U.S. patent application number 10/923605 was filed with the patent office on 2005-11-10 for prevention and treatment of amyloidogenic disease.
This patent application is currently assigned to Neuralab Limited. Invention is credited to Schenk, Dale B..
Application Number | 20050249727 10/923605 |
Document ID | / |
Family ID | 26748211 |
Filed Date | 2005-11-10 |
United States Patent
Application |
20050249727 |
Kind Code |
A1 |
Schenk, Dale B. |
November 10, 2005 |
Prevention and treatment of amyloidogenic disease
Abstract
The invention provides compositions and methods for treatment of
amyloidogenic diseases. Such methods entail administering an agent
that induces a beneficial immune response against an amyloid
deposit in the patient. The methods are particularly useful for
prophylactic and therapeutic treatment of Alzheimer's disease. In
such methods, a suitable agent is A.beta. peptide, active fragments
thereof or an antibody thereto.
Inventors: |
Schenk, Dale B.;
(Burlingame, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Neuralab Limited
Flatts
BM
|
Family ID: |
26748211 |
Appl. No.: |
10/923605 |
Filed: |
August 20, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10923605 |
Aug 20, 2004 |
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09724319 |
Nov 27, 2000 |
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09724319 |
Nov 27, 2000 |
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09322289 |
May 28, 1999 |
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09322289 |
May 28, 1999 |
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09201430 |
Nov 30, 1998 |
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6787523 |
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60080970 |
Apr 7, 1998 |
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60067740 |
Dec 2, 1997 |
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Current U.S.
Class: |
424/141.1 ;
424/146.1 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 2039/55577 20130101; A61K 9/0019 20130101; A61K 47/646
20170801; A61P 25/28 20180101; A61P 37/00 20180101; A61K 31/00
20130101; A61K 9/7023 20130101; A61K 2039/605 20130101; A61K
2039/55505 20130101; A61K 2039/505 20130101; A61K 9/2009 20130101;
A61K 31/739 20130101; A61K 2039/55572 20130101; A61K 39/0007
20130101; A61K 2039/53 20130101; A61K 2039/55566 20130101; C07K
2317/24 20130101; A61K 38/1709 20130101; C07K 2317/567 20130101;
A61K 9/2054 20130101; A61K 39/00 20130101; A61K 2300/00 20130101;
A61K 2039/55555 20130101; A61P 25/00 20180101; A61K 9/2031
20130101; A61K 9/4866 20130101; C07K 2319/00 20130101; A61K
2039/6037 20130101; C07K 16/18 20130101; A61K 38/193 20130101; C07K
14/4711 20130101; A61K 38/193 20130101 |
Class at
Publication: |
424/141.1 ;
424/146.1 |
International
Class: |
A61K 039/395 |
Claims
1-163. (canceled)
164. A method to inhibit the formation of amyloid plaques in
humans, comprising administering to a human subject in need of such
inhibition an effective amount of a humanized antibody or fragment
thereof that specifically immunoreacts with an epitope contained in
positions 13-28 of A.beta..
165. A method to reduce amyloid plaques in humans, comprising
administering to a human subject in need of such reduction an
effective amount of a humanized antibody or fragment thereof which
specifically immunoreacts with an epitope contained in positions
13-28 of A.beta..
166. A method to inhibit the formation of amyloid plaques in
humans, comprising administering to a human subject in need of such
inhibition an effective amount of a humanized antibody or fragment
thereof that binds to soluble A.beta. peptide.
167. A method to inhibit the formation of amyloid plaques in
humans, comprising administering to a human subject in need of such
inhibition an effective amount of a humanized antibody or fragment
thereof that sequesters A.beta. peptide from its bound, circulating
form in blood.
168. A method to reduce amyloid plaques in humans, comprising
administering to a human subject in need of such reduction an
effective amount of a humanized antibody or fragment thereof which
binds to soluble A.beta. peptide.
169. A method to reduce amyloid plaques in humans, comprising
administering to a human subject in need of such reduction an
effective amount of a humanized antibody or fragment thereof which
sequesters A.beta. peptide from its bound, circulating form in
blood.
170. The method of any one of claims 165-169, wherein the subject
is diagnosed with clinical or pre-clinical Alzheimer's disease or
Down's syndrome.
171. The method of any one of claims 170, wherein the antibody is
administered by a peripheral route.
172. The method of claim 171, wherein the antibody is administered
by an oral, intraperitoneal, subcutaneous, intramuscular, or
intravenous route.
173. The method of any one of claims 164-169, wherein the subject
is not diagnosed with clinical or pre-clinical Alzheimer's disease
or Down's syndrome.
174. The method of claim 173, wherein the antibody is administered
by a peripheral route.
175. The method of claim 174, wherein the antibody is administered
by an oral, intraperitoneal, subcutaneous, intramuscular, or
intravenous route.
176. The method of any one of claims 164-169, wherein the antibody
or fragment, when administered peripherally to humans, does not
need to cross the blood-brain barrier to inhibit the formation of
amyloid plaques.
177. The method of claim 176, wherein the subject is diagnosed with
clinical or pre-clinical Alzheimer's disease or Down's
syndrome.
178. The method of claim 177, wherein the antibody is administered
by a peripheral route.
179. The method of claim 178, wherein the antibody is administered
by an oral, intraperitoneal, subcutaneous, intramuscular, or
intravenous route.
180. The method of claim 176, wherein the subject is not diagnosed
with clinical or pre-clinical Alzheimer's disease or Down's
syndrome.
181. The method of claim 180, wherein the antibody is administered
by a peripheral route.
182. The method of claim 181, wherein the antibody is administered
by an oral, intraperitoneal, subcutaneous, intramuscular, or
intravenous route.
183. The method of any one of claims 164-169, wherein the antibody
or fragment, when administered peripherally to humans, does not
elicit cellular responses to inhibit the formation of amyloid
plaques.
184. The method of claim 183, wherein the subject is diagnosed with
clinical or pre-clinical Alzheimer's disease or Down's
syndrome.
185. The method of claim 184, wherein the antibody is administered
by a peripheral route.
186. The method of claim 185, wherein the antibody is administered
by an oral, intraperitoneal, subcutaneous, intramuscular, or
intravenous route.
187. The method of claim 183, wherein the subject is not diagnosed
with clinical or pre-clinical Alzheimer's disease or Down's
syndrome.
188. The method of claim 187, wherein the antibody is administered
by a peripheral route.
189. The method of claim 188, wherein the antibody is administered
by an oral, intraperitoneal, subcutaneous, intramuscular, or
intravenous route.
190. The method of any of claims 165-169, wherein the antibody or
fragment, when administered peripherally to humans, does not
substantially bind aggregated A.beta. in the brain.
191. The method of claim 190, wherein the subject is diagnosed with
clinical or pre-clinical Alzheimer's disease or Down's
syndrome.
192. The method of claim 191, wherein the antibody is administered
by a peripheral route.
193. The method of claim 192, wherein the antibody is administered
by an oral, intraperitoneal, subcutaneous, intramuscular, or
intravenous route.
194. The method of claim 190, wherein the subject is not diagnosed
with clinical or pre-clinical Alzheimer's disease or Down's
syndrome.
195. The method of claim 194, wherein the antibody is administered
by a peripheral route.
196. The method of claim 195, wherein the antibody is administered
by an oral, intraperitoneal, subcutaneous, intramuscular, or
intravenous route.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 09/724,319, filed Nov. 27, 2000, which is a continuation of
U.S. application Ser. No. 09/322,289, filed May 28, 1999, which is
a continuation-in-part of U.S. application Ser. No. 09/201,430,
filed Nov. 30, 1998 which claims the benefit under 35 U.S.C. 119(e)
of U.S. Application No. 60/080,970, filed Apr. 7, 1998, and U.S.
Application 60/067,740, filed Dec. 2, 1997, all of which are
incorporated by reference in their 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.559-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 A.beta., other active immunogen or antibody to
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.
SUMMARY OF THE CLAIMED INVENTION
[0007] In one aspect, the invention provides methods of preventing
or treating a disease characterized by amyloid deposit in a
patient. Such methods entail administering an effective dosage of
an antibody that specifically binds to the amyloid deposit or a
component thereof to the patient. Such methods are particularly
useful for preventing or treating Alzheimer's disease in which case
the amyloid deposit is A.beta.. The methods can be used on both
asymtomatic patients and those currently showing symptoms of
disease.
[0008] 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.
[0009] In some methods, the antibody used binds to an epitope
within residues 1-28 of A.beta.. In some methods the antibody binds
to an epitope within residues 1-10, and in some methods within
residues 1-5. In some methods, the antibody specifically binds to
A.beta. peptide without binding to full-length amyloid precursor
protein (APP).
[0010] In some methods antibody is administered at a dosage of at
least 1 mg/kg body weight antibody: In some methods, the antibody
is administered in multiple dosages over a period 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 or intravenously.
[0011] 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.
[0012] 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 Alzheimer's disease. These methods entail
administering an effective dosage of a polypeptide comprising an
active fragment of A.beta. that induces an immune response to
A.beta. in the patient. In some methods, the fragment comprises an
epitope within amino acids 1-12 of A.beta.. In some method, the
fragment comprises an epitope within amino acids 1-16 of A.beta..
In some methods, the fragment comprises an epitope within amino
acids 13-28 of A.beta.. 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 20 contiguous amino acids from
A.beta.. Fragments are typically administered at greater than 10
micrograms per dose per patient.
[0014] 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 of
together as a composition. The adjuvant can be, for example, alum,
MPL, QS-21 or incomplete Freund's adjuvant.
[0015] The invention further provides pharmaceutical compositions
comprising active fragments of A.beta., such as described above,
and an adjuvant.
[0016] The invention further provides methods of screening an
antibody to A.beta. or an active fragment of A.beta. for use in
treatment of Alzheimer's disease. Such methods entail administering
an antibody that specifically binds to A.beta. or a fragment of
A.beta. to a transgenic animal disposed to develop characteristics
of Alzheimer's disease. One then detects a reduction in the extent
or rate of development of the characteristics relative to a control
transgenic animal as a measure of the efficacy of the antibody or
fragment. Optionally, antibodies can also be screened for capacity
to bind an epitope within amino acids 1-28 or other epitope of
A.beta..
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1: Antibody titer after injection of transgenic mice
with A.beta.1-42.
[0018] 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 distribition.
[0019] 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.
[0020] 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.
[0021] 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.
[0022] FIG. 6: Kinetics of antibody response to AN1792
immunization. Titers are expressed as geometric means of values for
the 6 animals in each group.
[0023] FIG. 7: Quantitative image analysis of the cortical amyloid
burden in PBS- and AN1792-treated mice.
[0024] FIG. 8: Quantitative image analysis of the neuritic plaque
burden in PBS- and AN1792-treated mice.
[0025] FIG. 9: Quantitative image analysis of the percent of the
retrosplenial cortex occupied by astrocytosis in PBS- and
AN1792-treated mice.
[0026] FIG. 10: Lymphocyte Proliferation Assay on spleen cells from
AN1792-treated (FIG. 10A) or PBS-treated (FIG. 10B).
[0027] 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.
[0028] 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.
[0029] 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.
[0030] 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.
[0031] 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.
[0032] FIG. 15A: The values for mice for the PBS-treated control
group and the untreated control group.
[0033] FIG. 15B: The values for mice in the AN1528/alum and
AN1528/MPL-treatment groups.
[0034] FIG. 15C: The values for mice in the AN1528/QS21 and
AN1792/Freund's adjuvant treatment groups.
[0035] FIG. 15D: The values for mice in the AN1792/Thimerosol and
AN1792/alum treatment groups.
[0036] FIG. 15E: The values for mice in the AN1792/MPL and
AN1792/QS21 treatment groups.
[0037] FIG. 16: Mean titer of mice treated with polyclonal antibody
to A.beta..
[0038] FIG. 17: Mean titer of mice treated with monoclonal antibody
10D5 to A.beta..
[0039] FIG. 18: Mean titer of mice treated with monoclonal antibody
2F12 to A.beta..
DEFINITIONS
[0040] 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.
[0041] 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.
[0042] 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
(http://www.ncbi.nlm.nih.gov/). 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))
[0043] 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, gin, his, lys, arg; Group V (residues
influencing chain orientation): gly, pro; and Group VI (aromatic
side chains): trp, tyr, phe. Conservative substitutions involve
substitutions between amino acids in the same class.
Non-conservative substitutions constitute exchanging a member of
one of these classes for a member of another.
[0044] 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.
[0045] 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.
[0046] 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 fragments including
separate heavy chains, light chains Fab, Fab' F(ab')2, Fabc, and
Fv. Fragments are produced by recombinant DNA techniques, or by
enzymatic or chemical separation of intact immunoglobulins. The
term "antibody" also includes one or more immunoglobulin chain 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).
[0047] 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.
[0048] 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.
[0049] 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.
[0050] An "immunogenic agent" or "immunogen" is capable of inducing
an immunological response against itself on administration to a
patient, optionally in conjunction with an adjuvant.
[0051] The term "naked polynucleotide" refers to a polynucleotide
not complexed with colloidal materials. Naked polynucleotides are
sometimes cloned in a plasmid vector.
[0052] 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.
[0053] The term "patient" includes human and other mammalian
subjects that receive either prophylactic or therapeutic
treatment.
[0054] Disaggregated or monomeric A.beta. means soluble, monomeric
peptide units of A.beta.. One method to prepare monomeric A.beta.
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.
[0055] 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 I-125 label (see Morel
et al., Molec. Immunol. 25(1):7-15 (1988)); solid phase direct
biotin-avidin EIA (Cheung et al., Virology 176:546-552 (1990)); and
direct labeled RIA (Moldenhauer et al., Scand. J. Immunol. 32:77-82
(1990)). Typically, such an assay involves the use of purified
antigen bound to a solid surface or cells bearing either of these,
an unlabelled test immunoglobulin and a 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%.
[0056] 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
[0057] I. General
[0058] The invention provides pharmaceutical compositions and
methods for prophylactic and therapeutic treatment of diseases
characterized by accumulation of amyloid deposits. Amyloid deposits
comprise a peptide aggregated to an insoluble mass. The nature of
the peptide varies in different diseases but in most cases, the
aggregate has a .beta.-pleated sheet structure and stains with
Congo Red dye. Diseases characterized by amyloid deposits include
Alzheimer's disease (AD), both late and early onset. In both
diseases, the amyloid deposit comprises a peptide termed A.beta.,
which accumulates in the brain of affected individuals. Examples of
some other diseases characterized by amyloid deposits are SAA
amyloidosis, hereditary Icelandic syndrome, multiple myeloma, and
spongiform encephalopathies, including mad cow disease, Creutzfeldt
Jakob disease, sheep scrapie, and mink spongiform encephalopathy
(see Weissmann et al., Curr. Opin. Neurobiol. 7, 695-700 (1997);
Smits et al., Veterinary Quarterly 19, 101-105 (1997); Nathanson et
al., Am. J. Epidemiol. 145, 959-969 (1997)). The peptides forming
the aggregates in these diseases are serum amyloid A, cystantin C,
IgG kappa light chain respectively for the first three, and prion
protein for the others.
[0059] II. Therapeutic Agents
[0060] A. Alzheimer's Disease
[0061] 1. Agents Inducing Active Immune Response
[0062] Therapeutic agents for use in the present invention induce
an immune response against A.beta. peptide. These agents include
A.beta. peptide itself and variants thereof, analogs and mimetics
of A.beta. peptide that induce and/or crossreact with antibodies to
A.beta. peptide, and antibodies or T-cells reactive with A.beta.
peptide. Induction of an immune response can be active as when an
immunogen is administered to induce antibodies or T-cells reactive
with A.beta. in a patient, or passive, as when an antibody is
administered that itself binds to A.beta. in patient.
[0063] 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.
[0064] 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.
[0065] The therapeutic agent used in the claimed methods can be any
of the naturally occurring forms of A.beta. peptide, and
particularly the human forms (i.e., A.beta.39, A.beta.40,
A.beta.41, A.beta.42 or 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:
1 (SEQ ID NO:1) H.sub.2N-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gl-
y-Tyr-Glu- Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-As- p-
Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-GIy-Leu-Met-
Val-Gly-Gly-Val-Val-IIe-Ala-OH.
[0066] A.beta.41, A.beta.40 and A.beta.39 differ from A.beta.42 by
the omission of Ala, Ala-Ile, and Ala-Ile-Val respectively from the
C-terminal end. A.beta.43 differs from A.beta.42 by the presence of
a threonine residue at the C-terminus. The therapeutic agent can
also be an active fragment or analog of a natural A.beta. peptide
that contains an epitope that induces a similar protective or
therapeutic immune response on administration to a human.
Immunogenic fragments typically have a sequence of at least 3, 5,
6, 10 or 20 contiguous amino acids from a natural peptide.
Immunogenic fragments include A.beta.1-5, 1-6, 1-12, 13-28, 17-28,
1-28, 25-35, 35-40 and 35-42. Fragments lacking at least one, and
sometimes at least 5 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 AB43 includes the first 38 amino acids from the N-terminal end
of AB. Fragments from the N-terminal half of A.beta. are preferred
in some methods. Analogs include allelic, species and induced
variants. Analogs typically differ from naturally occurring
peptides at one 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. Examples of unnatural amino acids are .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, .omega.-N-methylarginine.
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.
[0067] A.beta., its fragments, analogs and other amyloidogenic
peptides 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.).
[0068] Therapeutic agents also include longer polypeptides that
include, for example, an A.beta. peptide, active fragment or analog
together with other amino acids. For example, A.beta. peptide can
be present as intact APP protein or a segment thereof, such as the
C-100 fragment that begins at the N-terminus of A.beta. and
continues to the end of APP. 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 form (i.e., as an .beta.-amyloid
peptide) or in dissociated form. Therapeutic agents also include
multimers of monomeric immunogenic agents.
[0069] In a further variation, an immunogenic peptide, such as
A.beta., can be presented as a viral or bacterial vaccine. 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 outersurface 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, vaccinia and fowl pox.
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).
[0070] 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.
[0071] 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 other amyloidogenic peptide. Compounds identified by
such screens are then further analyzed for capacity to induce
antibodies or reactive lymphocytes to A.beta. or other
amyloidogenic peptide. For example, multiple dilutions of sera can
be tested on microtiter plates that have been precoated with
A.beta. peptide and a standard ELISA can be performed to test for
reactive antibodies to A.beta.. 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 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
such as fragments of A.beta., analogs of A.beta. and longer
peptides including A.beta., described above.
[0072] 2. Agents Inducing Passive Immune Response
[0073] 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. 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.
[0074] Polyclonal sera typically contain mixed populations of
antibodies binding to several epitopes along the length of A.beta..
Monoclonal antibodies bind to a specific epitope within A.beta.
that can be a conformational or nonconformational epitope. Some
monoclonal antibodies bind to an epitope within residues 1-28 of
A.beta. (with the first N terminal residue of natural A.beta.
designated 1). Some monoclonal antibodies bind to an epitope within
residues 1-10 of A.beta. Some monoclonal antibodies bind to an
epitope within residues 1-16 of A.beta.. Some monoclonal antibodies
bind to an epitope within residues 1-25 of A.beta.. Some monoclonal
antibodies bind to an epitope within amino acids 1-5, 5-10, 10-15,
15-20, 25-30, 10-20, 20-30, or 10-25 of A.beta.. Prophylactic and
therapeutic efficacy of antibodies can be tested using the
transgenic animal model procedures described in the Examples.
[0075] i. General Characteristics of Immunoglobulins
[0076] 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.
[0077] 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" ion 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).
[0078] The variable regions of each light/heavy chain pair form the
antibody binding site. Thus, and 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).
[0079] ii. Production of Nonhuman Antibodies
[0080] The production of non-human monoclonal antibodies, e.g.,
murine, guinea pig, 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 A.beta.. 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.
[0081] iii. Chimeric and Humanized Antibodies
[0082] 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. 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.
[0083] 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.
[0084] 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:
[0085] (1) noncovalently binds antigen directly,
[0086] (2) is adjacent to a CDR region,
[0087] (3) otherwise interacts with a CDR region (e.g. is within
about 6 A of a CDR region), or
[0088] (4) participates in the VL-VH interface.
[0089] 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.
[0090] iv. Human Antibodies
[0091] 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..
[0092] (1) Trioma Methodology
[0093] 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.
[0094] 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 A.beta., 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.
[0095] 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, 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.
[0096] 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.
[0097] (2) Transgenic Non-Human Mammals
[0098] 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.beta. 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.
[0099] (3) Phage Display Methods
[0100] 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.
[0101] 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.
[0102] v. Selection of Constant Region
[0103] 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.
Light chain constant regions can be lambda or kappa. Antibodies can
be expressed as tetramers containing two light and two heavy
chains, as separate heavy chains, light chains, as Fab, Fab'
F(ab')2, and Fv, or as single chain antibodies in which heavy and
light chain variable domains are linked through a spacer.
[0104] vi. Expression of Recombinant Antibodies
[0105] 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.
[0106] 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.
[0107] 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.
[0108] 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).
[0109] 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.
[0110] 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.
[0111] 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)).
[0112] 4. Other Therapeutic Agents
[0113] Therapeutic agents for use in the present methods also
include T-cells that bind to A.beta. peptide. For example, T-cells
can be activated against A.beta. peptide by expressing a human MHC
class I gene and a human .beta.-2-microglobulin gene from an insect
cell line, whereby an empty complex is formed on the surface of the
cells and can bind to A.beta. peptide. T-cells contacted with the
cell line become specifically activated against the peptide. See
Peterson et al., U.S. Pat. No. 5,314,813. Insect cell lines
expressing an MHC class II antigen can similarly be used to
activate CD4 T cells.
[0114] B. Other Diseases
[0115] The same or analogous principles determine production of
therapeutic or preventative agents for amyloidogenic diseases. In
general, the agents noted above for use in treatment of Alzheimer's
disease can also be used for treatment early onset Alzheimer's
disease associated with Down's syndrome. In mad cow disease, prion
peptide, active fragments, and analogs, and antibodies to prion
peptide are used in place of A.beta. peptide, active fragments,
analogs and antibodies to A.beta. peptide in treatment of
Alzheimer's disease. In treatment of multiple myeloma, IgG light
chain and analogs and antibodies thereto are used, and so forth in
other diseases.
[0116] 1. Carrier Proteins
[0117] 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 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.
[0118] 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-thi- o) propionate (SPDP) and
succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-c- arboxylate
(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 .ang.-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)cy- clohexane-1-carboxylic acid. The carboxyl
groups can be activated by combining them with succinimide or
1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt.
[0119] Immunogenic peptides can also be expressed as fusion
proteins with carriers. The immunogenic peptide can be linked at
the amino terminus, the carboxyl terminus, or internally to the
carrier. Optionally, multiple repeats of the immunogenic peptide
can be present in the fusion protein.
[0120] The same or similar carrier proteins and methods of linkage
can be used for generating immunogens to be used in generation of
antibodies against A.beta. 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..
[0121] 4. Nucleic Acid Encoding Therapeutic Agents
[0122] Immune responses against amyloid deposits can also be
induced by administration of nucleic acids encoding A.beta.
peptide, 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.
[0123] 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)), 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)).
[0124] 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).
[0125] 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). 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).
[0126] 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.
[0127] III. Patients Amenable to Treatment
[0128] 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 be or
she lives long enough. Therefore, the present methods can be
administered prophylactically to the general population without 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.
[0129] 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.
[0130] IV. Treatment Regimes
[0131] In prophylactic applications, pharmaceutical compositions or
medicants are administered to a patient susceptible to, or
otherwise at risk of, a particular disease in an amount sufficient
to eliminate or reduce the risk or delay the outset 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 and its
complications. An amount adequate to accomplish this is defined as
a therapeutically- or pharmaceutically-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
fade.
[0132] 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 in some diseases, such as mad
cow disease, the patient can be a nonhuman mammal, such as a
bovine. 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 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 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.
[0133] 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. 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. 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.
[0134] 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
10-109, or more, virions per dose.
[0135] Agents for inducing an immune response can be administered
by parenteral, topical, intravenous, oral, subcutaneous,
intraperitoneal, intranasal or intramuscular means for prophylactic
and/or therapeutic treatment. The most typical route of
administration of an immunogenic agent is subcutaneous although
others can be equally effective. The next most common is
intramuscular injection. This type of injection is most typically
performed in the arm or leg muscles. Intravenous injections as well
as intraperitoneal injections, intraarterial, intracranial, or
intradermal injections are also effective in generating an immune
response. 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.
[0136] 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.
[0137] 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 alum, 3
De-O-acylated monophosphoryl lipid A (MPL) (see GB 2220211). 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 Ajuvant Approach (eds.
Powell & Newman, Plenum Press, NY, 1995); U.S. Pat. No.
5,057,540). 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 (Bioworld
Today, Nov. 15, 1998). 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.
[0138] 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% Squalane,
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 monophosphorylipid 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, Worcester, 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).
[0139] 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 vaccine 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.
[0140] 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. However, some reagents suitable for
administration to animals, such as Complete Freund's adjuvant are
not typically included in compositions for human use.
[0141] 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, 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).
[0142] 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.
[0143] 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.
[0144] Additional formulations suitable for other modes of
administration include oral, intranasal, and pulmonary
formulations, suppositories, and transdermal applications.
[0145] 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%.
[0146] 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.
[0147] 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)).
[0148] V. Methods of Diagnosis
[0149] 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).
[0150] 1. Active Immunization
[0151] 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.
[0152] 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.
[0153] 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.
[0154] 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 patient can be compared with a
control value (mean plus standard deviation) determined in
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.
[0155] 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).
[0156] 2. Passive Immunization
[0157] 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.
[0158] In some methods, a baseline measurement of antibody to
A.beta. 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.
[0159] 3. Diagnostic Kits
[0160] 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. or reacts with T-cells-specific for 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. For detection of reactive T-cells, the label can be supplied
as 3H-thymidine to measure a proliferative response. Kits also
typically contain labelling providing directions for use of the
kit. The labelling may also include a chart or other correspondence
regime correlating levels of measured label with levels of
antibodies to A.beta. or T-cells reactive with A.beta.. The term
labelling 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
labelling encompasses advertising leaflets and brochures, packaging
materials, instructions, audio or video cassettes, computer discs,
as well as writing imprinted directly on kits.
EXAMPLES
[0161] I. Prophylactic Efficacy of A.beta. Against AD
[0162] 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..
[0163] A. Methods
[0164] 1. Source of Mice
[0165] Thirty PDAPP heterogenic female mice were randomly divided
into the following groups: 10 mice to be injected with aggregated
A.beta.42 (one died in transit), 5 mice to be injected with
PBS/adjuvant or PBS, and 10 uninjected controls. Five mice were
injected with peptides dervied from the sequence of serum amyloid
protein (SAP).
[0166] 2. Preparation of Immunogens
[0167] Preparation of aggregated A.beta.42: two milligrams of
A.beta.42 (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.
[0168] 3. Preparation of Injections
[0169] 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.).
[0170] 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.
[0171] 4. Titration of Mouse Bleeds, Tissue Preparation and
Immunohistochemistry
[0172] The above methods are described infra in General Materials
and Methods.
[0173] B. Results
[0174] 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.
[0175] The PBS-treated mice were titered against aggregated
A.beta.42 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.
[0176] 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.
[0177] 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.
[0178] 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).
[0179] 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.
[0180] 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)
[0181] Evidence from a subset of the A.beta.1-42- and PBS-injected
mice indicated plaque-associated MHC II immunoreactivity was absent
in the A.beta.I-42 injected mice, consistent with lack of an
A.beta.-related inflammatory response.
[0182] 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.
[0183] C. Conclusion
[0184] 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.
[0185] II. Dose Response Study
[0186] 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 11, 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 vaccine doses. 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.
[0187] 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).
[0188] 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.
[0189] 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.
[0190] 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.
[0191] III. Screen for Therapeutic Efficacy Against Established
AD
[0192] 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.
[0193] 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.
[0194] 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.
[0195] A. Materials and Methods
[0196] 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 .about.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.
[0197] 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.
[0198] For immunizations, 100.mu.g of AN1792 in 200 .mu.l 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.
[0199] B. Results
[0200] 1. Effects of AN1792 Treatment on Amyloid Burden
[0201] 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.
[0202] 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.
[0203] 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.
[0204] 2. AN1792 Treatment-Associated Cellular and
Morphological
[0205] Changes
[0206] 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.
[0207] 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.
[0208] 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.
[0209] 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.
[0210] These results are indicative of active, cell-mediated
removal of amyloid in plaque-bearing brain regions.
[0211] 3. AN1792 Effects on A.beta. Levels: ELISA
Determinations
[0212] (a) Cortical Levels
[0213] In untreated PDAPP mice, the median level of total A.beta.
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 AN 1792-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).
2TABLE 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
[0214] (b) Hippocampal Levels
[0215] 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).
3TABLE 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
[0216] (c) Cerebellar Levels
[0217] 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 A.beta. 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).
4TABLE 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
[0218] 4. Effects of AN1792 Treatment on APP Levels
[0219] APP-.alpha. and the full-length APP molecule both contain
all or part of the A.beta. 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-/FL (full length) or APP-.alpha. were
essentially unchanged by treatment with the exception that APP- 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.
[0220] 5. Effects of AN1792 Treatment on Neurodegenerative and
Gliotic Pathology
[0221] 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.
[0222] 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.
[0223] 6. Antibody Responses
[0224] 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.
[0225] 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.
[0226] 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.
[0227] 7. Cellular-Mediated Immune Responses
[0228] 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).
[0229] C. Conclusion
[0230] 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.
[0231] IV. Screen of A.beta. Fragments
[0232] 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 are euthanized at the end
of 4 months of injections. Histochemistry, A.beta. levels, and
toxicology analysis was determined post mortem.
[0233] A. Materials and Methods
[0234] 1. Preparation of Immunogens
[0235] 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.beta.13-28 peptide
derivative also had two glycine residues added prior to the
carboxyl terminal cysteine as indicated.
5 A.beta.1-12 peptide NH.sub.2-DAEFRHDSGYEVC-COOH (SEQ ID NO:2)
A.beta.1-5 peptide NH.sub.2-DAEFRC-COOH (SEQ ID NO:3) A.beta.33-42
peptide NH.sub.2-C-amino-heptanoic acid-GLMVGGVVIA-COOH (SEQ ID
NO:4) A.beta.13-28 peptide Ac-NH-HHQKLVFFAEDVGSNKGGC-COOH (SEQ ID
NO:5)
[0236] 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
[0237] [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.
[0238] 2. Preparation of Aggregated A.beta. Peptides
[0239] 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.
[0240] 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%.
[0241] B. Results and Discussion
[0242] 1. Study Design
[0243] 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.
[0244] For each immunization, 100 .mu.g of each A.beta. peptide in
200 .mu.l 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.
[0245] 2. A.beta. and APP Levels in the Brain
[0246] 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.
[0247] 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).
[0248] For the cortex, a subset of treatment groups had median
total A.beta. 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 discemable
correlations between A.beta.-specific antibody titers and A.beta.
levels in the cortical region of the brain for any of the
groups.
[0249] 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).
[0250] 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.
[0251] 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-/FL, recognizes
both APP-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..
[0252] 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.
[0253] 2. Histochemical Analyses
[0254] 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 A.beta.1-5 peptide conjugate (67%, p=0.02).
[0255] 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..
[0256] 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.
[0257] 3. Measurement of Antibody Titers
[0258] 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 vaccines
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), A.beta.33-42
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, A.beta.25-35,
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.
[0259] 4. Lymphoproliferative Responses
[0260] 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.
[0261] 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 A.beta.1-12 conjugate, A.beta.13-28
conjugate and A.beta.25-35 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.
6 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 .sup.
42-mer 8/8 pBx6 0/8 PBS 0-mer 0/8
[0262] 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.
[0263] 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.
[0264] V. Preparation of Polyclonal Antibodies for Passive
Protection
[0265] 125 non-transgenic mice were immunized with A.beta., plus
adjuvant, and euthanized at 4-5months. 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.
[0266] VI. Passive Immunization with Antibodies to A.beta.
[0267] 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-A.beta.
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 cell line producing the antibody 266
has the ATCC accession number ______, having been deposited on Jul.
20, 2004.
[0268] 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..
7 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
[0269] 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 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.
[0270] VII. Comparison of Different Adjuvants
[0271] This example compares CFA, alum, an oil-in water emulsion
and MPL for capacity to stimulate an immune response.
[0272] A. Materials and Methods
[0273] 1. Study Design
[0274] 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 therof 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.
[0275] 2. Preparation of Immunogens
[0276] 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.
[0277] 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.
[0278] To prepare vaccine 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).
[0279] To prepare vaccine 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.
[0280] To prepare vaccine 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.
[0281] To prepare vaccine 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.
[0282] 3. Measurement of Antibody Titers
[0283] 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.
[0284] 4. Tissue Preparation
[0285] After about 14 weeks, all guinea pigs were administered CO2.
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.
[0286] B. Results
[0287] 1. Antibody Responses
[0288] 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 vaccine 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 vaccines, MPL and high dose AN1792/alum.
[0289] 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.
[0290] 2. A.beta. Levels in the Brain
[0291] 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 vaccines. 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..
[0292] VIII. Immune Response to Different Adjuvants in Mice
[0293] 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.
8TABLE 7 Experimental Design of Study 010 Group N.sup.a
Adjuvant.sup.b Dose Antigen Dose(.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 + 25 .mu.g/2 mg AN1792 33 Alum 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.
[0294] The ELISA titers of antibodies against A.beta.42 in each
group are shown in Table 8 below.
9TABLE 8 Geometric Mean Antibody Titers Treatment Week of Bleed
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
[0295] 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.
[0296] IX. Therapeutic Efficacy of Different Adjuvants
[0297] 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.
[0298] 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.
[0299] 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 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 suspensions were vortexed again and incubated
overnight at 37.degree. C. for use the next day.
[0300] To prepare vaccine doses with alum, 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 .mu.l in 1.times.PBS. The suspension was
then gently mixed for approximately 4 hr at RT prior to
injection.
[0301] To prepare vaccine 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.
[0302] To prepare vaccine 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 50 .mu.l 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.
[0303] To prepare vaccine doses with Freund's Adjuvant (Group 4),
100 .mu.g of AN1792 in 200 .mu.l PBS was emulsified 1:1 (vol:vol)
with Complete Freund's Adjuvant (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). For the vaccines containing the adjuvants alum, MPL or
QS-21, 100 .mu.g per dose of AN1792 or AN1528 was combined with
alum (2 mg per dose) or MPL (50 .mu.g per dose) or QS-21 (25 .mu.g
per dose) in a final volume of 200 .mu.l PBS and delivered by
subcutaneous inoculation on the back between the shoulder blades.
For the group receiving FA, 100 .mu.g of AN1792 was emulsified 1:1
(vol:vol) with Complete Freund's adjuvant (CFA) in a final volume
of 400 .mu.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.mu.g
AN1792 was combined with 5 .mu.g thimerosal in a final volume of 50
.mu.l PBS and delivered subcutaneously. The ninth, control group
received only 200 .mu.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 A.beta. and
APP levels in brain and by immunohistochemical evaluation of the
presence of amyloid plaques in brain sections. In addition,
A.beta.-specific antibody titers, and A.beta.-dependent
proliferative and cytokine responses were determined.
[0304] 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.
10TABLE 9 Geometric Mean Antibody Titers.sup.a Week of Bleed
Treatment 3.3 5.0 9.0 13.0 17.0 Alum/ 102 1,081 2,366 1,083 572
AN1792 (12/21).sup.b (17/20) (21/21) (19/21) (18/21) MPL/ 6241
28,867 1,1242 5,665 8,204 AN1792 (21/21) (21/21) (21/21) (20/20)
(20/20) QS-21/ 30 227 327 1,511 1,188 AN1792 (1/20) (10/19) (10/19)
(17/18) (14/18) CFA/ 10,076 61,279 75,386 41,628 30,574 AN1792
(15/15) (15/15) (15/15) (15/15) (15/15) Alum/ 25 33 39 37 31 AN1528
(0/21) (1/21) (3/20) (1/20) (2/20) MPL/ 184 2,591 1,653 1,156 3,099
AN1528 (15/21) (20/21) (21/21) (20/20) (20/20) QS-21/ 29 221 51 820
2,994 AN1528 (1/22) (13/22) (4/22) (20/22) (21/22) PBS plus 25 33
39 37 47 Thimerosal (0/16) (2/16) (4/16) (3/16) (4/16) PBS 25 25 25
25 25 (0/16) (0/16) (0/15) (0/12) (0/16) Footnotes: .sup.aGeometric
mean antibody titers measured against A.beta.1-42 .sup.bNumber of
responders per group
[0305] 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
A42 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.
[0306] X. Toxicity Analysis
[0307] 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.
[0308] XI. Therapeutic Treatment with Anti-A.beta. Antibodies
[0309] 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.
[0310] 1. Study Design
[0311] 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 AN1 792)
(details of the deposit of 266 are discussed in Example VI, supra),
21F12 (directed to A residues 33-42). A fifth group was treated
with an A.beta.-specific polyclonal antibody fraction (raised by
immunization with aggregated AN1792). The negative control group
received the diluent, PBS, alone without antibody.
[0312] 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.
11TABLE 10 EXPERIMENTAL DESIGN OF STUDY 006 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
[0313] 2. Materials and Methods
[0314] a. Preparation of the Antibodies
[0315] 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.
[0316] The anti-A.beta. monoclonal antibodies were prepared from
ascities 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.
[0317] 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.
[0318] 3. A.beta. and APP Levels in the Brain:
[0319] 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.
[0320] 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-Woods
et al., 1997).
[0321] 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 11. 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).
12TABLE 11 CORTEX Medians Means Treatment Total A.beta. A.beta.42
Total A.beta. A.beta.42 Group N.sup.a ELISA 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.fNumber of animals per group at the end of the experiment
.sup.gng/g tissue .sup.hMann Whitney analysis .sup.iNA: not
applicable .sup.jStandard Deviation
[0322] 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 12). 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.
13TABLE 12 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
[0323] Total A.beta. was also measured in the cerebellum (Table
13). 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).
14TABLE 13 CEREBELLUM Medians Means Total A.beta. Total A.beta.
Treatment ELISA P % ELISA Group N.sup.a value.sup.b value.sup.c
Change 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
[0324] 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.
[0325] 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.
[0326] 4. Histochemical Analyses:
[0327] The morphology of A.beta.-immunoreactive plaques in subsets
of brains from mice in the PBS, polyclonal A.beta.42, 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.
[0328] The largest alteration in both the extent and appearance of
amyloid plaques occurred in the animals immunized with the
polyclonal A.beta.42 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.
[0329] 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. Major
differences were not seen when the 21F12 and 266 groups were
compared with the PBS controls.
[0330] 5. Measurement of Antibody Titers:
[0331] 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.
[0332] 6. Lymphoproliferative Responses:
[0333] 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.
[0334] 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.
[0335] XII. Prevention and Treatment of Subjects
[0336] A single-dose phase I trial is performed to determine
safety. 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.
[0337] 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.
[0338] 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.
[0339] 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.
[0340] XIII. General Materials and Methods
[0341] 1. Measurement of Antibody Titers
[0342] 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.
[0343] 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 (3,340 ,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).
[0344] 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.
[0345] 2. Lymphocyte Proliferation Assay
[0346] 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.degree.
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 (Coming, 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 peptides, A.beta.1-16,
A.beta.1-40, 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.
[0347] 4. Brain Tissue Preparation
[0348] 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 A.beta. proteins and APP forms using specific ELISAs
(Johnson-Wood et al., supra).
[0349] 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).
[0350] 5. Measurement of A.beta. Levels
[0351] 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.degree. 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 monoclonal antibody
266, specific for amino acids 13-28 of A.beta. (Seubert, et al.),
as the capture antibody, and biotinylated monoclonal antibody 3D6,
specific for amino acids 1-5 of A.beta. (Johnson-Wood, et al), as
the reporter antibody. The 3D6 monoclonal antibody does not
recognize secreted APP or full-length APP, but detects only A.beta.
species with an amino-terminal aspartic acid. This assay has a
lower limit of sensitivity of .about.50 g/ml (11 M) and shows no
cross-reactivity to the endogenous murine A.beta. protein at
concentrations up to 1 ng/ml (Johnson-Wood et al., supra).
[0352] 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 producing the antibody 3D6 has the ATCC accession number
PTA-5130, having been deposited on Apr. 8, 2003. This assay has a
lower limit of sensitivity of .about.50 pg/ml (11 pM) and shows no
cross-reactivity to the endogenous murine A.beta. protein at
concentrations up to 1 ng/ml (Johnson-Wood et al., supra).
[0353] 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 N H2SO4. The reaction product was quantified using a
Molecular Devices Vmax measuring the difference in absorbance at
450 nm and 650 nm.
[0354] 6. Measurement of APP Levels
[0355] 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-.alpha. 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-.alpha. 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-.alpha.
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.
[0356] 7. Immunohistochemistry
[0357] Brains were fixed for three days at 4.degree. 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.
[0358] 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.).
[0359] 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 11 min, respectively.
[0360] 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.
[0361] 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.
[0362] 8. Image Analysis
[0363] 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).times.100. 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).times.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.
[0364] 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.
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 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 5 Xaa His Gln Lys Leu Val Phe
Phe Ala Glu Asp Val Gly Ser Asn Lys 1 5 10 15 Gly Gly Cys
* * * * *
References