28, 1-28, 25-35, 35-40 and 35-42. These fragments require
screening for activity in clearing or preventing amyloid deposits
as described in the Examples before use. Fragments lacking at least
one, and sometimes at least or 10 C-terminal amino acid present in
a naturally occurring forms of A.beta. are used in some methods.
For example, a fragment lacking 5 amino acids from the C-terminal
end of A.beta.43 includes the first 38 amino acids from the
N-terminal end of A.beta.. Other components of amyloid plaques, for
example, synuclein, and epitopic fragments thereof can also be used
to induce an immunogenic response.
[0076] Unless otherwise indicated, reference to A.beta. includes
the natural human amino acid sequences indicated above as well as
analogs including allelic, species and induced variants. 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 a one, two or a
few positions. For example, the natural aspartic acid residue at
position 1 and/or 7 of A.beta. can be replaced with iso-aspartic
acid. Examples of unnatural amino acids are D-amino acids,
.alpha.,.alpha.-disubstituted amino acids, N-alkyl amino acids,
lactic acid, 4-hydroxyproline, .gamma.-carboxyglutamate,
.gamma.-N,N,N-trimethyllysine, .gamma.-N-acetyllysine,
O-phosphoserine, N-acetylserine, N-formylmethionine,
3-methylhistidine, 5-hydroxylysine, co-N-methylarginine, and
isoaspartic acid. 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.
[0077] A.beta., 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 A.beta. peptide are also available
commercially (e.g., American Peptides Company, Inc., Sunnyvale,
Calif. and California Peptide Research, Inc. Napa, Calif.).
[0078] Therapeutic agents also include longer polypeptides that
include, for example, an active fragment of A.beta. peptide,
together with other amino acids. For example, preferred agents
include fusion proteins comprising a segment of A.beta. 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 A.beta. 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 A.beta. 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.
[0079] In a further variation, an immunogenic peptide, such as a
fragment of A.beta., can be presented by a virus or a 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 a 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. Therapeutic agents also include
peptides and other compounds that do not necessarily have a
significant amino acid sequence similarity with A.beta. but
nevertheless serve as mimetics of A.beta. 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 A.beta.
or other amyloidogenic peptides 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, 6th ed.), p. 181). Agents other than
A.beta. peptides should induce an immunogenic response against one
or more of the preferred segments of A.beta. listed above (e.g.,
1-10, 1-7, 1-3, and 3-7). Preferably, such agents induce an
immunogenic response that is specifically directed to one of these
segments without being directed to other segments of A.beta..
[0080] 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 by reference for all
purposes). Peptide libraries can also be generated by phage display
methods. See, e.g., Devlin, WO 91/18980.
[0081] 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 A.beta.
or other amyloidogenic peptides. For example, initial screens can
be performed with any polyclonal sera or monoclonal antibody to
A.beta. or a fragment thereof. Compounds can then be screened for
binding to a specific epitope within A.beta. (e.g., 1-10, 1-7, 1-3,
1-4, 1-5 and 3-7). 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 A.beta. or
fragments thereof. For example, multiple dilutions of sera can be
tested on microtiter plates that have been precoated with A.beta.
or a fragment thereof and a standard ELISA can be performed to test
for reactive antibodies to A.beta. or the fragment. Compounds can
then be tested for prophylactic and therapeutic efficacy in
transgenic animals predisposed to an amyloidogenic disease, as
described in the Examples. Such animals include, for example, mice
bearing a 717 mutation of APP described by Games et al., supra, 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)). The same screening approach can be used on other
potential agents analogs of A.beta. and longer peptides including
fragments of A.beta., described above.
[0082] 2. Agents Inducing Passive Immune Response
[0083] Therapeutic agents of the invention also include antibodies
that specifically bind to A.beta. or other component of amyloid
plaques. Such antibodies can be monoclonal or polyclonal. Some such
antibodies bind specifically to the aggregated form of A.beta.
without binding to the dissociated form. Some bind specifically to
the dissociated form without binding to the aggregated form. Some
bind to both aggregated and dissociated forms. Some such antibodies
bind to a naturally occurring short form of A.beta. (i.e.,
A.beta.39, 40 or 41) without binding to a naturally occurring long
form of A.beta. (i.e., A.beta.42 and A.beta.43). Some antibodies
bind to a long form without binding to a short form. Some
antibodies bind to A.beta. without binding to full-length amyloid
precursor protein. 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 A.beta., and the other for an Fc receptor. Some
antibodies bind to A.beta. 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.
[0084] Polyclonal sera typically contain mixed populations of
antibodies binding to several epitopes along the length of A.beta..
However, polyclonal sera can be specific to a particular segment of
A.beta., such as A.beta.1-10. Monoclonal antibodies bind to a
specific epitope within A.beta. 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 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.. Less
preferred antibodies include those binding to epitopes with
residues 10-15, 15-20, 25-30, 10-20, 20-30, or 10-25 of A.beta.. It
is recommended that such antibodies be screened for activity in the
mouse model described in the Examples before use. For example, it
has been found that certain antibodies to epitopes within residues
10-18, 16-24, 18-21 and 33-42 lack activity. In some methods,
multiple monoclonal antibodies having binding specificities to
different epitopes are used. Such antibodies can be administered
sequentially or simultaneously. Antibodies to amyloid components
other than A.beta. can also be used. For example, antibodies can be
directed to the amyloid associated protein synuclein.
[0085] When an antibody is said to bind to an epitope within
specified residues, such as A.beta. 1-5 for example, what is meant
is that the antibody specifically binds to a polypeptide containing
the specified residues (i.e., A.beta.1-5 in this an example). Such
an antibody does not necessarily contact every residue within
A.beta.1-5. Nor does every single amino acid substitution or
deletion with in A.beta.1-5 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 A.beta..
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. For
example, antibodies that compete with the 3D6 antibody for binding
to A.beta. bind to the same or similar epitope as 3D6, i.e., within
residues A.beta.1-5. Likewise antibodies that compete with the 10D5
antibody bind to the same or similar epitope, i.e, within residues
A.beta. 3-6. Screening antibodies for epitope specificity is a
useful predictor of therapeutic efficacy. For example, an antibody
determined to bind to an epitope within residues 1-7 of A.beta. is
likely to be effective in preventing and treating Alzheimer's
disease.
[0086] Monoclonal or polyclonal antibodies that specifically bind
to a preferred segment of A.beta. without binding to other regions
of A.beta. have a number of advantages relative to monoclonal
antibodies binding to other regions or polyclonal sera to intact
A.beta.. 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 amyloid deposits without inducing a
clearing response against intact APP polypeptide, thereby reducing
the potential for side effects.
[0087] i. General Characteristics of Immunoglobulins
[0088] 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.
[0089] 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).
[0090] 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), or Chothia & Lesk, J. Mol. Biol.
196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989).
[0091] ii. Production of Nonhuman Antibodies
[0092] The production of non-human monoclonal antibodies, e.g.,
murine, guinea pig, primate, rabbit or rat, can be accomplished by,
for example, immunizing the animal with A.beta.. A longer
polypeptide comprising A.beta. or an immunogenic fragment of
A.beta. or anti-idiotypic antibodies to an antibody to AD can also
be used. See Harlow & Lane, Antibodies, A Laboratory Manual
(CSHP NY, 1988) (incorporated by reference for all purposes). Such
an immunogen can be obtained from a natural source, by peptide
synthesis or by recombinant expression. Optionally, the immunogen
can be administered fused or otherwise complexed with a carrier
protein, as described below. Optionally, the immunogen can be
administered with an adjuvant. Several types of adjuvant can be
used as described below. Complete Freund's adjuvant followed by
incomplete adjuvant is preferred for immunization of laboratory
animals. Rabbits or guinea pigs are typically used for making
polyclonal antibodies. Mice are typically used for making
monoclonal antibodies. Antibodies are screened for specific binding
to A.beta.. Optionally, antibodies are further screened for binding
to a specific region of A.beta.. The latter screening can be
accomplished by determining binding of an antibody to a collection
of deletion mutants of an A.beta. peptide and determining which
deletion mutants bind to the antibody. Binding can be assessed, for
example, by Western blot or ELISA. The smallest fragment to show
specific binding to the antibody defines the epitope of the
antibody. Alternatively, epitope specificity can be determined by a
competition assay is which a test and reference antibody compete
for binding to A.beta.. If the test and reference antibodies
compete, then they bind to the same epitope or epitopes
sufficiently proximal that binding of one antibody interferes with
binding of the other. The preferred isotype for such antibodies is
mouse isotype IgG2a or equivalent isotype in other species. Mouse
isotype IgG2a is the equivalent of human isotype IgG1.
[0093] iii. Chimeric and Humanized Antibodies
[0094] Chimeric and humanized antibodies have the same or similar
binding specificity and affinity as a mouse or other nonhuman
antibody that provides the starting material for construction of a
chimeric or humanized antibody. Chimeric antibodies are antibodies
whose light and heavy chain genes have been constructed, typically
by genetic engineering, from immunoglobulin gene segments belonging
to different species. For example, the variable (V) segments of the
genes from a mouse monoclonal antibody may be joined to human
constant (C) segments, such as IgG1 and IgG4. Human isotype IgG1 is
preferred. A typical chimeric antibody is thus a hybrid protein
consisting of the V or antigen-binding domain from a mouse antibody
and the C or effector domain from a human antibody.
[0095] Humanized antibodies have variable region framework residues
substantially from a human antibody (termed an acceptor antibody)
and complementarity determining regions substantially from a
mouse-antibody, (referred to as the donor immunoglobulin). See,
Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989) and
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 (incorporated by reference in their 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.
[0096] 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: [0097] (1) noncovalently binds antigen directly, [0098] (2)
is adjacent to a CDR region, [0099] (3) otherwise interacts with a
CDR region (e.g. is within about 6 A of a CDR region), or [0100]
(4) participates in the VL-VH interface.
[0101] 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.
[0102] iv. Human Antibodies
[0103] Human antibodies against A.beta. 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 A.beta. as the immunogen, and/or by screening
antibodies against a collection of deletion mutants of A.beta..
Human antibodies preferably have isotype specificity human
IgG1.
[0104] (1) Trioma Methodology
[0105] 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.
[0106] 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 AD, a fragment thereof, larger polypeptide containing A.beta.
or fragment, or an anti-idiotypic antibody to an antibody to
A.beta.. 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.
[0107] 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 A.beta. 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 A.beta. or a
fragment thereof.
[0108] 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.
[0109] (2) Transgenic Non-Human Mammals
[0110] Human antibodies against A.beta. 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., WO93/12227 (1993); 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
(1991) (each of which is incorporated by reference in its entirety
for all purposes). Transgenic mice are particularly suitable.
Anti-A.beta. antibodies are obtained by immunizing a transgenic
nonhuman mammal, such as described by Lonberg or Kucherlapati,
supra, with A 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 A.beta. or other amyloid peptide as an affinity
reagent.
[0111] (3) Phage Display Methods
[0112] A further approach for obtaining human anti-A.beta.
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 A.beta.,
fragments, longer polypeptides containing A.beta. 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 A.beta. or a fragment thereof are selected.
Sequences encoding such antibodies (or a 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 A.beta. peptide or fragment thereof.
[0113] 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
A.beta. (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 A.beta. are selected.
These phage display the variable regions of completely human
anti-A.beta. antibodies. These antibodies usually have the same or
similar epitope specificity as the murine starting material.
[0114] v. Selection of Constant Region
[0115] 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').sub.2, and Fv, or as
single chain antibodies in which heavy and light chain variable
domains are linked through a spacer.
[0116] vi. Expression of Recombinant Antibodies
[0117] 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.
[0118] 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.
[0119] 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.
[0120] 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 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).
[0121] 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.
[0122] 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.
[0123] 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)).
[0124] 3. Carrier Proteins
[0125] Some agents for inducing an immune response contain the
appropriate epitope for inducing an immune response against amyloid
deposits but are too small to be immunogenic. In this situation, a
peptide immunogen can be linked to a suitable carrier to help
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. Other carriers include
T-cell epitopes that bind to multiple MHC alleles, e.g., at least
75% of all human MHC alleles. Such carriers are sometimes known in
the art as "universal T-cell epitopes." Examples of universal
T-cell epitopes include:
Influenza Hemagluttinin: HA.sub.307-319 PKYVKQNTLKLAT (SEQ ID
NO:43)
PADRE (common residues bolded) AKXVAAWTLKAAA (SEQ ID NO:44)
Malaria CS: T3 epitope EKKIAKMEKASSVFNV (SEQ ID NO:45)
Hepatitis B surface antigen: HBsAg.sub.19-28 FFLLTRILTI (SEQ ID
NO:46)
Heat Shock Protein 65: hsp65.sub.153-171 DQSIGDLIAEAMDKVGNEG (SEQ
ID NO:47)
bacille Calmette-Guerin QVHFQPLPPAVVKL (SEQ ID NO:48)
Tetanus toxoid: TT.sub.830-844 QYIKANSKFIGITEL (SEQ ID NO:49)
Tetanus toxoid: TT.sub.947-967 FNNFTVSFWLRVPKVSASHLE (SEQ ID
NO:50)
HIV gp120 T1: KQIINMWQEVGKAMYA (SEQ ID NO:51).
[0126] Other carriers for stimulating or enhancing an immune
response include cytokines such as IL-1, IL-1 .alpha. and .beta.
peptides, IL-2, .gamma.INF, IL-10, GM-CSF, and chemokines, such as
MIP1.alpha. 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.
[0127] 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.
[0128] 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.
[0129] Some agents of the invention comprise a fusion protein in
which an N-terminal fragment of A.beta. is linked at its C-terminus
to a carrier peptide. In such agents, the N-terminal residue of the
fragment of A.beta. 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 A.beta. to be in free form. Some agents of
the invention comprises a plurality of repeats of an N-terminal
segment of A.beta. linked at the C-terminus to one or more copy of
a carrier peptide. The N-terminal fragment of A.beta. incorporated
into such fusion proteins sometimes begins at A.beta.1-3 and ends
at A.beta.7-11. A.beta.1-7, A.beta.1-3, 1-4, 1-5, and 3-7 are
preferred N-terminal fragment of A.beta.. Some fusion proteins
comprise different N-terminal segments of A.beta. in tandem. For
example, a fusion protein can comprise A.beta.1-7 followed by
A.beta.1-3 followed by a heterologous peptide.
[0130] In some fusion proteins, an N-terminal segment of A.beta. is
fused at its N-terminal end to a heterologous carrier peptide. The
same variety of N-terminal segments of A.beta. can be used as with
C-terminal fusions. Some fusion proteins comprise a heterologous
peptide linked to the N-terminus of an N-terminal segment of
A.beta., which is in turn linked to one or more additional
N-terminal segments of A.beta. in tandem.
[0131] Some examples of fusion proteins suitable for use in the
invention are shown below. Some of these fusion proteins comprise
segments of A.beta. 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 comprises segments of A.beta. linked to
carrier peptides described in U.S. Pat. No. 5,736,142. Some
heterologous peptides are universal T-cell epitopes. In some
methods, the agent for administration is simply a single fusion
protein with an A.beta. segment linked to a heterologous segment in
linear configuration. 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). Epitopes of A.beta. are underlined.
[0132] 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. ##STR1##
AN90549 (A.beta.1-7/Tetanus toxoid 830-844 in a MAP4
configuration): DAEFRHDQYIKANSKFIGITEL (SEQ ID NO:52) AN90550
(A.beta.1-7/Tetanus toxoid 947-967 in a MAP4 configuration):
DAEFRHDFNNFTVSFWLRVPKVSASHLE (SEQ ID NO:53) AN90542
(A.beta.1-7/Tetanus toxoid 830-844+947-967 in a linear
configuration): DAEFRHDQYIKANSKFIGITELFNNFTVSFWLRVPKVSASHLE (SEQ ID
NO:54) AN90576: (A.beta. 3-9)/Tetanus toxoid 830-844 in a MAP4
configuration): EFRHDSGQYIKANSKFIGITEL (SEQ ID NO:55) Peptide
described in U.S. Pat. No. 5,736,142 (all in linear
configurations): AN90562 (A.beta.1-7/peptide) AKXVAAWTLKAAADAEFRHD
(SEQ ID NO:56) AN90543 (A.beta.1-7 x 3/peptide):
DAEFRHDDAEFRHDDAEFRHDAKXVAAWTLKAAA (SEQ ID NO:57)
[0133] Other examples of fusion proteins (immunogenic epitope of
A.beta. bolded) include AKXVAAWTLKAAA-DAEFRHD-DAEFRHD-DAEFRHD (SEQ
ID NO:58) [0134] DAEFRHD-AKXVAAWTLKAAA (SEQ ID NO:59) [0135]
DAEFRHD-ISQAVHAAHAEINEAGR (SEQ ID NO:60) [0136]
FRHDSGY-ISQAVHAAHAEINEAGR (SEQ ID NO:61) [0137]
EFRHDSG-ISQAVHAAHAEINEAGR (SEQ ID NO:62) [0138]
PKYVKQNTLKLAT-DAEFRHD-DAEFRHD-DAEFRHD (SEQ ID NO:63) [0139]
DAEFRHD-PKYVKQNTLKLAT-DAEFRHD (SEQ ID NO:64) [0140]
DAEFRHD-DAEFRHD-DAEFRHD-PKYVKQNTLKLAT (SEQ ID NO:65) [0141]
DAEFRHD-DAEFRHD-PKYVKQNTLKLAT (SEQ ID NO:66) [0142]
DAEFRHD-PKYVKQNTLKLAT-EKKIAKMEKASSVFNV-QYIKANSKFIGITEL-FNNFTVSFWLRVPKVSAS-
HLE-DAEFRHD (SEQ ID NO:67) [0143]
DAEFRHD-DAEFRHD-DAEFRHD-QYIKANSKFIGITEL-FNNFTVSFWLRVPKVSASHLE (SEQ
ID NO:68) [0144] DAEFRHD-QYIKANSKFIGITELCFNNFTVSFWLRVPKVSASHLE (SEQ
ID NO:69) DAEFRHD-QYIKANSKFIGITELCFNNFTVSFWLRVPKVSASHLE-DAEFRHD
(SEQ ID NO:70) [0145] DAEFRHD-QYIKANSKFIGITEL (SEQ ID NO:77)_on a 2
branched resin ##STR2## [0146] EQVTNVGGAISQAVHAAHAEINEAGR (SEQ ID
NO:71) (Synuclein fusion protein in MAP-4 configuration).
[0147] The same or similar carrier proteins and methods of linkage
can be used for generating immunogens to be used in generation of
antibodies against AD for use in passive immunization. For example,
A.beta. or a fragment linked to a carrier can be administered to a
laboratory animal in the production of monoclonal antibodies to
A.beta..
[0148] 4. Nucleic Acid Encoding Therapeutic Agents
[0149] Immune responses against amyloid deposits can also be
induced by administration of nucleic acids encoding segments of
A.beta. 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.
[0150] 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)).
[0151] 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. Nos. 5,208,036, 5,264,618, 5,279,833 and
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).
[0152] 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
(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).
[0153] 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,
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. Screening Antibodies for Clearing Activity
[0154] The invention provides methods of screening an antibody for
activity in clearing an amyloid deposit or any other antigen, or
associated biological entity, for which clearing activity is
desired. To screen for activity against an amyloid deposit, a
tissue sample from a brain of a patient with Alzheimer's disease or
an animal model having characteristic Alzheimer'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 pagocytic 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 A.beta. can be used. Preferably, a series of
measurements is made of the amount of amyloid deposit 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 labelled antibody to A.beta. 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 amyloid deposits indicates that the antibody under test has
clearing activity. Such antibodies are likely to be useful in
preventing or treating Alzheimer's and other amyloidogenic
diseases.
[0155] 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
A.beta., 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 (see, e.g., Example 14).
IV. Patients Amenable to Treatment
[0156] Patients amenable to treatment include individuals at risk
of disease but not showing symptoms, as well as patients presently
showing symptoms. In the case of Alzheimer's disease, virtually
anyone is at risk of suffering from Alzheimer's disease if he or
she lives long enough. Therefore, the present methods can be
administered prophylactically to the general population without the
need for any assessment of the risk of the subject patient. The
present methods are especially useful for individuals who do have a
known genetic risk of Alzheimer's disease. 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 Alzheimer's disease include
mutations in the APP gene, particularly mutations at position 717
and positions 670 and 671 referred to as the Hardy and Swedish
mutations respectively (see Hardy, TINS, supra). Other markers of
risk are mutations in the presenilin genes, PS1 and PS2, and ApoE4,
family history of AD, hypercholesterolemia or atherosclerosis.
Individuals presently suffering from Alzheimer's disease can be
recognized from characteristic dementia, as well as the presence of
risk factors described above. In addition, a number of diagnostic
tests are available for identifying individuals who have AD. These
include measurement of CSF tau and A.beta.42 levels. Elevated tau
and decreased A.beta.42 levels signify the presence of AD.
Individuals suffering from Alzheimer's disease can also be
diagnosed by ADRDA criteria as discussed in the Examples
section.
[0157] In asymptomatic patients, treatment can begin at any age
(e.g., 10, 20, 30). Usually, however, it is not necessary to begin
treatment until a patient reaches 40, 50, 60 or 70. Treatment
typically entails multiple dosages over a period of time. Treatment
can be monitored by assaying antibody, or activated T-cell or
B-cell responses to the therapeutic agent (e.g., A.beta. peptide)
over time. If the response falls, a booster dosage is indicated. In
the case of potential Down's syndrome patients, treatment can begin
antenatally by administering therapeutic agent to the mother or
shortly after birth.
V. Treatment Regimes
[0158] In prophylactic applications, pharmaceutical compositions or
medicaments are administered to a patient susceptible to, or
otherwise at risk of, Alzheimer's disease in an amount sufficient
to eliminate or reduce the risk, lessen the severity, or delay the
outset of the disease, including 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
medicants are administered to a patient suspected of, or already
suffering from such a disease in an amount sufficient to cure, or
at least partially arrest, the symptoms of the disease
(biochemical, histologic and/or behavioral), including its
complications and intermediate pathological phenotypes in
development of the disease. In some methods, administration of
agent reduces or eliminates myocognitive impairment in patients
that have not yet developed characteristic Alzheimer's pathology.
An amount adequate to accomplish therapeutic or prophylactic
treatment is defined as a therapeutically- or
prophylactically-effective dose. 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.
[0159] 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.
[0160] 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.
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 A.beta. 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.
[0161] 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.
[0162] Agents for inducing an immune response can be administered
by parenteral, topical, intravenous, oral, subcutaneous,
intraarterial, intracranial, 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 on 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.
[0163] Agents of the invention can optionally be administered in
combination with other agents that are at least partly effective in
treatment of amyloidogenic disease. In the case of Alzheimer's and
Down's syndrome, in which amyloid deposits 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.
[0164] 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
A.beta., 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)). Another adjuvant is CpG (WO 98/40100).
Alternatively, A.beta. can be coupled to an adjuvant. However, such
coupling should not substantially change the conformation of
A.beta. 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.
[0165] A preferred class of adjuvants is aluminum salts (alum),
such as aluminum hydroxide, aluminum phosphate, aluminum 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.). 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 Complete Freund's Adjuvant (CFA) and Incomplete
Freund's Adjuvant (IFA). Other adjuvants include cytokines, such as
interleukins (IL-1, IL-2, and IL-12), macrophage colony stimulating
factor (M-CSF), tumor necrosis factor (TNF).
[0166] 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, 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.
[0167] 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.
[0168] 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).
[0169] 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.
[0170] 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.
[0171] Additional formulations suitable for other modes of
administration include oral, intranasal, and pulmonary
formulations, suppositories, and transdermal applications.
[0172] 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%.
[0173] 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.
[0174] 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)).
VI. Methods of Diagnosis
[0175] The invention provides methods of detecting an immune
response against A.beta. peptide in a patient suffering from or
susceptible to Alzheimer's disease. 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).
[0176] 1. Active Immunization
[0177] 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.
[0178] 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.
[0179] 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 plateaued 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.
[0180] 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.
[0181] 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
A.beta. peptide, typically A.beta.42. The immune response can be
determined from the presence of, e.g., antibodies or T-cells that
specifically bind to A.beta. peptide. ELISA methods of detecting
antibodies specific to A.beta. 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 sample from a patient being tested
is contacted with amyloid deposits (e.g., from a PDAPP mouse) and
phagocytic cells bearing Fc receptors. Subsequent clearing of the
amyloid deposit is then monitored. The existence and extent of
clearing response provides an indication of the existence and level
of antibodies effective to clear A.beta. in the tissue sample of
the patient under test.
[0182] 2. Passive Immunization
[0183] 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.
[0184] In some methods, a baseline measurement of antibody to AD 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.
[0185] 3. Diagnostic Kits
[0186] 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
A.beta.. The kit can also include a label. For detection of
antibodies to A.beta., the label is typically in the form of
labelled 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 A.beta.. 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.
[0187] The invention also provides diagnostic kits for performing
in vivo imaging. Such kits typically contain an antibody binding to
an epitope of A.beta., preferably within residues 1-10. Preferably,
the antibody is labelled or a secondary labeling reagent is
included in the kit. Preferably, the kit is labelled with
instructions for performing an in vivo imaging assay.
VII. In Vivo Imaging
[0188] The invention provides methods of in vivo imaging amyloid
deposits in a patient. Such methods are useful to diagnose or
confirm diagnosis of Alzheimer's disease, or susceptibility
thereto. For example, the methods can be used on a patient
presenting with symptoms of dementia. If the patient has abnormal
amyloid deposits, then the patient is likely suffering from
Alzheimer's disease. The methods can also be used on asymptomatic
patients. Presence of abnormal deposits of amyloid indicates
susceptibility to future symptomatic disease. The methods are also
useful for monitoring disease progression and/or response to
treatment in patients who have been previously diagnosed with
Alzheimer's disease.
[0189] The methods work by administering a reagent, such as
antibody, that binds to A.beta. in the patient and then detecting
the agent after it has bound. Preferred antibodies bind to A.beta.
deposits in a patient without binding to full length APP
polypeptide. Antibodies binding to an epitope of A.beta. within
amino acids 1-10 are particularly preferred. In some methods, the
antibody binds to an epitope within amino acids 7-10 of A.beta..
Such antibodies typically bind without inducing a substantial
clearing response. In other methods, the antibody binds to an
epitope within amino acids 1-7 of A.beta.. Such antibodies
typically bind and induce a clearing response to A.beta.. However,
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
C-terminal of residue 10 of A.beta. do not show as strong signal as
antibodies binding to epitopes within residues 1-10, presumably
because the C-terminal epitopes are inaccessible in amyloid
deposits. Accordingly, such antibodies are less preferred.
[0190] 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 labelled, although in
some methods, the primary reagent with affinity for AD 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.
[0191] Diagnosis is performed by comparing the number, size and/or
intensity of labelled 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. Prophylactic Efficacy of A.beta. Against AD
[0192] These examples describe administration of A.beta.42 peptide
to transgenic mice overexpressing APP with a mutation at position
717 (APP.sub.717V.fwdarw.F) that predisposes them to develop
Alzheimer's-like neuropathology. Production and characteristics of
these mice (PDAPP mice) is described in Games et al., Nature,
supra. These animals, in their heterozygote form, begin to deposit
A.beta. at six months of age forward. By fifteen months of age they
exhibit levels of A.beta. deposition equivalent to that seen in
Alzheimer's disease. PDAPP mice were injected with aggregated
A.beta..sub.42 (aggregated A.beta..sub.42) or phosphate buffered
saline. Aggregated A.beta..sub.42 was chosen because of its ability
to induce antibodies to multiple epitopes of A.beta..
[0193] A. Methods
[0194] 1. Source of Mice
[0195] Thirty PDAPP heterogenic female mice were randomly divided
into the following groups: 10 mice to be injected with aggregated A
P42 (one died in transit), 5 mice to be injected with PBS/adjuvant
or PBS, and 10 uninjected controls. Five mice were injected with
peptides derived from the sequence of serum amyloid protein
(SAP).
[0196] 2. Preparation of Immunogens
[0197] Preparation of aggregated A.beta.42: two milligrams of A042
(US Peptides Inc, lot K-42-12) was dissolved in 0.9 ml water and
made up to 1 ml by adding 0.1 ml 10.times.PBS. This was vortexed
and allowed to incubate overnight 37.degree. C., under which
conditions the peptide aggregated. Any unused A.beta. was stored as
a dry lyophilized powder at -20.degree. C. until the next
injection.
[0198] 3. Preparation of Injections
[0199] For each injection, 100 .mu.g of aggregated A.beta.42 in PBS
per mouse was emulsified 1:1 with Complete Freund's adjuvant (CFA)
in a final volume of 400 .mu.l emulsion for the first immunization,
followed by a boost of the same amount of immunogen in Incomplete
Freund's adjuvant (IFA) at 2 weeks. Two additional doses in IFA
were given at monthly intervals. The subsequent immunizations were
done at monthly intervals in 500 .mu.l of PBS. Injections were
delivered intraperitoneally (i.p.).
[0200] PBS injections followed the same schedule and mice were
injected with a 1:1 mix of PBS/Adjuvant at 400 .mu.l per mouse, or
500 .mu.l of PBS per mouse. SAP injections likewise followed the
same schedule using a dose of 100 .mu.g per injection.
[0201] 4. Titration of Mouse Bleeds, Tissue and
Immunohistochemistry
[0202] The above methods are described infra in General Materials
and Methods.
[0203] B. Results
[0204] PDAPP mice were injected with either aggregated A.beta.42
(aggregated A.beta.42), SAP peptides, or phosphate buffered saline.
A group of PDAPP mice were also left as uninjected, positive
controls. The titers of the mice to aggregated A.beta.42 were
monitored every other month from the fourth boost until the mice
were one year of age. Mice were sacrificed at 13 months. At all
time points examined, eight of the nine aggregated A.beta.42 mice
developed a high antibody titer, which remained high throughout the
series of injections (titers greater than 1/10000). The ninth mouse
had a low, but measurable titer of approximately 1/1000 (FIG. 1,
Table 1). SAPP-injected mice had titers of 1:1,000 to 1:30,000 for
this immunogen with only a single mouse exceeding 1:10,0000.
[0205] The PBS-treated mice were titered against aggregated A042 at
six, ten and twelve months. At a 1/100 dilution the PBS mice, when
titered against aggregated A.beta.42, only exceeded 4 times
background at one data point, otherwise, they were less than 4
times background at all time points (Table 1). The SAP-specific
response was negligible at these time points with all titers less
than 300.
[0206] Seven out of the nine mice in the aggregated A.beta.1-42
treated group had no detectable amyloid in their brains. In
contrast, brain tissue from mice in the SAP and PBS groups
contained numerous amyloid deposits in the hippocampus, as well as
in the frontal and cingulate cortices. The pattern of deposition
was similar to that of untreated controls, with characteristic
involvement of vulnerable subregions, such as the outer molecular
layer of the hippocampal dentate gyrus. One mouse from the
A.beta.1-42-injected group had a greatly reduced amyloid burden,
confined to the hippocampus. An isolated plaque was identified in
another A.beta.1-42-treated mouse.
[0207] Quantitative image analyses of the amyloid burden in the
hippocampus verified the dramatic reduction achieved in the
A.beta.42(AN1792)-treated animals (FIG. 2). The median values of
the amyloid burden for the PBS group (2.22%), and for the untreated
control group (2.65%) were significantly greater than for those
immunized with AN1792 (0.00%, p=0.0005). In contrast, the median
value for the group immunized with SAP peptides (SAPP) was 5.74%.
Brain tissue from the untreated, control mice contained numerous
A.beta. amyloid deposits visualized with the A.beta.-specific
monoclonal antibody (mAb) 3D6 in the hippocampus, as well as in the
retrosplenial cortex. A similar pattern of amyloid deposition was
also seen in mice immunized with SAPP or PBS (FIG. 2). In addition,
in these latter three groups there was a characteristic involvement
of vulnerable subregions of the brain classically seen in AD, such
as the outer molecular layer of the hippocampal dentate gyrus, in
all three of these groups.
[0208] The brains that contained no A.beta. deposits were also
devoid of neuritic plaques that are typically visualized in PDAPP
mice with the human APP antibody 8E5. All of brains from the
remaining groups (SAP-injected, PBS and uninjected mice) had
numerous neuritic plaques typical of untreated PDAPP mice. A small
number of neuritic plaques were present in one mouse treated with
AN1792, and a single cluster of dystrophic neurites was found in a
second mouse treated with AN1792. Image analyses of the
hippocampus, and shown in FIG. 3, demonstrated the virtual
elimination of dystrophic neurites in AN1792-treated mice (median
0.00%) compared to the PBS recipients (median 0.28%, p=0.0005).
[0209] Astrocytosis characteristic of plaque-associated
inflammation was also absent in the brains of the A.beta.1-42
injected group. The brains from the mice in the other groups
contained abundant and clustered GFAP-positive astrocytes typical
of A.beta. plaque-associated gliosis. A subset of the GFAP-reacted
slides were counter-stained with Thioflavin S to localize the
A.beta. deposits. The GFAP-positive astrocytes were associated with
A.beta. plaques in the SAP, PBS and untreated controls. No such
association was found in the plaque-negative A.beta.1-42 treated
mice, while minimal plaque-associated gliosis was identified in one
mouse treated with AN1792.
[0210] Image analyses, shown in FIG. 4 for the retrosplenial
cortex, verified that the reduction in astrocytosis was significant
with a median value of 1.56% for those treated with AN1792 versus
median values greater than 6% for groups immunized with SAP
peptides, PBS or untreated (p=0.0017)
[0211] Evidence from a subset of the A.beta.1-42- and PBS-injected
mice indicated plaque-associated MHC II immunoreactivity was absent
in the API-42 injected mice, consistent with lack of an
A.beta.-related inflammatory response.
[0212] Sections of the mouse brains were also reacted with a mAb
specific with a monoclonal antibody specific for MAC-1, a cell
surface protein. MAC-1 (CD11b) is an integrin family member and
exists as a heterodimer with CD18. The CD11b/CD18 complex is
present on monocytes, macrophages, neutrophils and natural killer
cells (Mak and Simard). The resident MAC-1-reactive cell type in
the brain is likely to be microglia based on similar phenotypic
morphology in MAC-1 immunoreacted sections. Plaque-associated MAC-1
labeling was lower in the brains of mice treated with AN1792
compared to the PBS control group, a finding consistent with the
lack of an A.beta.-induced inflammatory response.
[0213] C. Conclusion
[0214] The lack of A.beta. plaques and reactive neuronal and
gliotic changes in the brains of the A.beta.1-42-injected mice
indicate that no or extremely little amyloid was deposited in their
brains, and pathological consequences, such as gliosis and neuritic
pathology, were absent. PDAPP mice treated with A.beta.1-42 show
essentially the same lack of pathology as control nontransgenic
mice. Therefore, A.beta.1-42 injections are highly effective in the
prevention of deposition or clearance of human A.beta. from brain
tissue, and elimination of subsequent neuronal and inflammatory
degenerative changes. Thus, administration of A.beta. peptide can
have both preventative and therapeutic benefit in prevention of
AD.
II. Dose Response Study
[0215] Groups of five-week old, female Swiss Webster mice (N=6 per
group) were immunized with 300, 100, 33, 11, 3.7, 1.2, 0.4, or 0.13
ug of A.beta. formulated in CFA/IFA administered intraperitoneally.
Three doses were given at biweekly intervals followed by a fourth
dose one month later. The first dose was emulsified with CFA and
the remaining doses were emulsified with IFA. Animals were bled 4-7
days following each immunization starting after the second dose for
measurement of antibody titers. Animals in a subset of three
groups, those immunized with II, 33, or 300 .mu.g of antigen, were
additionally bled at approximately monthly intervals for four
months following the fourth immunization to monitor the decay of
the antibody response across a range of doses of immunogenic
formulations. These animals received a final fifth immunization at
seven months after study initiation. They were sacrificed one week
later to measure antibody responses to AN1792 and to perform
toxicological analyses.
[0216] A declining dose response was observed from 300 to 3.7 .mu.g
with no response at the two lowest doses. Mean antibody titers are
about 1:1000 after 3 doses and about 1:10,000 after 4 doses of
11-300 .mu.g of antigen (see FIG. 5).
[0217] Antibody titers rose dramatically for all but the lowest
dose group following the third immunization with increases in GMTs
ranging from 5- to 25-fold. Low antibody responses were then
detectable for even the 0.4 .mu.g recipients. The 1.2 and 3.7 .mu.g
groups had comparable titers with GMTs of about 1000 and the
highest four doses clustered together with GMTs of about 25,000,
with the exception of the 33 .mu.g dose group with a lower GMT of
3000. Following the fourth immunization, the titer increase was
more modest for most groups. There was a clear dose response across
the lower antigen dose groups from 0.14 .mu.g to 11 .mu.g ranging
from no detectable antibody for recipients of 0.14 .mu.g to a GMT
of 36,000 for recipients of 11 .mu.g. Again, titers for the four
highest dose groups of 11 to 300 .mu.g clustered together. Thus
following two immunizations, the antibody titer was dependent on
the antigen dose across the broad range from 0.4 to 300 .mu.g. By
the third immunization, titers of the highest four doses were all
comparable and they remained at a plateau after an additional
immunization.
[0218] One month following the fourth immunization, titers were 2-
to 3-fold higher in the 300 .mu.g group than those measured from
blood drawn five days following the immunization (FIG. 6). This
observation suggests that the peak anamnestic antibody response
occurred later than 5 days post-immunization. A more modest (50%)
increase was seen at this time in the 33 .mu.g group. In the 300
.mu.g dose group at two months following the last dose, GMTs
declined steeply by about 70%. After another month, the decline was
less steep at 45% (100 .mu.g) and about 14% for the 33 and 11 .mu.g
doses. Thus, the rate of decline in circulating antibody titers
following cessation of immunization appears to be biphasic with a
steep decline the first month following peak response followed by a
more modest rate of decrease thereafter.
[0219] The antibody titers and the kinetics of the response of
these Swiss Webster mice are similar to those of young heterozygous
PDAPP transgenic mice immunized in a parallel manner. Dosages
effective to induce an immune response in humans are typically
similar to dosages effective in mice.
III. Screen for Therapeutic Efficacy Against Established AD
[0220] This assay is designed to test immunogenic agents for
activity in arresting or reversing neuropathologic characteristics
of AD in aged animals. Immunizations with 42 amino acid long
A.beta. (AN1792) were begun at a time point when amyloid plaques
are already present in the brains of the PDAPP mice.
[0221] Over the time course used in this study, untreated PDAPP
mice develop a number of neurodegenerative changes that resemble
those found in AD (Games et al., supra and Johnson-Wood et al.,
Proc. Natl. Acad. Sci. USA 94, 1550-1555 (1997)). The deposition of
A.beta. into amyloid plaques is associated with a degenerative
neuronal response consisting of aberrant axonal and dendritic
elements, called dystrophic neurites. Amyloid deposits that are
surrounded by and contain dystrophic neurites called neuritic
plaques. In both AD and the PDAPP mouse, dystrophic neurites have a
distinctive globular structure, are immunoreactive with a panel of
antibodies recognizing APP and cytoskeletal components, and display
complex subcellular degenerative changes at the ultrastructural
level. These characteristics allow for disease-relevant, selective
and reproducible measurements of neuritic plaque formation in the
PDAPP brains. The dystrophic neuronal component of PDAPP neuritic
plaques is easily visualized with an antibody specific for human
APP (monoclonal antibody 8E5), and is readily measurable by
computer-assisted image analysis. Therefore, in addition to
measuring the effects of AN1792 on amyloid plaque formation, we
monitored the effects of this treatment on the development of
neuritic dystrophy.
[0222] Astrocytes and microglia are non-neuronal cells that respond
to and reflect the degree of neuronal injury. GFAP-positive
astrocytes and MHC II-positive microglia are commonly observed in
AD, and their activation increases with the severity of the
disease. Therefore, we also monitored the development of reactive
astrocytosis and microgliosis in the AN1792-treated mice.
[0223] A. Materials and Methods
[0224] Forty-eight, heterozygous female PDAPP mice, 11 to 11.5
months of age, obtained from Charles River, were randomly divided
into two groups: 24 mice to be immunized with 100 .mu.g of AN1792
and 24 mice to be immunized with PBS, each combined with Freund's
adjuvant. The AN1792 and PBS groups were again divided when they
reached 15 months of age. At 15 months of age approximately half of
each group of the AN1792- and PBS-treated animals were euthanized
(n=10 and 9, respectively), the remainder continued to receive
immunizations until termination at .about.18 months (n=9 and 12,
respectively). A total of 8 animals (5 AN1792, 3 PBS) died during
the study. In addition to the immunized animals, one-year old
(n=10), 15-month old (n=10) and 18-month old (n=10) untreated PDAPP
mice were included for comparison in the ELISAs to measure A.beta.
and APP levels in the brain; the one-year old animals were also
included in the immunohistochemical analyses.
[0225] Methodology was as in Example 1 unless otherwise indicated.
US Peptides lot 12 and California Peptides lot ME0339 of AN1792
were used to prepare the antigen for the six immunizations
administered prior to the 15-month time point. California Peptides
lots ME0339 and ME0439 were used for the three additional
immunizations administered between 15 and 18 months.
[0226] For immunizations, 100 .mu.g of AN1792 in 2001 PBS or PBS
alone was emulsified 1:1 (vol:vol) with Complete Freund's adjuvant
(CFA) or Incomplete Freund's adjuvant (IFA) or PBS in a final
volume of 400 .mu.l. The first immunization was delivered with CFA
as adjuvant, the next four doses were given with IFA and the final
four doses with PBS alone without added adjuvant. A total of nine
immunizations were given over the seven-month period on a two-week
schedule for the first three doses followed by a four-week interval
for the remaining injections. The four-month treatment group,
euthanized at 15 months of age, received only the first 6
immunizations.
[0227] B. Results
[0228] 1. Effects of AN1792 Treatment on Amyloid Burden
[0229] The results of AN1792 treatment on cortical amyloid burden
determined by quantitative image analysis are shown in FIG. 7. The
median value of cortical amyloid burden was 0.28% in a group of
untreated 12-month old PDAPP mice, a value representative of the
plaque load in mice at the study's initiation. At 18 months, the
amyloid burden increased over 17-fold to 4.87% in PBS-treated mice,
while AN1792-treated mice had a greatly reduced amyloid burden of
only 0.01%, notably less than the 12-month untreated and both the
15- and 18-month PBS-treated groups. The amyloid burden was
significantly reduced in the AN1792 recipients at both 15 (96%
reduction; p=0.003) and 18 (>99% reduction; p=0.0002)
months.
[0230] Typically, cortical amyloid deposition in PDAPP mice
initiates in the frontal and retrosplenial cortices (RSC) and
progresses in a ventral-lateral direction to involve the temporal
and entorhinal cortices (EC). Little or no amyloid was found in the
EC of 12 month-old mice, the approximate age at which AN1792 was
first administered. After 4 months of AN1792 treatment, amyloid
deposition was greatly diminished in the RSC, and the progressive
involvement of the EC was entirely eliminated by AN1792 treatment.
The latter observation showed that AN1792 completely halted the
progression of amyloid that would normally invade the temporal and
ventral cortices, as well as arrested or possibly reversed
deposition in the RSC.
[0231] The profound effects of AN1792 treatment on developing
cortical amyloid burden in the PDAPP mice are further demonstrated
by the 18-month group, which had been treated for seven months. A
near complete absence of cortical amyloid was found in the
AN1792-treated mouse, with a total lack of diffuse plaques, as well
as a reduction in compacted deposits.
[0232] 2. AN1792 Treatment-associated Cellular and Morphological
Changes
[0233] A population of A.beta.-positive cells was found in brain
regions that typically contain amyloid deposits. Remarkably, in
several brains from AN1792 recipients, very few or no extracellular
cortical amyloid plaques were found. Most of the A.beta.
immunoreactivity appeared to be contained within cells with large
lobular or clumped soma. Phenotypically, these cells resembled
activated microglia or monocytes. They were immunoreactive with
antibodies recognizing ligands expressed by activated monocytes and
microglia (MHC II and CD11b) and were occasionally associated with
the wall or lumen of blood vessels. Comparison of near-adjacent
sections labeled with A.beta. and MHC II-specific antibodies
revealed that similar patterns of these cells were recognized by
both classes of antibodies. Detailed examination of the
AN1792-treated brains revealed that the MHC II-positive cells were
restricted to the vicinity of the limited amyloid remaining in
these animals. Under the fixation conditions employed, the cells
were not immunoreactive with antibodies that recognize T cell (CD3,
CD3e) or B cell (CD45RA, CD45RB) ligands or leukocyte common
antigen (CD45), but were reactive with an antibody recognizing
leukosialin (CD43) which cross-reacts with monocytes. No such cells
were found in any of the PBS-treated mice.
[0234] PDAPP mice invariably develop heavy amyloid deposition in
the outer molecular layer of the hippocampal dentate gyrus. The
deposition forms a distinct streak within the perforant pathway, a
subregion that classically contains amyloid plaques in AD. The
characteristic appearance of these deposits in PBS-treated mice
resembled that previously characterized in untreated PDAPP mice.
The amyloid deposition consisted of both diffuse and compacted
plaques in a continuous band. In contrast, in a number of brains
from AN1792-treated mice this pattern was drastically altered. The
hippocampal amyloid deposition no longer contained diffuse amyloid,
and the banded pattern was completely disrupted. Instead, a number
of unusual punctate structures were present that are reactive with
anti-A.beta. antibodies, several of which appeared to be
amyloid-containing cells.
[0235] MHC II-positive cells were frequently observed in the
vicinity of extracellular amyloid in AN1792-treated animals. The
pattern of association of A.beta.-positive cells with amyloid was
very similar in several brains from AN1792-treated mice. The
distribution of these monocytic cells was restricted to the
proximity of the deposited amyloid and was entirely absent from
other brain regions devoid of A.beta. plaques. Confocal microscopy
of MHCII- and A.beta.-labelled sections revealed that plaque
material was contained within many of the monocytic cells.
[0236] Quantitative image analysis of MHC II and MAC I-labeled
sections revealed a trend towards increased immunoreactivity in the
RSC and hippocampus of AN1792-treated mice compared to the PBS
group which reached significance with the measure of MAC 1
reactivity in hippocampus.
[0237] These results are indicative of active, cell-mediated
clearance of amyloid in plaque-bearing brain regions.
[0238] 3. AN1792 Effects on A.beta. Levels: ELISA
Determinations
[0239] (a) Cortical Levels
[0240] In untreated PDAPP mice, the median level of total AD in the
cortex at 12 months was 1,600 ng/g, which increased to 8,700 ng/g
by 15 months (Table 2). At 18 months the value was 22,000 ng/g, an
increase of over 10-fold during the time course of the experiment.
PBS-treated animals had 8,600 ng/g total A.beta. at 15 months which
increased to 19,000 ng/g at 18 months. In contrast, AN1792-treated
animals had 81% less total A.beta. at 15 months (1,600 ng/g) than
the PBS-immunized group. Significantly less (p=0.0001) total
A.beta. (5,200 ng/g) was found at 18 months when the AN1792 and PBS
groups were compared (Table 2), representing a 72% reduction in the
A.beta. that would otherwise be present. Similar results were
obtained when cortical levels of A.beta.42 were compared, namely
that the AN1792-treated group contained much less A.beta.42, but in
this case the differences between the AN1792 and PBS groups were
significant at both 15 months (p=0.04) and 18 months (p=0.0001,
Table 2). TABLE-US-00001 TABLE 2 Median A.beta. Levels (ng/g) in
Cortex UNTREATED PBS AN1792 Age Total A.beta.42 (n) Total A.beta.42
(n) Total A.beta.42 (n) 12 1,600 1,300 (10) 15 8,700 8,300 (10)
8,600 7,200 (9) 1,600 1,300* (10) 18 22,200 18,500 (10) 19,000
15,900 (12) 5,200** 4,000** (9) *p = 0.0412 **p = 0.0001 (b)
Hippocampal Levels
[0241] In untreated PDAPP mice, median hippocampal levels of total
A.beta. at twelve months of age were 15,000 ng/g which increased to
51,000 ng/g at 15 months and further to 81,000 ng/g at 18 months
(Table 3). Similarly, PBS immunized mice showed values of 40,000
ng/g and 65,000 ng/g at 15 months and 18 months, respectively.
AN1792 immunized animals exhibited less total A.beta., specifically
25,000 ng/g and 51,000 ng/g at the respective 15-month and 18-month
timepoints. The 18-month AN1792-treated group value was
significantly lower than that of the PBS treated group (p=0.0105;
Table 3). Measurement of A.beta.42 gave the same pattern of
results, namely that levels in the AN1792-treated group were
significantly lower than in the PBS group (39,000 ng/g vs. 57,000
ng/g, respectively; p=0.002) at the 18-month evaluation (Table 3).
TABLE-US-00002 TABLE 3 Median A.beta. Levels (ng/g) in Hippocampus
UNTREATED PBS AN1792 Age Total A.beta.42 (n) Total A.beta.42 (n)
Total A.beta.42 (n) 12 15,500 11,100 (10) 15 51,500 44,400 (10)
40,100 35,70 (9) 24,50 22,100 (10) 18 80,800 64,200 (10) 65,400
57,10 (12) 50,90 38.900** (9) *p = 0.0105 **p = 0.0022
[0242] (c) Cerebellar Levels
[0243] In 12-month untreated PDAPP mice, the median cerebellar
level of total A.beta. was 15 ng/g (Table 4). At 15 months, this
median increased to 28 ng/g and by 18 months had risen to 35 ng/g.
PBS-treated animals displayed median total A.beta. values of 21
ng/g at 15 months and 43 ng/g at 18 months. AN1792-treated animals
were found to have 22 ng/g total AD at 15 months and significantly
less (p=0.002) total A.beta. at 18 months (25 ng/g) than the
corresponding PBS group (Table 4). TABLE-US-00003 TABLE 4 Median
A.beta. Levels (ng/g) in Cerebellum UNTREATED PBS AN1792 Age Total
A.beta. (n) Total A.beta. (n) Total A.beta. (n) 12 15.6 (10) 15
27.7 (10) 20.8 (9) 21.7 (10) 18 35.0 (10) 43.1 (12) 24.8* (9) *p =
0.0018
[0244] 4. Effects of AN1792 Treatment on APP Levels
[0245] APP-.alpha. and the full-length APP molecule both contain
all or part of the AD sequence and thus could be potentially
impacted by the generation of an AN1792-directed immune response.
In studies to date, a slight increase in APP levels has been noted
as neuropathology increases in the PDAPP mouse. In the cortex,
levels of either APP-.alpha./FL (full length) or APP-.alpha. were
essentially unchanged by treatment with the exception that
APP-.alpha. was reduced by 19% at the 18-month timepoint in the
AN1792-treated vs. the PBS-treated group. The 18-month
AN1792-treated APP values were not significantly different from
values of the 12-month and 15-month untreated and 15-month PBS
groups. In all cases the APP values remained within the ranges that
are normally found in PDAPP mice.
[0246] 5. Effects of AN1792 Treatment on Neurodegenerative and
Gliotic Pathology
[0247] Neuritic plaque burden was significantly reduced in the
frontal cortex of AN1792-treated mice compared to the PBS group at
both 15 (84%; p=0.03) and 18 (55%; p=0.01) months of age (FIG. 8).
The median value of the neuritic plaque burden increased from 0.32%
to 0.49% in the PBS group between 15 and 18 months of age. This
contrasted with the greatly reduced development of neuritic plaques
in the AN1792 group, with median neuritic plaque burden values of
0.05% and 0.22%, in the 15 and 18 month groups, respectively.
[0248] Immunizations with AN1792 seemed well tolerated and reactive
astrocytosis was also significantly reduced in the RSC of
AN1792-treated mice when compared to the PBS group at both 15 (56%;
p=0.011) and 18 (39%; p=0.028) months of age (FIG. 9). Median
values of the percent of astrocytosis in the PBS group increased
between 15 and 18 months from 4.26% to 5.21%. AN1792-treatment
suppressed the development of astrocytosis at both time points to
1.89% and 3.2%, respectively. This suggests the neuropil was not
being damaged by the clearance process.
[0249] 6. Antibody Responses
[0250] As described above, eleven-month old, heterozygous PDAPP
mice (N=24) received a series of 5 immunizations of 100 .mu.g of
AN1792 emulsified with Freund's adjuvant and administered
intraperitoneally at weeks 0, 2, 4, 8, and 12, and a sixth
immunization with PBS alone (no Freund's adjuvant) at week 16. As a
negative control, a parallel set of 24 age-matched transgenic mice
received immunizations of PBS emulsified with the same adjuvants
and delivered on the same schedule. Animals were bled within three
to seven days following each immunization starting after the second
dose. Antibody responses to AN1792 were measured by ELISA.
Geometric mean titers (GMT) for the animals that were immunized
with AN1792 were approximately 1,900, 7,600, and 45,000 following
the second, third and last (sixth) doses respectively. No
A.beta.-specific antibody was measured in control animals following
the sixth immunization.
[0251] Approximately one-half of the animals were treated for an
additional three months, receiving immunizations at about 20, 24
and 27 weeks. Each of these doses was delivered in PBS vehicle
alone without Freund's adjuvant. Mean antibody titers remained
unchanged over this time period. In fact, antibody titers appeared
to remain stable from the fourth to the eighth bleed corresponding
to a period covering the fifth to the ninth injections.
[0252] To determine if the A.beta.-specific antibodies elicited by
immunization that were detected in the sera of AN1792-treated mice
were also associated with deposited brain amyloid, a subset of
sections from the AN1792- and PBS-treated mice were reacted with an
antibody specific for mouse IgG. In contrast to the PBS group,
A.beta. plaques in AN1792-treated brains were coated with
endogenous IgG. This difference between the two groups was seen in
both 15- and 18-month groups. Particularly striking was the lack of
labeling in the PBS group, despite the presence of a heavy amyloid
burden in these mice. These results show that immunization with a
synthetic A.beta. protein generates antibodies that recognize and
bind in vivo to the A.beta. in amyloid plaques.
[0253] 7. Cellular-Mediated Immune Responses
[0254] Spleens were removed from nine AN1792-immunized and 12
PBS-immunized 18-month old PDAPP mice 7 days after the ninth
immunization. Splenocytes were isolated and cultured for 72 h in
the presence of A.beta.40, A.beta.42, or A.beta.40-1 (reverse order
protein). The mitogen Con A served as a positive control. Optimum
responses were obtained with >1.7 .mu.M protein. Cells from all
nine AN1792-treated animals proliferated in response to either
A.beta.1-40 or A.beta.1-42 protein, with equal levels of
incorporation for both proteins (FIG. 10A). There was no response
to the A.beta.40-1 reverse protein. Cells from control animals did
not respond to any of the A.beta. proteins (FIG. 10B).
[0255] C. Conclusion
[0256] The results of this study show that AN1792 immunization of
PDAPP mice possessing existing amyloid deposits slows and prevents
progressive amyloid deposition and retard consequential
neuropathologic changes in the aged PDAPP mouse brain.
Immunizations with AN1792 essentially halted amyloid developing in
structures that would normally succumb to amyloidosis. Thus,
administration of A.beta. peptide has therapeutic benefit in the
treatment of AD.
IV. Screen of A.beta. Fragments
[0257] 100 PDAPP mice age 9-11 months were immunized with 9
different regions of APP and A.beta. 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 A.beta. peptide conjugates 1-12, 13-28, 32-42,
1-5, all coupled to sheep anti-mouse IgG via a cystine link; an APP
polypeptide amino acids 592-695, aggregated human A.beta.1-40, and
aggregated human A.beta. 25-35, and aggregated rodent A.beta.42.
Aggregated A.beta.42 and PBS were used as positive and negative
controls, respectively. Ten mice were used per treatment group.
Titers were monitored as above and mice were euthanized at the end
of 4 months of injections. Histochemistry, A.beta. levels, and
toxicology analysis was determined post mortem.
[0258] A. Materials and Methods
[0259] 1. Preparation of Immunogens
[0260] Preparation of Coupled A.beta. Peptides: Four Human A.beta.
Peptide Conjugates (Amino Acid residues 1-5, 1-12, 13-28, and
33-42, each conjugated to sheep anti-mouse IgG) were prepared by
coupling through an artificial cysteine added to the A.beta.
peptide using the crosslinking reagent sulfo-EMCS. The A.beta.
peptide derivatives were synthesized with the following final amino
acid sequences. In each case, the location of the inserted cysteine
residue is indicated by underlining. The A 13-28 peptide derivative
also had two glycine residues added prior to the carboxyl terminal
cysteine as indicated. TABLE-US-00004 A.beta.1-12 peptide (SEQ ID
NO:74) NH2-DAEFRHDSGYEVC-COOH A.beta.1-5 peptide (SEQ ID NO:73)
NH2-DAEFRC-COOH A.beta.33-42 peptide (SEQ ID NO:74)
NH2-C-amino-heptanoic acid-GLMVGGVVIA-COOH A.beta.13-28 peptide
(SEQ ID NO:75) Ac-NH-HHQKLVFFAEDVGSNKGGC-COOH
[0261] To prepare for the coupling reaction, ten mg of sheep
anti-mouse IgG (Jackson ImmunoResearch Laboratories) was dialyzed
overnight against 10 mM sodium borate buffer, pH 8.5. The dialyzed
antibody was then concentrated to a volume of 2 mL using an Amicon
Centriprep tube. Ten mg sulfo-EMCS
[0262] [N (.gamma.-maleimidocuproyloxy)succinimide] (Molecular
Sciences Co.) was dissolved in one mL deionized water. A 40-fold
molar excess of sulfo-EMCS was added dropwise with stirring to the
sheep anti-mouse IgG and then the solution was stirred for an
additional ten min. The activated sheep anti-mouse IgG was 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, were 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 A.beta. peptide
was dissolved in 20 mL of 10 mM NaPO4, pH 8.0, with the exception
of the A.beta.33-42 peptide for which 10 mg was first dissolved in
0.5 mL of DMSO and then diluted to 20 mL with the 10 mM NaPO4
buffer. The peptide solutions were each added to 10 mL of activated
sheep anti-mouse IgG and rocked at room temperature for 4 hr. The
resulting conjugates were 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 were 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 were
determined using the BCA protein assay (Pierce Chemicals) with
horse IgG for the standard curve. Conjugation was documented by the
molecular weight increase of the conjugated peptides relative to
that of the activated sheep anti-mouse IgG. The A.beta.1-5 sheep
anti-mouse conjugate was a pool of two conjugations, the rest were
from a single preparation.
[0263] 2. Preparation of Aggregated A.beta. Peptides
[0264] Human 1-40 (AN1528; California Peptides Inc., Lot ME0541),
human 1-42 (AN1792; California Peptides Inc., Lots ME0339 and
ME0439), human 25-35, and rodent 1-42 (California Peptides Inc.,
Lot ME0218) peptides were freshly solubilized for the preparation
of each set of injections from lyophilized powders that had been
stored desiccated at -20.degree. C. For this purpose, two mg of
peptide were added to 0.9 ml of deionized water and the mixture was
vortexed to generate a relatively uniform solution or suspension.
Of the four, AN1528 was the only peptide soluble at this step. A
100 .mu.l aliquot of 10.times.PBS (1.times.PBS: 0.15 M NaCl, 0.01 M
sodium phosphate, pH 7.5) was then added at which point AN1528
began to precipitate. The suspension was vortexed again and
incubated overnight at 37.degree. C. for use the next day.
[0265] Preparation of the pBx6 protein: An expression plasmid
encoding pBx6, a fusion protein consisting of the 100-amino acid
bacteriophage MS-2 polymerase N-terminal leader sequence followed
by amino acids 592-695 of APP (.beta.APP) was constructed as
described by Oltersdorf et al., J. Biol. Chem. 265, 4492-4497
(1990). The plasmid was transfected into E. coli and the protein
was expressed after induction of the promoter. The bacteria were
lysed in 8M urea and pBx6 was partially purified by preparative SDS
PAGE. Fractions containing pBx6 were identified by Western blot
using a rabbit anti-pBx6 polyclonal antibody, pooled, concentrated
using an Amicon Centriprep tube and dialysed against PBS. The
purity of the preparation, estimated by Coomassie Blue stained SDS
PAGE, was approximately 5 to 10%.
[0266] B. Results and Discussion
[0267] 1. Study Design
[0268] One hundred male and female, nine- to eleven-month old
heterozygous PDAPP transgenic mice were obtained from Charles River
Laboratory and Taconic Laboratory. The mice were sorted into ten
groups to be immunized with different regions of A.beta. or APP
combined with Freund's adjuvant. Animals were distributed to match
the gender, age, parentage and source of the animals within the
groups as closely as possible. The immunogens included four A.beta.
peptides derived from the human sequence, 1-5, 1-12, 13-28, and
33-42, each conjugated to sheep anti-mouse IgG; four aggregated
A.beta. peptides, human 1-40 (AN1528), human 1-42 (AN1792), human
25-35, and rodent 1-42; and a fusion polypeptide, designated as
pBx6, containing APP amino acid residues 592-695. A tenth group was
immunized with PBS combined with adjuvant as a control.
[0269] For each immunization, 100 .mu.g of each A.beta. peptide in
2001 PBS or 200 .mu.g of the APP derivative pBx6 in the same volume
of PBS or PBS alone was emulsified 1:1 (vol:vol) with Complete
Freund's adjuvant (CFA) in a final volume of 400 .mu.l for the
first immunization, followed by a boost of the same amount of
immunogen in Incomplete Freund's adjuvant (IFA) for the subsequent
four doses and with PBS for the final dose. Immunizations were
delivered intraperitoneally on a biweekly schedule for the first
three doses, then on a monthly schedule thereafter. Animals were
bled four to seven days following each immunization starting after
the second dose for the measurement of antibody titers. Animals
were euthanized approximately one week after the final dose.
[0270] 2. AD and APP Levels in the Brain
[0271] Following about four months of immunization with the various
A.beta. peptides or the APP derivative, brains were removed from
saline-perfused animals. One hemisphere was prepared for
immunohistochemical analysis and the second was used for the
quantitation of A.beta. and APP levels. To measure the
concentrations of various forms of beta amyloid peptide and amyloid
precursor protein, the hemisphere was dissected and homogenates of
the hippocampal, cortical, and cerebellar regions were prepared in
5 M guanidine. These were diluted and the level of amyloid or APP
was quantitated by comparison to a series of dilutions of standards
of A.beta. peptide or APP of known concentrations in an ELISA
format.
[0272] The median concentration of total A.beta. for the control
group immunized with PBS was 5.8-fold higher in the hippocampus
than in the cortex (median of 24,318 ng/g hippocampal tissue
compared to 4,221 ng/g for the cortex). The median level in the
cerebellum of the control group (23.4 ng/g tissue) was about
1,000-fold lower than in the hippocampus. These levels are similar
to those that we have previously reported for heterozygous PDAPP
transgenic mice of this age (Johnson-Woods et al., 1997,
supra).
[0273] For the cortex, a subset of treatment groups had median
total Ad and A.beta.1-42 levels which differed significantly from
those of the control group (p<0.05), those animals receiving
AN1792, rodent A.beta.1-42 or the A.beta.1-5 peptide conjugate as
shown in FIG. 11. The median levels of total A.beta. were reduced
by 75%, 79% and 61%, respectively, compared to the control for
these treatment groups. There were no discernable correlations
between A.beta.-specific antibody titers and AD levels in the
cortical region of the brain for any of the groups.
[0274] In the hippocampus, the median reduction of total A.beta.
associated with AN1792 treatment (46%, p=0.0543) was not as great
as that observed in the cortex (75%, p=0.0021). However, the
magnitude of the reduction was far greater in the hippocampus than
in the cortex, a net reduction of 11,186 ng/g tissue in the
hippocampus versus 3,171 ng/g tissue in the cortex. For groups of
animals receiving rodent A.beta.1-42 or A.beta.1-5, the median
total A.beta. levels were reduced by 36% and 26%, respectively.
However, given the small group sizes and the high variability of
the amyloid peptide levels from animal to animal within both
groups, these reductions were not significant. When the levels of
A.beta.1-42 were measured in the hippocampus, none of the
treatment-induced reductions reached significance. Thus, due to the
smaller A.beta. burden in the cortex, changes in this region are a
more sensitive indicator of treatment effects. The changes in
A.beta. levels measured by ELISA in the cortex are similar, but not
identical, to the results from the immunohistochemical analysis
(see below).
[0275] Total A.beta. was also measured in the cerebellum, a region
typically minimally affected with AD pathology. None of the median
A.beta. concentrations of any of the groups immunized with the
various A.beta. peptides or the APP derivative differed from that
of the control group in this region of the brain. This result
suggests that non-pathological levels of A.beta. are unaffected by
treatment.
[0276] APP concentration was also determined by ELISA in the cortex
and cerebellum from treated and control mice. Two different APP
assays were utilized. The first, designated APP-.alpha./FL,
recognizes both APP-alpha (.alpha., the secreted form of APP which
has been cleaved within the A.beta. sequence), and full-length
forms (FL) of APP, while the second recognizes only APP-.alpha.. In
contrast to the treatment-associated diminution of A.beta. in a
subset of treatment groups, the levels of APP were unchanged in all
of the treated compared to the control animals. These results
indicate that the immunizations with A.beta. peptides are not
depleting APP; rather the treatment effect is specific to
A.beta..
[0277] In summary, total A.beta. and A.beta.1-42 levels were
significantly reduced in the cortex by treatment with AN1792,
rodent A.beta.1-42 or A.beta.1-5 conjugate. In the hippocampus,
total A.beta. was significantly reduced only by AN1792 treatment.
No other treatment-associated changes in A.beta. or APP levels in
the hippocampal, cortical or cerebellar regions were
significant.
[0278] 2. Histochemical Analyses
[0279] Brains from a subset of six groups were prepared for
immunohistochemical analysis, three groups immunized with the
A.beta. peptide conjugates A.beta.1-5, A.beta.1-12, and
A.beta.13-28; two groups immunized with the full length A.beta.
aggregates AN1792 and AN1528 and the PBS-treated control group. The
results of image analyses of the amyloid burden in brain sections
from these groups are shown in FIG. 12. There were significant
reductions of amyloid burden in the cortical regions of three of
the treatment groups versus control animals. The greatest reduction
of amyloid burden was observed in the group receiving AN1792 where
the mean value was reduced by 97% (p=0.001). Significant reductions
were also observed for those animals treated with AN1528 (95%,
p=0.005) and the API-5 peptide conjugate (67%, p=0.02).
[0280] The results obtained by quantitation of total A.beta. or
A.beta.1-42 by ELISA and amyloid burden by image analysis differ to
some extent. Treatment with AN1528 had a significant impact on the
level of cortical amyloid burden when measured by quantitative
image analysis but not on the concentration of total A.beta. in the
same region when measured by ELISA. The difference between these
two results is likely to be due to the specificities of the assays.
Image analysis measures only insoluble A.beta. aggregated into
plaques. In contrast, the ELISA measures all forms of A.beta., both
soluble and insoluble, monomeric and aggregated. Since the disease
pathology is thought to be associated with the insoluble
plaque-associated form of A.beta., the image analysis technique may
have more sensitivity to reveal treatment effects. However since
the ELISA is a more rapid and easier assay, it is very useful for
screening purposes. Moreover it may reveal that the
treatment-associated reduction of A.beta. is greater for
plaque-associated than total A.beta..
[0281] To determine if the A.beta.-specific antibodies elicited by
immunization in the treated animals reacted with deposited brain
amyloid, a subset of the sections from the treated animals and the
control mice were reacted with an antibody specific for mouse IgG.
In contrast to the PBS group, A.beta.-containing plaques were
coated with endogenous IgG for animals immunized with the A.beta.
peptide conjugates A.beta.1-5, A.beta.1-12, and A.beta.13-28; and
the full length A.beta. aggregates AN1792 and AN1528. Brains from
animals immunized with the other A.beta. peptides or the APP
peptide pBx6 were not analyzed by this assay.
[0282] 3. Measurement of Antibody Titers
[0283] Mice were bled four to seven days following each
immunization starting after the second immunization, for a total of
five bleeds. Antibody titers were measured as A.beta.1-42-binding
antibody using a sandwich ELISA with plastic multi-well plates
coated with A.beta.1-42. As shown in FIG. 13, peak antibody titers
were elicited following the fourth dose for those four immunogenic
formulations which elicited the highest titers of AN1792-specific
antibodies: AN1792 (peak GMT: 94,647), AN1528 (peak GMT: 88,231),
A.beta.1-12 conjugate (peak GMT: 47,216) and rodent A.beta.1-42
(peak GMT: 10,766). Titers for these groups declined somewhat
following the fifth and sixth doses. For the remaining five
immunogens, peak titers were reached following the fifth or the
sixth dose and these were of much lower magnitude than those of the
four highest titer groups: A.beta.1-5 conjugate (peak GMT: 2,356),
pBx6 (peak GMT: 1,986), A.beta.13-28 conjugate (peak GMT: 1,183),
A033-42 conjugate (peak GMT: 658), A.beta.25-35 (peak GMT: 125).
Antibody titers were also measured against the homologous peptides
using the same ELISA sandwich format for a subset of the
immunogens, those groups immunized with A.beta.1-5, A.beta.13-28,
A025-35, A.beta.33-42 or rodent A.beta.1-42. These titers were
about the same as those measured against A.beta.1-42 except for the
rodent A.beta.1-42 immunogen in which case antibody titers against
the homologous immunogen were about two-fold higher. The magnitude
of the AN1792-specific antibody titer of individual animals or the
mean values of treatment groups did not correlate with efficacy
measured as the reduction of A.beta. in the cortex.
[0284] 4. Lymphoproliferative Responses
[0285] A.beta.-dependent lymphoproliferation was measured using
spleen cells harvested approximately one week following the final,
sixth, immunization. Freshly harvested cells, 105 per well, were
cultured for 5 days in the presence of A.beta.1-40 at a
concentration of 5 .mu.M for stimulation. Cells from a subset of
seven of the ten groups were also cultured in the presence of the
reverse peptide, A.beta.40-1. As a positive control, additional
cells were cultured with the T cell mitogen, PHA, and, as a
negative control, cells were cultured without added peptide.
[0286] Lymphocytes from a majority of the animals proliferated in
response to PHA. There were no significant responses to the
A.beta.40-1 reverse peptide. Cells from animals immunized with the
larger aggregated A.beta. peptides, AN1792, rodent A.beta.1-42 and
AN1528 proliferated robustly when stimulated with A.beta.1-40 with
the highest cpm in the recipients of AN1792. One animal in each of
the groups immunized with API-12 conjugate, A.beta.13-28 conjugate
and A25-35 proliferated in response to A.beta.1-40. The remaining
groups receiving A.beta.1-5 conjugate, A.beta.33-42 conjugate pBx6
or PBS had no animals with an A.beta.-stimulated response. These
results are summarized in Table 5 below. TABLE-US-00005 TABLE 5
Immunogen Conjugate A.beta. Amino Acids Responders A.beta.1-5 Yes
5-mer 0/7 A.beta.1-12 Yes 12-mer 1/8 A.beta.13-28 Yes 16-mer 1/9
A.beta.25-35 11-mer 1/9 A.beta.33-42 Yes 10-mer 0/10 A.beta.1-40
40-mer 5/8 A.beta.1-42 42-mer 9/9 r A.beta.1-42 42-mer 8/8 pBx6 0/8
PBS 0-mer 0/8
[0287] These results show that AN1792 and AN1528 stimulate strong T
cell responses, most likely of the CD4+ phenotype. The absence of
an A.beta.-specific T cell response in animals immunized with
A.beta.1-5 is not surprising since peptide epitopes recognized by
CD4+ T cells are usually about 15 amino acids in length, although
shorter peptides can sometimes function with less efficiency. Thus
the majority of helper T cell epitopes for the four conjugate
peptides are likely to reside in the IgG conjugate partner, not in
the A.beta. region. This hypothesis is supported by the very low
incidence of proliferative responses for animals in each of these
treatment groups. Since the A.beta.1-5 conjugate was effective at
significantly reducing the level of A.beta. in the brain, in the
apparent absence of A.beta.-specific T cells, the key effector
immune response induced by immunization with this peptide appears
to be antibody.
[0288] Lack of T-cell and low antibody response from fusion peptide
pBx6, encompassing APP amino acids 592-695 including all of the
A.beta. residues may be due to the poor immunogenicity of this
particular preparation. The poor immunogenicity of the A.beta.25-35
aggregate is likely due to the peptide being too small to be likely
to contain a good T cell epitope to help the induction of an
antibody response. If this peptide were conjugated to a carrier
protein, it would probably be more immunogenic.
V. Preparation of Polyclonal Antibodies for Passive Protection
[0289] 125 non-transgenic mice were immunized with 100 .mu.g
A.beta.1-42, plus CFA/IFA adjuvant, and euthanized at 4-5 months.
Blood was collected from immunized mice. IgG was separated from
other blood components. Antibody specific for the immunogen may be
partially purified by affinity chromatography. An average of about
0.5-1 mg of immunogen-specific antibody is obtained per mouse,
giving a total of 60-120 mg.
VI. Passive Immunization with Antibodies to A.beta.
[0290] Groups of 7-9 month old PDAPP mice each are injected with
0.5 mg in PBS of polyclonal anti-A.beta. or specific anti-AD
monoclonals as shown below. The cell line designated
RB44-10D5.19.21 producing the antibody 10D5 has the American Type
Culture Collection (ATCC) accession number PTA-5129, having been
deposited on Apr. 8, 2003. The hydridoma resulting from fusion of
SP2/0 with A/J mouse spleen: 266.2 producing the antibody 266 has
the ATCC accession number PTA-6123, having been deposited on Jul.
20, 2004. The address of the American Type Culture Collection is
10801 University Boulevard, Manassas, Va. 20110-2209. 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 A.beta. into a mouse, preparing hybridomas and
screening the hybridomas for an antibody that specifically binds to
a desired fragment of A.beta. without binding to other
nonoverlapping fragments of A.beta.. TABLE-US-00006 TABLE 6
Antibody Epitope 2H3 A.beta. 1-12 10D5 A.beta. 1-12 266 A.beta.
13-28 21F12 A.beta. 33-42 Mouse polyclonal Anti-Aggregated
A.beta.42 anti-human A.beta.42
[0291] Mice were 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 A.beta.42 or other
immunogen. Titers were monitored as above and mice were euthanized
at the end of 6 months of injections. Histochemistry, A.beta.
levels and toxicology were performed post mortem. Ten mice were
used per group. Additional studies of passive immunization are
described in Examples XI and XII below.
VII. Comparison of Different Adjuvants
[0292] This example compares CFA, alum, an oil-in water emulsion
and MPL for capacity to stimulate an immune response.
[0293] A. Materials and Methods
[0294] 1. Study Design
[0295] One hundred female Hartley strain six-week old guinea pigs,
obtained from Elm Hill, were sorted into ten groups to be immunized
with AN1792 or a palmitoylated derivative thereof combined with
various adjuvants. Seven groups received injections of AN1792 (33
.mu.g unless otherwise specified) combined with a) PBS, b) Freund's
adjuvant, c) MPL, d) squalene, e) MPL/squalene f) low dose alum, or
g) high dose alum (300 .mu.g AN1792). Two groups received
injections of a palmitoylated derivative of AN1792 (33 .mu.g)
combined with a) PBS or b) squalene. A final, tenth group received
PBS alone without antigen or additional adjuvant. For the group
receiving Freund's adjuvant, the first dose was emulsified with CFA
and the remaining four doses with IFA. Antigen was administered at
a dose of 33 .mu.g for all groups except the high dose alum group,
which received 300 .mu.g of AN1792. Injections were administered
intraperitoneally for CFA/IFA and intramuscularly in the hind limb
quadriceps alternately on the right and left side for all other
groups. The first three doses were given on a biweekly schedule
followed by two doses at a monthly interval). Blood was drawn six
to seven days following each immunization, starting after the
second dose, for measurement of antibody titers.
[0296] 2. Preparation of Immunogens
[0297] Two mg A.beta.42 (California Peptide, Lot ME0339) was added
to 0.9 ml of deionized water and the mixture was vortexed to
generate a relatively uniform suspension. A 100 .mu.l aliquot of
10.times.PBS (1.times.PBS, 0.15 M NaCl, 0.01 M sodium phosphate, pH
7.5) was added. The suspension was vortexed again and incubated
overnight at 37.degree. C. for use the next day. Unused A.beta.1-42
was stored with desiccant as a lyophilized powder at -20.degree.
C.
[0298] A palmitoylated derivative of AN1792 was prepared by
coupling palmitic anhydride, dissolved in dimethyl formamide, to
the amino terminal residue of AN1792 prior to removal of the
nascent peptide from the resin by treatment with hydrofluoric
acid.
[0299] To prepare formulation doses with Complete Freund's adjuvant
(CFA) (group 2), 33 .mu.g of AN1792 in 200 .mu.l PBS was emulsified
1:1 (vol:vol) with CFA in a final volume of 400 .mu.l for the first
immunization. For subsequent immunizations, the antigen was
similarly emulsified with Incomplete Freund's adjuvant (IFA).
[0300] To prepare formulation doses with MPL for groups 5 and 8,
lyophilized powder (Ribi ImmunoChem Research, Inc., Hamilton,
Mont.) was added to 0.2% aqueous triethylamine to a final
concentration of 1 mg/ml and vortexed. The mixture was heated to 65
to 70.degree. C. for 30 sec to create a slightly opaque uniform
suspension of micelles. The solution was freshly prepared for each
set of injections. For each injection in group 5, 33 .mu.g of
AN1792 in 16.5 .mu.l PBS, 50 .mu.g of MPL (50 .mu.l) and 162 .mu.l
of PBS were mixed in a borosilicate tube immediately before
use.
[0301] To prepare formulation doses with the low oil-in-water
emulsion, AN1792 in PBS was added to 5% squalene, 0.5% Tween 80,
0.5% Span 85 in PBS to reach a final single dose concentration of
33 .mu.g AN1792 in 250 .mu.l (group 6). The mixture was emulsified
by passing through a two-chambered hand-held device 15 to 20 times
until the emulsion droplets appeared to be about equal in diameter
to a 1.0 .mu.m diameter standard latex bead when viewed under a
microscope. The resulting suspension was opalescent, milky white.
The emulsions were freshly prepared for each series of injections.
For group 8, MPL in 0.2% triethylamine was added at a concentration
of 50 .mu.g per dose to the squalene and detergent mixture for
emulsification as noted above. For the palmitoyl derivative (group
7), 33 .mu.g per dose of palmitoyl-NH-A.beta.1-42 was added to
squalene and vortexed. Tween 80 and Span 85 were then added with
vortexing. This mixture was added to PBS to reach final
concentrations of 5% squalene, 0.5% Tween 80, 0.5% Span 85 and the
mixture was emulsified as noted above.
[0302] To prepare formulation doses with alum (groups 9 and 10),
AN1792 in PBS was added to Alhydrogel (aluminum hydroxide gel,
Accurate, Westbury, N.Y.) to reach concentrations of 33 .mu.g (low
dose, group 9) or 300 .mu.g (high dose, group 10) AN1792 per 5 mg
of alum in a final dose volume of 250 .mu.l. The suspension was
gently mixed for 4 hr at RT.
[0303] 3. Measurement of Antibody Titers
[0304] Guinea pigs were bled six to seven days following
immunization starting after the second immunization for a total of
four bleeds. Antibody titers against A.beta.42 were measured by
ELISA as described in General Materials and Methods.
[0305] 4. Tissue Preparation
[0306] After about 14 weeks, all guinea pigs were euthanized by
administering CO.sub.2. Cerebrospinal fluid was collected and the
brains were removed and three brain regions (hippocampus, cortex
and cerebellum) were dissected and used to measure the
concentration of total A.beta. protein using ELISA.
[0307] B. Results
[0308] 1. Antibody Responses
[0309] There was a wide range in the potency of the various
adjuvants when measured as the antibody response to AN1792
following immunization. As shown in FIG. 14, when AN1792 was
administered in PBS, no antibody was detected following two or
three immunizations and negligible responses were detected
following the fourth and fifth doses with geometric mean titers
(GMTs) of only about 45. The o/w emulsion induced modest titers
following the third dose (GMT 255) that were maintained following
the fourth dose (GMT 301) and fell with the final dose (GMT 54).
There was a clear antigen dose response for AN1792 bound to alum
with 300 .mu.g being more immunogenic at all time points than 33
.mu.g. At the peak of the antibody response, following the fourth
immunization, the difference between the two doses was 43% with
GMTs of about 1940 (33 .mu.g) and 3400 (300 .mu.g). The antibody
response to 33 .mu.g AN1792 plus MPL was very similar to that
generated with almost a ten-fold higher dose of antigen (300 .mu.g)
bound to alum. The addition of MPL to an o/w emulsion decreased the
potency of the formulations relative to that with MPL as the sole
adjuvant by as much as 75%. A palmitoylated derivative of AN1792
was completely non-immunogenic when administered in PBS and gave
modest titers when presented in an o/w emulsion with GMTs of 340
and 105 for the third and fourth bleeds. The highest antibody
titers were generated with Freund's adjuvant with a peak GMT of
about 87,000, a value almost 30-fold greater than the GMTs of the
next two most potent formulations, MPL and high dose
AN1792/alum.
[0310] The most promising adjuvants identified in this study are
MPL and alum. Of these two, MPL appears preferable because a
10-fold lower antigen dose was required to generate the same
antibody response as obtained with alum. The response can be
increased by increasing the dose of antigen and/or adjuvant and by
optimizing the immunization schedule. The o/w emulsion was a very
weak adjuvant for AN1792 and adding an o/w emulsion to MPL adjuvant
diminished the intrinsic adjuvant activity of MPL alone.
[0311] 2. A.beta. Levels in the Brain
[0312] At about 14 weeks the guinea pigs were deeply anesthetized,
the cerebrospinal fluid (CSF) was drawn and brains were excised
from animals in a subset of the groups, those immunized with
Freund's adjuvant (group 2), MPL (group 5), alum with a high dose,
300 .mu.g, of AN1792 (group 10) and the PBS immunized control group
(group 3). To measure the level of A.beta. peptide, one hemisphere
was dissected and homogenates of the hippocampal, cortical, and
cerebellar regions were prepared in 5 M guanidine. These were
diluted and quantitated by comparison to a series of dilutions of
A.beta. standard protein of known concentrations in an ELISA
format. The levels of A.beta. protein in the hippocampus, the
cortex and the cerebellum were very similar for all four groups
despite the wide range of antibody responses to A.beta. elicited by
these formulations. Mean A.beta. levels of about 25 ng/g tissue
were measured in the hippocampus, 21 ng/g in the cortex, and 12
ng/g in the cerebellum. Thus, the presence of a high circulating
antibody titer to A.beta. for almost three months in some of these
animals did not alter the total A.beta. levels in their brains. The
levels of A.beta. in the CSF were also quite similar between the
groups. The lack of large effect of AN1792 immunization on
endogenous A.beta. indicates that the immune response is focused on
pathological formations of A.beta..
VIII. Immune Response to Different Adjuvants in Mice
[0313] Six-week old female Swiss Webster mice were used for this
study with 10-13 animals per group. Immunizations were given on
days 0, 14, 28, 60, 90 and 20 administered subcutaneously in a dose
volume of 200 .mu.l. PBS was used as the buffer for all
formulations. Animals were bleed seven days following each
immunization starting after the second dose for analysis of
antibody titers by ELISA. The treatment regime of each group is
summarized in Table 7. TABLE-US-00007 TABLE 7 Experimental Design
Dose Group N.sup.a Adjuvant.sup.b Dose Antigen (.mu.g) 1 10 MPL
12.5 .mu.g AN1792 33 2 10 MPL 25 .mu.g AN1792 33 3 10 MPL 50 .mu.g
AN1792 33 4 13 MPL 125 .mu.g AN1792 33 5 13 MPL 50 .mu.g AN1792 150
6 13 MPL 50 .mu.g AN1528 33 7 10 PBS AN1792 33 8 10 PBS None 9 10
Squalene 5% AN1792 33 emulsified 10 10 Squalene 5% AN1792 33
admixed 11 10 Alum 2 mg AN1792 33 12 13 MPL + Alum 50 .mu.g/2 mg
AN1792 33 13 10 QS-21 5 .mu.g AN1792 33 14 10 QS-21 10 .mu.g AN1792
33 15 10 QS-21 25 AN1792 AN1792 33 16 13 QS-21 25 AN1792 AN1792 150
17 13 QS-21 25 AN1792 AN1528 33 18 13 QS-21 + MPL 25 .mu.g/50 .mu.g
AN1792 33 19 13 QS-21 + Alum 25 .mu.g/2 mg AN1792 33 Footnotes:
.sup.aNumber of mice in each group at the initiation of the
experiment. .sup.bThe adjuvants are noted. The buffer for all these
formulations was PBS. For group 8, there was no adjuvant and no
antigen.
[0314] The ELISA titers of antibodies against A.beta.42 in each
group are shown in Table 8 below. TABLE-US-00008 TABLE 8 Geometric
Mean Antibody Titers Week of Bleed Treatment Group 2.9 5.0 8.7 12.9
16.7 1 248 1797 2577 6180 4177 2 598 3114 3984 5287 6878 3 1372
5000 7159 12333 12781 4 1278 20791 14368 20097 25631 5 3288 26242
13229 9315 23742 6 61 2536 2301 1442 4504 7 37 395 484 972 2149 8
25 25 25 25 25 9 25 183 744 952 1823 10 25 89 311 513 817 11 29 708
2618 2165 3666 12 198 1458 1079 612 797 13 38 433 566 1080 626 14
104 541 3247 1609 838 15 212 2630 2472 1224 1496 16 183 2616 6680
2085 1631 17 28 201 375 222 1540 18 31699 15544 23095 6412 9059 19
63 243 554 299 441 The table shows that the highest titers were
obtained for groups 4, 5 and 18, in which the adjuvants were 125
.mu.g MPL, 50 .mu.g MPL and QS-21 plus MPL.
IX. Therapeutic Efficacy of Different Adjuvants
[0315] A therapeutic efficacy study was conducted in PDAPP
transgenic mice with a set of adjuvants suitable for use in humans
to determine their ability to potentiate immune responses to
A.beta. and to induce the immune-mediated clearance of amyloid
deposits in the brain.
[0316] One hundred eighty male and female, 7.5- to 8.5-month old
heterozygous PDAPP transgenic mice were obtained from Charles River
Laboratories. The mice were sorted into nine groups containing 15
to 23 animals per group to be immunized with AN1792 or AN1528
combined with various adjuvants. Animals were distributed to match
the gender, age, and parentage of the animals within the groups as
closely as possible. The adjuvants included alum, MPL, and QS-21,
each combined with both antigens, and Freund's adjuvant (FA)
combined with only AN1792. An additional group was immunized with
AN1792 formulated in PBS buffer plus the preservative thimerosal
without adjuvant. A ninth group was immunized with PBS alone as a
negative control.
[0317] Preparation of aggregated A.beta. peptides: human
A.beta.1-40 (AN1528; California Peptides Inc., Napa, Calif.; Lot
ME0541) and human A.beta.1-42 (AN1792; California Peptides Inc.,
Lot ME0439) peptides were freshly solubilized for the preparation
of each set of injections from lyophilized powders that had been
stored desiccated at -20.degree. C. For this purpose, two mg of
peptide were added to 0.9 ml of deionized water and the mixture was
vortexed to generate a relatively uniform solution or suspension.
AN1528 was soluble at this step, in contrast to AN1792. A 1001
aliquot of 10.times.PBS (1.times.PBS: 0.15 M NaCl, 0.01 M sodium
phosphate, pH 7.5) was then added at which point AN1528 began to
precipitate. The suspensions were vortexed again and incubated
overnight at 37.degree. C. for use the next day.
[0318] To prepare formulation doses with alum (Groups 1 and 5).
A.beta. peptide in PBS was added to Alhydrogel (two percent aqueous
aluminum hydroxide gel, Sargeant, Inc., Clifton, N.J.) to reach
concentrations of 100 .mu.g A.beta. peptide per 2 mg of alum.
10.times.PBS was added to a final dose volume of 200 ml in
1.times.PBS. The suspension was then gently mixed for approximately
4 hr at RT prior to injection.
[0319] To prepare formulation doses for with MPL (Groups 2 and 6),
lyophilized powder (Ribi ImmunoChem Research, Inc., Hamilton,
Mont.; Lot 67039-E0896B) was added to 0.2% aqueous triethylamine to
a final concentration of 1 mg/ml and vortexed. The mixture was
heated to 65 to 70.degree. C. for 30 sec to create a slightly
opaque uniform suspension of micelles. The solution was stored at
4.degree. C. For each set of injections, 100 .mu.g of peptide per
dose in 50 .mu.l PBS, 50 .mu.g of MPL per dose (50 .mu.l) and 100
.mu.l of PBS per dose were mixed in a borosilicate tube immediately
before use.
[0320] To prepare formulation doses with QS-21 (Groups 3 and 7),
lyophilized powder (Aquila, Framingham, Mass.; Lot A7018R) was
added to PBS, pH 6.6-6.7 to a final concentration of 1 mg/ml and
vortexed. The solution was stored at -20.degree. C. For each set of
injections, 100 .mu.g of peptide per dose in 501 PBS, 25 .mu.g of
QS-21 per dose in 25 .mu.l PBS and 125 .mu.l of PBS per dose were
mixed in a borosilicate tube immediately before use.
[0321] To prepare formulation doses with Freund's Adjuvant (Group
4), 100 g of AN1792 in 200 l PBS was emulsified 1:1 (vol:vol) with
Complete Freund's Adjuvant (CFA) in a final volume of 400 l for the
first immunization. For subsequent immunizations, the antigen was
similarly emulsified with Incomplete Freund's Adjuvant (IFA). For
the formulations containing the adjuvants alum, MPL or QS21, 100 g
per dose of AN1792 or AN1528 was combined with alum (2 mg per dose)
or MPL (50 g per dose) or QS21 (25 g per dose) in a final volume of
200 l PBS and delivered by subcutaneous inoculation on the back
between the shoulder blades. For the group receiving FA, 100 g of
AN1792 was emulsified 1:1 (vol:vol) with Complete Freund's adjuvant
(CFA) in a final volume of 400 l and delivered intraperitoneally
for the first immunization, followed by a boost of the same amount
of immunogen in Incomplete Freund's adjuvant (IFA) for the
subsequent five doses. For the group receiving AN1792 without
adjuvant, 10 g AN1792 was combined with 5 g thimerosal in a final
volume of 50 l PBS and delivered subcutaneously. The ninth, control
group received only 200 l PBS delivered subcutaneously.
Immunizations were given on a biweekly schedule for the first three
doses, then on a monthly schedule thereafter on days 0, 16, 28, 56,
85 and 112. Animals were bled six to seven days following each
immunization starting after the second dose for the measurement of
antibody titers. Animals were euthanized approximately one week
after the final dose. Outcomes were measured by ELISA assay of
.beta.A and APP levels in brain and by immunohistochemical
evaluation of the presence of amyloid plaques in brain sections. In
addition, .beta.-specific antibody titers, and .beta.-dependent
proliferative and cytokine responses were determined.
[0322] Table 9 shows that the highest antibody titers to
A.beta.1-42 were elicited with FA and AN1792, titers which peaked
following the fourth immunization (peak GMT: 75,386) and then
declined by 59% after the final, sixth immunization. The peak mean
titer elicited by MPL with AN1792 was 62% lower than that generated
with FA (peak GMT: 28,867) and was also reached early in the
immunization scheme, after 3 doses, followed by a decline to 28% of
the peak value after the sixth immunization. The peak mean titer
generated with QS-21 combined with AN1792 (GMT: 1,511) was about
5-fold lower than obtained with MPL. In addition, the kinetics of
the response were slower, since an additional immunization was
required to reach the peak response. Titers generated by alum-bound
AN1792 were marginally greater than those obtained with QS-21 and
the response kinetics were more rapid. For AN1792 delivered in PBS
with thimerosal the frequency and size of titers were barely
greater than that for PBS alone. The peak titers generated with MPL
and AN1528 (peak GMT 3099) were about 9-fold lower than those with
AN1792. Alum-bound AN1528 was very poorly immunogenic with low
titers generated in only some of the animals. No antibody responses
were observed in the control animals immunized with PBS alone.
TABLE-US-00009 TABLE 9 Geometric Mean Antibody Titers.sup.a Week of
Bleed Treatment 3.3 5.0 9.0 13.0 17.0 Alum/ 102 (12/21).sup.b 1,081
(17/20) 2,366 (21/21) 1,083 (19/21) 572 (18/21) AN1792 MPL/ 6241
(21/21) 28,867 (21/21) 1,1242 (21/21) 5,665 (20/20) 8,204 (20/20)
AN1792 QS-21/ 30 (1/20) 227 (10/19) 327 (10/19) 1,511 (17/18) 1,188
(14/18) AN1792 CFA/ 10,076 (15/15) 61,279 (15/15) 75,386 (15/15)
41,628 (15/15) 30,574 (15/15) AN1792 Alum/ 25 (0/21) 33 (1/21) 39
(3/20) 37 (1/20) 31 (2/20) AN1528 MPL/ 184 (15/21) 2,591 (20/21)
1,653 (21/21) 1,156 (20/20) 3,099 (20/20) AN1528 QS-21/ 29 (1/22)
221 (13/22) 51 (4/22) 820 (20/22) 2,994 (21/22) AN1528 PBS plus 25
(0/16) 33 (2/16) 39 (4/16) 37 (3/16) 47 (4/16) Thimerosal PBS 25
(0/16) 25 (0/16) 25 (0/15) 25 (0/12) 25 (0/16) Footnotes:
.sup.aGeometric mean antibody titers measured against A.beta.1-42
.sup.bNumber of responders per group
[0323] The results of AN1792 or AN1592 treatment with various
adjuvants, or thimerosal on cortical amyloid burden in 12-month old
mice determined by ELISA are shown in FIGS. 15A-15E. In PBS control
PDAPP mice (FIG. 15A), the median level of total A in the cortex at
12 months was 1,817 ng/g. Notably reduced levels of A were observed
in mice treated with AN1792 plus CFA/IFA (FIG. 15C), AN1792 plus
alum (FIG. 15D), AN1792 plus MPL (FIG. 15E) and QS21 plus AN1792
(FIG. 15E). The reduction reached statistical significance
(p<0.05) only for AN1792 plus CFA/IFA (FIG. 15C). However, as
shown in Examples I and III, the effects of immunization in
reducing A levels become substantially greater in 15 month and 18
month old mice. Thus, it is expected that at least the AN1792 plus
alum, AN1792 plus MPL and AN1792 plus QS21 compositions will
achieve statistical significance in treatment of older mice. By
contrast, the AN1792 plus the preservative thimerosal (FIG. 15D)
showed a median level of A about the same as that in the PBS
treated mice. Similar results were obtained when cortical levels of
A.beta.42 were compared. The median level of A42 in PBS controls
was 1624 ng/g. Notably reduced median levels of 403, 1149, 620 and
714 were observed in the mice treated with AN1792 plus CFA/IFA,
AN1792 plus alum, AN1792 plus MPL and AN1792 plus QS21
respectively, with the reduction achieving statistical significance
(p=0.05) for the AN1792 CFA/IFA treatment group. The median level
in the AN1792 thimerosal treated mice was 1619 ng/g A42.
[0324] A further therapeutic adjuvant/immunogen efficacy study was
performed in 9-10.5 month old male and female heterozygous PDAPP
transgenic mice. The duration of the study was 25 weeks with 29-40
animals per treatment group; therefore the animals were 15-16.5
months old at termination. The treatment groups are identified in
Table 10 below. TABLE-US-00010 Dilution Adminis- Adjuvant Immunogen
Buffer tration Group 1: MPL-SE AN1792-GCS (75 .mu.g) PBS SC (250
.mu.l) Group 2: ISA 51 AN1792-GCS (75 .mu.g) PBS IP (400 .mu.l)
Group 3: QS21 AN1792-GCS (75 .mu.g) PBS SC (250 .mu.l) Group 4:
QS21 AN1792-GCS (75 .mu.g) PBS SC (250 .mu.l) abbrev. Group 5: PBS
-- -- SC (250 .mu.l) Table 10 abbreviations: MAP--multi-antigenic
peptide; TT--tetanus toxoid t-cell epitope (830-844);
SQ--subcutaneous; IP--intraperitoneally; PBS--phosphate, buffered
saline; ISA-51 is a commercially available adjuvant similar to IFA;
GCS is a glycine/citrate/sucrose formulation, MPL-SE is MPL in a
stabilized water and oil emulsion.
[0325] The immunization schedule was identical for all of the
treatment groups except for Group 3, the QS21/AN1792 abbreviated
schedule group. The mice were injected on weeks 0, 2, 4, 8, 12, 16,
20, 24, with bleeds on weeks 3, 5, 9, 13, 17, 21 and 25. Groups 1,
2, received eight injections and Group 3 received four injections
during the 25-week period of the study. Group 4, the QS21/AN1792
abbreviated schedule, received injections on weeks 0, 2, 4, and 8
only. This group was not injected for the remainder of the study,
although they were bled on the same bleed schedule as the rest of
the study to follow titer decay. Groups 3 and 5, QS21/AN1792 and
PBS respectively, served as the positive and negative controls for
this study.
[0326] The titers were determined by the anti-A13 antibody titer
assay.
[0327] Group 1, the MPL-SE/AN1792 group, raised a peak geometric
mean titer (GMT) of 17,100 at 9 weeks falling to a GMT of 10,000 at
25 weeks. Initially, the MPL-SE titers rose at a somewhat higher
rate than the QS21/AN1792 control group (Group 4).
[0328] Group 2, the ISA 51/AN1792 group, produced high titers
throughout the study reaching a GMT of over 100,000 for the last 9
weeks of the study.
[0329] Group 3, the QS21/AN1792 control group, reached its peak
titer at 17 weeks with a GMT of 16,000. The titer then fell over
the next 8 weeks to finish with a GMT of 8,700. One animal in this
group failed to raise a titer over the entire course of the
experiment.
[0330] Group 4, the QS21/AN1792 abbreviated injection schedule
group, reached a peak titer of 7,300 at 13 weeks, five weeks after
its final injection. The titer then fell to a GMT of 2,100 at the
final bleed (25 weeks). As in the control group, one animal failed
to raise a detectable titer, while another animal lost all titer by
the end of the decay period.
[0331] Group 5, the PBS alone group, had no titers.
[0332] To evaluate the cortical A.beta. levels, total A.beta. and
A.beta..sub.1-42 were measured by ELISA. Briefly, one brain
hemisphere was dissected for cortical, hippocampal, and cerebellar
tissue followed by homogenization in 5M guanidine buffer and
assayed for brain A.beta.. The cortical total A.beta. and
A.beta.342 results are similar. A Mann-Whitney statistical analysis
was performed to determine significance between the groups with a p
value of <0.05 indicating a significant change in A.beta..
[0333] All treatment groups significantly lowered total A.beta.
levels as compared to the PBS control group (see Table 11). The
MPL-SE/AN1792 group, showed the greatest change in A.beta., and it
is significantly better than the other treatment groups. The
QS21/AN1792 abbreviated group, was similar in its overall change of
A.beta. to the QS21 control group that received all eight
injections. The A.beta. levels in the ISA 51/AN1792 group, were
similarly lowered compared to the CFA/IFA:MAP(A.beta..sub.1-7)
group. TABLE-US-00011 TABLE 11 Cortical A.beta. levels PBS MPL-SE
ISA QS-21 QS-21 (4) MEDIAN 7,335 1,236 3,026 2,389 2,996 (ng/g
tissue) RANGE 550-18,358 70-3,977 23-9,777 210-11,167 24-16,834
(ng/g tissue) p value -- <0.0001 <0.0001 <0.0001
<0.0001 N 38 29 36 34 40
[0334] In conclusion, MPL-SE, ISA-51 and QS21 adjuvants combined
with AN1792 are effective in inducing a sufficient immune response
significantly to retard A.beta. deposition in the cortex.
X. Toxicity Analysis
[0335] Tissues were collected for histopathologic examination at
the termination of studies described in Examples 2, 3 and 7. In
addition, hematology and clinical chemistry were performed on
terminal blood samples from Examples 3 and 7. Most of the major
organs were evaluated, including brain, pulmonary, lymphoid,
gastrointestinal, liver, kidney, adrenal and gonads. Although
sporadic lesions were observed in the study animals, there were no
obvious differences, either in tissues affected or lesion severity,
between AN1792 treated and untreated animals. There were no unique
histopathological lesions noted in AN-1528-immunized animals
compared to PBS-treated or untreated animals. There were also no
differences in the clinical chemistry profile between adjuvant
groups and the PBS treated animals in Example 7. Although there
were significant increases in several of the hematology parameters
between animals treated with AN1792 and Freund's adjuvant in
Example 7 relative to PBS treated animals, these type of effects
are expected from Freund's adjuvant treatment and the accompanying
peritonitis and do not indicate any adverse effects from AN1792
treatment. Although not part of the toxicological evaluation, PDAPP
mouse brain pathology was extensively examined as part of the
efficacy endpoints. No sign of treatment related adverse effect on
brain morphology was noted in any of the studies. These results
indicate that AN1792 treatment is well tolerated and at least
substantially free of side effects.
XI. Therapeutic Treatment with Anti-A.beta. antibodies
[0336] This examples tests the capacity of various monoclonal and
polyclonal antibodies to A.beta. to inhibit accumulation of A.beta.
in the brain of heterozygotic transgenic mice.
[0337] 1. Study Design
[0338] Sixty male and female, heterozygous PDAPP transgenic mice,
8.5 to 10.5 months of age were obtained from Charles River
Laboratory. The mice were sorted into six groups to be treated with
various antibodies directed to A.beta.. Animals were distributed to
match the gender, age, parentage and source of the animals within
the groups as closely as possible. As shown in Table 10, the
antibodies included four murine A.beta.-specific monoclonal
antibodies, 2H3 (directed to A.beta. residues 1-12), 10D5 (directed
to A.beta. residues 1-16) (details of the deposit of 10D5 are
discussed in Example VI, supra), 266 (directed to A.beta. residues
13-28 and binds to monomeric but not to aggregated AN1792), 21F12
(directed to A.beta. residues 33-42). A fifth group was treated
with an AD-specific polyclonal antibody fraction (raised by
immunization with aggregated AN1792). The negative control group
received the diluent, PBS, alone without antibody.
[0339] The monoclonal antibodies were injected at a dose of about
10 mg/kg (assuming that the mice weighed 50 g). Injections were
administered intraperitoneally every seven days on average to
maintain anti-A.beta. titers above 1000. Although lower titers were
measured for mAb 266 since it does not bind well to the aggregated
AN1792 used as the capture antigen in the assay, the same dosing
schedule was maintained for this group. The group receiving
monoclonal antibody 2H3 was discontinued within the first three
weeks since the antibody was cleared too rapidly in vivo. Animals
were bled prior to each dosing for the measurement of antibody
titers. Treatment was continued over a six-month period for a total
of 196 days. Animals were euthanized one week after the final dose.
TABLE-US-00012 TABLE 12 EXPERIMENTAL DESIGN Treatment Treatment
Antibody Antibody Group N.sup.a Antibody Specificity Isotype 1 9
none NA.sup.b NA (PBS alone) 2 10 Polyclonal A.beta.1-42 mixed 3 0
mAb.sup.c 2H3 A.beta.1-12 IgG1 4 8 mAb 10D5 A.beta.1-16 IgG1 5 6
mAb 266 A.beta.13-28 IgG1 6 8 mAb 21F12 A.beta.33-42 IgG2a
Footnotes .sup.aNumber of mice in group at termination of the
experiment. All groups started with 10 animals per group. .sup.bNA:
not applicable .sup.cmAb: monoclonal antibody
[0340] 2. Materials and Methods
[0341] a. Preparation of the Antibodies
[0342] The anti-A.beta. polyclonal antibody was prepared from blood
collected from two groups of animals. The first group consisted of
100 female Swiss Webster mice, 6 to 8 weeks of age. They were
immunized on days 0, 15, and 29 with 100 .mu.g of AN1792 combined
with CFA/IFA. A fourth injection was given on day 36 with one-half
the dose of AN1792. Animals were exsanguinated upon sacrifice at
day 42, serum was prepared and the sera were pooled to create a
total of 64 ml. The second group consisted of 24 female mice
isogenic with the PDAPP mice but nontransgenic for the human APP
gene, 6 to 9 weeks of age. They were immunized on days 0, 14, 28
and 56 with 100 .mu.g of AN1792 combined with CFA/IFA. These
animals were also exsanguinated upon sacrifice at day 63, serum was
prepared and pooled for a total of 14 ml. The two lots of sera were
pooled. The antibody fraction was purified using two sequential
rounds of precipitation with 50% saturated ammonium sulfate. The
final precipitate was dialyzed against PBS and tested for
endotoxin. The level of endotoxin was less than 1 EU/mg.
[0343] The anti-A.beta. monoclonal antibodies were prepared from
ascites fluid. The fluid was first delipidated by the addition of
concentrated sodium dextran sulfate to ice-cold ascites fluid by
stirring on ice to a reach a final concentration of 0.238%.
Concentrated CaCl.sub.2 was then added with stirring to reach a
final concentration of 64 mM. This solution was centrifuged at
10,000.times.g and the pellet was discarded. The supernatant was
stirred on ice with an equal volume of saturated ammonium sulfate
added dropwise. The solution was centrifuged again at
10,000.times.g and the supernatant was discarded. The pellet was
resuspended and dialyzed against 20 mM Tris-HCl, 0.4 M NaCl, pH
7.5. This fraction was applied to a Pharmacia FPLC Sepharose Q
Column and eluted with a reverse gradient from 0.4 M to 0.275 M
NaCl in 20 mM Tris-HCl, pH 7.5.
[0344] The antibody peak was identified by absorbance at 280 nm and
appropriate fractions were pooled. The purified antibody
preparation was characterized by measuring the protein
concentration using the BCA method and the purity using SDS-PAGE.
The pool was also tested for endotoxin. The level of endotoxin was
less than 1 EU/mg. titers, titers less than 100 were arbitrarily
assigned a titer value of 25.
[0345] 3. AD and APP Levels in the Brain:
[0346] Following about six months of treatment with the various
anti-A.beta. antibody preparations, brains were removed from the
animals following saline perfusion. One hemisphere was prepared for
immunohistochemical analysis and the second was used for the
quantitation of A.beta. and APP levels. To measure the
concentrations of various forms of beta amyloid peptide and amyloid
precursor protein (APP), the hemisphere was dissected and
homogenates of the hippocampal, cortical, and cerebellar regions
were prepared in 5M guanidine. These were serially diluted and the
level of amyloid peptide or APP was quantitated by comparison to a
series of dilutions of standards of A.beta. peptide or APP of known
concentrations in an ELISA format.
[0347] The levels of total A.beta. and of A.beta.1-42 measured by
ELISA in homogenates of the cortex, and the hippocampus and the
level of total A.beta. in the cerebellum are shown in Tables 11,
12, and 13, respectively. The median concentration of total A.beta.
for the control group, inoculated with PBS, was 3.6-fold higher in
the hippocampus than in the cortex (median of 63,389 ng/g
hippocampal tissue compared to 17,818 ng/g for the cortex). The
median level in the cerebellum of the control group (30.6 ng/g
tissue) was more than 2,000-fold lower than in the hippocampus.
These levels are similar to those that we have previously reported
for heterozygous PDAPP transgenic mice of this age (Johnson-Wood et
al., 1997).
[0348] For the cortex, one treatment group had a median A.beta.
level, measured as A.beta.1-42, which differed significantly from
that of the control group (p<0.05), those animals receiving the
polyclonal anti-A.beta. antibody as shown in Table 13. The median
level of A.beta.1-42 was reduced by 65%, compared to the control
for this treatment group. The median levels of A.beta.1-42 were
also significantly reduced by 55% compared to the control in one
additional treatment group, those animals dosed with the mAb 10D5
(p=0.0433). TABLE-US-00013 TABLE 13 CORTEX Medians Total A.beta.
Means Treatment ELISA A.beta.42 Total A.beta. A.beta.42 Group
N.sup.a value.sup.b P value.sup.c % Change ELISA value P value %
Change ELISA value ELISA value PBS 9 17818 NA.sup.d NA 13802 NA NA
16150 +/- 7456.sup.e.sup. 12621 +/- 5738 Polyclonal 10 6160 0.0055
-65 4892 0.0071 -65 5912 +/- 4492 4454 +/- 3347 anti-A.beta.42 mAb
10D5 8 7915 0.1019 -56 6214 0.0433 -55 9695 +/- 6929 6943 +/- 3351
mAb 266 6 9144 0.1255 -49 8481 0.1255 -39 9204 +/- 9293 7489 +/-
6921 mAb 21F12 8 15158 0.2898 -15 13578 0.7003 -2 12481 +/- 7082
11005 +/- 6324 Footnotes: .sup.aNumber of animals per group at the
end of the experiment .sup.bng/g tissue .sup.cMann Whitney analysis
.sup.dNA: not applicable .sup.eStandard Deviation
[0349] In the hippocampus, the median percent reduction of total
A.beta. associated with treatment with polyclonal anti-A.beta.
antibody (50%, p=0.0055) was not as great as that observed in the
cortex (65%) (Table 14). However, the absolute magnitude of the
reduction was almost 3-fold greater in the hippocampus than in the
cortex, a net reduction of 31,683 ng/g tissue in the hippocampus
versus 11,658 ng/g tissue in the cortex. When measured as the level
of the more amyloidogenic form of A.beta., A.beta.1-42, rather than
as total A.beta., the reduction achieved with the polyclonal
antibody was significant (p=0.0025). The median levels in groups
treated with the mAbs 10D5 and 266 were reduced by 33% and 21%,
respectively. TABLE-US-00014 TABLE 14 HIPPOCAMPUS Medians Total
A.beta. A.beta.42 Means Treatment ELISA ELISA Total A.beta.
A.beta.42 Group N.sup.a value.sup.b P value.sup.c % Change value P
value % Change ELISA value ELISA value PBS 9 63389 NA.sup.d NA
54429 NA NA .sup. 58351 +/- 13308.sup.e 52801 +/- 14701 Polyclonal
10 31706 0.0055 -50 27127 0.0025 -50 30058 +/- 22454 24853 +/-
18262 anti-A.beta.42 mAb 10D5 8 46779 0.0675 -26 36290 0.0543 -33
44581 +/- 18632 36465 +/- 17146 mAb 266 6 48689 0.0990 -23 43034
0.0990 -21 36419 +/- 27304 32919 +/- 25372 mAb 21F12 8 51563 0.7728
-19 47961 0.8099 -12 57327 +/- 28927 50305 +/- 23927 Footnotes:
.sup.aNumber of animals per group at the end of the experiment
.sup.bng/g tissue .sup.cMann Whitney analysis .sup.dNA: not
applicable .sup.eStandard Deviation
[0350] Total A.beta. was also measured in the cerebellum (Table
15). Those groups dosed with the polyclonal anti-A.beta. and the
266 antibody showed significant reductions of the levels of total
A.beta. (43% and 46%, p=0.0033 and p=0.0184, respectively) and that
group treated with 10D5 had a near significant reduction (29%,
p=0.0675). TABLE-US-00015 TABLE 15 CEREBELLUM Medians Total A.beta.
Means Treatment ELISA P % Total A.beta. Group N.sup.a value.sup.b
value.sup.c Change ELISA value PBS 9 30.64 NA.sup.d NA 40.00 +/-
31.89.sup.e Polyclonal 10 17.61 0.0033 -43 18.15 +/- 4.36
anti-A.beta.42 mAb 10D5 8 21.68 0.0675 -29 27.29 +/- 19.43 mAb 266
6 16.59 0.0184 -46 19.59 +/- 6.59 mAb 21F12 8 29.80 >0.9999 -3
32.88 +/- 9.90 Footnotes: .sup.aNumber of animals per group at the
end of the experiment .sup.bng/g tissue .sup.cMann Whitney analysis
.sup.dNA: not applicable .sup.eStandard Deviation
[0351] APP concentration was also determined by ELISA in the cortex
and cerebellum from antibody-treated and control, PBS-treated mice.
Two different APP assays were utilized. The first, designated
APP-.alpha./FL, recognizes both APP-alpha (.alpha., the secreted
form of APP which has been cleaved within the A.beta. sequence),
and full-length forms (FL) of APP, while the second recognizes only
APP-.alpha.. In contrast to the treatment-associated diminution of
A.beta. in a subset of treatment groups, the levels of APP were
virtually unchanged in all of the treated compared to the control
animals. These results indicate that the immunizations with A.beta.
antibodies deplete A.beta. without depleting APP.
[0352] In summary, A.beta. levels were significantly reduced in the
cortex, hippocampus and cerebellum in animals treated with the
polyclonal antibody raised against AN1792. To a lesser extent
monoclonal antibodies to the amino terminal region of A.beta.1-42,
specifically amino acids 1-16 and 13-28 also showed significant
treatment effects.
[0353] 4. Histochemical Analyses:
[0354] The morphology of A.beta.-immunoreactive plaques in subsets
of brains from mice in the PBS, polyclonal A1342, 21F12, 266 and
10D5 treatment groups was qualitatively compared to that of
previous studies in which standard immunization procedures with
A.beta.42 were followed.
[0355] The largest alteration in both the extent and appearance of
amyloid plaques occurred in the animals immunized with the
polyclonal A.beta.342 antibody. The reduction of amyloid load,
eroded plaque morphology and cell-associated A.beta.
immunoreactivity closely resembled effects produced by the standard
immunization procedure. These observations support the ELISA
results in which significant reductions in both total A.beta. and
A.beta.42 were achieved by administration of the polyclonal
A.beta.42 antibody.
[0356] In similar qualitative evaluations, amyloid plaques in the
10D5 group were also reduced in number and appearance, with some
evidence of cell-associated A.beta. immunoreactivity. Relative to
control-treated animals, the polyclonal Ig fraction against A.beta.
and one of the monoclonal antibodies (10D5) reduced plaque burden
by 93% and 81%, respectively (p<0.005). 21F12 appeared to have a
relatively modest effect on plaque burden. Micrographs of brain
after treatment with pabA.beta..sub.1-42 show diffuse deposits and
absence of many of the larger compacted plaques in the
pabA.beta..sub.1-42 treated group relative to control treated
animals.
[0357] 5. Measurement of Antibody Titers:
[0358] A subset of three randomly chosen mice from each group were
bled just prior to each intraperitoneal inoculation, for a total of
30 bleeds. Antibody titers were measured as A.beta.1-42-binding
antibody using a sandwich ELISA with plastic multi-well plates
coated with A.beta.1-42 as described in detail in the General
Materials and Methods. Mean titers for each bleed are shown in
FIGS. 16-18 for the polyclonal antibody and the monoclonals 10D5
and 21F12, respectively. Titers averaged about 1000 over this time
period for the polyclonal antibody preparation and were slightly
above this level for the 10D5- and 21F12-treated animals.
[0359] 6. Lymphoproliferative Responses:
[0360] A.beta.-dependent lymphoproliferation was measured using
spleen cells harvested eight days following the final antibody
infusion. Freshly harvested cells, 10.sup.5 per well, were cultured
for 5 days in the presence of A.beta.1-40 at a concentration of 5
.mu.M for stimulation. As a positive control, additional cells were
cultured with the T cell mitogen, PHA, and, as a negative control,
cells were cultured without added peptide.
[0361] Splenocytes from aged PDAPP mice passively immunized with
various anti-A.beta. antibodies were stimulated in vitro with
AN1792 and proliferative and cytokine responses were measured. The
purpose of these assays was to determine if passive immunization
facilitated antigen presentation, and thus priming of T cell
responses specific for AN1792. No AN1792-specific proliferative or
cytokine responses were observed in mice passively immunized with
the anti-A.beta. antibodies.
XII: Further Study of Passive Immunization
[0362] In a second study, treatment with 10D5 was repeated and two
additional anti-A.beta. antibodies were tested, monoclonals 3D6
(A.beta..sub.1-5) and 16C11 (A.beta..sub.33-42). Control groups
received either PBS or an irrelevant isotype-matched antibody
(TM2a). The mice were older (11.5-12 month old heterozygotes) than
in the previous study, otherwise the experimental design was the
same. Once again, after six months of treatment, 10D5 reduced
plaque burden by greater than 80% relative to either the PBS or
isotype-matched antibody controls (p=0.003). One of the other
antibodies against A.beta., 3D6, was equally effective, producing
an 86% reduction (p=0.003). In contrast, the third antibody against
the peptide, 16C11, failed to have any effect on plaque burden.
Similar findings were obtained with A.beta..sub.42 ELISA
measurements. These results demonstrate that an antibody response
against A.beta. peptide, in the absence of T cell immunity, is
sufficient to decrease amyloid deposition in PDAPP mice, but that
not all anti-A.beta. antibodies are efficacious. Antibodies
directed to epitopes comprising amino acids 1-5 or 3-7 of A.beta.
are particularly efficacious.
[0363] In summary, we have shown that passively administered
antibodies against A.beta. reduced the extent of plaque deposition
in a mouse model of Alzheimer's disease. When held at modest serum
concentrations (25-70 .mu.g/ml), the antibodies gained access to
the CNS at levels sufficient to decorate .beta.-amyloid plaques.
Antibody entry into the CNS was not due to abnormal leakage of the
blood-brain barrier since there was no increase in vascular
permeability as measured by Evans Blue in PDAPP mice. In addition,
the concentration of antibody in the brain parenchyma of aged PDAPP
mice was the same as in non-transgenic mice, representing 0.1% of
the antibody concentration in serum (regardless of isotype).
XIII: Monitoring of Antibody Binding
[0364] To determine whether antibodies against A.beta. could be
acting directly within the CNS, brains taken from saline-perfused
mice at the end of the Example XII, were examined for the presence
of the peripherally-administered antibodies. Unfixed cryostat brain
sections were exposed to a fluorescent reagent against mouse
immunoglobulin (goat anti-mouse IgG-Cy3). Plaques within brains of
the 10D5 and 3D6 groups were strongly decorated with antibody,
while there was no staining in the 16C11 group. To reveal the full
extent of plaque deposition, serial sections of each brain were
first immunoreacted with an anti-A.beta. antibody, and then with
the secondary reagent. 10D5 and 3D6, following peripheral
administration, gained access to most plaques within the CNS. The
plaque burden was greatly reduced in these treatment groups
compared to the 16C11 group. These data indicate that peripherally
administered antibodies can enter the CNS where they can directly
trigger amyloid clearance. It is likely that 16C11 also had access
to the plaques but was unable to bind.
XIV: Ex Vivo Screening Assay for Activity of an Antibody Against
Amyloid Deposits
[0365] To examine the effect of antibodies on plaque clearance, we
established an ex vivo assay in which primary microglial cells were
cultured with unfixed cryostat sections of either PDAPP mouse or
human AD brains. Microglial cells were obtained from the cerebral
cortices of neonate DBA/2N mice (1-3 days). The cortices were
mechanically dissociated in HBSS.sup.- (Hanks' Balanced Salt
Solution, Sigma) with 50 .mu.g/ml DNase I (Sigma). The dissociated
cells were filtered with a 100 .mu.m cell strainer (Falcon), and
centrifuged at 1000 rpm for 5 minutes. The pellet was resuspended
in growth medium (high glucose DMEM, 10% FBS, 25 ng/ml rmGM-CSF),
and the cells were plated at a density of 2 brains per T-75 plastic
culture flask. After 7-9 days, the flasks were rotated on an
orbital shaker at 200 rpm for 2 h at 37.degree. C. The cell
suspension was centrifuged at 1000 rpm and resuspended in the assay
medium.
[0366] 10-.mu.m cryostat sections of PDAPP mouse or human AD brains
(post-mortem interval<3 hr) were thaw mounted onto poly-lysine
coated round glass coverslips and placed in wells of 24-well tissue
culture plates. The coverslips were washed twice with assay medium
consisting of H-SFM (Hybridoma-serum free medium, Gibco BRL) with
1% FBS, glutamine, penicillin/streptomycin, and 5 ng/ml rmGM-CSF
(R&D). Control or anti-A.beta. antibodies were added at a
2.times. concentration (5 .mu.g/ml final) for 1 hour. The
microglial cells were then seeded at a density of
0.8.times.10.sup.6 cells/ml assay medium. The cultures were
maintained in a humidified incubator (37.degree. C., 5% CO.sub.2)
for 24 hr or more. At the end of the incubation, the cultures were
fixed with 4% paraformaldehyde and permeabilized with 0.1%
Triton-X100. The sections were stained with biotinylated 3D6
followed by a streptavidin/Cy3 conjugate (Jackson ImmunoResearch).
The exogenous microglial cells were visualized by a nuclear stain
(DAPI). The cultures were observed with an inverted fluorescent
microscope (Nikon, TE300) and photomicrographs were taken with a
SPOT digital camera using SPOT software (Diagnostic instruments).
For Western blot analysis, the cultures were extracted in 8M urea,
diluted 1:1 in reducing tricine sample buffer and loaded onto a 16%
tricine gel (Novex). After transfer onto immobilon, blots were
exposed to 5 .mu.g/ml of the pabAB42 followed by an HRP-conjugated
anti-mouse antibody, and developed with ECL (Amersham)
[0367] When the assay was performed with PDAPP brain sections in
the presence of 16C11 (one of the antibodies against A.beta. that
was not efficacious in vivo), .beta.-amyloid plaques remained
intact and no phagocytosis was observed. In contrast, when adjacent
sections were cultured in the presence of 10D5, the amyloid
deposits were largely gone and the microglial cells showed numerous
phagocytic vesicles containing A.beta.. Identical results were
obtained with AD brain sections; 10D5 induced phagocytosis of AD
plaques, while 16C11 was ineffective. In addition, the assay
provided comparable results when performed with either mouse or
human microglial cells, and with mouse, rabbit, or primate
antibodies against A.beta..
[0368] Table 16 shows whether binding and/or phagocytosis was
obtained for several different antibody binding specificities. It
can be seen that antibodies binding to epitopes within aa 1-7 both
bind and clear amyloid deposits, whereas antibodies binding to
epitopes within amino acids 4-10 bind without clearing amyloid
deposits. Antibodies binding to epitopes C-terminal to residue 10
neither bind nor clear amyloid deposits. TABLE-US-00016 TABLE 16
Analysis of Epitope Specificity Antibody epitope isotype Staining
Phagocytosis N-Term mab 3D6 1-5 IgG2b + + 10D5 3-6 IgG1 + + 22C8
3-7 IgG2a + + 6E10 5-10 IgG1 + - 14A8 4-10 rat IgG1 + - 13-28 18G11
10-18 rat IgG1 - - 266 16-24 IgG1 - - 22D12 18-21 IgG2b - - C-Term
2G3 -40 IgG1 - - 16C11 -40/-42 IgG1 - - 21F12 -42 IgG2a - - Immune
serum rabbit (CFA) 1-6 + + mouse (CFA) 3-7 + + mouse (QS-21) 3-7 +
+ monkey (QS-21) 1-5 + + mouse (MAP1-7) + +
[0369] Table 17 shows results obtained with several antibodies
against A13, comparing their abilities to induce phagocytosis in
the ex vivo assay and to reduce in vivo plaque burden in passive
transfer studies. Although 16C11 and 21F12 bound to aggregated
synthetic A.beta. peptide with high avidity, these antibodies were
unable to react with .beta.-amyloid plaques in unfixed brain
sections, could not trigger phagocytosis in the ex vivo assay, and
were not efficacious in vivo. 10D5, 3D6, and the polyclonal
antibody against A.beta. were active by all three measures. The
22C8 antibody binds more strongly to an analog form of natural
A.beta. in which aspartic acid at positions 1 and 7 is replaced
with iso-aspartic acid. These results show that efficacy in vivo is
due to direct antibody mediated clearance of the plaques within the
CNS, and that the ex vivo assay is predictive of in vivo
efficacy.
[0370] The same assay has been used to test clearing of an antibody
against a fragment of synuclein referred to as NAC. Synuclein has
been shown to be an amyloid plaque-associated protein. An antibody
to NAC was contacted with a brain tissue sample containing amyloid
plaques, an microglial cells, as before. Rabbit serum was used as a
control. Subsequent monitoring showed a marked reduction in the
number and size of plaques indicative of clearing activity of the
antibody. TABLE-US-00017 TABLE 17 The ex vivo assay as predictor of
in vivo efficacy. Avidity for Binding to aggregated .beta.-amyloid
Ex vivo In vivo Antibody Isotype A.beta. (pM) plaques efficacy
efficacy monoclonal 3D6 IgG2b 470 + + + 10D5 IgG1 43 + + + 16C11
IgG1 90 - - - 21F12 IgG2a 500 - - - TM2a IgG1 - - - - polyclonal
1-42 mix 600 + + +
[0371] Confocal microscopy was used to confirm that A.beta. was
internalized during the course of the ex vivo assay. In the
presence of control antibodies, the exogenous microglial cells
remained in a confocal plane above the tissue, there were no
phagocytic vesicles containing A.beta., and the plaques remained
intact within the section. In the presence of 10D5, nearly all
plaque material was contained in vesicles within the exogenous
microglial cells. To determine the fate of the internalized
peptide, 10D5 treated cultures were extracted with 8M urea at
various time-points, and examined by Western blot analysis. At the
one hour time point, when no phagocytosis had yet occurred,
reaction with a polyclonal antibody against A.beta. revealed a
strong 4 kD band (corresponding to the A.beta. peptide). A.beta.
immunoreactivity decreased at day 1 and was absent by day 3. Thus,
antibody-mediated phagocytosis of A.beta. leads to its
degradation.
[0372] To determine if phagocytosis in the ex vivo assay was
Fc-mediated, F(ab')2 fragments of the anti-A.beta. antibody 3D6
were prepared. Although the F(ab')2 fragments retained their full
ability to react with plaques, they were unable to trigger
phagocytosis by microglial cells. In addition, phagocytosis with
the whole antibody could be blocked by a reagent against murine Fc
receptors (anti-CD16/32). These data indicate that in vivo
clearance of A.beta. occurs through Fc-receptor mediated
phagocytosis.
XV: Passage of Antibodies through Blood Brain Barrier
[0373] This example determines the concentration of antibody
delivered to the brain following intravenous injection into a
peripheral tissue of either normal or PDAPP mice. PDAPP or control
normal mice were perfused with 0.9% NaCl. Brain regions
(hippocampus or cortex) were dissected and rapidly frozen. Brain
were homogenized in 0.1% triton+protease inhibitors. Immunoglobulin
was detected in the extracts by ELISA. Fab'2 Goat Anti-mouse IgG
were coated onto an RIA plate as capture reagent. The serum or the
brain extracts were incubated for 1 hr. The isotypes were detected
with anti-mouse IgG1-HRP or IgG2a-HRP or IgG2b-HRP (Caltag).
Antibodies, regardless of isotype, were present in the CNS at a
concentration that is 1:1000 that found in the blood. For example,
when the concentration of IgG1 was three times that of IgG2a in the
blood, it was three times IgG2a in the brain as well, both being
present at 0.1% of their respective levels in the blood. This
result was observed in both transgenic and nontransgenic mice--so
the PDAPP does not have a uniquely leak blood brain barrier.
XVI: Therapeutic Efficacy of an A.beta. Peptide in MAP
Configuration
[0374] A therapeutic adjuvant/immunogen efficacy study was
performed in 9-10.5 month old male and female heterozygous PDAPP
transgenic mice to test the efficacy of a fusion protein comprising
A.beta.1-7 in tetrameric MAP configuration as described above. The
duration of the study was 25 weeks with 29-40 animals per treatment
group; therefore the animals were 15-16.5 months old at
termination. The methodology used in this study is the same as that
in the therapeutic study of different adjuvants in Example VIII
above. The treatment groups are identified in Table 18 below.
TABLE-US-00018 TABLE 18 Dilution Adminis- Adjuvant Immunogen Buffer
tration Group 1: CFA/IFA MAP(A.beta. 1-7: TT) PBS IP (400 .mu.l)
(100 .mu.g) Group 2: QS21 AN1792-GCS (75 .mu.g) PBS SC (250 .mu.l)
Group 3: PBS -- -- SC (250 .mu.l) Table abbreviations:
MAP--multi-antigenic peptide; TT--tetanus toxoid t-cell epitope
(830-844); SC--subcutaneous; IP--intraperitoneally; PBS--phosphate
buffered saline; GCS is a glycine/citrate/sucrose formulation.
[0375] The immunization schedule was identical for all of the
treatment groups. The mice were injected on weeks 0, 2, 4, 8, 12,
16, 20, 24, with bleeds on week 3, 5, 9, 13, 17, 21 and 25. Groups
1, 2, 3, 4, and 6 received eight injections Groups 2 and 3,
QS21/AN1792 and PBS respectively, served as the positive and
negative controls for this study.
[0376] The titers were determined by the anti-A.beta. antibody
titer assay.
[0377] Group 1, CFA/IFA:MAP(A.beta.1-7:TT) group, had low titer
levels. The peak GMT reached was only 1,200 at 13 weeks, falling to
a GMT of 600 by week 25. There were 3 of the 30 mice that did not
raise any titer and another 7 mice that did not exceed a titer of
400 by the end of the study.
[0378] Group 2, the QS21/AN1792 control group, reached its peak
titer at 17 weeks with a GMT of 16,000. The titer then fell over
the next 8 weeks to finish with a GMT of 8,700. One animal in this
group failed to raise a titer over the entire course of the
experiment.
[0379] Group 3, the PBS alone group, had no titers.
[0380] Both treatment groups showed a significant lowing in
cortical A.beta. levels as compared to the PBS control group (see
Table 19). The CFA/IFA:MAP(A.beta.1-7) group, significantly lowered
A13 as compared to the PBS control group in spite of the relatively
low titers of anti-A.beta. antibodies. TABLE-US-00019 TABLE 19
Cortical A.beta. levels PBS MAP QS-21 MEDIAN 7,335 3,692 2,389
(ng/g tissue) RANGE 550-18,358 240-10,782 210-11,167 (ng/g tissue)
p value -- 0.0003 <0.0001 N 38 30 34
[0381] In conclusion, the A.beta.1-7MAP immunogen is effective in
inducing a sufficient immune response significantly to retard
A.beta. deposition in the cortex.
XVII. Epitope Mapping of Immunogenic Response to A.beta. in
Monkeys
[0382] This example analyzes the response of a primate to
immunization with AN1792 (i.e., A.beta.1-42). Eleven groups of
monkeys (4/sex/group) were immunized with AN1792 (75 or 300
.mu.g/dose) in combination with QS-21 adjuvant (50 or 100
.mu.g/dose) or 5% sterile dextrose in water (D5W, control group).
All animals received IM injections on one of three injection
schedules as shown in Table 20 for a total of 4, 5 or 8 doses.
Serum samples (from 4 monkeys/sex/group) collected on Day 175 of
the study and CSF samples (from 3 monkeys/sex/group) collected on
Day 176 of the study (at the 6 month necropsy) were evaluated for
their ability to bind to A.beta.1-40 peptide and APP.
TABLE-US-00020 TABLE 20 Group Assignments and Dose Levels Group
Sched- # Monkeys AN1792 Dose QS-21 Dose Dose No. ule.sup.a (M/F)
(.mu.g/dose) (.mu.g/dose) Route 1.sup.b 1 4/4 0 0 IM 2 1 4/4
Vehicle.sup.c 50 IM 3 1 4/4 Vehicle 100 IM 4 1 4/4 75 50 IM 5 1 4/4
300 50 IM 6 1 4/4 75 100 IM 7 1 4/4 300 100 IM 8 2 4/4 75 100 IM 9
2 4/4 300 100 IM 10 3 4/4 75 100 IM 11 3 4/4 300 100 IM
.sup.aSchedule 1, Dose Days 1, 15, 29, 57, 85, 113, 141, 169;
Schedule 2, Dose Days 1, 29, 57, 113, 169; Schedule 3, Dose Days 1,
43, 85, 169 .sup.bD5W injection control group .sup.cVehicle
consists of the glycine/citrate/sucrose buffer which is the
excipient for AN1792.
[0383] The exact array of linear peptides recognized by the
antibodies in the serum samples from animals immunized with AN1792
was determined by an ELISA that measured the binding of these
antibodies to overlapping peptides that covered the entire
A.beta.1-42 sequence. Biotinylated peptides with partial sequences
of AN1792 were obtained from Chiron Technologies as 10 amino acid
peptides with an overlap of 9 residues and a step of one residue
per peptide (synthesis No. 5366, No. 5331 and No. 5814). The first
32 peptides (from the eight amino acid position upstream of the
N-terminal of AN1792 down to the twenty-fourth amino acid of
AN1792) are biotinylated on the C-terminal with a linker of GGK.
The last 10 peptides (repeating the thirty-second peptide from the
previous series) are biotinylated on the N-terminal with a linker
consisting of EGEG (SEQ ID NO:76). The lyophilized biotinylated
peptides were dissolved at a concentration of 5 mM in DMSO. These
peptide stocks were diluted to 5 .mu.M in TTBS (0.05% Tween 20, 25
mM Tris HCl, 137 mM NaCl, 5.1 mM KCl, pH=7.5). 100 .mu.l aliquots
of this 5 .mu.M solution were added in duplicate to streptavidin
pre-coated 96-well plates (Pierce). Plates were incubated for one
hour at room temperature, then washed four times with TTBS. Serum
samples were diluted in specimen diluent without azide to normalize
titers, and 100 .mu.l was added per well. These plates were
incubated one hour at room temperature and then washed four times
with TTBS. HRP-conjugated goat anti-human antibody (Jackson
ImmunoResearch) was diluted 1:10,000 in specimen diluent without
azide and 100 .mu.l was added per well. The plates were again
incubated and washed. To develop the color reaction, TMB (Pierce),
was added at 100 .mu.l per well and incubated for 15 min prior to
the addition of 30 .mu.l of 2 N H.sub.2SO.sub.4 to stop the
reaction. The optical density was measured at 450 nm on a Vmax or
Spectramax colorimetric plate reader.
[0384] Immunization with AN1792 resulted in the production of
antibodies in 100% of the animals in all of the dose groups by Day
175. Mean titers in the groups ranged from 14596-56084. There was a
trend for titers to be higher within an immunization schedule in
the presence of higher antigen and/or higher adjuvant
concentration, but no statistically significant differences could
be demonstrated due to the high variability in individual animal
responses to the immunizations.
[0385] Sera which were positive for antibodies to AN1792 were also
positive for antibodies to A.beta.1-40. Mean titers in the groups
ranged from 36867-165991, and as for anti-AN1792 titers, showed no
statistically significant differences between groups at Day 175.
Binding to AN1792 showed a highly positive correlation (Spearman
r=0.8671) with binding to A.beta.1-40.
[0386] Of the 48 monkeys immunized on various schedules with
AN1792, 33 yielded CSF samples of adequate volume and quality for
analysis. Thirty-two (97%) of these monkeys had positive titers to
AN1792. Titers ranged from 2-246, with a mean of 49.44.+-.21.34.
CSF anti-AN1792 levels were 0.18.+-.0.11% of what was measured in
the serum and demonstrated a highly positive correlation (Spearman
r=0.7840) with serum titers. No differences were seen across groups
or between sexes in the percentage of antibody in the CSF. The
level of antibody in the CSF is consistent with the passive
transfer of peripherally generated antibody across the
blood-brain-barrier into the central nervous system.
[0387] Testing of a subset of anti-AN1792 positive CSF samples
demonstrated that, like the antibody in serum samples, antibody in
the CSF cross-reacts with A.beta.1-40. Titers to A.beta.1-40 showed
a high correlation (Spearman r=0.9634) to their respective AN1792
titers. Testing of a subset of CSF samples with the highest titers
to AN1792 showed no binding to APP, as for the serum
antibodies.
[0388] When sera from Day 175 was tested against a series of
overlapping 10-mer peptides, antibodies from all of the monkeys
bound to the peptide whose sequence covered amino acids 1-10 of the
AN1792 peptide (amino acids 653-672 of APP). In some animals, this
was the only peptide to which binding could be measured (see FIG.
19).
[0389] In other animals, other reactivities could be measured, but
in all cases the reactivity to the N-terminal peptide sequence was
the predominant one. The additional reactivities fell into two
groups. First and most common, was the binding to peptides
centering around the N-terminal 1-10 AN1792 peptide (FIG. 20).
Binding of this type was directed to the peptides covering amino
acids-1-8, -1-9, and 2-11 of the AN1792 peptide. These
reactivities, combined with that to the 1-10 peptide, represent the
overwhelming majority of reactivity in all animals. Epitope mapping
of individual animals over time indicates that the antibody
reactivity to the 1-10 peptide proceeds the spread to the adjacent
peptides. This demonstrates a strong biasing of the immune response
to the N-terminus of the AN1792 peptide with its free terminal
aspartic acid residue. The second minor detectable activity in some
animals was binding to peptides located C-terminally to the major
area and centered around peptides covering amino acids 7-16, 11-20
and 16-25 of the AN1792 peptide. These reactivities were seen in
only 10-30% of the monkeys.
[0390] Variability in response between different animals (e.g.,
whether amino acids 1-10 were the exclusive or predominant reactive
epitope) did not correlate with antigen/adjuvant dose, dosing
schedule, or antibody titer, and is probably a reflection of each
individual animal's genetic make-up.
XVIII. Prevention and Treatment of Human Subjects
[0391] A single-dose phase I trial is performed to determine safety
in humans. A therapeutic agent is administered in increasing
dosages to different patients starting from about 0.01 the level of
presumed efficacy, and increasing by a factor of three until a
level of about 10 times the effective mouse dosage is reached.
[0392] A phase II trial is performed to determine therapeutic
efficacy. Patients with early to mid Alzheimer's Disease defined
using Alzheimer's disease and Related Disorders Association (ADRDA)
criteria for probable AD are selected. Suitable patients score in
the 12-26 range on the Mini-Mental State Exam (MMSE). Other
selection criteria are that patients are likely to survive the
duration of the study and lack complicating issues such as use of
concomitant medications that may interfere. Baseline evaluations of
patient function are made using classic psychometric measures, such
as the MMSE, and the ADAS, which is a comprehensive scale for
evaluating patients with Alzheimer's Disease status and function.
These psychometric scales provide a measure of progression of the
Alzheimer's condition. Suitable qualitative life scales can also be
used to monitor treatment. Disease progression can also be
monitored by MRI. Blood profiles of patients can also be monitored
including assays of immunogen-specific antibodies and T-cells
responses.
[0393] Following baseline measures, patients begin receiving
treatment. They are randomized and treated with either therapeutic
agent or placebo in a blinded fashion. Patients are monitored at
least every six months. Efficacy is determined by a significant
reduction in progression of a treatment group relative to a placebo
group.
[0394] A second phase II trial is performed to evaluate conversion
of patients from non-Alzheimer's Disease early memory loss,
sometimes referred to as age-associated memory impairment (AAMI) or
mild cognitive impairment (MCI), to probable Alzheimer's disease as
defined as by ADRDA criteria. Patients with high risk for
conversion to Alzheimer's Disease are selected from a non-clinical
population by screening reference populations for early signs of
memory loss or other difficulties associated with pre-Alzheimer's
symptomatology, a family history of Alzheimer's Disease, genetic
risk factors, age, sex, and other features found to predict
high-risk for Alzheimer's Disease. Baseline scores on suitable
metrics including the MMSE and the ADAS together with other metrics
designed to evaluate a more normal population are collected. These
patient populations are divided into suitable groups with placebo
comparison against dosing alternatives with the agent. These
patient populations are followed at intervals of about six months,
and the endpoint for each patient is whether or not he or she
converts to probable Alzheimer's Disease as defined by ADRDA
criteria at the end of the observation.
XIX. General Materials and Methods
[0395] 1. Measurement of Antibody Titers
[0396] Mice were bled by making a small nick in the tail vein and
collecting about 200 .mu.l of blood into a microfuge tube. Guinea
pigs were bled by first shaving the back hock area and then using
an 18 gauge needle to nick the metatarsal vein and collecting the
blood into microfuge tubes. Blood was allowed to clot for one hr at
room temperature (RT), vortexed, then centrifuged at 14,000.times.g
for 10 min to separate the clot from the serum. Serum was then
transferred to a clean microfuge tube and stored at 4.degree. C.
until titered.
[0397] Antibody titers were measured by ELISA. 96-well microtiter
plates (Costar EIA plates) were coated with 100 .mu.l of a solution
containing either 10 .mu.g/ml either A.beta.42 or SAPP or other
antigens as noted in each of the individual reports in Well Coating
Buffer (0.1 M sodium phosphate, pH 8.5, 0.1% sodium azide) and held
overnight at RT. The wells were aspirated and sera were added to
the wells starting at a 1/100 dilution in Specimen Diluent (0.014 M
sodium phosphate, pH 7.4, 0.15 M NaCl, 0.6% bovine serum albumin,
0.05% thimerosal). Seven serial dilutions of the samples were made
directly in the plates in three-fold steps to reach a final
dilution of 1/218,700. The dilutions were incubated in the
coated-plate wells for one hr at RT. The plates were then washed
four times with PBS containing 0.05% Tween 20. The second antibody,
a goat anti-mouse Ig conjugated to horseradish peroxidase (obtained
from Boehringer Mannheim), was added to the wells as 100 .mu.l of a
1/3000 dilution in Specimen Diluent and incubated for one hr at RT.
Plates were again washed four times in PBS, Tween 20. To develop
the chromogen, 100 .mu.l of Slow TMB (3,3',5,5'-tetramethyl
benzidine obtained from Pierce Chemicals) was added to each well
and incubated for 15 min at RT. The reaction was stopped by the
addition of 25 .mu.l of 2 M H.sub.2SO.sub.4. The color intensity
was then read on a Molecular Devices Vmax at (450 nm-650 nm).
[0398] Titers were defined as the reciprocal of the dilution of
serum giving one half the maximum OD. Maximal OD was generally
taken from an initial 1/100 dilution, except in cases with very
high titers, in which case a higher initial dilution was necessary
to establish the maximal OD. If the 50% point fell between two
dilutions, a linear extrapolation was made to calculate the final
titer. To calculate geometric mean antibody titers, titers less
than 100 were arbitrarily assigned a titer value of 25.
[0399] 2. Lymphocyte Proliferation Assay
[0400] Mice were anesthetized with isoflurane. Spleens were removed
and rinsed twice with 5 ml PBS containing 10% heat-inactivated
fetal bovine serum (PBS-FBS) and then homogenized in a 50 Centricon
unit (Dako A/S, Denmark) in 1.5 ml PBS-FBS for 10 sec at 100 rpm in
a Medimachine (Dako) followed by filtration through a 100 micron
pore size nylon mesh. Splenocytes were washed once with 15 ml
PBS-FBS, then pelleted by centrifugation at 200.times.g for 5 min.
Red blood cells were lysed by resuspending the pellet in 5 mL
buffer containing 0.15 M NH4Cl, 1 M KHCO3, 0.1 M NaEDTA, pH 7.4 for
five min at RT. Leukocytes were then washed as above. Freshly
isolated spleen cells (10.sup.5 cells per well) were cultured in
triplicate sets in 96-well U-bottomed tissue culture-treated
microtiter plates (Corning, Cambridge, Mass.) in RPMI 1640 medium
(JRH Biosciences, Lenexa, Kans.) supplemented with 2.05 mM L
glutamine, 1% Penicillin/Streptomycin, and 10% heat-inactivated
FBS, for 96 hr at 37.degree. C. Various A.beta. peptides,
A.beta.1-16, A1-40, A.beta.1-42 or A.beta.40-1 reverse sequence
protein were also added at doses ranging from 5 to 0.18 micromolar
in four steps. Cells in control wells were cultured with
Concanavalin A (Con A) (Sigma, cat. # C-5275, at 1 microgram/ml)
without added protein. Cells were pulsed for the final 24 hr with
3H-thymidine (1 .mu.Ci/well obtained from Amersham Corp., Arlington
Heights Ill.). Cells were then harvested onto UniFilter plates and
counted in a Top Count Microplate Scintillation Counter (Packard
Instruments, Downers Grove, Ill.). Results are expressed as counts
per minute (cpm) of radioactivity incorporated into insoluble
macromolecules.
[0401] 4. Brain Tissue Preparation
[0402] After euthanasia, the brains were removed and one hemisphere
was prepared for immunohistochemical analysis, while three brain
regions (hippocampus, cortex and cerebellum) were dissected from
the other hemisphere and used to measure the concentration of
various AD proteins and APP forms using specific ELISAs
(Johnson-Wood et al., supra).
[0403] Tissues destined for ELISAs were homogenized in 10 volumes
of ice-cold guanidine buffer (5.0 M guanidine-HCl, 50 mM Tris-HCl,
pH 8.0). The homogenates were mixed by gentle agitation using an
Adams Nutator (Fisher) for three to four hr at RT, then stored at
-20.degree. C. prior to quantitation of A.beta. and APP. Previous
experiments had shown that the analytes were stable under this
storage condition, and that synthetic A.beta. protein (Bachem)
could be quantitatively recovered when spiked into homogenates of
control brain tissue from mouse littermates (Johnson-Wood et al.,
supra).
[0404] 5. Measurement of A.beta. Levels
[0405] The brain homogenates were diluted 1:10 with ice cold Casein
Diluent (0.25% casein, PBS, 0.05% sodium azide, 20 .mu.g/ml
aprotinin, 5 mM EDTA pH 8.0, 10 .mu.g/ml leupeptin) and then
centrifuged at 16,000.times.g for 20 min at 4 C. The synthetic
A.beta. protein standards (1-42 amino acids) and the APP standards
were prepared to include 0.5 M guanidine and 0.1% bovine serum
albumin (BSA) in the final composition. The "total" A.beta.
sandwich ELISA utilizes monoclonal antibody (mAb) 266, specific for
amino acids 13-28 of A.beta. (Seubert, et al.), as the capture
antibody, and biotinylated mAb 3D6, specific for amino acids 1-5 of
A.beta. (Johnson-Wood, et al), as the reporter antibody. The 3D6
mAb does not recognize secreted APP or full-length APP, but detects
only A.beta. species with an amino-terminal aspartic acid. The cell
line RB96 3D6.32.2.4 producing the antibody 3D6 has the ATCC
accession number PTA-5130, having been deposited on Apr. 8, 2003.
The address of the American Type Culture Collection (ATCC) is 10801
University Boulevard, Manassas, Va. 20110-2209. This assay has a
lower limit of sensitivity of .about.50 ng/ml (11 nM) and shows no
cross-reactivity to the endogenous murine A.beta. protein at
concentrations up to 1 ng/ml (Johnson-Wood et al., supra).
[0406] The A.beta.1-42 specific sandwich ELISA employs mA.beta.
21F12, specific for amino acids 33-42 of A.beta. (Johnson-Wood, et
al.), as the capture antibody. Biotinylated mA.beta. 3D6 is also
the reporter antibody in this assay which has a lower limit of
sensitivity of about 125 .mu.g/ml (28 .mu.M, Johnson-Wood et al.).
For the A.beta. ELISAs, 100 .mu.l of either mA.beta. 266 (at 10
.mu.g/ml) or mA.beta. 21F12 at (5 .mu.g/ml) was coated into the
wells of 96-well immunoassay plates (Costar) by overnight
incubation at RT. The solution was removed by aspiration and the
wells were blocked by the addition of 200 .mu.l of 0.25% human
serum albumin in PBS buffer for at least 1 hr at RT. Blocking
solution was removed and the plates were stored desiccated at
4.degree. C. until used. The plates were rehydrated with Wash
Buffer [Tris-buffered saline (0.15 M NaCl, 0.01 M Tris-HCl, pH
7.5), plus 0.05% Tween 20] prior to use. The samples and standards
were added in triplicate aliquots of 100 .mu.l per well and then
incubated overnight at 4.degree. C. The plates were washed at least
three times with Wash Buffer between each step of the assay. The
biotinylated mA.beta. 3D6, diluted to 0.5 .mu.g/ml in Casein Assay
Buffer (0.25% casein, PBS, 0.05% Tween 20, pH 7.4), was added and
incubated in the wells for 1 hr at RT. An avidin-horseradish
peroxidase conjugate, (Avidin-HRP obtained from Vector, Burlingame,
Calif.), diluted 1:4000 in Casein Assay Buffer, was added to the
wells for 1 hr at RT. The colorimetric substrate, Slow TMB-ELISA
(Pierce), was added and allowed to react for 15 minutes at RT,
after which the enzymatic reaction was stopped by the addition of
25 .mu.l 2 NH2SO4. The reaction product was quantified using a
Molecular Devices Vmax measuring the difference in absorbance at
450 nm and 650 nm.
[0407] 6. Measurement of APP Levels
[0408] Two different APP assays were utilized. The first,
designated APP-.alpha./FL, recognizes both APP-alpha (.alpha.) and
full-length (FL) forms of APP. The second assay is specific for
APP-.alpha.. The APP-.alpha./FL assay recognizes secreted APP
including the first 12 amino acids of A.beta.. Since the reporter
antibody (2H3) is not specific to the .alpha.-clip-site, occurring
between amino acids 612-613 of APP695 (Esch et al., Science 248,
1122-1124 (1990)); this assay also recognizes full length APP
(APP-FL). Preliminary experiments using immobilized APP antibodies
to the cytoplasmic tail of APP-FL to deplete brain homogenates of
APP-FL suggest that approximately 30-40% of the APP-.alpha./FL APP
is FL (data not shown). The capture antibody for both the
APP-.alpha./FL and APP-A assays is mAb 8E5, raised against amino
acids 444 to 592 of the APP695 form (Games et al., supra). The
reporter mAb for the APP-.alpha./FL assay is mAb 2H3, specific for
amino acids 597-608 of APP695 (Johnson-Wood et al., supra) and the
reporter antibody for the APP-.alpha. assay is a biotinylated
derivative of mAb 16H9, raised to amino acids 605 to 611 of APP.
The lower limit of sensitivity of the APP-.alpha.FL assay is about
11 ng/ml (150 .rho.M) (Johnson-Wood et al.) and that of the APP-A
specific assay is 22 ng/ml (0.3 nM). For both APP assays, mAb 8E5
was coated onto the wells of 96-well EIA plates as described above
for mAb 266. Purified, recombinant secreted APP-.alpha. was used as
the reference standard for the APP-A assay and the APP-.alpha./FL
assay (Esch et al., supra). The brain homogenate samples in 5 M
guanidine were diluted 1:10 in ELISA Specimen Diluent (0.014 M
phosphate buffer, pH 7.4, 0.6% bovine serum albumin, 0.05%
thimerosal, 0.5 M NaCl, 0.1% NP40). They were then diluted 1:4 in
Specimen Diluent containing 0.5 M guanidine. Diluted homogenates
were then centrifuged at 16,000.times.g for 15 seconds at RT. The
APP standards and samples were added to the plate in duplicate
aliquots and incubated for 1.5 hr at RT. The biotinylated reporter
antibody 2H3 or 16H9 was incubated with samples for 1 hr at RT.
Streptavidin-alkaline phosphatase (Boehringer Mannheim), diluted
1:1000 in specimen diluent, was incubated in the wells for 1 hr at
RT. The fluorescent substrate 4-methyl-umbellipheryl-phosphate was
added for a 30-min RT incubation and the plates were read on a
Cytofluor.TM. 2350 fluorimeter (Millipore) at 365 nm excitation and
450 nm emission.
[0409] 7. Immunohistochemistry
[0410] Brains were fixed for three days at 40 C in 4%
paraformaldehyde in PBS and then stored from one to seven days at
4.degree. C. in 1% paraformaldehyde, PBS until sectioned.
Forty-micron-thick coronal sections were cut on a vibratome at RT
and stored in cryoprotectant (30% glycerol, 30% ethylene glycol in
phosphate buffer) at -20.degree. C. prior to immunohistochemical
processing. For each brain, six sections at the level of the dorsal
hippocampus, each separated by consecutive 240 .mu.m intervals,
were incubated overnight with one of the following antibodies: (1)
a biotinylated anti-A.beta. (mAb, 3D6, specific for human A.beta.)
diluted to a concentration of 2 .mu.g/ml in PBS and 1% horse serum;
or (2) a biotinylated mAb specific for human APP, 8E5, diluted to a
concentration of 3 .mu.g/ml in PBS and 1.0% horse serum; or (3) a
mAb specific for glial fibrillary acidic protein (GFAP; Sigma
Chemical Co.) diluted 1:500 with 0.25% Triton X-100 and 1% horse
serum, in Tris-buffered saline, pH 7.4 (TBS); or (4) a mAb specific
for CD11b, MAC-1 antigen, (Chemicon International) diluted 1:100
with 0.25% Triton X-100 and 1% rabbit serum in TBS; or (5) a mAb
specific for MHC II antigen, (Pharmingen) diluted 1:100 with 0.25%
Triton X-100 and 1% rabbit serum in TBS; or (6) a rat mAb specific
for CD 43 (Pharmingen) diluted 1:100 with 1% rabbit serum in PBS or
(7) a rat mAb specific for CD 45RA (Pharmingen) diluted 1:100 with
1% rabbit serum in PBS; or (8) a rat monoclonal A.beta. specific
for CD 45RB (Pharmingen) diluted 1:100 with 1% rabbit serum in PBS;
or (9) a rat monoclonal A.beta. specific for CD 45 (Pharmingen)
diluted 1:100 with 1% rabbit serum in PBS; or (10) a biotinylated
polyclonal hamster A.beta. specific for CD3e (Pharmingen) diluted
1:100 with 1% rabbit serum in PBS or (11) a rat mAb specific for
CD3 (Serotec) diluted 1:200 with 1% rabbit serum in PBS; or with
(12) a solution of PBS lacking a primary antibody containing 1%
normal horse serum.
[0411] Sections reacted with antibody solutions listed in 1, 2 and
6-12 above were pretreated with 1.0% Triton X-100, 0.4% hydrogen
peroxide in PBS for 20 min at RT to block endogenous peroxidase.
They were next incubated overnight at 4.degree. C. with primary
antibody. Sections reacted with 3D6 or 8E5 or CD3e mAbs were then
reacted for one hr at RT with a horseradish
peroxidase-avidin-biotin-complex with kit components "A" and "B"
diluted 1:75 in PBS (Vector Elite Standard Kit, Vector Labs,
Burlingame, Calif.). Sections reacted with antibodies specific for
CD 45RA, CD 45RB, CD 45, CD3 and the PBS solution devoid of primary
antibody were incubated for 1 hour at RT with biotinylated anti-rat
IgG (Vector) diluted 1:75 in PBS or biotinylated anti-mouse IgG
(Vector) diluted 1:75 in PBS, respectively. Sections were then
reacted for one hr at RT with a horseradish
peroxidase-avidin-biotin-complex with kit components "A" and "B"
diluted 1:75 in PBS (Vector Elite Standard Kit, Vector Labs,
Burlingame, Calif.).
[0412] Sections were developed in 0.01% hydrogen peroxide, 0.05%
3,3'-diaminobenzidine (DAB) at RT. Sections destined for incubation
with the GFAP-, MAC-1-AND MHC II-specific antibodies were
pretreated with 0.6% hydrogen peroxide at RT to block endogenous
peroxidase then incubated overnight with the primary antibody at
4.degree. C. Sections reacted with the GFAP antibody were incubated
for 1 hr at RT with biotinylated anti-mouse IgG made in horse
(Vector Laboratories; Vectastain Elite ABC Kit) diluted 1:200 with
TBS. The sections were next reacted for one hr with an
avidin-biotin-peroxidase complex (Vector Laboratories; Vectastain
Elite ABC Kit) diluted 1:1000 with TBS. Sections incubated with the
MAC-1- or MHC II-specific monoclonal antibody as the primary
antibody were subsequently reacted for 1 hr at RT with biotinylated
anti-rat IgG made in rabbit diluted 1:200 with TBS, followed by
incubation for one hr with avidin-biotin-peroxidase complex diluted
1:1000 with TBS. Sections incubated with GFAP-, MAC-1- and MHC
II-specific antibodies were then visualized by treatment at RT with
0.05% DAB, 0.01% hydrogen peroxide, 0.04% nickel chloride, TBS for
4 and II min, respectively.
[0413] Immunolabeled sections were mounted on glass slides (VWR,
Superfrost slides), air dried overnight, dipped in Propar (Anatech)
and overlaid with coverslips using Permount (Fisher) as the
mounting medium.
[0414] To counterstain A.beta. plaques, a subset of the
GFAP-positive sections were mounted on Superfrost slides and
incubated in aqueous 1% Thioflavin S (Sigma) for 7 min following
immunohistochemical processing. Sections were then dehydrated and
cleared in Propar, then overlaid with coverslips mounted with
Permount.
[0415] 8. Image Analysis
[0416] A Videometric 150 Image Analysis System (Oncor, Inc.,
Gaithersburg, Md.) linked to a Nikon Microphot-FX microscope
through a CCD video camera and a Sony Trinitron monitor was used
for quantification of the immunoreactive slides. The image of the
section was stored in a video buffer and a color- and
saturation-based threshold was determined to select and calculate
the total pixel area occupied by the immunolabeled structures. For
each section, the hippocampus was manually outlined and the total
pixel area occupied by the hippocampus was calculated. The percent
amyloid burden was measured as: (the fraction of the hippocampal
area containing A.beta. deposits immunoreactive with mAb
3D6).times.100. Similarly, the percent neuritic burden was measured
as: (the fraction of the hippocampal area containing dystrophic
neurites reactive with monoclonal antibody 8E5) x100. The C-Imaging
System (Compix, Inc., Cranberry Township, Pa.) operating the Simple
32 Software Application program was linked to a Nikon Microphot-FX
microscope through an Optronics camera and used to quantitate the
percentage of the retrospenial cortex occupied by GFAP-positive
astrocytes and MAC-1- and MHC II-positive microglia. The image of
the immunoreacted section was stored in a video buffer and a
monochrome-based threshold was determined to select and calculate
the total pixel area occupied by immunolabeled cells. For each
section, the retrosplenial cortex (RSC) was manually outlined and
the total pixel area occupied by the RSC was calculated. The
percent astrocytosis was defined as: (the fraction of RSC occupied
by GFAP-reactive astrocytes).times.100. Similarly, percent
microgliosis was defined as: (the fraction of the RSC occupied by
MAC-1- or MHC II-reactive microglia) X 100. For all image analyses,
six sections at the level of the dorsal hippocampus, each separated
by consecutive 240 .mu.m intervals, were quantitated for each
animal. In all cases, the treatment status of the animals was
unknown to the observer.
[0417] Although the foregoing invention has been described in
detail for purposes of clarity of understanding, it will be obvious
that certain modifications may be practiced within the scope of the
appended claims. All publications and patent documents cited herein
are hereby incorporated by reference in their entirety for all
purposes to the same extent as if each were so individually
denoted.
[0418] From the foregoing it will be apparent that the invention
provides for a number of uses. For example, the invention provides
for the use of any of the antibodies to A.beta. described above in
the treatment, prophylaxis or diagnosis of amyloidogenic disease,
or in the manufacture of a medicament or diagnostic composition for
use in the same. Likewise, the invention provides for the use of
any of the epitopic fragments of A.beta. described above for the
treatment or prophylaxis of amyloidogenic disease or in the
manufacture of a medicament for use in the same. TABLE-US-00021
TABLE 1 TITER AT 50% MAXIMAL O.D. Aggregated AP injected mice Age
of mouse mouse mouse mouse mouse mouse mouse mouse mouse PDAPP 100
101 102 103 104 105 106 107 108 4 70000 150000 15000 120000 1000
15000 50000 80000 100000 6 15000 65000 30000 55000 300 15000 15000
50000 60000 8 20000 55000 50000 50000 400 15000 18000 50000 60000
10 40000 20000 60000 50000 900 15000 50000 20000 40000 12 25000
30000 60000 40000 2700 20000 70000 25000 20000 PBS injected mice on
both Immunogens at 1/100 Age of mouse mouse mouse mouse mouse PDAPP
113 114 115 116 117 6 <4x <4x <4x <4x <4x bkg bkg
bkg bkg bkg 10 5x <4x <4x <4x <4x bkg bkg bkg bkg bkg
12 <4x <4x <4x <4x <4x bkg bkg bkg bkg bkg
[0419]
Sequence CWU 1
1
5 1 42 PRT Homo sapiens human Abeta42 beta-amyloid peptide 1 Asp
Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 10
15 Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile
20 25 30 Gly Leu Met Val Gly Gly Val Val Ile Ala 35 40 2 13 PRT
Artificial Sequence Description of Artificial SequenceAbeta1-12
peptide with carboxyl terminal Cys residue inserted 2 Asp Ala Glu
Phe Arg His Asp Ser Gly Tyr Glu Val Cys 1 5 10 3 6 PRT Artificial
Sequence Description of Artificial SequenceAbeta1-5 peptide with
carboxyl terminal Cys residue inserted 3 Asp Ala Glu Phe Arg Cys 1
5 4 12 PRT Artificial Sequence Description of Artificial
SequenceAbeta33-42 peptide with carboxyl terminal Cys residue
inserted MOD_RES (2) Xaa = amino hepatanoic acid 4 Cys Xaa Gly Leu
Met Val Gly Gly Val Val Ile Ala 1 5 10 5 19 PRT Artificial Sequence
Description of Artificial SequenceAbeta13-28 peptide with carboxyl
terminal Cys residue inserted and two added Gly residues MOD_RES
(1) Xaa = acetyl histidine 5 Xaa His Gln Lys Leu Val Phe Phe Ala
Glu Asp Val Gly Ser Asn Lys 1 5 10 15 Gly Gly Cys
* * * * *