U.S. patent application number 10/223809 was filed with the patent office on 2003-08-21 for novel method for down-regulation of amyloid.
Invention is credited to Degan, Florence Dal, Jensen, Martin Roland, Koefoed, Peter, Nielsen, Klaus Gregorius, Rasmussen, Peter Birk.
Application Number | 20030157117 10/223809 |
Document ID | / |
Family ID | 32870659 |
Filed Date | 2003-08-21 |
United States Patent
Application |
20030157117 |
Kind Code |
A1 |
Rasmussen, Peter Birk ; et
al. |
August 21, 2003 |
Novel method for down-regulation of amyloid
Abstract
Disclosed are novel methods for combatting diseases
characterized by deposition of amyloid. The methods generally rely
on immunization against amyloid precursor protien (APP) or beta
amyloid (A.beta.). Immunization is preferably effected by
administration of analogues of autologous APP or A.beta., said
analogues being capable of inducing antibody production against the
autologous amyloidogenic polypeptides. Especially preferred as an
immunogen is autologous A.beta. which has been modified by
introduction of one single or a few foreign, immunodominant and
promiscuous T-cell epitopes. Also disclosed are nucleic acid
vaccination against APP or A.beta. and vaccination using live
vaccines as well as methods and means useful for the vaccination.
Such methods and means include methods for the preparation of
analogues and pharmaceutical formulations, as well as nucleic acid
fragments, vectors, transformed cells, polypeptides and
pharmaceutical formulations.
Inventors: |
Rasmussen, Peter Birk;
(Horsholm, DK) ; Jensen, Martin Roland; (Horsholm,
DK) ; Nielsen, Klaus Gregorius; (Horsholm, DK)
; Koefoed, Peter; (Horsholm, DK) ; Degan, Florence
Dal; (Horsholm, DK) |
Correspondence
Address: |
FROMMER LAWRENCE & HAUG
745 FIFTH AVENUE- 10TH FL.
NEW YORK
NY
10151
US
|
Family ID: |
32870659 |
Appl. No.: |
10/223809 |
Filed: |
August 20, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60337543 |
Oct 22, 2001 |
|
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60373027 |
Apr 16, 2002 |
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Current U.S.
Class: |
424/185.1 ;
435/226 |
Current CPC
Class: |
A61P 25/00 20180101;
A01K 2227/105 20130101; A01K 2267/0312 20130101; A61K 2039/53
20130101; A61K 2039/6068 20130101; A61P 37/04 20180101; A61K
2039/6087 20130101; A61K 39/385 20130101; A61K 45/06 20130101; A01K
2217/05 20130101; C07K 14/4711 20130101; A61P 3/10 20180101; A61P
25/16 20180101; A61K 2039/6037 20130101; A61P 25/14 20180101; A61P
25/28 20180101; A01K 67/0275 20130101; A61K 2039/64 20130101; A61K
39/0007 20130101; Y02A 50/30 20180101; A61K 2039/6093 20130101;
A61P 43/00 20180101 |
Class at
Publication: |
424/185.1 ;
435/226 |
International
Class: |
A61K 039/00; C12N
009/64 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 20, 2001 |
DK |
PA 2001 01231 |
Apr 16, 2002 |
DK |
PA 2002 0058 |
Claims
1. A method for in vivo down-regulation of amyloid precursor
protein (APP) or beta amyloid (A.beta.) in an animal, including a
human being, the method comprising effecting presentation to the
animal's immune system of an immunogenically effective amount of at
least one analogue of APP or A.beta. that incorporates into the
same molecule at least one B-cell epitope of APP and/or A.beta. and
at least one foreign T-helper epitope (T.sub.H epitope) so that
immunization of the animal with the analogue induces production of
antibodies against the animal's autologous APP or A.beta., wherein
the analogue a) is a polyamino acid that consists of at least one
copy of a subsequence of residues 672-714 in SEQ ID NO: 2, wherein
the foreign T.sub.H epitope is incorporated by means of amino acid
addition and/or insertion and/or deletion and/or substitution,
wherein the subsequence is selected from the group consisting of
residues 1-42, residues 1-40, residues 1-39, residues 1-35,
residues 1-34, residues 1-28, residues 1-12, residues 1-5, residues
13-28, residues 13-35, residues 17-28, residues 25-35, residues
35-40, residues 36-42 and residues 35-42 of the amino acid sequence
consisting of amino acid residues 673-714 of SEQ ID NO: 2; and/or
b) is a polyamino acid that contains the foreign T.sub.H epitopes
and a disrupted APP or A.beta. sequence so that the analogue does
not include any subsequence of SEQ ID NO: 2 that binds productively
to MHC class II molecules initiating a T-cell response; and/or c)
is a polyamino acid that comprises the foreign T.sub.H epitope and
APP or A.beta. derived amino acids, and comprises 1 single
methionine residue located in the C-terminus of the analogue,
wherein other methionine residues in APP or A.beta. and in the
foreign T.sub.H epitope have been substituted or deleted, and
preferably have been substituted by leucin or isoleucine; and/or d)
is a conjugate comprising a polyhydroxypolymer backbone to which is
separately coupled a polyamino acid as defined in a) and/or a
polyamino acid as defined in b) and/or a polyamino acid as defined
in c); and/or e) is a conjugate comprising a polyhydroxypolymer
backbone to which is separately coupled 1) the foreign T.sub.H
epitope and 2) a polyamino acid selected from the group consisting
of a subsequence as defined in a), a disrupted sequence of APP or
A.beta. as defined in b), and an APP or A.beta. derived amino acid
sequence that comprises 1 single methionine residue located in the
C-terminus, wherein other methionine residues in APP or A.beta. and
in the foreign T.sub.H epitope have been substituted or deleted,
and preferably have been substituted by leucin or isoleucine.
2. The method according to claim 1, wherein a substantial fraction
of B-cell epitopes of APP or A.beta. are preserved in the analogue
and wherein at least one first moiety is introduced which effects
targeting of the analogue to an antigen presenting cell (APC) or a
B-lymphocyte, and/or at least one second moiety is introduced which
stimulates the immune system, and/or at least one third moiety is
introduced which optimizes presentation of the analogue to the
immune system.
3. The method according to claim 2, wherein the first and/or of the
second and/or of the third moiety is/are attached as side groups by
covalent or non-covalent binding to suitable chemical groups in the
APP or A.beta. sequence.
4. The method according to claim 1, wherein the analogue comprises
a fusion polypeptide.
5. The method according to claim 1, wherein introduction of the
amino acid substitution and/or deletion and/or insertion and/or
addition results in a substantial preservation of the overall
tertiary structure of APP or A.beta..
6. The method according to claim 1, wherein the analogue includes
duplication of at least one B-cell epitope of APP or A.beta. and/or
introduction of a hapten.
7. The method according to claim 1, wherein the foreign T-cell
epitope is immunodominant in the animal.
8. The method according to claim 1, wherein the foreign T-cell
epitope is promiscuous, such as a foreign T-cell epitope which is
selected from a natural promiscuous T-cell epitope and an
artificial MHC-II binding peptide sequence.
9. The method according to claim 8, wherein the natural T-cell
epitope is selected from a Tetanus toxoid epitope such as P2 or
P30, a diphtheria toxoid epitope, an influenza virus hemagluttinin
epitope, and a P. falciparum CS epitope.
10. The method according to claim 1, wherein the analogue comprises
B-cell epitopes which are not exposed to the extracellular phase
when present in a cell-bound form of the precursor polypeptide
A.beta..
11. The method according to claim 1, wherein the analogue lacks at
least one B-cell epitope which is exposed to the extracellular
phase when present in a cell-bound form of the precursor
polypeptide.
12. The method according to claim 1, wherein the analogue comprises
at most 9 consecutive amino acids of SEQ ID NO: 2.
13. The method according to claim 12, wherein the analogue
comprises at least one subsequence of SEQ ID NO: 2 so that each
such at least one subsequence of SEQ ID NO: 2 independently
consists of amino acid stretches selected from the group consisting
of 9 consecutive amino acids of SEQ ID NO: 2, 8 consecutive amino
acids of SEQ ID NO: 2, 7 consecutive amino acids of SEQ ID NO: 2, 6
consecutive amino acids of SEQ ID NO: 2, 5 consecutive amino acids
of SEQ ID NO: 2, 4 consecutive amino acids of SEQ ID NO: 2, and 3
consecutive amino acids of SEQ ID NO: 2.
14. The method according to claim 12, wherein the consecutive amino
acids begin at an amino acid residue selected from the group
consisting of residue 672, 673, 674, 675, 676, 677, 678, 679, 680,
681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693,
694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706,
707, 708, 709, 710, 711, 712, 713, and 714.
15. The method according to claim 1, wherein presentation to the
immune system is effected by having at least two copies of an
A.beta. derived fragment or the analogue covalently of
non-covalently linked to a carrier molecule capable of effecting
presentation of multiple copies of antigenic determinants.
16. The method according to claim 1, wherein the polyamino acid and
T.sub.H epitope are attached to the polyhydroxypolymer by means of
an amide bond.
17. The method according to claim 1, wherein the the
polyhydroxypolymer is a polysaccharide.
18. The method according to claim 1, wherein the analogue has been
formulated with an adjuvant which facilitates breaking of
autotolerance to autoantigens.
19. The method according to claim 1, wherein an effective amount of
the analogue is administered to the animal via a route selected
from a parenteral route, and the intramuscular routes; the
peritoneal route; the oral route; the buccal route; the sublinqual
route; the epidural route; the spinal route; the anal route; and
the intracranial route.
20. The method according to claim 19, wherein the parenteral route
is intracutaneous or subcutaneous administration.
21. The method according to claim 19, wherein the effective amount
is between 0.5 .mu.g and 2,000 .mu.g of the analogue.
22. The method according to claim 1, wherein presentation of the
analogue to the immune system is effected by introducing one or
more nucleic acids encoding the analogue into the animal's cells
and thereby obtaining in vivo expression by the cells of the one or
more nucleic acids introduced.
23. The method according to claim 22, wherein the one or more
nucleic acids introduced are selected from naked DNA, DNA
formulated with charged or uncharged lipids, DNA formulated in
liposomes, DNA included in a viral vector, DNA formulated with a
transfection-facilitating protein or polypeptide, DNA formulated
with a targeting protein or polypeptide, DNA formulated with
Calcium precipitating agents, DNA coupled to an inert carrier
molecule, DNA encapsulated in chitin or chitosan, and DNA
formulated with an adjuvant.
24. The method according to claim 19, wherein an effective amount
of the analogue is administered at a frequency of at least one
administration or introduction per year.
25. A method for treating and/or preventing and/or ameliorating
Alzheimer's disease or other diseases and conditions characterized
by amyloid deposits, the method comprising down-regulating APP or
A.beta. according to the method of any one of the preceding claims
to such an extent that the total amount of amyloid is decreased or
that the rate of amyloid formation is reduced with clinical
significance.
26. An analogue of APP or A.beta. which is derived from an animal
APP or A.beta. wherein is introduced a modification which has as a
result that immunization of the animal with the analogue induces
production of antibodies against the animal's autologous APP or
A.beta., and wherein the analogue is as defined claim 1.
27. An immunogenic composition comprising an immunogenically
effective amount of an analogue according to claim 26, the
composition further comprising a pharmaceutically and
immunologically acceptable carrier and/or vehicle and optionally an
adjuvant.
28. A nucleic acid fragment which encodes an analogue according to
claim 26.
29. A vector carrying the nucleic acid fragment according to claim
28, such as a vector that is capable of autonomous replication.
30. The vector according to claim 29 which is selected from the
group consisting of a plasmid, a phage, a cosmid, a
mini-chromosome, and a virus.
31. The vector according to claim 29, comprising, in the
5'.fwdarw.3' direction and in operable linkage, a promoter for
driving expression of the nucleic acid fragment according to claim
28, optionally a nucleic acid sequence encoding a leader peptide
enabling secretion of or integration into the membrane of the
polypeptide fragment, the nucleic acid fragment according to claim
28, and optionally a terminator.
32. The vector according to claim 29 wherein, when introduced into
a host cell, the vector is capable or incapable of being integrated
in the host cell genome.
33. The vector according to claim 31, wherein the promoter drives
expression in a eukaryotic cell and/or in a prokaryotic cell.
34. A transformed cell carrying the vector of claim 29.
35. The transformed cell of claim 34, wherein the cell is capable
of replicating the nucleic acid fragment according to claim 28.
36. The transformed cell according to claim 34, wherein the cell is
a microorganism selected from a bacterium, a yeast, a protozoan, or
a cell derived from a multicellular organism selected from a
fungus, an insect cell, an insect S.sub.2 or SF cell, a plant cell,
and a mammalian cell.
37. The transformed cell according to claim 34, wherein the cell
expresses the nucleic acid fragment according to claim 29.
38. The transformed cell of claim 37, wherein the cell secretes or
carries on its surface the analogue according to claim 26.
39. The method according to claim 1, wherein presentation to the
immune system is effected by administering a non-pathogenic
microorganism or virus which is carrying a nucleic acid fragment
which encodes and expresses the analogue.
40. A composition for inducing production of antibodies against
amyloid, the composition comprising a nucleic acid fragment
according to claim 28 or a vector according to claim 29, and a
pharmaceutically and immunologically acceptable carrier and/or
vehicle and/or adjuvant.
41. A stable cell line which carries the vector according to claim
29 and which expresses the nucleic acid fragment according to claim
28, and which optionally secretes or carries the analogue according
to claim 26 on its surface.
42. The method of claim 1, wherein the animal is a human.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to improvements in therapy and
prevention of Alzheimer's disease (AD) and other diseases
characterized by deposition of amyloid, e.g. characterized by
amyloid deposits in the central nervous system (CNS). More
specifically, the present invention provides a method for
down-regulating (undesired) deposits of amyloid by enabling the
production of antibodies against a relevant protein (APP or
A.beta.) or components thereof in subjects suffering from or in
danger of suffering from diseases having a pathology involving
amyloid deposition. The invention also provides for methods of
producing polypeptides useful in this method as well as for the
modified polypeptides as such. Also encompassed by the present
invention are nucleic acid fragments encoding the modified
polypeptides as well as vectors incorporating these nucleic acid
fragments and host cells and cell lines transformed therewith.
Finally, the present invention also provides for a new type of
conjugate peptide immunogen.
BACKGROUND OF THE INVENTION
[0002] Amyloidosis is the extracellular deposition of insoluble
protein fibrils leading to tissue damage and disease (Pepys, 1996;
Tan et al., 1995; Kelly, 1996). The fibrils form when normally
soluble proteins and peptides self-associate in an abnormal manner
(Kelly, 1997).
[0003] Amyloid is associated with serious diseases including
systemic amyloidosis, AD, maturity onset diabetes, Parkinson's
disease, Huntington's disease, fronto-temporal dementia and the
prion-related transmissible spongiform encephalopathies (kuru and
Creutzfeldt-Jacob disease in humans and scrapie and BSE in sheep
and cattle, respectively) and the amyloid plaque formation in for
instance Alzheimer's seems to be closely associated with the
progression of human disease. In animal models over-expression, or
the expression of modified forms, of proteins found in deposits,
like the .beta.-amyloid protein, has been shown to induce various
symptoms of disease, e.g. Alzheimer's-like symptoms. There is no
specific treatment for amyloid deposition and these diseases are
usually fatal.
[0004] The subunits of amyloid fibrils may be wild-type, variant or
truncated proteins, and similar fibrils can be formed in vitro from
oligopeptides and denatured proteins (Bradbury et al., 1960;
Filshie et al., 1964; Burke & Rougvie, 1972). The nature of the
polypeptide component of the fibrils defines the character of the
amyloidosis. Despite large differences in the size, native
structure and function of amyloid proteins, all amyloid fibrils are
of indeterminate length, unbranched, 70 to 120 .ANG. in diameter,
and display characteristic staining with Congo Red (Pepys, 1996).
They are characteristic of a cross-.beta. structure (Pauling &
Corey, 1951) in which the polypeptide chain is organized in
.beta.-sheets. Although the amyloid proteins have very different
precursor structures, they can all undergo a structural conversion,
perhaps along a similar pathway, to a misfolded form that is the
building block of the .beta.-sheet helix protofilament.
[0005] This distinctive fibre pattern led to the amyloidoses being
called the .beta.-fibrilloses (Glenner, 1980a,b), and the fibril
protein of AD was named the .beta.-protein before its secondary
structure was known (Glenner & Wong, 1984). The characteristic
cross-.beta. diffraction pattern, together with the fibril
appearance and tinctorial properties are now the accepted
diagnostic hallmarks of amyloid, and suggest that the fibrils,
although formed from quite different protein precursors, share a
degree of structural similarity and comprise a structural
super-family, irrespective of the nature of their precursor
proteins (Sunde M, Serpell L C, Bartlam M, Fraser P E, Pepys M B,
Blake CCFJ Mol Biol Oct. 31, 1997; 273(3):729-739).
[0006] One of the most widespread and well-known diseases where
amyloid deposits in the central nervus system are suggested to have
a central role in the progression of the disease, is AD.
[0007] AD
[0008] Alzheimer's disease (AD) is an irreversible, progressive
brain disorder that occurs gradually and results in memory loss,
behavioural and personality changes, and a decline in mental
abilities. These losses are related to the death of brain cells and
the breakdown of the connections between them. The course of this
disease varies from person to person, as does the rate of decline.
On average, AD patients live for 8 to 10 years after they are
diagnosed, though the disease can last for up to 20 years.
[0009] AD advances by stages, from early, mild forgetfulness to a
severe loss of mental function. This loss is known as dementia. In
most people with AD, symptoms first appear after the age of 60, but
earlier onsets are not infrequent. The earliest symptoms often
include loss of recent memory, faulty judgment, and changes in
personality. Often, people in the initial stages of AD think less
clearly and forget the names of familiar people and common objects.
Later in the disease, they may forget how to do even simple tasks.
Eventually, people with AD lose all reasoning ability and become
dependent on other people for their everyday care. Ultimately, the
disease becomes so debilitating that patients are bedridden and
likely to develop other illnesses and infections. Most commonly,
people with AD die from pneumonia.
[0010] Although the risk of developing AD increases with age, AD
and dementia symptoms are not a part of normal aging. AD and other
dementing disorders are caused by diseases that affect the brain.
In normal aging, nerve cells in the brain are not lost in large
numbers. In contrast, AD disrupts three key processes: Nerve cell
communication, metabolism, and repair. This disruption ultimately
causes many nerve cells to stop functioning, lose connections with
other nerve cells, and die.
[0011] At first, AD destroys neurons in parts of the brain that
control memory, especially in the hippocampus and related
structures. As nerve cells in the hippocampus stop functioning
properly, short-term memory fails, and often, a person's ability to
do easy and familiar tasks begins to decline. AD also attacks the
cerebral cortex, particularly the areas responsible for language
and reasoning. Eventually, many other areas of the brain are
involved, all these brain regions atrophy (shrink), and the AD
patient becomes bedridden, incontinent, totally helpless, and
unresponsive to the outside world (source: National Institute on
Aging Progress Report on Alzheimer's Disease, 1999).
[0012] The Impact of AD
[0013] AD is the most common cause of dementia among people age 65
and older. It presents a major health problem because of its
enormous impact on individuals, families, the health care system,
and society as a whole. Scientists estimate that up to 4 million
people currently suffer from the disease, and the prevalence
doubles every 5 years beyond age 65. It is also estimated that
approximately 360,000 new cases (incidence) will occur each year,
though this number will increase as the population ages (Brookmeyer
et al., 1998).
[0014] AD puts a heavy economic burden on society. A recent study
in the United States estimated that the annual cost of caring for
one AD patient is $18,408 for a patient with mild AD, $30,096 for a
patient with moderate AD, and $36,132 for a patient with severe AD.
The annual national cost of caring for AD patients in the US is
estimated to be slightly over $50 billion (Leon et al., 1998).
[0015] Approximately 4 million Americans are 85 or older, and in
most industrialized countries, this age group is one of the fastest
growing segments of the population. It is estimated that this group
will number nearly 8.5 million by the year 2030 in the US; some
experts who study population trends suggest that the number could
be even greater. As more and more people live longer, the number of
people affected by diseases of aging, including AD, will continue
to grow. For example, some studies show that nearly half of all
people age 85 and older have some form of dementia. (National
Institute on Aging Progress Report on Alzheimer's Disease,
1999)
[0016] The Main Characteristics of AD
[0017] Two abnormal structures in the brain are the hallmarks of
AD: amyloid plaques and neurofibrillary tangles (NFT). Plaques are
dense, largely insoluble deposits of protein and cellular material
outside and around the brain's neurons. Tangles are insoluble
twisted fibres that build up inside neurons.
[0018] Two types of AD exist: familial AD (FAD), which follows a
certain pattern of inheritance, and sporadic AD, where no obvious
pattern of inheritance is seen. Because of differences in the age
at onset, AD is further described as early-onset (occurring in
people younger than 65) or late-onset (occurring in those 65 and
older). Early-onset AD is rare (about 10 percent of cases) and
generally affects people aged 30 to 60. Some forms of early-onset
AD are inherited and run in families. Early-onset AD also often
progresses faster than the more common, late-onset form.
[0019] All FADs known so far have an early onset, and as many as 50
percent of FAD cases are now known to be caused by defects in three
genes located on three different chromosomes. These are mutations
in the APP gene on chromosome 21; mutations in a gene on
chromosomefl4, called presenilin 1; and mutations in a gene on
chromosome 1, called presenilin 2. There is as yet no evidence,
however, that any of these mutations play a major role in the more
common, sporadic or non-familial form of late-onset AD. (National
Institute on Aging Progress Report on Alzheimer's Disease,
1999)
[0020] Amyloid Plaques
[0021] In AD, amyloid plaques develop first in areas of the brain
used for memory and other cognitive functions. They consist of
largely insoluble deposits of beta amyloid (hereinafter designated
A.beta.)--a protein fragment of a larger protein called amyloid
precursor protein (APP, the amino acid sequence of which is set
forth in SEQ ID NO: 2)--intermingled with portions of neurons and
with non-nerve cells such as microglia and astrocytes. It is not
known whether amyloid plaques themselves constitute the main cause
of AD or whether they are a by-product of the AD process.
Certainly, changes in the APP protein can cause AD, as shown in the
inherited form of AD caused by mutations in the APP gene, and
A.beta. plaque formation seems to be closely associated with the
progression of the human disease (Lippa C. F. et al. 1998).
[0022] APP
[0023] APP is one of many proteins that are associated with cell
membranes. After it is made, APP becomes embedded in the nerve
cell's membrane, partly inside and partly outside the cell. Recent
studies using transgenic mice demonstrate that APP appears to play
an important role in the growth and survival of neurons. For
example, certain forms and amounts of APP may protect neurons
against both short- and long-term damage and may render damaged
neurons better able to repair themselves and help parts of neurons
grow after brain injury.
[0024] While APP is embedded in the cell membrane, proteases act on
particular sites in APP, cleaving it into protein fragments. One
protease helps cleave APP to form A.beta., and another protease
cleaves APP in the middle of the amyloid fragment so that A.beta.
cannot be formed. The A.beta. formed is of two different lengths, a
shorter 40 (or 41) amino acids A.beta. that is relatively soluble
and aggregates slowly, and a slightly longer, 42 amino acids
"sticky" A.beta. that rapidly forms insoluble clumps. While A.beta.
is being formed, it is not yet known exactly how it moves through
or around nerve cells. In the final stages of this process, the
"sticky" A.beta. aggregates into long filaments outside the cell
and, along with fragments of dead and dying neurons and the
microglia and astrocytes, forms the plaques that are characteristic
of AD in brain tissue.
[0025] Some evidence exists that the mutations in APP render more
likely that A.beta. will be snipped out of the APP precursor, thus
causing either more total A.beta. or relatively more of the
"sticky" form to be made. It also appears that mutations in the
presenilin genes may contribute to the degeneration of neurons in
at least two ways: By modifying A.beta. production or by triggering
the death of cells more directly. Other researchers suggest that
mutated presenilins 1 and 2 may be involved in accelerating the
pace of apoptosis.
[0026] It is to be expected that as the disease progresses, more
and more plaques will be formed, filling more and more of the
brain. Studies suggest that it may be that the A.beta. is
aggregating and disaggregating at the same time, in a sort of
dynamic equilibrium. This raises the hope that it may be possible
to break down the plaques even after they have formed. (National
Institute on Aging Progress Report on Alzheimer's Disease,
1999).
[0027] It is believed that A.beta. is toxic to neurons. In tissue
culture studies, researchers observed an increase in death of
hippocampal neurons cells engineered to over-express mutated forms
of human APP compared to neurons over-expressing the normal human
APP (Luo et al., 1999).
[0028] Furthermore, overexpression or the expression of modified
forms of the A.beta. protein has in animal models been demonstrated
to induce Alzheimer-like symptoms, (Hsiao K. et al., 1998)
[0029] Given that increased A.beta. generation, its aggregation
into plaques, and the resulting neurotoxicity may lead to AD, it is
of therapeutic interest to investigate conditions under which
A.beta. aggregation into plaques might be slowed down or even
blocked.
[0030] Presenilins
[0031] Mutations in presenilin-1 (S-180) account for almost 50% of
all cases of early-onset familial AD (FAD). Around 30 mutations
have been identified that give rise to AD. The onset of AD varies
with the mutations. Mutations in presenilin-2 account for a much
smaller part of the cases of FAD, but is still a significant
factor. It is not known whether presenilins are involved in
sporadic non-familial AD. The function of the presenilins is not
known, but they appear to be involved in the processing of APP to
give A.beta.-42 (the longer stickier form of the peptide, SEQ ID
NO: 2, residues 673-714), since AD patients with presenilin
mutations have increased levels of this peptide. It is unclear
whether the presenilins also have a role in causing the generation
of NFT's. Some suggest that presenilins could also have a more
direct role in the degeneration of neurons and neuron death.
Presenilin-1 is located at chromosome 14 while presenilin-2 is
linked to chromosome 1. If a person harbours a mutated version of
just one of these genes he or she is almost certain to develop
early onset AD.
[0032] There is some uncertainty to whether presenilin-1 is
identical to the hypothetical gamma-secretase involved in the
processing of APP (Naruse et al., 1998).
[0033] Apolipoprotein E
[0034] Apolipoprotein E is usually associated with cholesterol, but
is also found in plaques and tangles of AD brains. While alleles
1-3 do not seem to be involved in AD there is a significant
correlation between the presence of the APOE-.epsilon.4 allele and
development of late AD (Strittmatter et al., 1993). It is, however,
a risk factor and not a direct cause as is the case for the
presenilin and APP mutations and it is not limited to familial
AD.
[0035] The ways in which the ApoE .epsilon.4 protein increases the
likelihood of developing AD are not known with certainty, but one
possible theory is that it facilitates A.beta. buildup and this
contributes to lowering the age of onset of AD, or the presence or
absence of particular APOE alleles may affect the way neurons
respond to injury (Buttini et al., 1999).
[0036] Also Apo A1 has been shown to be amyloigenic. Intact apo A1
can itself form amyloid-like fibrils in vitro that are Congo red
positive (Am J Pathol 147 (2): 238-244 (Aug 1995), Wisniewski T,
Golabek A A, Kida E, Wisniewski K E, Frangione B).
[0037] There seem to be some contradictory results indicating that
there is a positive effect of the APOE-.epsilon.4 allele in
decreasing symptoms of mental loss, compared to other alleles
(Stern, Brandt, 1997, Annals of Neurology 41).
[0038] Neurofibrillary Tangles
[0039] This second hallmark of AD consists of abnormal collections
of twisted threads found inside nerve cells. The chief component of
tangles is one form of a protein called tau (.tau.). In the central
nervous system, tau proteins are best known for their ability to
bind and help stabilize microtubules, which are one constituent of
the cell's internal support structure, or skeleton. However, in AD
tau is changed chemically, and this altered tau can no longer
stabilize the microtubules, causing them to fall disintegrate. This
collapse of the transport system may at first result in
malfunctions in communication between nerve cells and may later
lead to neuronal death.
[0040] In AD, chemically altered tau twists into paired helical
filaments--two threads of tau that are wound around each other.
These filaments are the major substance found in neurofibrillary
tangles. In one recent study, researchers found neurofibrillary
changes in fewer than 6 percent of the neurons in a particular part
of the hippocampus in healthy brains, in more than 43 percent of
these neurons in people who died with mild AD, and in 71 percent of
these neurons in people who died with severe AD. When the loss of
neurons was studied, a similar progression was found. Evidence of
this type supports the idea that the formation of tangles and the
loss of neurons progress together over the course of AD. (National
Institute on Aging Progress Report on Alzheimer's Disease,
1999).
[0041] Tauopathies and Tangles
[0042] Several neurodegenerative diseases, other than AD, are
characterized by the aggregation of tau into insoluble filaments in
neurons and glia, leading to dysfunction and death. Very recently,
several groups of researchers, who were studying families with a
variety of hereditary dementias other than AD, found the first
mutations in the tau gene on chromosome 17 (Clark et al., 1998;
Hutton et al., 1998; Poorkaj et al., 1998; Spillantini et al.,
1998). In these families, mutations in the tau gene cause neuronal
cell death and dementia. These disorders which share some
characteristics with AD but differ in several important respects,
are collectively called "fronto temporal dementia and parkinsonism
linked to chromosome 17" (FTDP-17). They are diseases such as
Parkinson's disease, some forms of amyotrophic lateral sclerosis
(ALS), corticobasal degeneration, progressive supranuclear palsy,
and Pick's disease, and are all characterized by abnormal
aggregation of tau protein.
[0043] Other AD-Like Neurological Diseases.
[0044] There are important parallels between AD and other
neurological diseases, including prion diseases (such as kuru,
Creutzfeld-Jacob disease and bovine spongiform encephalitis),
Parkinson's disease, Huntington's disease, and fronto-temporal
dementia. All involve deposits of abnormal proteins in the brain.
AD and prion diseases cause dementia and death, and both are
associated with the formation of insoluble amyloid fibrils, but
from membrane proteins that are different from each other.
[0045] Scientists studying Parkinson's disease, the second most
common neurodegenerative disorder after AD, discovered the first
gene linked to the disease. This gene codes for a protein called
synuclein, which, intriguingly, is also found in the amyloid
plaques of AD patients' brains (Lavedan C, 1998, Genome Res. 8(9):
871-80). Investigators have also discovered that genetic defects in
Huntington's disease, another progressive neurodegenerative
disorder that causes dementia, cause the Huntington protein to form
into insoluble fibrils very similar to the A.beta. fibrils of AD
and the protein fibrils of prion disease, (Scherzinger E, et al.,
1999, PNAS U.S.A. 96(8): 4604-9).
[0046] Scientists have also discovered a novel gene, which when
mutated, is responsible for familial British dementia (FBD), a rare
inherited disease that causes severe movement disorders and
progressive dementia similar to that seen in AD. In a biochemical
analysis of the amyloid fibrils found in the FBD plaques, a unique
peptide named ABri was found (Vidal et al., 1999). A mutation at a
particular point along this gene resuits in the production of a
longer-than-normal Bri protein. The ABri peptide, which is snipped
from the mutated end of the Bri protein is deposited as amyloid
fibrils. These plaques are thought to lead to the neuronal
dysfunction and dementia that characterizes FBD.
[0047] Immunization with A.beta.
[0048] The immune system will normally take part in the clearing of
foreign protein and proteinaceous particles in the organism but the
deposits associated with the above-mentioned diseases consist
mainly of self-proteins, thereby rendering the role of the immune
system in the control of these diseases less obvious. Further, the
deposits are located in a compartment (the CNS) normally separated
from the immune system, both facts suggesting that any vaccine or
immunotherapeutical approach would be unsuccessful.
[0049] Nevertheless, scientists have recently attempted immunizing
mice with a vaccine composed of heterologous human A.beta. and a
substance known to excite the immune system (Schenk et al., 1999
and WO 99/27944). The vaccine was tested in a partial transgenic
mouse model of AD with a human mutated gene for APP inserted into
the DNA of the mouse. The mice produced the modified APP protein
and developed amyloid plaques as they grew older. This mouse model
was used to test whether vaccination against the modified
transgenic human APP had an effect on plaque build-up. In a first
experiment, one group of transgenic mice was given monthly
injections of the vaccine starting at 6 weeks of age and ending at
11 months. A second group of transgenic mice received no injections
and served as a control group. By 13 months of age, the mice in the
control group had plaques covering 2 to 6 percent of their brains.
In contrast, the immunized mice had virtually no plaques.
[0050] In a second experiment, the researchers began the injections
at 11 months, when some plaques had already developed. Over a
7-month period, the control transgenic mice had a 17-fold increase
in the amount of plaque in their brains, whereas those who received
the vaccine had a 99-percent decrease compared to the 18-month-old
control transgenic mice. In some mice, some of the pre-existing
plaque deposits appeared to have been removed by the treatment. It
was also found that other plaque-associated damage, such as
inflammation and abnormal nerve cell processes, lessened as a
result of the immunization.
[0051] The above is thus a preliminary study in mice and for
example, scientists need to find out whether vaccinated mice remain
healthy in other respects and whether memory of those vaccinated
remains normal. Furthermore, because the mouse model is not a
complete representation of AD (the animals do not develop
neurofibrillary tangles nor do many of their neurons die),
additional studies will be necessary to determine whether humans
have a similar or different reaction from mice. Another issue to
consider is that the method may perhaps "cure" amyloid deposition
but fail to stop development of dementia.
[0052] Technical issues present major challenges as well. For
example it is unlikely that it is even possible, using this
technology, to create a vaccine which enables humans to raise
antibodies against their own proteins. So numerous issues of safety
and effectiveness will need to be resolved before any tests in
humans can be considered.
[0053] The work by Schenk et al. thus shows that if it was possible
to generate a strong immune response towards self-proteins in
proteinaceous deposits in the central nervus system such as the
plaques formed in AD, it is possible to both prevent the formation
of the deposits and possibly also clear already formed plaques.
[0054] Recently, clinical trials using the above-discussed A.beta.
vaccines have been terminated due to adverse effects: A number of
the vaccinated subjects developed chronic encephalitis that may be
due to an uncontrolled autoimmunity against A.beta. in the CNS.
OBJECT OF THE INVENTION
[0055] The object of the present invention is to provide novel
therapies against conditions characterized by deposition of
amyloid, such as AD. A further object is to develop an autovaccine
against amyloid, in order to obtain a novel treatment for AD and
for other pathological disorders involving amyloid deposition.
SUMMARY OF THE INVENTION
[0056] Described herein is the use of an autovaccination technology
for generating strong immune responses against otherwise
non-immunogenic APP and A.beta. Described is also the preparation
of such vaccines for the prevention, possible cure or alleviation
of the symptoms of such diseases associated with amyloid
deposits.
[0057] Thus, in its broadest and most general scope, the present
invention relates to a method for in vivo down-regulation of
amyloid precursor protein (APP) or beta amyloid (A.beta.) in an
animal, including a human being, the method comprising effecting
presentation to the animal's immune system of an immunogenically
effective amount of at least one analogue of APP or A.beta. that
incorporates into the same molecule at least one B-cell epitope of
APP and/or A.beta. and at least one foreign T-helper epitope
(T.sub.H epitope) so that immunization of the animal with the
analogue induces production of antibodies against the animal's
autologous APP or A.beta., wherein the analogue
[0058] a) is a polyamino acid that consists of at least one copy of
a subsequence of residues 672-714 in SEQ ID NO: 2, wherein the
foreign T.sub.H epitope is incorporated by means of amino acid
addition and/or insertion and/or deletion and/or substitution,
wherein the subsequence is selected from the group consisting of
residues 1-42, residues 1-40, residues 1-39, residues 1-35,
residues 1-34, residues 1-28, residues 1-12, residues 1-5, residues
13-28, residues 13-35, residues 17-28, residues 25-35, residues
35-40, residues 36-42 and residues 35-42 of the amino acid sequence
consisting of amino acid residues 673-714 of SEQ ID NO: 2;
and/or
[0059] b) is a polyamino acid that contains the foreign T.sub.H
epitopes and a disrupted APP or A.beta. sequence so that the
analogue does not include any subsequence of SEQ ID NO: 2 that
binds productively to MHC class II molecules initiating a T-cell
response; and/or
[0060] c) is a polyamino acid that comprises the foreign T.sub.H
epitope and APP or A.beta. derived amino acids, and comprises 1
single methionine residue located in the C-terminus of the
analogue, wherein other methionine residues in APP or A.beta. and
in the foreign TH epitope have been substituted or deleted, and
preferably have been substituted by leucin or isoleucine;
and/or
[0061] d) is a conjugate comprising a polyhydroxypolymer backbone
to which is separately coupled a polyamino acid as defined in a)
and/or a polyamino acid as defined in b) and/or a polyamino acid as
defined in c); and/or
[0062] e) is a conjugate comprising a polyhydroxypolymer backbone
to which is separately coupled 1) the foreign T.sub.H epitope and
2) a polyamino acid selected from the group consisting of a
subsequence as defined in a), a disrupted sequence of APP or
A.beta. as defined in b), and an APP or A.beta. derived amino acid
sequence that comprises 1 single methionine residue located in the
C-terminus, wherein other methionine residues in APP or A.beta. and
in the foreign T.sub.H epitope have been substituted or deleted,
and preferably have been substituted by leucin or isoleucine.
[0063] The present assignee has previously filed an international
patent application directed to safe vaccination strategies against
amyloidogenic polypeptides such as APP and A.beta., cf. WO
01/62284. This application was not published on the filing date of
the present application and further does not contain details
concerning the above-mentioned useful analogues of APP and
A.beta..
[0064] The invention also relates to analogues of the APP and
A.beta. as well as to nucleic acid fragments encoding a subset of
these. Also immunogenic compositions comprising the analogues or
the nucleic acid fragments are part of the invention.
LEGEND TO THE FIGURE
[0065] FIG. 1: Schematic depiction of Autovac variants derived from
the amyloid precursor protein with the purpose of generating
antibody responses against the A.beta. protein A.beta.-43 (or
C-100). The APP is shown schematically at the top of the figure and
the remaining schematic constructs show that the model epitopes P2
and P30 are substituted or inserted into various truncations of
APP. In the figure, the black pattern indicates the APP signal
sequence, two-way cross-hatching is the extracellular part of APP,
dark vertical hatching is the transmembrane domain of APP, light
vertical hatching is the intracellular domain of APP, coarse
cross-hatching indicates the P30 epitope, and fine cross-hatching
indicates the P2 epitope. The full line box indicates A.beta.-42/43
and the full-line box and the dotted line box together indicate
C-100. "Abeta" denotes A.beta..
[0066] FIG. 2: Schematic depiction of an embodiment of the
synthesis of generally applicable immunogenic conjugates. Peptide A
(any antigenic sequence, e.g. an A.beta. sequence described herein)
and peptide B (an amino acid sequence including a foreign T-helper
epitope are synthesized and mixed. After that they are contacted
with a suitable activated polyhydroxypolymer, peptides A and B are
attached via the activation group in a ration corresponding to the
initial ratio between these two substances in the peptide mixture.
Cf. Example 4 for details.
DETAILED DISCLOSURE OF THE INVENTION
[0067] Definitions
[0068] In the following a number of terms used in the present
specification and claims will be defined and explained in detail in
order to clarify the metes and bounds of the invention.
[0069] The terms "amyloid" and "amyloid protein" which are used
interchangeably herein denote a class of proteinaceous unbranched
fibrils of indeterminate length. Amyloid fibrils display
characteristic staining with Congo Red and share a cross-.beta.
structure in which the polypeptide chain is organized in
.beta.-sheets. Amyloid is generally derived from amyloidogenic
proteins which have very different precursor structures but which
can all undergo a structural conversion to a misfolded form that is
the building block of the .beta.-sheet helix protofilament.
Normally, the diameter of amyloid fibrils varies between about 70
to about 120 .ANG..
[0070] The term "amyloidogenic protein" is intended to denote a
polypeptide which is involved in the formation of amyloid deposits,
either by being part of the deposits as such or by being part of
the biosynthetic pathway leading to the formation of the deposits.
Hence, examples of amyloidogenic proteins are APP and A.beta., but
also proteins involved in the metabolism of these may be
amyloidogenic proteins.
[0071] An "amyloid polypeptide" is herein intended to denote
polypeptides comprising the amino acid sequence of the
above-discussed amyloidogenic proteins derived from humans or other
mammals (or truncates thereof sharing a substantial amount of
B-cell epitopes with an intact amyloidogenic protein)--an
amyloidogenic polypeptide can therefore e.g. comprise substantial
parts of a precursor for the amyloidogenic polypeptide (in the case
of A.beta., one possible amyloid polypeptide could be APP derived).
Also unglycosylated forms of amyloidogenic polypeptides which are
prepared in prokaryotic system are included within the boundaries
of the term as are forms having varying glycosylation patterns due
to the use of e.g. yeasts or other non-mammalian eukaryotic
expression systems. It should, however, be noted that when using
the term "an amyloidogenic polypeptide" it is intended that the
polypeptide in question is normally non-immunogenic when presented
to the animal to be treated. In other words, the amyloidogenic
polypeptide is a self-protein or is an analogue of such a
self-protein which will not normally give rise to an immune
response against the amyloidogenic of the animal in question.
[0072] An "analogue" is an APP or A.beta. derived molecule that
incorporates one or several changes in its molecular structure.
Such a change can e.g. be in the form of fusion of APP or A.beta.
poly-amino acids to a suitable fusion partner (i.e. a change in
primary structure exclusively involving C- and/or N-terminal
additions of amino acid residues) and/or it can be in the form of
insertions and/or deletions and/or substitutions in the
polypeptide's amino acid sequence. Also encompassed by the term are
derivatized APP or A.beta. derived molecules, cf. the discussion
below of modifications of APP or A.beta.. In some cases the
analogue may be constructed so as to be less able or even unable to
elicit antibodies against the normal precursor protein(s) of the
amyloid, thereby avoiding undesired interference with the
(physiologically normal) non-aggregated form of the polypeptide
being a precursor of the amyloid protein.
[0073] It should be noted that the use as a vaccine in a human of a
xeno-analogue (e.g. a canine or porcine analogue) of a human APP or
A.beta.can be imagined to produce the desired immunity against the
APP or A.beta.. Such use of an xeno-analogue for immunization is
also considered part of the invention.
[0074] The term "polypeptide" is in the present context intended to
mean both short peptides of from 2 to 10 amino acid residues,
oligopeptides of from 11 to 100 amino acid residues, and
polypeptides of more than 100 amino acid residues. Furthermore, the
term is also intended to include proteins, i.e. functional
biomolecules comprising at least one polypeptide; when comprising
at least two polypeptides, these may form complexes, be covalently
linked, or may be non-covalently linked. The polypeptide(s) in a
protein can be glycosylated and/or lipidated and/or comprise
prosthetic groups. Also, the term "polyamino acid" is an equivalent
to the term "polypeptide"
[0075] The terms "T-lymphocyte" and "T-cell" will be used
interchangeably for lymphocytes of thymic origin which are
responsible for various cell mediated immune responses as well as
for helper activity in the humoral immune response. Likewise, the
terms "B-lymphocyte" and "B-cell" will be used interchangeably for
antibody-producing lymphocytes.
[0076] The term "subsequence" means any consecutive stretch of at
least 3 amino acids or, when relevant, of at least 3 nucleotides,
derived directly from a naturally occurring amyloid amino acid
sequence or nucleic acid sequence, respectively.
[0077] The term "animal" is in the present context in general
intended to denote an animal species (preferably mammalian), such
as Homo sapiens, Canis domesticus, etc. and not just one single
animal. However, the term also denotes a population of such an
animal species, since it is important that the individuals
immunized according to the method of the invention all harbour
substantially the same APP or A.beta. allowing for immunization of
the animals with the same immunogen(s). It will be clear to the
skilled person that an animal in the present context is a living
being which has an immune system. It is preferred that the animal
is a vertebrate, such as a mammal.
[0078] By the term "in vivo down-regulation of APP or A.beta." is
herein meant reduction in the living organism of the total amount
of deposited amyloid protein (or amyloid as such) of the relevant
type. The down-regulation can be obtained by means of several
mechanisms: of these, simple interference with amyloid by antibody
binding so as to prevent misaggregation is the most simple.
However, it is also within the scope of the present invention that
the antibody binding results in removal of amyloid by scavenger
cells (such as macrophages and other phagocytic cells) and that the
antibodies interfer with other amyloidogenic polypeptides which
lead to amyloid formation. A further possibility is that antibodies
bind A.beta. outside the CNS, thereby effectively removing A.beta.
from the CNS via a simple mass action principle.
[0079] The expression "effecting presentation . . . to the immune
system" is intended to denote that the animal's immune system is
subjected to an immunogenic challenge in a controlled manner. As
will appear from the disclosure below, such challenge of the immune
system can be effected in a number of ways of which the most
important are vaccination with polypeptide containing
"pharmaccines" (i.e. a vaccine which is administered to treat or
ameliorate ongoing disease) or nucleic acid "pharmaccine"
vaccination. The important result to achieve is that immune
competent cells in the animal are confronted with the antigen in an
immunologically effective manner, whereas the precise mode of
achieving this result is of less importance to the inventive idea
underlying the present invention.
[0080] The term "immunogenically effective amount" has its usual
meaning in the art, i.e. an amount of an immunogen, which is
capable of inducing an immune response that significantly engages
pathogenic agents sharing immunological features with the
immunogen.
[0081] When using the expression that the APP or A.beta. has been
"modified" is herein meant that a chemical modification of the
polypeptide has been performed on APP or A.beta.. Such a
modification can e.g. be derivatization (e.g. alkylation) of
certain amino acid residues in the sequence, but as will be
appreciated from the disclosure below, the preferred modifications
comprise changes of the primary structure of the amino acid
sequence.
[0082] When discussing "autotolerance towards APP or A.beta." it is
understood that since the polypeptide is a self-protein in the
population to be vaccinated, normal individuals in the population
do not mount an immune response against the polypeptide; it cannot
be excluded, though, that occasional individuals in an animal
population might be able to produce antibodies against the native
polypeptide, e.g. as part of an autoimmune disorder. At any rate,
an animal will normally only be autotolerant towards its own APP or
A.beta., but it cannot be excluded that analogues derived from
other animal species or from a population having a different
phenotype would also be tolerated by said animal.
[0083] A "foreign T-cell epitope" (or: "foreign T-lymphocyte
epitope") is a peptide which is able to bind to an MHC molecule and
which stimulates T-cells in an animal species. Preferred foreign
T-cell epitopes in the invention are "promiscuous" epitopes, i.e.
epitopes which bind to a substantial fraction of a particular class
of MHC molecules in an animal species or population. Only a very
limited number of such promiscuous T-cell epitopes are known, and
they will be discussed in detail below. Promiscuous T-cell epitopes
are also denoted "universal" T-cell epitopes. It should be noted
that in order for the immunogens which are used according to the
present invention to be effective in as large a fraction of an
animal population as possible, it may be necessary to 1) insert
several foreign T-cell epitopes in the same analogue or 2) prepare
several analogues wherein each analogue has a different promiscuous
epitope inserted. It should be noted also that the concept of
foreign T-cell epitopes also encompasses use of cryptic T-cell
epitopes, i.e. epitopes which are derived from a self-protein and
which only exerts immunogenic behaviour when existing in isolated
form without being part of the self-protein in question.
[0084] A "foreign T helper lymphocyte epitope" (a foreign T.sub.H
epitope) is a foreign T cell epitope, which binds an MHC Class II
molecule and can be presented on the surface of an antigen
presenting cell (APC) bound to the MHC Class II molecule.
[0085] A "functional part" of a (bio)molecule is in the present
context intended to mean the part of the molecule which is
responsible for at least one of the biochemical or physiological
effects exerted by the molecule. It is well-known in the art that
many enzymes and other effector molecules have an active site which
is responsible for the effects exerted by the molecule in question.
Other parts of the molecule may serve a stabilizing or solubility
enhancing purpose and can therefore be left out if these purposes
are not of relevance in the context of a certain embodiment of the
present invention. For instance it is possible to use certain
cytokines as a modifying moiety in APP or A.beta. (cf. the detailed
discussion below), and in such a case, the issue of stability may
be irrelevant since the coupling to the APP or Apmay provide the
stability necessary.
[0086] The term "adjuvant" has its usual meaning in the art of
vaccine technology, i.e. a substance or a composition of matter
which is 1) not in itself capable of mounting a specific immune
response against the immunogen of the vaccine, but which is 2)
nevertheless capable of enhancing the immune response against the
immunogen. Or, in other words, vaccination with the adjuvant alone
does not provide an immune response against the immunogen,
vaccination with the immunogen may or may not give rise to an
immune response against the immunogen, but the combined vaccination
with immunogen and adjuvant induces an immune response against the
immunogen which is stronger than that induced by the immunogen
alone.
[0087] "Targeting" of a molecule is in the present context intended
to denote the situation where a molecule upon introduction in the
animal will appear preferentially in certain tissue(s) or will be
preferentially associated with certain cells or cell types. The
effect can be accomplished in a number of ways including
formulation of the molecule in composition facilitating targeting
or by introduction in the molecule of groups, which facilitate
targeting. These issues will be discussed in detail below.
[0088] "Stimulation of the immune system" means that a substance or
composition of matter exhibits a general, non-specific
immunostimulatory effect. A number of adjuvants and putative
adjuvants (such as certain cytokines) share the ability to
stimulate the immune system. The result of using an
immunostimulating agent is an increased "alertness" of the immune
system meaning that simultaneous or subsequent immunization with an
immunogen induces a significantly more effective immune response
compared to isolated use of the immunogen.
[0089] "Productive binding" means binding of a peptide to the MHC
molecule (Class I or II) so as to be able to stimulate T-cells that
engage a cell that present the peptide bound to the MHC molecule.
For instance, a peptide bound to an MHC Class II molecule on the
surface of an APC is said to be productively bound if this APC will
stimulate a T.sub.H cell that binds to the presented peptide-MHC
Class II complex.
[0090] Preferred Embodiments of Amyloid Down-Regulation
[0091] It is preferred that the analogue used as an immunogen in
the method of the invention is a modified APP or A.beta. molecule
wherein at least one change is present in the amino acid sequence
of the APP or A.beta., since the chances of obtaining the
all-important breaking of autotolerance is greatly facilitated that
way--this is e.g. evident from the results presented in Example 2
herein, where immunization with wild-type A.beta. is compared to
immunization with an A.beta. variant molecule. It has been shown
(in Dalum I et al., 1996, J. Immunol. 157: 4796-4804) that
potentially self-reactive B-lymphocytes recognizing self-proteins
are physiologically present in normal individuals. However, in
order for these B-lymphocytes to be induced to actually produce
antibodies reactive with the relevant self-proteins, assistance is
needed from cytokine producing T-helper lymphocytes (T.sub.H-cells
or T.sub.H-lymphocytes). Normally this help is not provided because
T-lymphocytes in general do not recognize T-cell epitopes derived
from self-proteins when presented by antigen presenting cells
(APCs). However, by providing an element of "foreignness" in a
self-protein (i.e. by introducing an immunologically significant
modification), T-cells recognizing the foreign element are
activated upon recognizing the foreign epitope on an APC (such as,
initially, a mononuclear cell). Polyclonal B-lymphocytes (which are
also APCs) capable of recognising self-epitopes on the modified
self-protein also internalise the antigen and subsequently presents
the foreign T-cell epitope(s) thereof, and the activated
T-lymphocytes subsequently provide cytokine help to these
self-reactive polyclonal B-lymphocytes. Since the antibodies
produced by these polyclonal B-lymphocytes are reactive with
different epitopes on the modified polypeptide, including those
which are also present in the native polypeptide, an antibody
cross-reactive with the non-modified self-protein is induced. In
conclusion, the T-lymphocytes can be led to act as if the
population of polyclonal B-lymphocytes have recognised an entirely
foreign antigen, whereas in fact only the inserted epitope(s)
is/are foreign to the host. In this way, antibodies capable of
cross-reacting with non-modified self-antigens are induced.
[0092] Several ways of modifying a peptide self-antigen in order to
obtain breaking of autotolerance are known in the art. It is
nevertheless preferred that the analogue according to the present
invention includes
[0093] at least one first moiety is introduced which effects
targeting of the modified molecule to an antigen presenting cell
(APC), and/or
[0094] at least one second moiety is introduced which stimulates
the immune system, and/or
[0095] at least one third moiety is introduced which optimizes
presentation of the analogue to the immune system.
[0096] However, all these modifications should be carried out while
maintaining a substantial fraction of the original B-lymphocyte
epitopes in the APP or A.beta. since the B-lymphocyte recognition
of the native molecule is thereby enhanced.
[0097] In one preferred embodiment, side groups (in the form of the
foreign T-cell epitopes or the above-mentioned first, second and
third moieties) are covalently or non-covalently introduced. This
is to mean that stretches of amino acid residues derived from the
APP or AD are derivatized without altering the primary amino acid
sequence, or at least without introducing changes in the peptide
bonds between the individual amino acids in the chain.
[0098] An alternative, and preferred, embodiment utilises amino
acid substitution and/or deletion and/or insertion and/or addition
(which may be effected by recombinant means or by means of peptide
synthesis; modifications which involves longer stretches of amino
acids can give rise to fusion polypeptides). One especially
preferred version of this embodiment is the technique described in
WO 95/05849, which discloses a method for down-regulating
self-proteins by immunising with analogues of the self-proteins
wherein a number of amino acid sequence(s) has been substituted
with a corresponding number of amino acid sequence(s) which each
comprise a foreign immunodominant T-cell epitope, while at the same
time maintaining the overall tertiary structure of the self-protein
in the analogue. For the purposes of the present invention, it is
however sufficient if the modification (be it an insertion,
addition, deletion or substitution) gives rise to a foreign T-cell
epitope and at the same time preserves a substantial number of the
B-cell epitopes in the APP or A.beta.. However, in order to obtain
maximum efficacy of the immune response induced, it is preferred
that the overall tertiary structure of the APP or A.beta. is
maintained in the modified molecule.
[0099] In some cases, it is preferred that the APP or A.beta. or
fragments thereof are mutated. Especially preferred are
substitution variants where the methionine in position 35 in
A.beta.-43 has been substituted, preferably with leucine or
isoleucine, or simply deleted. Especially preferred analogues
contain one single methionine that is located in the C-terminus,
either because it is naturally occurring in the amyloidogenic
polypeptide or foreign T.sub.H epitope, or because it has been
inserted or added. Hence, it also preferred that the part of the
analogue that includes the foreign T.sub.H epitope is free from
methionine, except from the possible C-terminal location of a
methionine.
[0100] The main reason for removing all but one methionine is that
it becomes possible to recombinantly prepare multimeric analogues
that can be subsequently cleaved by cyanogenbromide to leave the
single analogues. The advantage is, that recombinant production
becomes facilitated this way.
[0101] In fact, it is generally preferred that all analogues of APP
or A.beta. that are used according to the present invention share
the characteristic of merely including one single methionine that
is positioned as the C-terminal amino acid in the analogue and that
other methionines in either the amyloidogenic polypeptide or the
foreign T.sub.H epitope are deleted or substituted for another
amino acid.
[0102] One further interesting mutation is a deletion or
substitution of the phenylalanine in position 19 in A.beta.-43, and
it is especially preferred that the mutation is a substitution of
this phenylalanine residue with a proline.
[0103] Other interesting polyamino acids to be used in the
analogues are truncated parts of the A.beta.-43 protein. These can
also be employed in immunogenic analogues according to the present
invention. Especially preferred are the truncates A.beta.(1-42),
A.beta.(1-40), A.beta.(1-39), A.beta.(1-35), A.beta.(1-34),
A.beta.(1-34), A.beta.(1-28), A.beta.(1-12), A.beta.(1-5),
A.beta.(13-28), A.beta.(13-35), A.beta.(17-28), A.beta.(25-35),
A.beta.(35-40), A.beta.(36-42), and A.beta.(35-42) (where the
numbers in the parentheses indicate the amino acid stretches of
A.beta.-43 that constitute the relevant fragment--A.beta.(35-40) is
e.g. identical to amino acids 706-711 in SEQ ID NO: 2). All these
variants with truncated parts of A.beta.-43 can be made with the
A.beta. fragments described herein, in particular with variants 9,
10, 11, 12, and 13 mentioned in Example 1.
[0104] The following formula describes the molecular constructs
generally covered by the invention:
(MOD.sub.1).sub.s1(amyloid.sub.e1).sub.n1(MOD.sub.2).sub.s2(amyloid.sub.e2-
).sub.n2 . . . (MOD.sub.x).sub.sx(amyloid.sub.ex).sub.nx (I)
[0105] where amyloid.sub.e1-amyloid.sub.ex are x B-cell epitope
containing sub-sequences of APP or A.beta. which independently are
identical or non-identical and which may contain or not contain
foreign side groups, x is an integer .gtoreq.3, n1-nx are x
integers .gtoreq.0 (at least one is .gtoreq.1), MOD.sub.1-MOD.sub.x
are x modifications introduced between the preserved B-cell
epitopes, and s.sub.1-s.sub.x are x integers .gtoreq.0 (at least
one is .gtoreq.1 if no side groups are introduced in the
amyloid.sub.ex sequences). Thus, given the general functional
restraints on the immunogenicity of the constructs, the invenion
allows for all kinds of permutations of the original seuence of the
APP or A.beta., and all kinds of modifications therein. Thus,
included in the invention are modified APP or A.beta. obtained by
omission of parts of the sequence which e.g. exhibit adverse
effects in vivo or omission of parts which are normally
intracellular and thus could give rise to undesired immunological
reactions.
[0106] One preferred version of the constructs outlined above are,
when applicable, those where the B-cell epitope containing
subsequence of an amyloid protein is not extracellularly exposed in
the precursor polypeptide from which the amyloid is derived. By
making such a choice of the epitopes, it is ensured that antibodies
are not generated which would be reactive with the cells producing
the precursor and thereby the immune response which is generated
becomes limited to an immune response against the undesired amyloid
deposits. In this case it will e.g. be feasible to induce immunity
against epitopes of APP or A.beta. which are only exposed to the
extracellular phase when being free from any coupling to the cells
from which they are produced.
[0107] Maintenance of a substantial fraction of B-cell epitopes or
even the overall tertiary structure of a protein which is subjected
to modification as described herein can be achieved in several
ways. One is simply to prepare a polyclonal antiserum directed
against the polypeptide in question (e.g. an antiserum prepared in
a rabbit) and thereafter use this antiserum as a test reagent (e.g.
in a competitive ELISA) against the modified proteins which are
produced. Modified versions (analogues) which react to the same
extent with the antiserum as does the APP or A.beta. must be
regarded as having the same overall tertiary structure as APP or
A.beta. whereas analogues exhibiting a limited (but still
significant and specific) reactivity with such an antiserum are
regarded as having maintained a substantial fraction of the
original B-cell epitopes.
[0108] Alternatively, a selection of monoclonal antibodies reactive
with distinct epitopes on the APP or A.beta. can be prepared and
used as a test panel. This approach has the advantage of allowing
1) an epitope mapping of the APP or A.beta. and 2) a mapping of the
epitopes which are maintained in the analogues prepared.
[0109] Of course, a third approach would be to resolve the
3-dimensional structure of the APP or A.beta. or of a biologically
active truncate thereof (cf. above) and compare this to the
resolved three-dimensional structure of the analogues prepared.
Three-dimensional structure can be resolved by the aid of X-ray
diffraction studies and NMR-spectroscopy. Further information
relating to the tertiary structure can to some extent be obtained
from circular dichroism studies which have the advantage of merely
requiring the polypeptide in pure form (whereas X-ray diffraction
requires the provision of crystallized polypeptide and NMR requires
the provision of isotopic variants of the polypeptide) in order to
provide useful information about the tertiary structure of a given
molecule. However, ultimately X-ray diffraction and/or NMR are
necessary to obtain conclusive data since circular dichroism can
only provide indirect evidence of correct 3-dimensional structure
via information of secondary structure elements.
[0110] One preferred embodiment of the invention utilises multiple
presentations of B-lymphocyte epitopes of APP or A.beta. (i.e.
formula I wherein at least one B-cell epitope is present in two
positions). This effect can be achieved in various ways, e.g. by
simply preparing fusion polypeptides comprising the structure (APP
or A.beta. derived polypeptide).sub.m, where m is an integer
.gtoreq.2 and then introduce the modifications discussed herein in
at least one of the APP or A.beta. sequences. It is preferred that
the modifications introduced includes at least one duplication of a
B-lymphocyte epitope and/or the introduction of a hapten. These
embodiments including multiple presentations of selected epitopes
are especially preferred in situations where merely minor parts of
the APP or A.beta. are useful as constituents in a vaccine
agent.
[0111] As mentioned above, the introduction of a foreign T-cell
epitope can be accomplished by introduction of at least one amino
acid insertion, addition, deletion, or substitution. Of course, the
normal situation will be the introduction of more than one change
in the amino acid sequence (e.g. insertion of or substitution by a
complete T-cell epitope) but the important goal to reach is that
the analogue, when processed by an antigen presenting cell (APC),
will give rise to such a foreign immunodominant T-cell epitope
being presented in context of an MCH Class II molecule on the
surface of the APC. Thus, if the amino acid sequence of the APP or
A.beta. in appropriate positions comprises a number of amino acid
residues which can also be found in a foreign TH epitope then the
introduction of a foreign TH epitope can be accomplished by
providing the remaining amino acids of the foreign epitope by means
of amino acid insertion, addition, deletion and substitution. In
other words, it is not necessary to introduce a complete TH epitope
by insertion or substitution in order to fulfill the purpose of the
present invention.
[0112] It is preferred that the number of amino acid insertions,
deletions, substitutions or additions is at least 2, such as 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 25
insertions, substitutions, additions or deletions. It is
furthermore preferred that the number of amino acid insertions,
substitutions, additions or deletions is not in excess of 150, such
as at most 100, at most 90, at most 80, and at most 70. It is
especially preferred that the number of substitutions, insertions,
deletions, or additions does not exceed 60, and in particular the
number should not exceed 50 or even 40. Most preferred is a number
of not more than 30. With respect to amino acid additions, it
should be noted that these, when the resulting construct is in the
form of a fusion polypeptide, is often considerably higher than
150.
[0113] Preferred embodiments of the invention includes modification
by introducing at least one foreign immunodominant T-cell epitope.
It will be understood that the question of immune dominance of a
T-cell epitope depends on the animal species in question. As used
herein, the term "immunodominance" simply refers to epitopes which
in the vaccinated individual/population gives rise to a significant
immune response, but it is a well-known fact that a T-cell epitope
which is immunodominant in one individual/population is not
necessarily immunodominant in another individual of the same
species, even though it may be capable of binding MHC-II molecules
in the latter individual. Hence, for the purposes of the present
invention, an immune dominant T-cell epitope is a T-cell epitope
which will be effective in providing T-cell help when present in an
antigen. Typically, immune dominant T-cell epitopes has as an
inherent feature that they will substantially always be presented
bound to an MHC Class II molecule, irrespective of the polypeptide
wherein they appear.
[0114] Another important point is the issue of MHC restriction of
T-cell epitopes. In general, naturally occurring T-cell epitopes
are MHC restricted, i.e. a certain peptides constituting a T-cell
epitope will only bind effectively to a subset of MHC Class II
molecules. This in turn has the effect that in most cases the use
of one specific T-cell epitope will result in a vaccine component
which is only effective in a fraction of the population, and
depending on the size of that fraction, it can be necessary to
include more T-cell epitopes in the same molecule, or alternatively
prepare a multi-component vaccine wherein the components are
variants of APP or AP which are distinguished from each other by
the nature of the T-cell epitope introduced.
[0115] If the MHC restriction of the T-cells used is completely
unknown (for instance in a situation where the vaccinated animal
has a poorly defined MHC composition), the fraction of the
population covered by a specific vaccine composition can be
approximated by means of the following formula 1 f population = 1 -
i = 1 n ( 1 - p i ) ( II )
[0116] where p.sub.i is the frequency in the population of
responders to the i.sup.th foreign T-cell epitope present in the
vaccine composition, and n is the total number of foreign T-cell
epitopes in the vaccine composition. Thus, a vaccine composition
containing 3 foreign T-cell epitopes having response frequencies in
the population of 0.8, 0.7, and 0.6, respectively, would give
1-0.2.times.0.3.times.0.4=0.976
[0117] i.e. 97.6 percent of the population will statistically mount
an MHC-II mediated response to the vaccine.
[0118] The above formula does not apply in situations where a more
or less precise MHC restriction pattern of the peptides used is
known. If, for instance a certain peptide only binds the human
MHC-II molecules encoded by HLA-DR alleles DR1, DR3, DR5, and DR7,
then the use of this peptide together with another peptide which
binds the remaining MHC-II molecules encoded by HLA-DR alleles will
accomplish 100% coverage in the population in question. Likewise,
if the second peptide only binds DR3 and DR5, the addition of this
peptide will not increase the coverage at all. If one bases the
calculation of population response purely on MHC restriction of
T-cell epitopes in the vaccine, the minimum fraction of the
population covered by a specific vaccine composition can be
determined by means of the following formula: 2 f population = 1 -
j = 1 3 ( 1 - j ) 2 ( III )
[0119] wherein .phi..sub.j is the sum of frequencies in the
population of allelic haplotypes encoding MHC molecules which bind
any one of the T-cell epitopes in the vaccine and which belong to
the j.sup.th of the 3 known HLA loci (DP, DR and DQ); in practice,
it is first determined which MHC molecules will recognize each
T-cell epitope in the vaccine and thereafter these are listed by
type (DP, DR and DQ)--then, the individual frequencies of the
different listed allelic haplotypes are summed for each type,
thereby yielding .phi..sub.1, .phi..sub.2, and .phi..sub.3.
[0120] It may occur that the value Pi in formula II exceeds the
corresponding theoretical value .pi..sub.i: 3 i = 1 - j = 1 3 ( 1 -
j ) 2 ( IV )
[0121] wherein .nu..sub.j is the sum of frequencies in the
population of allelic haplotype encoding MHC molecules which bind
the i.sup.th T-cell epitope in the vaccine and which belong to the
j.sup.th of the 3 known HLA loci (DP, DR and DQ). This means that
in 1-.pi..sub.i of the population is a frequency of responders of
f.sub.residual.sub..sub.--.sub-
.i=(p.sub.i-.pi..sub.i)/(1-.pi..sub.i). Therefore, formula III can
be adjusted so as to yield formula V: 4 f population = 1 - j = 1 3
( 1 - j ) 2 + ( 1 - i = 1 n ( 1 - f residual_i ) ) ( V )
[0122] where the term 1-f.sub.residual-i is set to zero if
negative. It should be noted that formula V requires that all
epitopes have been haplotype mapped against identical sets of
haplotypes.
[0123] Therefore, when selecting T-cell epitopes to be introduced
in the analogue, it is important to include all knowledge of the
epitopes which is available: 1) The frequency of responders in the
population to each epitope, 2) MHC restriction data, and 3)
frequency in the population of the relevant haplotypes.
[0124] There exist a number of naturally occurring "promiscuous"
T-cell epitopes which are active in a large proportion of
individuals of an animal species or an animal population and these
are preferably introduced in the vaccine thereby reducing the need
for a very large number of different analogues in the same
vaccine.
[0125] The promiscuous epitope can according to the invention be a
naturally occurring human T-cell epitope such as epitopes from
tetanus toxoid (e.g. the P2 and P30 epitopes), diphtheria toxoid,
Influenza virus hemagluttinin (HA), and P. falciparum CS
antigen.
[0126] Over the years a number of other promiscuous T-cell epitopes
have been identified. Especially peptides capable of binding a
large proportion of HLA-DR molecules encoded by the different
HLA-DR alleles have been identified and these are all possible
T-cell epitopes to be introduced in the analogues used according to
the present invention. Cf. also the epitopes discussed in the
following references which are hereby all incorporated by reference
herein: WO 98/23635 (Frazer I H et al., assigned to The University
of Queensland); Southwood S et. al, 1998, J. Immunol. 160:
3363-3373; Sinigaglia F et al., 1988, Nature 336: 778-780; Chicz R
M et al., 1993, J. Exp. Med 178: 27-47; Hammer J et al., 1993, Cell
74: 197-203; and Falk K et al., 1994, Immunogenetics 39: 230-242.
The latter reference also deals with HLA-DQ and -DP ligands. All
epitopes listed in these 5 references are relevant as candidate
natural epitopes to be used in the present invention, as are
epitopes which share common motifs with these.
[0127] Alternatively, the epitope can be any artificial T-cell
epitope which is capable of binding a large proportion of MHC Class
II molecules. In this context the pan DR epitope peptides ("PADRE")
described in WO 95/07707 and in the corresponding paper Alexander J
et al., 1994, Immunity 1: 751-761 (both disclosures are
incorporated by reference herein) are interesting candidates for
epitopes to be used according to the present invention. It should
be noted that the most effective PADRE peptides disclosed in these
papers carry D-amino acids in the C- and N-termini in order to
improve stability when administered. However, the present invention
primarily aims at incorporating the relevant epitopes as part of
the analogue which should then subsequently be broken down
enzymatically inside the lysosomal compartment of APCs to allow
subsequent presentation in the context of an MHC-II molecule and
therefore it is not expedient to incorporate D-amino acids in the
epitopes used in the present invention.
[0128] One especially preferred PADRE peptide is the one having the
amino acid sequence AKFVAAWTLKAAA (SEQ ID NO: 17) or an
immuologically effective subsequence thereof. This, and other
epitopes having the same lack of MHC restriction are preferred
T-cell epitopes which should be present in the analogues used in
the inventive method. Such super-promiscuous epitopes will allow
for the most simple embodiments of the invention wherein only one
single analogue is presented to the vaccinated animal's immune
system.
[0129] As mentioned above, the modification of the APP or A.beta.
can also include the introduction of a first moiety which targets
the modified amyloidogenic polypeptide to an APC or a B-lymphocyte.
For instance, the first moiety can be a specific binding partner
for a B-lymphocyte specific surface antigen or for an APC specific
surface antigen. Many such specific surface antigens are known in
the art. For instance, the moiety can be a carbohydrate for which
there is a receptor on the B-lymphocyte or the APC (e.g. mannan or
mannose). Alternatively, the second moiety can be a hapten. Also an
antibody fragment which specifically recognizes a surface molecule
on APCs or lymphocytes can be used as a first moiety (the surface
molecule can e.g. be an FC.gamma. receptor of macrophages and
monocytes, such as FC.gamma.RI or, alternatively any other specific
surface marker such as CD40 or CTLA-4). It should be noted that all
these exemplary targeting molecules can be used as part of an
adjuvant also, cf. below.
[0130] As an alternative or supplement to targeting the analogue to
a certain cell type in order to achieve an enhanced immune
response, it is possible to increase the level of responsiveness of
the immune system by including the above-mentioned second moiety
which stimulates the immune system. Typical examples of such second
moieties are cytokines, and heat-shock proteins or molecular
chaperones, as well as effective parts thereof.
[0131] Suitable cytokines to be used according to the invention are
those which will normally also function as adjuvants in a vaccine
composition, i.e. for instance interferon .gamma. (IFN-.gamma.),
interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 4 (IL-4),
interleukin 6 (IL-6), interleukin 12 (IL-12), interleukin 13
(IL-13), interleukin 15 (IL-15), and granulocyte-macrophage colony
stimulating factor (GM-CSF); alternatively, the functional part of
the cytokine molecule may suffice as the second moiety. With
respect to the use of such cytokines as adjuvant substances, cf.
the discussion below.
[0132] According to the invention, suitable heat-shock proteins or
molecular chaperones used as the second moiety can be HSP70, HSP90,
HSC70, GRP94 (also known as gp96, cf. Wearsch P A et al. 1998,
Biochemistry 37: 5709-19), and CRT (calreticulin).
[0133] Alternatively, the second moiety can be a toxin, such as
listeriolycin (LLO), lipid A and heat-labile enterotoxin. Also, a
number of mycobacterial derivatives such as MDP (muramyl
dipeptide), CFA (complete Freund's adjuvant) and the trehalose
diesters TDM and TDE are interesting possibilities.
[0134] Also the possibility of introducing a third moiety which
enhances the presentation of the analogue to the immune system is
an important embodiment of the invention. The art has shown several
examples of this principle. For instance, it is known that the
palmitoyl lipidation anchor in the Borrelia burgdorferi protein
OspA can be utilised so as to provide self-adjuvating polypeptides
(cf. e.g. WO 96/40718)--it seems that the lipidated proteins form
up micelle-like structures with a core consisting of the lipidation
anchor parts of the polypeptides and the remaining parts of the
molecule protruding therefrom, resulting in multiple presentations
of the antigenic determinants. Hence, the use of this and related
approaches using different lipidation anchors (e.g. a myristyl
group, a myristyl group, a farnesyl group, a geranyl-geranyl group,
a GPI-anchor, and an N-acyl diglyceride group) are preferred
embodiments of the invention, especially since the provision of
such a lipidation anchor in a recombinantly produced protein is
fairly straightforward and merely requires use of e.g. a naturally
occurring signal sequence as a fusion partner for the analogue.
Another possibility is use of the C3d fragment of complement factor
C3 or C3 itself (cf. Dempsey et al., 1996, Science 271, 348-350 and
Lou & Kohler, 1998, Nature Biotechnology 16, 458-462).
[0135] An alternative embodiment of the invention which also
results in the preferred presentation of multiple (e.g. at least 2)
copies of the important epitopic regions of APP or A.beta. to the
immune system is the covalent coupling of the analogue to certain
molecules, i.e. variants d and e mentioned above. For instance,
polymers can be used, e.g. carbohydrates such as dextran, cf. e.g.
Lees A et al., 1994, Vaccine 12: 1160-1166; Lees A et al., 1990, J.
Immunol. 145: 3594-3600, but also mannose and mannan are useful
alternatives. Integral membrane proteins from e.g. E. coli and
other bacteria are also useful conjugation partners. The
traditional carrier molecules such as keyhole limpet hemocyanin
(KLH), tetanus toxoid, diphtheria toxoid, and bovine serum albumin
(BSA) are also preferred and useful conjugation partners.
[0136] Preferred embodiments of covalent coupling of the APP or AP
derived material to polyhydroxypolymers such as carbohydrates
involve the use of at least one APP or A.beta. derived peptide and
at least one foreign T-helper epitope which are coupled separately
to the polyhydroxypolymer (i.e. the foreign T-helper epitope and
the APP or A.beta. derived amino acid sequence are not fused to
each other but rather bound to the polyhydroxypolymer which then
serves as a carrier backbone). Again, such an embodiment is most
preferred when the suitable B-cell epitope carrying regions of the
APP or A.beta. derived peptides are constituted by short peptide
stretches--this is because this approach is one very convenient way
to achieve multiple presentations of selected epitopes in the
resulting immunogenic agent. It is, however, also possible to
simply coupled analogues already described herein to the
polyhydroxypolymer backbone, i.e. that the APP or A.beta. derived
material is not attached to the backbone separately from the
foreign T.sub.H epitopes.
[0137] It is especially preferred that the coupling of the foreign
T-helper epitope and the APP or A.beta. derived (poly)peptide is by
means of an amide bond which can be cleaved by a peptidase. This
strategy has the effect that APCs will be able to take up the
conjugate and at the same time be able to process the conjugate and
subsequently present the foreign T-cell epitope in an MHC Class II
context.
[0138] One way of achieving coupling of peptides (both the APP or
A.beta. derived peptide of interest as well as the foreign epitope)
is to activate a suitable polyhydroxypolymer with tresyl
(trifluoroethylsulphonyl) groups or other suitable activation
groups such as maleimido, p-Nitrophenyl cloroformate (for
activation of OH groups and formation of a peptide bond between
peptide and polyhydroxypolymer), and tosyl (p-toluenesulfonyl). It
is e.g. possible to prepare activated polysaccharides as described
in WO 00/05316 and U.S. Pat. No. 5,874,469 (both incorporated by
reference herein) and couple these to APP or A.beta. derived
peptides or polyamino acids as well as to T-cell epitopes prepared
by means of conventional solid or liquid phase peptide synthesis
techniques. The resulting product consists of a polyhydroxypolymer
backbone (e.g. a dextran backbone) that has, attached thereto by
their N-termini or by other available nitrogen moieties, polyamino
acids derived from APP or A.beta. and from foreign T-cell epitopes.
If desired, it is possible to synthesise the APP or A.beta.
peptides so as to protect all available amino groups but the one at
the N-terminus, subsequently couple the resulting protected
peptides to the tresylated dextran moiety, and finally
de-protecting the resulting conjugate. A specific example of this
approach is described in the examples below.
[0139] Instead of using the water-soluble polysaccharide molecules
as taught in WO 00/05316 and U.S. Pat. No. 5,874,469, it is equally
possible to utilise cross-linked polysaccharide molecules, thereby
obtaining a particulate conjugate between polypeptides and
polysaccharide--this is believed to lead to an improved
presentation to the immune system of the polypeptides, since two
goals are reached, namely to obtain a local deposit effect when
injecting the conjugate and to obtain particles which are
attractive targets for APCs. The approach of using such particulate
systems is also detailed in the examples.
[0140] Considerations underlying chosen areas of introducing
modifications in APP or A.beta. are a) preservation of known and
predicted B-cell epitopes, b) preservation of tertiary structure,
c) avoidance of B-cell epitopes present on "producer cells" etc. At
any rate, as discussed above, it is fairly easy to screen a set of
analogues which have all been subjected to introduction of a T-cell
epitope in different locations.
[0141] Since the most preferred embodiments of the present
invention involve down-regulation of human A.beta., it is
consequently preferred that the APP or A.beta.polypeptide discussed
above is a human A.beta. polypeptide. In this embodiment, it is
especially preferred that the APP or A.beta. polypeptide has been
modified by substituting at least one amino acid sequence in SEQ ID
NO: 2 with at least one amino acid sequence of equal or different
length and containing a foreign T.sub.H epitope. Preferred examples
of modified amyloidogenic APP and A.beta. are shown schematically
in FIG. 1 using the P2 and P30 epitopes as examples. The rationale
behind such constructs is discussed in detail in the examples.
[0142] More specifically, a T.sub.H containing (or completing)
amino acid sequence which is introduced into SEQ ID NO: 2 may be
introduced at any amino acid in SEQ ID NO: 2. That is, the
introduction is possible after any of amino acids 1-770, but
preferably after any of amino acids 671, 672, 673, 674, 675, 676,
677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689,
690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702,
703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, and 714 in
SEQ ID NO: 2. This may be combined with deletion of any or all of
amino acids 1-671, or any of all of amino acids 715-770.
Furthermore, when utilising the technique of substitution, any one
of amino acids 671, 672, 673, 674, 675, 676, 677, 678, 679, 680,
681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693,
694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706,
707, 708, 709, 710, 711, 712, 713, and 714 in SEQ ID NO: 2 may be
deleted in combination with the introduction.
[0143] Another embodiment of the present invention is the
presentation of the analogues which do not include any subsequence
of of SEQ ID NO: 2 that binds productively to MHC class II
molecules initiating a T-cell response.
[0144] The rationale behind such a strategy for design of the
immunogen that engages the immune system to induce e.g. an
anti-A.beta. immune response is the following: It has been noted
that when immunizing with abundant autologous proteins such as
A.beta. formulated in an adjuvant which is sufficiently strong to
break the body's tolerance towards the autologous protein, there is
a danger that in some vaccinated individuals the immune response
induced cannot be discontinued simply by discontinueing the
immunisation. This is because the induced immune response in such
individuals is most likely driven by a native T.sub.H epitope of
the autologous protein, and this has the adverse effect that the
vaccinated individual's own protein will be able to function as an
immunizing agent in its own right: An autoimmune condition has thus
been established.
[0145] The preferred methods including use of foreign T.sub.H
epitopes have to the best of the inventors' knowledge never been
observed to produce this effect, because the anti-self immune
response is driven by a foreign T.sub.H epitope, and it has been
repeatedly demonstrated by the inventors that the induced immune
response invoked by the preferred technology indeed declines after
discontinuation of immunizations. However, in theory it could
happen in a few individuals that the immune response would also be
driven by an autologous T.sub.H epitope of the relevant
self-protein one immunises against)--this is especially relevant
when considering self-proteins that are relatively abundant, such
as A.beta., whereas other therapeutically relevant self-proteins
are only present locally or in so low amounts in the body, that a
"self-immunization effect" is not a possibility. One very simple
way of avoiding this is hence to altogether avoid inclusion in the
immunogen of peptide sequences that could serve as T.sub.H epitopes
(and since peptides shorter than about 9 amino acids cannot serve
as T.sub.H epitopes, the use of shorter fragments is one simple and
feasible approach). Therefore, this embodiment of the invention
also serves to ensure that the immunogen does not indlude peptide
sequences of the target APP or A.beta. that could serve as
"self-stimulating T.sub.H epitopes" including sequences that merely
contain conservative substitutions in a sequence of the target
protein that might otherwise function as a T.sub.H epitope.
[0146] Preferred embodiments of the immune system presentation of
the analogues of the APP or A.beta. involve the use of a chimeric
peptide comprising at least one APP or A.beta. derived peptide,
which does not bind productively to MHC class II molecules, and at
least one foreign T-helper epitope. Moreover, it is preferred that
the APP or A.beta. derived peptide harbours a B-cell epitope. It is
especially advantageous if the immunogenic analogue is one, wherein
the amino acid sequences comprising one or more B-cell epitopes are
represented either as a continuous sequence or as a sequence
including inserts, wherein the inserts comprise foreign T-helper
epitopes.
[0147] Again, such an embodiment is most preferred when the
suitable B-cell epitope carrying regions of the APP or A.beta. are
constituted by short peptide stretches that in no way would be able
to bind productively to an MHC Class II molecule. The selected
B-cell epitope or epitopes of the amyloidogenic polypeptide should
therefore comprise at most 9 consecutive amino acids of SEQ ID NO:
2. Shorter peptides are preferred, such as those having at most 8,
7, 6, 5, 4, or 3 consecutive amino acids from the amyloidogenic
polypeptide's amino acid sequence.
[0148] It is preferred that the analogue comprises at least one
subsequence of SEQ ID NO: 2 so that each such at least one
subsequence independently consists of amino acid stretches from the
APP or A.beta. selected from the group consisting of 9 consecutive
amino acids, 8 consecutive amino acids, 7 consecutive amino acids,
6 consecutive amino acids, 5 consecutive amino acids, 4 consecutive
amino acids, and 3 consecutive amino acids.
[0149] It is especially preferred that the consecutive amino acids
begins at an amino acid residue selected from the group consisting
of residue 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682,
683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695,
696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708,
709, 710, 711, 712, 713, and 714 of SEQ ID NO: 2.
[0150] Protein/Peptide Vaccination; Formulation and Administration
of the Analogues
[0151] When effecting presentation of the analogue to an animal's
immune system by means of administration thereof to the animal, the
formulation of the polypeptide follows the principles generally
acknowledged in the art.
[0152] Preparation of vaccines which contain peptide sequences as
active ingredients is generally well understood in the art, as
exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231;
4,599,230; 4,596,792; and 4,578,770, all incorporated herein by
reference. Typically, such vaccines are prepared as injectables
either as liquid solutions or suspensions; solid forms suitable for
solution in, or suspension in, liquid prior to injection may also
be prepared. The preparation may also be emulsified. The active
immunogenic ingredient is often mixed with excipients which are
pharmaceutically acceptable and compatible with the active
ingredient. Suitable excipients are, for example, water, saline,
dextrose, glycerol, ethanol, or the like, and combinations thereof.
In addition, if desired, the vaccine may contain minor amounts of
auxiliary substances such as wetting or emulsifying agents, pH
buffering agents, or adjuvants which enhance the effectiveness of
the vaccines; cf. the detailed discussion of adjuvants below.
[0153] The vaccines are conventionally administered parenterally,
by injection, for example, either subcutaneously, intracutaneously,
or intramuscularly. Additional formulations which are suitable for
other modes of administration include suppositories and, in some
cases, oral, buccal, sublingual, intraperitoneal, intravaginal,
anal, epidural, spinal, and intracranial formulations. For
suppositories, traditional binders and carriers may include, for
example, polyalkalene glycols or triglycerides; such suppositories
may be formed from mixtures containing the active ingredient in the
range of 0.5% to 10%, preferably 1-2%. Oral formulations include
such normally employed excipients as, for example, pharmaceutical
grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate, and the like. 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%. For oral
formulations, cholera toxin is an interesting formulation partner
(and also a possible conjugation partner).
[0154] The polypeptides may be formulated into the vaccine as
neutral or salt forms. Pharmaceutically acceptable salts include
acid addition salts (formed with the free amino groups of the
peptide) and which are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids as
acetic, oxalic, tartaric, mandelic, and the like. Salts formed with
the free carboxyl groups may also be derived from inorganic bases
such as, for example, sodium, potassium, ammonium, calcium, or
ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the
like.
[0155] The vaccines are administered in a manner compatible with
the dosage formulation, and in such amount as will be
therapeutically effective and immunogenic. The quantity to be
administered depends on the subject to be treated, including, e.g.,
the capacity of the individual's immune system to mount an immune
response, and the degree of protection desired. Suitable dosage
ranges are of the order of several hundred micrograms active
ingredient per vaccination with a preferred range from about 0.1
.mu.g to 2,000 .mu.g (even though higher amounts in the 1-10 mg
range are contemplated), such as in the range from about 0.5 .mu.g
to 1,000 .mu.g, preferably in the range from 1 .mu.g to 500 .mu.g
and especially in the range from about 10 .mu.g to 100 .mu.g.
Suitable regimens for initial administration and booster shots are
also variable but are typified by an initial administration
followed by subsequent inoculations or other administrations.
[0156] The manner of application may be varied widely. Any of the
conventional methods for administration of a vaccine are
applicable. These include oral application on a solid
physiologically acceptable base or in a physiologically acceptable
dispersion, parenterally, by injection or the like. The dosage of
the vaccine will depend on the route of administration and will
vary according to the age of the person to be vaccinated and the
formulation of the antigen.
[0157] Some of the polypeptides of the vaccine are sufficiently
immunogenic in a vaccine, but for some of the others the immune
response will be enhanced if the vaccine further comprises an
adjuvant substance.
[0158] Various methods of achieving adjuvant effect for the vaccine
are known. General principles and methods are detailed in "The
Theory and Practical Application of Adjuvants", 1995, Duncan E. S.
Stewart-Tull (ed.), John Wiley & Sons Ltd, ISBN 0-471-95170-6,
and also in "Vaccines: New Generationn Immunological Adjuvants",
1995, Gregoriadis G et al. (eds.), Plenum Press, New York, ISBN
0-306-45283-9, both of which are hereby incorporated by reference
herein.
[0159] It is especially preferred to use an adjuvant which can be
demonstrated to facilitate breaking of the autotolerance to
autoantigens; in fact, this is essential in cases where unmodified
amyloidogenic polypeptide is used as the active ingredient in the
autovaccine. Non-limiting examples of suitable adjuvants are
selected from the group consisting of an immune targeting adjuvant;
an immune modulating adjuvant such as a toxin, a cytokine, and a
mycobacterial derivative; an oil formulation; a polymer; a micelle
forming adjuvant; a saponin; an immunostimulating complex matrix
(ISCOM matrix); a particle; DDA; aluminium adjuvants; DNA
adjuvants; .gamma.-inulin; and an encapsulating adjuvant. In
general it should be noted that the disclosures above which relate
to compounds and agents useful as first, second and third moieties
in the analogues also refer mutatis mutandis to their use in the
adjuvant of a vaccine of the invention.
[0160] The application of adjuvants include use of agents such as
aluminum hydroxide or phosphate (alum), commonly used as 0.05 to
0.1 percent solution in buffered saline, admixture with synthetic
polymers of sugars (e.g. Carbopol.RTM.) used as 0.25 percent
solution, aggregation of the protein in the vaccine by heat
treatment with temperatures ranging between 700 to 101.degree. C.
for 30 second to 2 minute periods respectively and also aggregation
by means of cross-linking agents are possible. Aggregation by
reactivation with pepsin treated antibodies (Fab fragments) to
albumin, mixture with bacterial cells such as C. parvum or
endotoxins or lipopolysaccharide components of gram-negative
bacteria, emulsion in physiologically acceptable oil vehicles such
as mannide mono-oleate (Aracel A) or emulsion with 20 percent
solution of a perfluorocarbon (Fluosol-DA) used as a block
substitute may also be employed. Admixture with oils such as
squalene and IFA is also preferred.
[0161] According to the invention DDA (dimethyldioctadecylammonium
bromide) is an interesting candidate for an adjuvant as is DNA and
.gamma.-inulin, but also Freund's complete and incomplete adjuvants
as well as quillaja saponins such as QuilA and QS21 are interesting
as is RIBI. Further possibilities are monophosphoryl lipid A (MPL),
the above mentioned C3 and C3d, and muramyl dipeptide (MDP).
[0162] Liposome formulations are also known to confer adjuvant
effects, and therefore liposome adjuvants are preferred according
to the invention.
[0163] Also immunostimulating complex matrix type (ISCOM.RTM.
matrix) adjuvants are preferred choices according to the invention,
especially since it has been shown that this type of adjuvants are
capable of up-regulating MHC Class II expression by APCs. An ISCOMO
matrix consists of (optionally fractionated) saponins
(triterpenoids) from Quillaja saponaria, cholesterol, and
phospholipid. When admixed with the immunogenic protein, the
resulting particulate formulation is what is known as an ISCOM
particle where the saponin constitutes 60-70% w/w, the cholesterol
and phospholipid 10-15% w/w, and the protein 10-15% w/w. Details
relating to composition and use of immunostimulating complexes can
e.g. be found in the above-mentioned text-books dealing with
adjuvants, but also Morein B et al., 1995, Clin. Immunother. 3:
461-475 as well as Barr I G and Mitchell G F, 1996, Immunol. and
Cell Biol. 74: 8-25 (both incorporated by reference herein) provide
useful instructions for the preparation of complete
immunostimulating complexes.
[0164] Another highly interesting (and thus, preferred) possibility
of achieving adjuvant effect is to employ the technique described
in Gosselin et al., 1992 (which is hereby incorporated by reference
herein). In brief, the presentation of a relevant antigen such as
an antigen of the present invention can be enhanced by conjugating
the antigen to antibodies (or antigen binding antibody fragments)
against the Fc.gamma. receptors on monocytes/macrophages.
Especially conjugates between antigen and anti-Fc.gamma.RI have
been demonstrated to enhance immunogenicity for the purposes of
vaccination.
[0165] Other possibilities involve the use of the targeting and
immune modulating substances (i.a. cytokines) mentioned above as
candidates for the first and second moieties in the modified
versions of amyloidogenic polypeptides. In this connection, also
synthetic inducers of cytokines like poly I:C are
possibilities.
[0166] Suitable mycobacterial derivatives are selected from the
group consisting of muramyl dipeptide, complete Freund's adjutant,
RIBI, and a diester of trehalose such as TDM and TDE.
[0167] Suitable immune targeting adjuvants are selected from the
group consisting of CD40 ligand and CD40 antibodies or specifically
binding fragments thereof (cf. the discussion above), mannose, a
Fab fragment, and CTLA-4.
[0168] Suitable polymer adjuvants are selected from the group
consisting of a carbohydrate such as dextran, PEG, starch, mannan,
and mannose; a plastic polymer such as; and latex such as latex
beads.
[0169] Yet another interesting way of modulating an immune response
is to include the immunogen (optionally together with adjuvants and
pharmaceutically acceptable carriers and vehicles) in a "virtual
lymph node" (VLN) (a proprietary medical device developed by
ImmunoTherapy, Inc., 360 Lexington Avenue, New York, N.Y.
10017-6501). The VLN (a thin tubular device) mimics the structrue
and function of a lymph node. Insertion of a VLN under the skin
creates a site of sterile inflammation with an upsurge of cytokines
and chemokines. T- and B-cells as well as APCs rapidly respond to
the danger signals, home to the inflamed site and accumulate inside
the porous matrix of the VLN. It has been shown that the necessary
antigen dose required to mount an immune response to an antigen is
reduced when using the VLN and that immune protection conferred by
vaccination using a VLN surpassed conventional immunization using
Ribi as an adjuvant. The technology is i.a. described briefly in
Gelber C et al., 1998, "Elicitation of Robust Cellular and Humoral
Immune Responses to Small Amounts of Immunogens Using a Novel
Medical Device Designated the Virtual Lymph Node", in: "From the
Laboratory to the Clinic, Book of Abstracts, October 12-15th 1998,
Seascape Resort, Aptos, Calif.
[0170] Microparticle formulation of vaccines has been shown in many
cases to increase the immunogenicity of protein antigens and is
therefore another preferred embodiment of the invention.
Microparticles are made either as co-formulations of antigen with a
polymer, a lipid, a carbohydrate or other molecules suitable for
making the particles, or the microparticles can be homogeneous
particles consisting of only the antigen itself.
[0171] Examples of polymer based microparticles are PLGA and PVP
based particles (Gupta, R. K. et. al. 1998) where the polymer and
the antigen are condensed into a solid particle. Lipid based
particles can be made as micelles of the lipid (so-called
liposomes) entrapping the antigen within the micelle (Pietrobon, P.
J. 1995). Carbohydrate based particles are typically made of a
suitable degradable carbohydrate such as starch or chitosan. The
carbohydrate and the antigen are mixed and condensed into particles
in a process similar to the one used for polymer particles (Kas, H.
S. et. al. 1997).
[0172] Particles consisting only of the antigen can be made by
various spraying and freeze-drying techniques. Especially suited
for the purporses of the present invention is the super critical
fluid technology that is used to make very uniform particles of
controlled size (York, P. 1999 & Shekunov, B. et. al.
1999).
[0173] It is expected that the vaccine should be administered 1-6
times per year, such as 1, 2, 3, 4, 5, or 6 times a year to an
individual in need thereof. It has previously been shown that the
memory immunity induced by the use of the preferred autovaccines
according to the invention is not permanent, and therefore the
immune system needs to be periodically challenged with the
amyloidogenic polypeptide or modified amyloidogenic
polypeptides.
[0174] Due to genetic variation, different individuals may react
with immune responses of varying strength to the same polypeptide.
Therefore, the vaccine according to the invention may comprise
several different polypeptides in order to increase the immune
response, cf. also the discussion above concerning the choice of
foreign T-cell epitope introductions. The vaccine may comprise two
or more polypeptides, where all of the polypeptides are as defined
above.
[0175] The vaccine may consequently comprise 3-20 different
modified or unmodified polypeptides, such as 3-10 different
polypeptides.
[0176] Nucleic Acid Vaccination
[0177] As an alternative to classic administration of a
peptide-based vaccine, the technology of nucleic acid vaccination
(also known as "nucleic acid immunisation", "genetic immunisation",
and "gene immunisation") offers a number of attractive
features.
[0178] First, in contrast to the traditional vaccine approach,
nucleic acid vaccination does not require resource consuming
large-scale production of the immunogenic agent (e.g. in the form
of industrial scale fermentation of microorganisms producing
modified amyloidogenic polypeptides). Furthermore, there is no need
to device purification and refolding schemes for the immunogen. And
finally, since nucleic acid vaccination relies on the biochemical
apparatus of the vaccinated individual in order to produce the
expression product of the nucleic acid introduced, the optimum
post-translational processing of the expression product is expected
to occur; this is especially important in the case of
autovaccination, since, as mentioned above, a significant fraction
of the original B-cell epitopes should be preserved in the modified
molecule, and since B-cell epitopes in principle can be constituted
by parts of any (bio)molecule (e.g. carbohydrate, lipid, protein
etc.). Therefore, native glycosylation and lipidation patterns of
the immunogen may very well be of importance for the overall
immunogenicity and this is best ensured by having the host
producing the immunogen.
[0179] Hence, a preferred embodiment of the invention's variants
a-c comprises effecting presentation of the analogue to the immune
system by introducing nucleic acid(s) encoding the analogue into
the animal's cells and thereby obtaining in vivo expression by the
cells of the nucleic acid(s) introduced.
[0180] In this embodiment, the introduced nucleic acid is
preferably DNA which can be in the form of naked DNA, DNA
formulated with charged or uncharged lipids, DNA formulated in
liposomes, DNA included in a viral vector, DNA formulated with a
transfection-facilitating protein or polypeptide, DNA formulated
with a targeting protein or polypeptide, DNA formulated with
Calcium precipitating agents, DNA coupled to an inert carrier
molecule, DNA encapsulated in a polymer, e.g. in PLGA (cf. the
microencapsulation technology described in WO 98/31398) or in
chitin or chitosan, and DNA formulated with an adjuvant. In this
context it is noted that practically all considerations pertaining
to the use of adjuvants in traditional vaccine formulation apply
for the formulation of DNA vaccines. Hence, all disclosures herein
which relate to use of adjuvants in the context of polypeptide
based vaccines apply mutatis mutandis to their use in nucleic acid
vaccination technology.
[0181] As for routes of administration and administration schemes
of polypeptide based vaccines which have been detailed above, these
are also applicable for the nucleic acid vaccines of the invention
and all discussions above pertaining to routes of administration
and administration schemes for polypeptides apply mutatis mutandis
to nucleic acids. To this should be added that nucleic acid
vaccines can suitably be administered intraveneously and
intraarterially. Furthermore, it is well-known in the art that
nucleic acid vaccines can be administered by use of a so-called
gene gun, and hence also this and equivalent modes of
administration are regarded as part of the present invention.
Finally, also the use of a VLN in the administration of nucleic
acids has been reported to yield good results, and therefore this
particular mode of administration is particularly preferred.
[0182] Furthermore, the nucleic acid(s) used as an immunization
agent can contain regions encoding the 1.sup.st, 2.sup.nd and/or
3.sup.rd moieties, e.g. in the form of the immunomodulating
substances described above such as the cytokines discussed as
useful adjuvants. A preferred version of this embodiment
encompasses having the coding region for the analogue and the
coding region for the immunomodulator in different reading frames
or at least under the control of different promoters. Thereby it is
avoided that the analogue or epitope is produced as a fusion
partner to the immunomodulator. Alternatively, two distinct
nucleotide fragments can be used, but this is less preferred
because of the advantage of ensured co-expression when having both
coding regions included in the same molecule.
[0183] Accordingly, the invention also relates to a composition for
inducing production of antibodies against APP or A.beta., the
composition comprising
[0184] a nucleic acid fragment or a vector of the invention (cf.
the discussion of vectors below), and
[0185] a pharmaceutically and immunologically acceptable vehicle
and/or carrier and/or adjuvant as discussed above.
[0186] Under normal circumstances, the variant-encoding nucleic
acid is introduced in the form of a vector wherein expression is
under control of a viral promoter. For more detailed discussions of
vectors according to the invention, cf. the discussion below. Also,
detailed disclosures relating to the formulation and use of nucleic
acid vaccines are available, cf. Donnelly J J et al, 1997, Annu.
Rev. Immunol. 15: 617-648 and Donnelly J J et al., 1997, Life
Sciences 60: 163-172. Both of these references are incorporated by
reference herein.
[0187] Live Vaccines
[0188] A third alternative for effecting presentation of the
analogues as these are defined in variants a-c to the immune system
is the use of live vaccine technology. In live vaccination,
presentation to the immune system is effected by administering, to
the animal, a non-pathogenic microorganism which has been
transformed with a nucleic acid fragment encoding an analogue or
with a vector incorporating such a nucleic acid fragment. The
non-pathogenic microorganism can be any suitable attenuated
bacterial strain (attenuated by means of passaging or by means of
removal of pathogenic expression products by recombinant DNA
technology), e.g. Mycobacterium bovis BCG., non-pathogenic
Streptococcus spp., E. coli, Salmonella spp., Vibrio cholerae,
Shigella, etc. Reviews dealing with preparation of state-of-the-art
live vaccines can e.g. be found in Saliou P, 1995, Rev. Prat. 45:
1492-1496 and Walker P D, 1992, Vaccine 10: 977-990, both
incorporated by reference herein.
[0189] For details about the nucleic acid fragments and vectors
used in such live vaccines, cf. the discussion below.
[0190] As an alternative to bacterial live vaccines, the nucleic
acid fragment of the invention discussed below can be incorporated
in a non-virulent viral vaccine vector such as a vaccinia strain or
any other suitable poxyirus.
[0191] Normally, the non-pathogenic microorganism or virus is
administered only once to the animal, but in certain cases it may
be necessary to administer the microorganism more than once in a
lifetime in order to maintain protective immunity. It is even
contemplated that immunization schemes as those detailed above for
polypeptide vaccination will be useful when using live or virus
vaccines.
[0192] Alternatively, live or virus vaccination is combined with
previous or subsequent polypeptide and/or nucleic acid vaccination.
For instance, it is possible to effect primary immunization with a
live or virus vaccine followed by subsequent booster immunizations
using the polypeptide or nucleic acid approach.
[0193] The microorganism or virus can be transformed with nucleic
acid(s) containing regions encoding the 1.sup.st, 2.sup.nd and/or
3.sup.rd moieties, e.g. in the form of the immunomodulating
substances described above such as the cytokines discussed as
useful adjuvants. A preferred version of this embodiment
encompasses having the coding region for the analogue and the
coding region for the immunomodulator in different reading frames
or at least under the control of different promoters. Thereby it is
avoided that the analogue or epitopes are produced as fusion
partners to the immunomodulator. Alternatively, two distinct
nucleotide fragments can be used as transforming agents. Of course,
having the 1.sup.st and/or 2.sup.nd and/or 3.sup.rd moieties in the
same reading frame can provide as an expression product, an
analogue of the invention, and such an embodiment is especially
preferred according to the present invention.
[0194] Use of the Method of the Invention in Disease Treatment
[0195] As will be appreciated from the discussions above, the
provision of the method of the invention allows for control of
diseases characterized by amyloid deposits. In this context, AD is
the key target for the inventive method but also other diseases
characterized by A.beta. containing amyloid deposits are feasible
targets. Hence, an important embodiment of the method of the
invention for down-regulating amyloid activity comprises treating
and/or preventing and/or ameliorating AD or other diseases
characterized by amyloid deposition, the method comprising
down-regulating APP or A.beta. according to the method of the
invention to such an extent that the amount of amyloid is
significantly decreased.
[0196] It is especially preferred that the reduction in amyloid
results in an inversion of the balance between amyloid formation
and amyloid degradation/removal, i.e. that the rate of amyloid
degradation/removal is brought to exceed the rate of amyloid
formation. By carefully controlling the number and immunological
impact of immunizations of the individual in need thereof it will
be possible to obtain a balance over time which results in a net
reduction of amyloid deposits without having excessive adverse
effects.
[0197] Alternatively, if in an individual the method of the
invention cannot remove or reduce existing amyloid deposits, the
method of the invention can be used to obtain a clinically
significant reduction in the formation of new amyloid, thereby
significantly prolonging the time where the disease condition is
non-debilitating. It should be possible to monitor the rate of
amyloid depositing by either measuring the serum concentration of
amyloid (which is believed to be in equilibrium with the deposited
material), or by using positron-emission tomography (PET) scanning,
cf. Small G W, et al., 1996, Ann NY Acad Sci 802: 70-78.
[0198] Other diseases and conditions where the present means and
methods may be used in treatment or amelioration in an analogous
way have been mentioned above in the "Background of the invention"
or are listed below in the section headed "other amyloidic diseases
and proteins associated therewith".
[0199] Peptides, Polypeptides, and Compositions of the
Invention
[0200] As will be apparent from the above, the present invention is
based on the concept of immunising individuals against the APP or
A.beta. antigen in order to obtain a reduced amount of
pathology-related amyloid deposits. The preferred way of obtaining
such an immunization is to use the analogues described herein,
thereby providing molecules which have not previously been
disclosed in the art.
[0201] It is believed that the analogues discussed herein are
inventive in their own right, and therefore an important part of
the invention pertains to an analogue as described above. Hence,
any disclosure presented herein pertaining to modified APP or
A.beta. are relevant for the purpose of describing the
amyloidogenic analogues of the invention, and any such disclosures
apply mutatis mutandis to the description of these analogues.
[0202] It should be noted that preferred modified APP or A.beta.
molecules comprise modifications which results in a polypeptide
having a sequence identity of at least 70% with APP or A.beta. or
with a subsequence thereof of at least 10 amino acids in length.
Higher sequence identities are preferred, e.g. at least 75% or even
at least 80, 85, 90, or 95%. The sequence identity for proteins and
nucleic acids can be calculated as
(N.sub.ref-N.sub.dif).multidot.100/N.sub.ref, wherein N.sub.dif is
the total number of non-identical residues in the two sequences
when aligned and wherein N.sub.ref is the number of residues in one
of the sequences. Hence, the DNA sequence AGTCAGTC will have a
sequence identity of 75% with the sequence AATCAATC (N.sub.dif=2
and N.sub.ref=8).
[0203] The invention also pertains to compositions useful in
exercising the method of the invention. Hence, the invention also
relates to an immunogenic composition comprising an immunogenically
effective amount of an analouge as described above, said
composition further comprising a pharmaceutically and
immunologically acceptable diluent and/or vehicle and/or carrier
and/or excipient and optionally an adjuvant. In other words, this
part of the invention concerns formulations of analogues,
essentially as described above. The choice of adjuvants, carriers,
and vehicles is accordingly in line with what has been discussed
above when referring to formulation of modified and unmodified
amyloidogenic polypeptide for use in the inventive method for the
down-regulation of APP or A.beta..
[0204] The polypeptides are prepared according to methods
well-known in the art. Longer polypeptides are normally prepared by
means of recombinant gene technology including introduction of a
nucleic acid sequence encoding the analogue into a suitable vector,
transformation of a suitable host cell with the vector, expression
by the host cell of the nucleic acid sequence, recovery of the
expression product from the host cells or their culture
supernatant, and subseqeunt purification and optional further
modification, e.g. refolding or derivatization.
[0205] Shorter peptides are preferably prepared by means of the
well-known techniques of solid- or liquid-phase peptide synthesis.
However, recent advances in this technology has rendered possible
the production of full-length polypeptides and proteins by these
means, and therefore it is also within the scope of the present
invention to prepare the long constructs by synthetic means.
[0206] Nucleic Acid Fragments and Vectors of the Invention
[0207] It will be appreciated from the above disclosure that
poly-amino acid analogues can be prepared by means of recombinant
gene technology but also by means of chemical synthesis or
semisynthesis; the latter two options are especially relevant when
the modification consists in coupling to protein carriers (such as
KLH, diphtheria toxoid, tetanus toxoid, and BSA) and
non-proteinaceous molecules such as carbohydrate polymers and of
course also when the modification comprises addition of side chains
or side groups to an APP or A.beta. derived peptide chain.
[0208] For the purpose of recombinant gene technology, and of
course also for the purpose of nucleic acid immunization, nucleic
acid fragments encoding analogues are important chemical products.
Hence, an important part of the invention pertains to a nucleic
acid fragment which encodes an analogue of the invention, i.e. an
APP or A.beta. derived polypeptide which either comprises the
natural sequence to which has been added or inserted a fusion
partner or, preferably an APP or AP derived polypeptide wherein has
been introduced a foreign T-cell epitope by means of insertion
and/or addition, preferably by means of substitution and/or
deletion. The nucleic acid fragments of the invention are either
DNA or RNA fragments.
[0209] The nucleic acid fragments of the invention will normally be
inserted in suitable vectors to form cloning or expression vectors
carrying the nucleic acid fragments of the invention; such novel
vectors are also part of the invention. Details concerning the
construction of these vectors of the invention will be discussed in
context of transformed cells and microorganisms below. The vectors
can, depending on purpose and type of application, be in the form
of plasmids, phages, cosmids, mini-chromosomes, or virus, but also
naked DNA which is only expressed transiently in certain cells is
an important vector. Preferred cloning and expression vectors of
the invention are capable of autonomous replication, thereby
enabling high copy-numbers for the purposes of high-level
expression or high-level replication for subsequent cloning.
[0210] The general outline of a vector of the invention comprises
the following features in the 5'.fwdarw.3' direction and in
operable linkage: a promoter for driving expression of the nucleic
acid fragment of the invention, optionally a nucleic acid sequence
encoding a leader peptide enabling secretion (to the extracellular
phase or, where applicable, into the periplasma) of or integration
into the membrane of the polypeptide fragment, the nucleic acid
fragment of the invention, and optionally a nucleic acid sequence
encoding a terminator. When operating with expression vectors in
producer strains or cell-lines it is for the purposes of genetic
stability of the transformed cell preferred that the vector when
introduced into a host cell is integrated in the host cell genome.
In contrast, when working with vectors to be used for effecting in
vivo expression in an animal (i.e. when using the vector in DNA
vaccination) it is for security reasons preferred that the vector
is incapable of being integrated in the host cell genome;
typically, naked DNA or non-integrating viral vectors are used, the
choices of which are well-known to the person skilled in the
art
[0211] The vectors of the invention are used to transform host
cells to produce the analogue of the invention. Such transformed
cells, which are also part of the invention, can be cultured cells
or cell lines used for propagation of the nucleic acid fragments
and vectors of the invention, or used for recombinant production of
the analogues of the invention. Alternatively, the transformed
cells can be suitable live vaccine strains wherein the nucleic acid
fragment (one single or multiple copies) have been inserted so as
to effect secretion or integration into the bacterial membrane or
cell-wall of the analogue.
[0212] Preferred transformed cells of the invention are
microorganisms such as bacteria (such as the species Escherichia
[e.g. E.coli ], Bacillus [e.g. Bacillus subtilis ], Salmonella, or
Mycobacterium [preferably non-pathogenic, e.g. M. bovis BCG]),
yeasts (such as Saccharomyces cerevisiae), and protozoans.
Alternatively, the transformed cells are derived from a
multi-cellular organism such as a fungus, an insect cell, a plant
cell, or a mammalian cell. Most preferred are cells derived from a
human being, cf. the discussion of cell lines and vectors below.
Recent results have shown great promise in the use of a
commercially available Drosophila melanogaster cell line (the
Schneider 2 (S.sub.2) cell line and vector system available from
Invitrogen) for the recombinant production of polypeptides in
applicants' lab, and therefore this expression system is
particularly preferred.
[0213] For the purposes of cloning and/or optimized expression it
is preferred that the transformed cell is capable of replicating
the nucleic acid fragment of the invention. Cells expressing the
nucleic fragment are preferred useful embodiments of the invention;
they can be used for small-scale or large-scale preparation of the
analogue of the invention or, in the case of non-pathogenic
bacteria, as vaccine constituents in a live vaccine.
[0214] When producing the analogues of the invention by means of
transformed cells, it is convenient, although far from essential,
that the expression product is either exported out into the culture
medium or carried on the surface of the transformed cell.
[0215] When an effective producer cell has been identified it is
preferred, on the basis thereof, to establish a stable cell line
which carries the vector of the invention and which expresses the
nucleic acid fragment encoding the modified amyloidogenic
polypeptide. Preferably, this stable cell line secretes or carries
the analogue of the invention, thereby facilitating purification
thereof.
[0216] In general, plasmid vectors containing replicon and control
sequences which are derived from species compatible with the host
cell are used in connection with the hosts. The vector ordinarily
carries a replication site, as well as marking sequences which are
capable of providing phenotypic selection in transformed cells. For
example, E. coli is typically transformed using pBR322, a plasmid
derived from an E. coli species (see, e.g., Bolivar et al., 1977).
The pBR322 plasmid contains genes for ampicillin and tetracycline
resistance and thus provides easy means for identifying transformed
cells. The pBR plasmid, or other microbial plasmid or phage must
also contain, or be modified to contain, promoters which can be
used by the prokaryotic microorganism for expression.
[0217] Those promoters most commonly used in recombinant DNA
construction include the B-lactamase (penicillinase) and lactose
promoter systems (Chang et al., 1978; Itakura et al., 1977; Goeddel
et al., 1979) and a tryptophan (trp) promoter system (Goeddel et
al., 1979; EP-A-0 036 776). While these are the most commonly used,
other microbial promoters have been discovered and utilized, and
details concerning their nucleotide sequences have been published,
enabling a skilled worker to ligate them functionally with plasmid
vectors (Siebwenlist et al., 1980). Certain genes from prokaryotes
may be expressed efficiently in E. coli from their own promoter
sequences, precluding the need for addition of another promoter by
artificial means.
[0218] In addition to prokaryotes, eukaryotic microbes, such as
yeast cultures may also be used, and here the promoter should be
capable of driving expression. Saccharomiyces cerevisiase, or
common baker's yeast is the most commonly used among eukaryotic
microorganisms, although a number of other strains are commonly
available. For expression in Saccharomyces, the plasmid YRp7, for
example, is commonly used (Stinchcomb et al., 1979; Kingsman et
al., 1979; Tschemper et al., 1980). This plasmid already contains
the trpl gene which provides a selection marker for a mutant strain
of yeast lacking the ability to grow in tryptophan for example ATCC
No. 44076 or PEP4-1 (Jones, 1977). The presence of the trpl lesion
as a characteristic of the yeast host cell genome then provides an
effective environment for detecting transformation by growth in the
absence of tryptophan.
[0219] Suitable promoting sequences in yeast vectors include the
promoters for 3-phosphoglycerate kinase (Hitzman et al., 1980) or
other glycolytic enzymes (Hess et al., 1968; Holland et al., 1978),
such as enolase, glyceraldehyde-3-phosphate dehydrogenase,
hexokinase, pyruvate decarboxylase, phosphofructokinase,
glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate
kinase, triosephosphate isomerase, phosphoglucose isomerase, and
glucokinase. In constructing suitable expression plasmids, the
termination sequences associated with these genes are also ligated
into the expression vector 3' of the sequence desired to be
expressed to provide polyadenylation of the mRNA and
termination.
[0220] Other promoters, which have the additional advantage of
transcription controlled by growth conditions are the promoter
region for alcohol dehydrogenase 2, isocytochrome C, acid
phosphatase, degradative enzymes associated with nitrogen
metabolism, and the aforementioned glyceraldehyde-3-phosphate
dehydrogenase, and enzymes responsible for maltose and galactose
utilization. Any plasmid vector containing a yeast-compatible
promoter, origin of replication and termination sequences is
suitable.
[0221] In addition to microorganisms, cultures of cells derived
from multicellular organisms may also be used as hosts. In
principle, any such cell culture is workable, whether from
vertebrate or invertebrate culture. However, interest has been
greatest in vertebrate cells, and propagation of vertebrate in
culture (tissue culture) has become a routine procedure in recent
years (Tissue Culture, 1973). Examples of such useful host cell
lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell
lines, and W138, BHK, COS-7 293, Spodoptera frugiperda (SF) cells
(commercially available as complete expression systems from i.a.
Protein Sciences, 1000 Research Parkway, Meriden, Conn. 06450,
U.S.A. and from Invitrogen), and MDCK cell lines. In the present
invention, an especially preferred cell line is S.sub.2 available
from Invitrogen, PO Box 2312, 9704 CH Groningen, The
Netherlands.
[0222] Expression vectors for such cells ordinarily include (if
necessary) an origin of replication, a promoter located in front of
the gene to be expressed, along with any necessary ribosome binding
sites, RNA splice sites, polyadenylation site, and transcriptional
terminator sequences.
[0223] For use in mammalian cells, the control functions on the
expression vectors are often provided by viral material. For
example, commonly used promoters are derived from polyoma,
Adenovirus 2, and most frequently Simian Virus 40 (SV40). The early
and late promoters of SV40 virus are particularly useful because
both are obtained easily from the virus as a fragment which also
contains the SV40 viral origin of replication (Fiers et al., 1978).
Smaller or larger SV40 fragments may also be used, provided there
is included the approximately 250 bp sequence extending from the
HindIII site toward the BglI site located in the viral origin of
replication. Further, it is also possible, and often desirable, to
utilize promoter or control sequences normally associated with the
desired gene sequence, provided such control sequences are
compatible with the host cell systems.
[0224] An origin of replication may be provided either by
construction of the vector to include an exogenous origin, such as
may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV,
BPV) or may be provided by the host cell chromosomal replication
mechanism. If the vector is integrated into the host cell
chromosome, the latter is often sufficient.
[0225] Identification of Useful Analogues
[0226] It will be clear to the skilled person that not all possible
variants or modifications of naturally occurring APP or A.beta.
will have the ability to elicit antibodies in an animal which are
cross-reactive with the natural form. It is, however, not difficult
to set up an effective standard screen for modified amyloidogenic
molecules which fulfill the minimum requirements for immunological
reactivity discussed herein. Hence, it is possible to utilise a
method for the identification of a modified amyloidogenic
polypeptide which is capable of inducing antibodies against
unmodified amyloidogenic polypeptide in an animal species where the
unmodified amyloidogenic polypeptide is a (non-immunogenic)
self-protein, the method comprising
[0227] preparing, by means of peptide synthesis or genetic
engineering techniques, a set of mutually distinct analogue of the
invention wherein amino acids have been added to, inserted in,
deleted from, or substituted into the amino acid sequence of an APP
or AP of the animal species thereby giving rise to amino acid
sequences in the set which comprise T-cell epitopes which are
foreign to the animal species, or preparing a set of nucleic acid
fragments encoding the set of mutually distinct analogues,
[0228] testing members of the set of analogues or nucleic acid
fragments for their ability to induce production of antibodies by
the animal species against the unmodified APP or A.beta., and
[0229] identifying and optionally isolating the member(s) of the
set of analogues which significantly induces antibody production
against unmodified APP or A.beta. in the species or identifying and
optionally isolating the polypeptide expression products encoded by
members of the set of nucleic acid fragments which significantly
induces antibody production against unmodified APP or A.beta. in
the animal species.
[0230] In this context, the "set of mutually distinct modified
amyloidogenic polypeptides" is a collection of non-identical
analouges which have e.g. been selected on the basis of the
criteria discussed above (e.g: in combination with studies of
circular dichroism, NMR spectra, and/or X-ray diffraction
patterns). The set may consist of only a few members but it is
contemplated that the set may contain several hundred members.
[0231] The test of members of the set can ultimately be performed
in vivo, but a number of in vitro tests can be applied which narrow
down the number of modified molecules which will serve the purpose
of the invention.
[0232] Since the goal of introducing the foreign T-cell epitopes is
to support the B-cell response by T-cell help, a prerequisite is
that T-cell proliferation is induced by the analogue. T-cell
proliferation can be tested by standardized proliferation assays in
vitro. In short, a sample enriched for T-cells is obtained from a
subject and subsequently kept in culture. The cultured T-cells are
contacted with APCs of the subject which have previously taken up
the modified molecule and processed it to present its T-cell
epitopes. The proliferation of T-cells is monitored and compared to
a suitable control (e.g. T-cells in culture contacted with APCs
which have processed intact, native amyloidogenic polypeptide).
Alternatively, proliferation can be measured by determining the
concentration of relevant cytokines released by the T-cells in
response to their recognition of foreign T-cells.
[0233] Having rendered highly probable that at least one analogue
of either type of set is capable of inducing antibody production
against APP or A.beta., it is possible to prepare an immunogenic
composition comprising at least one analouge which is capable of
inducing antibodies against unmodified APP or A.beta. in an animal
species where the unmodified APP or A.beta. is a self-protein, the
method comprising admixing the member(s) of the set which
significantly induces production of antibodies in the animal
species which are reactive with the APP or A.beta. with a
pharmaceutically and immunologically acceptable carrier and/or
vehicle and/or diluent and/or excipient, optionally in combination
with at least one pharmaceutically and immunologically acceptable
adjuvant.
[0234] The above-described tests of polypeptide sets are
conveniently carried out by initially preparing a number of
mutually distinct nucleic acid sequences or vectors of the
invention, inserting these into appropriate expression vectors,
transforming suitable host cells (or host animals) with the
vectors, and effecting expression of the nucleic acid sequences of
the invention. These steps can be followed by isolation of the
expression products. It is preferred that the nucleic acid
sequences and/or vectors are prepared by methods comprising
exercise of a molecular amplification technique such as PCR or by
means of nucleic acid synthesis.
[0235] Specific Amyloidogenic Targets
[0236] In addition to the proteins most often associated with
Alzheimer's, APP, ApoE4 and Tau, there is long list of other
proteins that have somehow been linked to AD, either by their
direct presence in plaques or tangles of AD brains or by their
apparent genetic association with increased risk of developing AD.
Most, if not all, of these antigens are together with the
above-discussed A.beta., APP, presenilin and ApoE4, putative target
proteins in certain embodiment of the present invention. These
putative targets are already discussed thoroughly in WO, 01/62284.
Hence, these putative targets will only be mentioned briefly here,
whereas a more thorough background discussion can be be found in WO
01/62282 which is hereby incorporated by reference herein:
[0237] Alpha1-antichymotrypsin (ACT); Alpha2-macroglobulin; ABAD
(A.beta.-peptide binding alcohol dehydrogenase); APLP1 and 2
(amyloid precursor like protein 1 and 2); AMY117; Bax; Bcl-2;
Bleomycin hydrolase; BR1/ABR1; Chromogranin A; Clusterin/apoJ; CRF
(corticotropin releasing factor) binding protein; EDTF
(endothelial-derived toxic factor); Heparan sulfate proteoglycans;
Human collapsin response mediator protein-2; Huntingtin
(Huntington's disease protein); ICAM-1; IL-6; Lysosome-associated
antigen CD68; P21 ras; PLC-delta 1 (phospholipase C isoenzyme delta
1); Serum amyloid P component (SAP); Synaptophysin; Synuclein
(alpha-synuclein or NACP); and TGF-bl (transforming growth factor
b1).
[0238] The presently described means and methods for
down-regulation of APP or A.beta. can be combined with therapies,
e.g. active specific immunotherapy, against any of these other
amyloidogenic polypeptides.
[0239] Apart from Alzheimer's disease, also cerebral amyloid
angiopathy is a disease that would be a suitable target for the
presently disclosed technology.
[0240] It is contemplated that most methods for immunizing against
APP or A.beta. should be restricted to immunization giving rise to
antibodies cross-reactive with the native APP or A.beta..
Nevertheless, in some cases it will be of interest to induce
cellular immunity in the form of CTL responses against cells which
present MHC Class I epitopes from the amyloidogenic
polypeptides--this can be expedient in those cases wherein
reduction in the number of cells producing APP or A.beta. does not
constitute a serious adverse effect. In such cases where CTL
responses are desired it is preferred to utilise the teachings of
Applicant's WO 00/20027. The disclosures of these two documents are
hereby incorporated by reference herein.
[0241] Immunogen Carriers
[0242] Molecules comprising a T helper epitope and APP or A.beta.
peptides representing or including B-cell epitopes linked
covalently to a non-immunogenic polymer molecule acting as a
vehicle, e.g. a multivalent activated poly-hydroxypolymer, will, as
mentioned above, function as a vaccine molecule that only contains
the immunologically relevant parts, can be obtained, and are
interesting embodiments in variants d and e disclosed above.
Promiscuous or so-called universal T-helper epitopes can be used if
e.g. the target for the vaccine is a self-antigen such as APP or
A.beta.. Furthermore, elements that enhance the immunological
response could be also co-coupled to the vehicle and thereby act as
an adjuvant. Such elements could be mannose, tuftsin, muramyl
dipeptide, CpG motifs etc. In that case, subsequent adjuvant
formulation of the vaccine product might be unnecessary and the
product could be administered in pure water or saline.
[0243] By coupling cytotoxic T cell (CTL) epitopes together with
the T-helper epitopes it will also be possible to generate CTL's
specific for the antigen from which the CTL epitope was derived.
Elements that promote uptake of the product to the cytosol, such as
mannose, of the APC, e.g. a macrophage, could also be co-coupled to
the vehicle together with the CTL- and the T helper epitope and
enhance the CTL response.
[0244] The ratio of B-cell epitopes and T-helper epitopes (P2 and
P30) in the final product can be varied by varying the
concentration of these peptides in the synthesis step. As mentioned
above, the immunogenic molecule can be tagged with e.g. mannose,
tuftsin, CpG-motifs or other immune stimulating substances
(described herein) by adding these, if necessary by using e.g.
aminated derivatives of the substances, to the carbonate buffer in
the synthesis step.
[0245] If an insoluble activated polyhydroxy polymer is used to
combine the peptides containing the APP or A.beta. B-cell epitope
and the T-helper epitopes it can, as mentioned above be performed
as a solid phase synthesis and the final product can be harvested
and purified by wash and filtration. The elements to be coupled to
a tresyl activated polyhydroxypolymer (peptides, tags etc) can be
added to the polyhydroxypolymer at low pH, e.g. pH 4-5, and allowed
to be equally distributed in the "gel" by passive diffusion.
Subsequently, the pH can be raised to pH 9-10 to start the reaction
of the primary amino groups on the peptides and tags to the tresyl
groups on the polyhydroxy polymer. After coupling of peptides and
e.g. immune stimulating elements the gel is grinded to form
particles of suitable size for immunization.
[0246] Such an immunogen therefore comprises
[0247] a) at least one first amino acid sequence derived from APP
or A.beta., wherein the at least one first amino acid sequence
contains at least one B-cell and/or at least one CTL epitope,
and
[0248] b) at least one second amino acid sequence that includes a
foreign T helper cell epitope,
[0249] wherein each of the at least first and at least second amino
acid sequences are coupled to a pharmaceutically acceptable
activated polyhydroxypolymer carrier.
[0250] In order for the amino acid sequences to couple to the
polyhydroxypolymer it is normally necessary to "activate" the
polyhydroxypolymer with a suitable reactive group that can form the
necessary link to the amino acid sequences.
[0251] The term "polyhydroxypolymer" is intended to have the same
meaning as in WO 00/05316, i.e. the polyhydroxypolymer can have
exactly the same characteristics as is specifically taught in that
application. Hence, the polyhydroxypolymer can be water soluble or
insoluble (thus requiring different synthesis steps during
preparation of the immunogen). The polyhydroxypolymer can be
selected from naturally occurring polyhydroxy compounds and
synthetic polyhydroxy compounds.
[0252] Specific and preferred polyhydroxypolymers are
polysaccharides selected from acetan, amylopectin, gum agar-agar,
agarose, alginates, gum Arabic, carregeenan, cellulose,
cyclodextrins, dextran, furcellaran, galactomannan, gelatin,
ghatti, glucan, glycogen, guar, karaya, konjac/A, locust bean gum,
mannan, pectin, psyllium, pullulan, starch, tamarine, tragacanth,
xanthan, xylan, and xyloglucan. Dextran is especially
preferred.
[0253] However, the polyhydroxypolymer can also be selected from
highly branched poly(ethyleneimine)(PEI), tetrathienylene vinylene,
Kevlar (long chains of poly-paraphenyl terephtalamide),
Poly(urethanes), Poly(siloxanes), polydimethylsiloxane, silicone,
Poly(methyl methacrylate) (PMMA), Poly(vinyl alcohol), Poly(vinyl
pyrrolidone), Poly(2-hydroxy ethyl methacrylate), Poly(N-vinyl
pyrrolidone), Poly(vinyl alcohol), Poly(acrylic acid),
Polytetrafluoroethylene (PTFE), Polyacrylamide,
Poly(ethylene-co-vinyl acetate), Poly(ethylene glycol) and
derivatives, Poly(methacrylic acid), Polylactides (PLA),
Polyglycolides (PGA), Poly(lactide-co-glycolides) (PLGA),
Polyanhydrides, and Polyorthoesters.
[0254] The (weight) average molecular weight of the
polyhydroxypolymer in question (i.e. before activation) is
typically at least 1,000, such as at least 2,000, preferably in the
range of 2,500-2,000,000, more preferably in the range of
3,000-1,000,000, in particular in the range of 5,000-500,000. It
has been shown in the examples that polyhydroxypolymers having an
average molecular weight in the range of 10,000-200,000 are
particularly advantageous.
[0255] The polyhydroxypolymer is preferably water soluble to an
extent of at least 10 mg/ml, preferably at least 25 mg/ml, such as
at least 50 mg/ml, in particular at least 100 mg/ml, such as at
least 150 mg/ml at room temperature. It is known that dextran, even
when activated as described herein, fulfils the requirements with
respect to water solubility.
[0256] For some of the most interesting polyhydroxypolymers, the
ratio between C (carbon atoms) and OH groups (hydroxy groups) of
the unactivated polyhydroxypolymers (i.e. the native
polyhydroxypolymer before activation) is in the range of 1.3 to
2.5, such as 1.5-2.3, preferably 1.6-2.1, in particular 1.85-2.05.
Without being bound to any specific theory, it is believed that
such as a C/OH ratio of the unactivated polyhydroxypolymer
represents a highly advantageous level of hydrophilicity.
Polyvinylalcohol and polysaccharides are examples of
polyhydroxypolymers which fulfil this requirement. It is believed
that the above-mentioned ratio should be roughly the same for the
activated polyhydroxypolymer as the activation ratio should be
rather low.
[0257] The term "polyhydroxypolymer carrier" is intended-to denote
the part of the immunogen that carries the amino acid sequences. As
a general rule, the polyhydroxypolymer carrier has its outer limits
where the amino acid sequences can be cleaved of by a peptidase,
e.g. in an antigen presenting cell that is processing the
immunogen. Hence, the polyhydroxypolymer carrier can be the
polyhydroxypolymer with an activation group, where the bond between
the activation group and the amino acid sequence is cleavable by a
peptidase in an APC, or the polyhydroxypolymer carrier can be a
polyhydroxypolymer with activation group and e.g. a linker such as
a single L-amino acid or a number of D-amino acids, where the last
part of the linker can bond to the amino acid sequences and be
cleaved by a peptidase in an APC.
[0258] As mentioned above, the polyhydroxypolymers carry functional
groups (activation groups), which facilitate the anchoring of
peptides to the carrier. A wide range of applicable functional
groups are known in the art, e.g. tresyl (trifluoroethylsulphonyl),
maleimido, p-nitrophenyl cloroformate, cyanogenbromide, tosyl
(p-toluenesulfonyl), triflyl (trifluoromethanesulfonyl),
pentafluorobenzenesulfonyl, and vinyl sulphone groups. Preferred
examples of functional groups within the present invention are
tresyl, maleimido, tosyl, triflyl, pentafluorobenzenesulfonyl,
p-nitrophenyl cloroformate, and vinylsulphone groups, among which
tresyl, maleimido, and tosyl groups are particularly relevant.
[0259] Tresyl activated polyhydroxypolymers can be prepared using
tresyl chloride as described for activation of dextran in Example 1
in WO 00/05316 or as described in Gregorius et al., J. Immunol.
Meth. 181 (1995) 65-73.
[0260] Maleimido activated polyhydroxypolymers can be prepared
using p-maleimidophenyl isocyanate as described for activation of
dextran in Example 3 of WO 00/05316. Alternatively, maleimido
groups could be introduced to a polyhydroxypolymer, such as
dextran, by derivatisation of a tresyl activated polyhydroxypolymer
(such as tresyl activated dextran (TAD)) with a diamine compound
(generally H.sub.2N--C,H.sub.2, --NH.sub.2, where n is 1-20,
preferably 1-8), e.g. 1,3-diaminopropane, in excess and
subsequently react the amino groups introduced in TAD with reagents
such as succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxyl-
ate (SMCC), sulfo-succinimidyl
4-(N-maleimidomethyl)-cyclohexane-1-carboxy- late (sulfo-SMCC),
succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB),
sulfo-succinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB),
N-.gamma.-maleimidobutyryloxy-succinimide ester (GMBS) or
N-.gamma.-maleimidobutyryloxy-sulfosuccinimide ester. Although the
different reagents and routes for activation formally results in
slightly different maleimide activated products with respect to the
linkage between the maleimide functionality and the remainder of
the parent hydroxy group on which activation is performed, all and
every are considered as "maleimide activated
polyhydroxypolymers".
[0261] Tosyl activated polyhydroxypolymers can be prepared using
tosyl chloride as described for activation of dextran in Example 2
in WO 00/05316. Triflyl and pentafluorobenzenesulfonyl activated
polyhydroxypolymers are prepared as the tosyl or tresyl activated
analogues, e.g. by using the corresponding acid chlorides.
[0262] Cyanogenbromide activated polyhydroxypolymer can be prepared
by reacting the polyhydroxypolymer with cyanogenbromide using
conventional methods. The resulting functional groups are normally
cyanate esters with two hydroxy groups of the
polyhydroxypolymer.
[0263] The degree of activation can be expressed as the ratio
between the free hydroxy groups and the activation groups (i.e.
functionalised hydroxy groups). It is believed that a ratio between
the free hydroxy groups of the polyhydroxypolymer and the
activation groups should be between 250:1 and 4:1 in order to
obtain an advantageous balance between the hydrophilicity and the
reactivity of the polyhydroxypolymer. Preferably the ratio is
between 100:1 and 6:1, more preferably between 60:1 and 8:1, in
particular between 40:1 and 10:1.
[0264] Especially interesting activated polyhydroxypolymers for use
in the method for producing the generally applicable immunogen
according to the invention are tresyl, tosyl and maleimido
activated polysaccharides, especially tresyl activated dextran
(TAD), tosyl activated dextran (TosAD), and maleimido activated
dextran (MAD).
[0265] It is preferred that the bond between the polyhydroxypolymer
carrier and the amino acid sequences attached thereto are cleavable
by a peptidase, e.g. as a peptidase active in the processing of
antigens in an APC. It is therefore preferred that the at least
first and at least second amino acid sequences are coupled to the
activated polyhydroxypolymer carrier via an amide bond or a peptide
bond. It is especially preferred that the at least first and at
least second amino acid sequences each provide for the nitrogen
moiety of their respective amide bond.
[0266] The polyhydroxypolymer carrier may be substantially free of
amino acid residues, necessitating that the activation group
provides for part of a peptidase cleavable bond, but as mentioned
above, the carrier may also simply include a spacer including at
least one L-amino acid. Nevertheless, the at least first and at
least second amino acid sequences are normally bound to the
activated version of the polyhydroxypolymer via the nitrogen at the
N-terminus of the amino acid sequence.
[0267] The above-described generally applicable immunogen of the
present invention can be used in immunization methods essentially
as described herein for polypeptide vaccines. That is, all
disclosures relating to dosages, mode of administration and
formulation of polypeptide vaccines for down-regulating the
amyloidogenic polypeptides discussed herein apply mutatis mutandis
to the generally applicable immunogens.
[0268] Generally Applicable Safe Vaccination Technology
[0269] As discussed above, one preferred embodiment of the present
invention entails the use of variants of amyloidogenic polypeptides
that are incapable of providing self-derived T.sub.H epitopes that
may drive an immune response against the amyloidogenic
polypeptide.
[0270] However, it is believed by the present inventors that this
strategy for designing anti-self vaccines and for effecting
anti-self immunity, is a generally applicable technology that is
inventive in its own right. It should prove especially suited in
cases where the self-antigen it is sought to down-regulate is
sufficiently abundant in the body so that it is possible that
self-stimulation of an immune response could happen. Hence, all
disclosures above of this embodiment insofar as it relates to the
provision of an anti-self immune response against APP or A.beta.
applies mutatis mutandis to immunization against other
self-polypeptides, especially those that are present in sufficient
amounts for them to maintain the immune response in the form of an
uncontrolled autoimmune condition because autologous TH epitopes of
the relevant self-poly-peptide are driving the immune response.
EXAMPLE 1
[0271] The Auto Vaccination Approach for Immunizing Against AD
[0272] The fact that A.beta. protein knock out mice does not show
any abnormalities or adverse side effects, suggest that removal or
lowering the amounts of AP will be safe, Zheng H. (1996).
[0273] Published experiments where transgenic animals are immunized
against the transgenic human A.beta. protein suggest that if it was
possible to break the self tolerance, down-regulation of AP could
be obtained by auto-reactive antibodies. These experiments further
suggest that such down regulation of A.beta. potentially would both
prevent the formation of plaques, and even clear already formed
A.beta. plaques from the brain, cf. Schenk et al. (1999). But,
traditionally it is not possible to raise antibodies against
self-proteins.
[0274] The published data does thus not provide the means for
breaking true self-tolerance towards true self-proteins. Nor does
the data provide information on how to ensure that the immune
reaction is directed solely or predominantly towards the A.beta.
deposits, and not towards the cell membrane bound A.beta. precursor
protein (APP), if this is deemed necessary. An immune response
generated using the existing technology would presumably generate
an immune response towards self-proteins in an unregulated way so
unwanted and excessive auto-reactivity towards parts the A.beta.
protein may be generated. Hence, using existing immunization
strategies will most likely be unable to generate strong immune
responses towards self-proteins and will furthermore be unsafe due
to potential strong cross-reactivity towards membrane bound APP
which is present on a large number of cells in the CNS.
[0275] The present invention provides the means of effectively
generating a strong regulated immune response towards true
self-proteins which potentially could form plaques and cause
serious disease in the CNS or in other compartments of the ody. A
safe and efficacious human A.beta. protein therapeutic vaccine will
be developed by using this technology for the treatent of AD.
[0276] In light of this, it is possible to anticipate that AD, a
disease predicted to cripple the health care system in the next
entury, could be cured, or such vaccines described could at least
constitute an effective therapeutical approach for treatment of the
symptoms and progression of this disease. This technique represents
a entirely new immunological approach to blocking amyloid
deposition in AD and other neurologic diseases as well.
[0277] In the following table, 35 contemplated constructs are
indicated. All positions given in the table are relative to the
starting Methionine of APP (first amino acid in SEQ ID NO: 2) and
include both the starting and ending amino acid, e.g. the 672-714
fragment includes both amino acid 672 and 714. The starting and
ending positions for P2 and P30 indicate that the epitope
substitutes a part of the APP fragment at the positions indicated
(both positions included in the substitution)--in most constructs,
the introduced epitopes substitutes a fragment of the length of the
epitope. The asterisks in the table have the following meaning:
[0278] *) Only one position for P2 and P30 indicates that the
epitope has been inserted into the APP derivative at the position
indicated (the epitope begins at the amino acid C-terminally
adjacent to the given position).
[0279] **) Construction 34 contains three identical APP fragments
separated by P30 and P2, respectively.
[0280] ***) Construction 35 contains nine identical APP fragments
separated by alternating P30 and P2 epitopes.
1 APP AutoVac constructions Start of End of Position of APP APP P2
epitope Position of segment segment relative to P30 epitope Var.
relative to relative to aa 1 of relative to Molecule No. aa 1 of
APP aa 1 of APP APP aa 1 of APP length 1 630 770 656-670 635-655
141 2 630 714 656-670 635-655 85 3 672 770 735-749 714-728 99 4 672
770 714-728 99 5 672 770 714-728 99 6 672 770 723* 723* 135 7 672
770 723* 120 8 672 770 723* 114 9 672 714 672* 64 10 672 714 714*
64 11 672 714 672* 58 12 672 714 714* 58 13 672 714 714* 672* 79 14
672 714 680-694 43 14 672 714 685-799 43 16 672 714 690-704 43 17
672 714 695-709 43 18 672 714 675-695 43 19 672 714 680-700 43 20
672 714 685-705 43 21 672 714 690-710 43 22 672 714 680* 680* 79 23
672 714 690* 690* 79 24 672 714 700* 700* 79 25 672 714 710* 710*
79 26 672 714 680* 64 27 672 714 690* 64 28 672 714 700* 64 29 672
714 710* 64 30 672 714 680* 58 31 672 714 690* 58 32 672 714 700*
58 33 672 714 710* 58 34 672 714 After rep. After rep. 165 1** 2**
35 672 714 34 .times. 3* 34 .times. 3*** 165
[0281] The part of APP, against which it most interesting to
generate a response, is the 43 amino acid A.beta. core peptide
(A.beta.-43, corresponding to SEQ ID NO: 2, residues 672-714) that
is the main constituent of amyloid plaques in AD brains. This APP
fragment is part of all constructions listed above.
[0282] Variants 1 and 2 comprise a portion of APP upstream of
A.beta.-43 where the model epitopes P2 and P30 have been placed.
Variants 1 and 3-8 all comprise the C-100 fragment which has been
shown to be neurotoxic--the C-100 fragment corresponds to amino
acid residues 714-770 of SEQ ID NO: 2. In variants 3-5 the epitopes
substitutes a part of the C-100 fragment while the in variants 6-8
have been inserted into C-100.
[0283] Variants 9-35 contain only the core A.beta.-43 protein. In
variants 9-13, P2 and P30 are fused to either end of A.beta.-43; in
14-21 P2 and P30 substitutes part of A.beta.-43; in 22-33 P2 and
P30 are inserted into A.beta.-43; 34 contains three identical
A.beta.-43 fragments spaced by P30 and P2, respectively; 35
contains 9 A.beta.-43 repeats spaced by alternating P2 and P30
epitopes.
[0284] Truncated parts of the above-discussed A.beta.-43 protein
can also be employed in immunogenic analogues according to the
present invention. Especially preferred are the truncates
A.beta.(1-42), A.beta.(1-40), A.beta.(1-39), A.beta.(1-35),
A.beta.(1-34), A.beta.(1-34), A.beta.(1-28), A.beta.(1-12),
A.beta.(1-5), A.beta.(13-28), A.beta.(13-35), A.beta.(17-28),
A.beta.(25-35), A.beta.(35-40), A.beta.(36-42), and A.beta.(35-42)
(where the numbers in the parentheses indicate the amino acid
stretches of A.beta.-43 that constitute the relevant
fragment--A.beta.(35-40) is e.g. identical to amino acids 706-711
in SEQ ID NO: 2). All these variants with truncated parts of
A.beta.-43 can be made with the A.beta. fragments described herein,
in particular with variants 9, 10, 11, 12, and 13.
[0285] In some cases, it is preferred that the A.beta.-43 or
fragments thereof are mutated. Especially preferred are
substitution variants where the methionine in position 35 in
A.beta.-43 has been substituted, preferably with leucine or
isoleucine, or simply deleted. Especially preferred analogues
contain one single methionine that is located in the C-terminus,
either because it is naturally occurring in the amyloidogenic
polypeptide or foreign T.sub.H epitope, or because it has been
inserted or added. Hence, it also preferred that the part of the
analogue that includes the foreign T.sub.H epitope is free from
methionine, except from the possible C-terminal location of a
methionine.
[0286] In fact, it is generally preferred that all analogues of APP
or A.beta. that are used according to the present invention share
the characteristic of merely including one single methionine that
is positioned as the C-terminal amino acid in the analogue and that
other methionines in either the amyloidogenic polypeptide or the
foreign T.sub.H epitope are deleted or substituted for another
amino acid.
[0287] One further interesting mutation is a deletion or
substitution of the phenylalanine in position 19 in A.beta.-43, and
it is especially preferred that the mutation is a substitution of
this phenylalanine residue with a proline.
[0288] The following table sets forth a group of especially
preferred constructs that operate with truncates or mutations of
A.beta.-43:
2 Position of Position of A.beta. segment used in Position of
A.beta. segment P2 epitope P30 epitope Total Variant molecole
relative to relative to aa 1 of relative to relative to length of
No. aa 1 of A.beta. (1-42/43) molecule aa 1 of molecule aa 1 of
molecule molecule (aa) 36 1-28 22-49 50-64 1-21 64 37 1-12 (a) +
13-28 (b) 1-12 (a) + 49-64 (b) 34-48 13-33 64 38 1-12 (x 3) 1-12,
34-45, 61-72 46-60 13-33 72 39 13-28 (x 3) 1-16, 39-53, 69-84 54-68
17-37 84 40 1-12 (a) + 13-35 (b) + 1-12 (a) + 34-56 (b) + 57-71
13-33 78 36-42 (c) 72-78 (c) 41 1-28 (x 3) 1-28, 50-77, 93-120
78-92 29-49 120 42 1-43 (F19P/M35K) 1-43 65-79 44-64 79
[0289] In this table, the A.beta. segment used in the molecule is
indicated by amino acid numbers relative to aa 1 of the
A.beta.(1-42/43) molecule, i.e. 1-28 means that fragment 1-28 of
A.beta.(1-42/43) is used in the molecule. If two or more different
segments are used, both are indicated in the table, i.e. 1-12
(a)+13-28 (b) means that both fragment 1-12 and fragment 13-28 of
A.beta.(1-42/43) are used in the molecule.
[0290] Also, if the same segment is present in more than one copy
in the construction it is indicated in the table, i.e. 1-12 (x3)
shows that fragment 1-12 of A.beta.(1-42/43) is present in three
copies in the construction.
[0291] Further, the position of the A.beta. segment in the molecule
is shown by amino acid positions relative to the first amino acid
of the molecule, i.e. 22-49 shows that the A.beta. fragment in
question is positioned from amino acid 22 to amino acid 49 in the
molecule, both positions included. Positions of the P2 and P30
epitopes are indicated equivalently. If two or more different
A.beta. fragments are used in the molecule, their positions are all
shown, i.e. 1-12 (a)+49-64 (b) means that fragment (a) is
positioned from aa 1-12 in the molecule and fragment (b) from aa
49-64.
[0292] Moreover, if more than one copy of the same fragment is
present in the molecule, positions for all copies are shown, i.e.
1-12, 34-45, 61-72 shows that the three copies of the A.beta.
fragment are placed from position 1-12, 34-45 and 61-72,
respectively, in the molecule.
[0293] Finally, the total length indication of each molecule
includes both the A.beta. fragment(s) and the P2 and P30
epitopes.
[0294] Variant 42 contains two amino acid substitutions at
positions 19 (phe to pro) and 35 (met to lys) as it is indicated in
the column showing the A.beta. fragments.
[0295] See FIG. 1 and the tables above for details on particular
points for introduction of the foreign T-cell epitopes.
[0296] One further type of construct is especially preferred. Since
one goal of the present invention is to avoid destruction of the
cells producing APP whereas removal of A.beta. is desired, it seems
feasible to prepare autovaccine constructs comprising only parts of
A.beta. which are not exposed to the extracellular phase when
present in APP. Thus, such constructs would need to contain at
least one B-cell epitope derived from the amino acid fragment
defined by amino acids 700-714 in SEQ ID NO: 2. Since such a short
polypeptide fragment is predicted to be only weakly immunogenic it
is preferred that such an autovaccine construct consists of several
copies of the B-cell epiope, e.g. in the form of a construct having
the structure hown in Formula I in the detailed disclosure of the
present nvention, cf. above. In that version of Formula I, the
terms amyloid.sub.e1-amyloid.sub.ex are x B-cell epitope containing
amino acid sequences derived from amino acids 700-714 of SEQ ID NO:
2. A preferred alternative is the above-detailed possibility of
coupling the amyloidogenic (poly)peptide and the selected foreign
T-helper epitope to via an amide bond to a polysaccharide carrier
molecule--in this way multiple presentaions of the "weak" epitope
constituted by amino acids 700-714 of SEQ ID NO: 2 become possible,
and it also becomes possible to select an optimum ratio between
B-cell and T-cell epitopes.
EXAMPLE 2
[0297] Immunisation of Transgenic Mice with A.beta. and Modified
Proteins According to the Invention
[0298] Construction of the hAB43+-34 encoding DNA. The hAB43+-34
gene was constructed in several steps. First a PCR fragment was
generated with primers ME#801 (SEQ ID NO: 10) and ME#802 (SEQ ID
NO: 11) using primer ME#800 (SEQ ID NO: 9) as template. ME#800
encodes the human abeta-43 fragment with E. coli optimised codons.
ME#801 and 802 adds appropriate restriction sites to the
fragment.
[0299] The PCR fragment was purified, digested with NcoI and
HindIII, purified again and cloned into NcoI-HindIII digested and
purified pET28b+E. coli expression vector. The resulting plasmid
encoding wildtype human A.beta.-43 is named pAB1.
[0300] In the next step the T-helper epitope, P2, is added to the
C-terminus of the molecule. Primer ME#806 (SEQ ID NO: 12) contains
the sequence encoding the P2 epitope, thus generating a fusion of
P2 and Abeta-43 by the PCR reaction.
[0301] The cloning was performed by making a PCR fragment with
primers ME#178 (SEQ ID NO: 8) and ME#806 using pAB1 as template.
The fragment was purified, digested with NcoI and HindIII, purified
again and cloned into an NcoI-HindIII digested and purified
pET28b+vector. The resulting plasmid is called pAB2.
[0302] In an analogous manner, another plasmid was made harbouring
the A.beta.-43 encoding sequence with another T helper epitope,
P30, added to the N-terminus. This was done by making a PCR
fragment with primers ME#105 (SEQ ID NO: 7) and ME#807 (SEQ ID NO:
13) using pAB1 as template.
[0303] The fragment was purified, digested with NcoI and HindIII,
purified again and cloned into an NcoI-HindIII digested and
purified pET28b+vector. The resulting plasmid is called pAB3.
[0304] In the third step, a second A.beta.-43 repeat is added
C-terminally to the P2 epitope of plasmid pAB2 by primer ME#809
(SEQ ID NO: 14). ME#809 at the same time creates a BamHI site
immediately after the A.beta.-43 repeat. A PCR fragment was made
with primers ME#178 and ME#809 using pAB2 as template. The fragment
was digested with NcoI and HindIII, purified and cloned into
NcoI-HindIII digested and purified pET28b+vector. This plasmid is
named pAB4.
[0305] Finally, the P30 epitope--A.beta.-43 repeat sequence from
pAB3 was cloned into pAB4 plasmid. This was done by making a PCR
fragment with primers ME#811 (SEQ ID NO: 16) and ME#105 using pAB3
as template. The fragment was purified and used as primer in a
subsequent PCR with ME#810 (SEQ ID NO: 15) using pAB3 as template.
The resulting fragment was purified, digested with BamHI and
HindIII and cloned into BamHI-HindIII digested and purified pAB4
plasmid. The resulting plasmid, pAB5, encodes the hAB43+-34
molecule.
[0306] All PCR and cloning procedures were done essentially as
described by Sambrook, J., Fritsch, E. F. & Maniatis, T. 1989
"Molecular cloning: a laboratory manual". 2nd. Ed. Cold Spring
Harbor Laboratory, N.Y.
[0307] For all cloning procedures E. coli K-12 cells, strain Top-10
F' (Stratagene, USA), were used. The pET28b+vector was purchased
from Novagen, USA. All primers were synthesised at DNA Technology,
Denmark.
[0308] Expression and purification of hAB43+-34. The hAB43+-34
protein encoded by pAB5 was expressed in BL21-Gold (Novagen) E.
coli cells as described by the suppliers of the pET28b+system
(Novagen).
[0309] The expressed hAB43+-34 protein was purified to more than
85% purity by washing of inclusion bodies followed by
cation-exchange chromatography using a BioCad purification
workstation (PerSeptive Biosystems, USA) in the presence of 6 M
urea. The urea was hereafter removed by stepwise dialysis against a
solution containing decreasing amounts of urea. The final buffer
was 10 mM Tris, pH 8.5.
[0310] Immunisation study. Mice transgenic for human APP
(Alzheimer's precursor protein) were used for the study. These
mice, called TgRND8+, express a mutated form of APP that results in
high concentration of A.beta.-40 and A.beta.-42 in the mouse brains
(Janus, C. et. al.)
[0311] The mice (8-10 mice per group) were immunised with either
Abeta-42 (SEQ ID NO: 2, residues 673-714, synthesised by means of a
standard Fmoc strategy) or the hAB43+-34 variant (construct 34 in
the table in Example 1, recombinantly produced) four times at
two-week intervals. Doses were either 100 mg for A.beta. or 50 mg
for hAB43+-34. Mice were bled at day 43 (after three injections)
and after day 52 (after four injections) and the sera were used to
determine the level of anti-A.beta.-42 specific titres using a
direct A.beta.-42 ELISA.
[0312] The following tabel shows the mean relative anti-Abeta-42
titres.
3 Day 43 Day 52 Immunogen (after 3 immunizations) (after 4
immunizations) A.beta.-42 4000 3000 hAB43+ -34 16000 23000
[0313] As will be clear, the antibody titers obtained when
immunizing with the hAB43+-34 A.beta. variant are approximately 4
times and 7.5 times higher after 3 and 4 immunizations,
respectively, than the titers obtained when using the unaltered
wild-type A.beta.-42 as an immunogen. This fact is put further in
perspective, when considering the fact that the amount of variant
used for immunization was only 50% of the amount of wild-type
sequence used for immunization.
EXAMPLE 3
[0314] Synthesis of an A.beta. Peptide Copolymer Vaccine Using
Activated Poly-Hydroxypolymer as the Cross-Linking Agent.
[0315] Introduction. A traditional conjugate vaccine consists of a
(poly)peptide coupled covalently to a carrier protein. The peptide
contains the B-cell epitope(s) and the carrier protein provides
T-helper epitopes. However, most of the carrier protein will
normally be irrelevant as a source for T-helper epitopes, since
only aminor part of the total sequence contains the relevant
T-helper epitopes. Such epitopes can be defined and synthesized as
peptides of e.g. 12-15 amino acids. If these peptides are linked
covalently to peptides containing the B-cell epitopes, e.g. via a
multivalent activated poly-hydroxypolymer, a vaccine molecule that
only contains the relevant parts can be obtained. It is further
possible to provide a vaccine conjugate that contains an optimized
ratio between B-cell and T-cell epitopes.
[0316] Synthesis of the activated poly-hydroxypolymer.
Poly-hydroxypolymers such as dextran, starch, agarose etc. can be
activated with 2,2,2-trifluoroethanesulfonyl chloride (tresyl
chloride), either by means of a homogenous synthesis (dextran)
dissolved in N-methylpyrrolidinone (NMP) or by means of a
heterogeneous synthesis (starch, agarose, cross-linked dextran) in
e.g. acetone.
[0317] 225 ml dry N-methylpyrrolidinone (NMP) is added under dry
conditions to freeze dried, water-soluble dextran (4.5 g, 83 mmol,
clinical grade, Mw(avg) 78000) in a 500 ml round bottom flask
supplied with a magnet for stirring. The flask is placed in a
60.degree. C. oil bath with magnetic stirring. The temperature is
raised to 92.degree. C. over a period of 20 min. When the dextran
is dissolved the flask is immediately removed from the oil bath and
the temperature in the bath is lowered to 40.degree. C. The flask
is placed into the oil bath agaom, still with magnetic stirring,
and tresyl chloride (2.764 ml, 25 mmol) is added dropwise. After 15
min, dry pyridine (anhydrous, 2.020 ml, 25 mmol) is added
drop-wise. The flask is removed from the oil bath and stirred for 1
hour at room temperature. The product (Tresyl Activated Dextran,
TAD) is precipitated in 1200 ml cold ethanol (99.9%). The
supernatant is decanted and the precipitate is harvested in 50 ml
polypropylene tubes in a centrifuge at 2000 rpm. The precipitate is
dissolved in 50 ml 0.5% acetic acid, dialyzed 2 times against 5000
ml 0.5% acetic acid and freeze dried. TAD can be stored as a freeze
dried powder at -20.degree. C.
[0318] An insoluble poly-hydroxypolymer, such as agarose or
croos-linked dextran can be tresyl activated by making a suspension
of the poly-hydroxypolymer in e.g. acetone and perform the
synthesis as a solid phase synthesis. The activated
poly-hydroxypolymer can be harvested by filtration. Suitable
methods are reported in e.g. Nilsson K and Mosbach K (1987),
Methods in Enzymology 135, p. 67, and in Hermansson G T et al.
(1992), in "Immobilized Affinity Ligand Techniques", Academic
Press, Inc., p. 87.
[0319] Synthesis of the A Beta Peptide Copolymers Vaccines. TAD (10
mg) is dissolved in 100 .mu.l H.sub.2O and 1000 .mu.l carbonate
buffer, pH 9.6, containing 5 mg A.beta.-42 (SEQ ID NO: 2, residues
673-714), 2.5 mg P2 (SEQ ID NO: 4) and 2.5 mg P30 (SEQ ID NO: 6) is
added. The A.beta.-42 and the P2 and P30 peptides all contain
protected lysine groups: these are in the form of
1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) protected
lysine groups. The peptides are prepared by means of a standard
Fmoc strategy, where the conventional Fmoc-Lys(Boc)-OH has been
substituted with Fmoc-Lys(Dde)-OH (obtained from Novabiochem, cat.
no. 04-12-1121), i.e. the F-amino group in lysine is protected with
Dde instead of Boc.
[0320] The pH value is measured and adjusted to 9.6 using 1 M HCl.
After 2.5 hours at room temperature, hydrazine from an 80% solution
is added to a final hydrazine koncentration of 8% and the solution
is incubated for another 30 min. at room temperature and
freeze-dried immediately hereafter. The freeze-dried product is
dissolved in H.sub.2O and dialysed extensively against H.sub.2O
before the final freeze-drying.
[0321] The ratio between B-cell epitopes (A.beta.) and T-helper
epitopes (P2 and P30) in the final product can be varied by using
different concentrations of these peptides in the synthesis step.
Furthermore, the final product can be tagged with e.g. mannose (so
as to target the conjugate to APCs) by adding aminated mannose to
the carbonate buffer in the synthesis step.
[0322] If an insoluble activated poly-hydroxypolymer is used to
combine the peptides containing the B-cell epitope and the T-helper
epitopes, the coupling to the polymer can be performed as a solid
phase synthesis and the final product is harvested and purified by
wash and filtration.
[0323] As mentioned in the general description, the presently
described approach for preparing a peptide based vaccine may be
applied to any other polypeptide antigen where it would be
convenient to prepare a purely synthetic peptide vaccine and where
the polypeptide antigen in question provides a sufficient
immunogenicity in one single peptide:
EXAMPLE 4
[0324] Synthesis Peptide Copolymer Vaccines
[0325] TAD (10 mg) is dissolved in 100 .mu.l H.sub.2O and 1000
.mu.l carbonate buffer, pH 9.6, containing 1-5 mg peptide A (any
immunogenic peptide of interest!), 1-5 mg P2 (diphtheria toxoid P2
epitope) and 1-5 mg P30 (diphtheria toxoid P30 epitope) is added.
The pH value is measured and adjusted to 9.6 using 0.1 M HCl. After
2.5 hours at room temperature the solution is freeze dried
immediately hereafter. The freeze-dried product is dissolved in
H.sub.2O and dialysed extensively against H.sub.2O or desalted on a
gelfiltration column before the final freeze-drying. In case the
peptides have lysine in the sequence the .epsilon.-amine in the
lysine side chain should be protected by Dde using the
Fmoc-Lys(Dde)-OH derivative in the synthesis (Gregorius and Theisen
2001, submitted). After coupling, hydrazine from an 80% solution is
added to a final hydrazine concentration between 1-20% and the
solution is incubated for another 30 min at room temperature,
freeze dried immediately hereafter and dialysed extensively against
H.sub.2O or desalted on a gelfiltration column before the final
freeze-drying. The principle is set forth in schematic form in FIG.
2.
[0326] Such immunogens have been utilised by the inventors with a
short C-terminal fragment of the Borrelia burgdorferi protein OspC
as "peptide A" and a diptheria toxoid epitope (P2 or P30) as a
peptide B. The results of immunization studies with this antigen
revealed that only the immunogen of the invention including the
OspC fragment and a foreign diptheria epitope matching the MHC
haplotype of the vaccinated mice were capable of inducing
antibodies reactive with OspC in these mice. In contrast, a
molecule containing only the OspC peptide was unable to induce
antibody production and the same was true for a mixture of 2
immunogens where one contained the OspC and the other the epitope.
It is therefore concluded that the inclusion in the same
polyhydroxypolymer carrier is superior, if not essential, in order
to induce antibody production against a short peptide hapten as
OspC.
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Dementia. Annals of Neurology, 43, 815-825.
[0340] Schenk, D.; Barbour, R.; Dunn, W.; Gordon, G.; Grajeda, H.;
Guido, T.; Hu, K.; Huang, J.; Johnson-Wood, K.; Khan, K.;
Kholodenko, D.; Lee, M.; Liao, Z.; Lieberburg, I.; Motter, R.;
Mutter, L.; Soriano, F.; Shopp, G.; Vasquez, N.; Vandevert, C.;
Walker, S.; Wogulis, M.; Yednock, T.; Games, D.; Seubert, P.
(1999). Immunization with A-beta Attenuates Alzheimer's
Disease-Like Pathology in the PDAPP Mouse. Nature, 400(6740),
173-177.
[0341] Shekunov, B. et. al. (1999), J. Crystal Growth 198/199:
1345-1351.
[0342] Spillantini, M. G.; Murrell, J. R.; Goedert, M.; Farlow, M.
R.; Klug, A.; Ghetti, B. (1998). Mutation in the Tau Gene in
Familial Multiple System Tauopathy with Presenile Dementia.
Proceedings of the National Academy of Sciences U.S.A., 95(13),
7737-7741.
[0343] Strittmatter, W. J.; Saunders, A. M.; Schmechel, D.;
Pericak-Vance, M.; Enghild, J.; Salvesen, G. S.; Roses, A. D.
(1993). Apolipoprotein E: High-Avidity Binding to A.beta. and
Increased Frequency of Type 4 Allele in Late-Onset Familial
Alzheimer Disease. Proceedings of the National Academy of Sciences
U.S.A., 90, 1977-1981.
[0344] Vidal, R.; Frangione, B.; Rostagno, A.; Mead, S.; Revesz,
T.; Plant, G.; Ghiso, J. (1999). A Stop-Codon Mutation in the BR1
Gene Associated with Familial British Dementia. Nature, 399:
776-781.
[0345] Zheng H. (1996) "Mice deficient for the amyloid precursor
protein gene. Ann. N Y Acad. Sci., 777, 421-426.
[0346] York, P. (1999), PSTT 11: 430-440
[0347] The invention will now be further described by the following
numbered paragraphs:
[0348] 1. A method for in vivo down-regulation of amyloid precursor
protein (APP) or beta amyloid (A.beta.) in an animal, including a
human being, the method comprising effecting presentation to the
animal's immune system of an immunogenically effective amount of at
least one analogue of APP or A.beta. that incorporates into the
same molecule at least one B-cell epitope of APP and/or A.beta. and
at least one foreign T-helper epitope (T.sub.H epitope) so that
immunization of the animal with the analogue induces production of
antibodies against the animal's autologous APP or A.beta., wherein
the analogue
[0349] a) is a polyamino acid that consists of at least one copy of
a subsequence of residues 672-714 in SEQ ID NO: 2, wherein the
foreign T.sub.H epitope is incorporated by means of amino acid
addition and/or insertion and/or deletion and/or substitution,
wherein the subsequence is selected from the group consisting of
residues 1-42, residues 1-40, residues 1-39, residues 1-35,
residues 1-34, residues 1-28, residues 1-12, residues 1-5, residues
13-28, residues 13-35, residues 17-28, residues 25-35, residues
35-40, residues 36-42 and residues 35-42 of the amino acid sequence
consisting of amino acid residues 673-714 of SEQ ID NO: 2;
and/or
[0350] b) is a polyamino acid that contains the foreign TH epitopes
and a disrupted APP or AP sequence so that the analogue does not
include any subsequence of SEQ ID NO: 2 that binds productively to
MHC class II molecules initiating a T-cell response; and/or
[0351] c) is a polyamino acid that comprises the foreign T.sub.H
epitope and APP or A.beta. derived amino acids, and comprises 1
single methionine residue located in the C-terminus of the
analogue, wherein other methionine residues in APP or A.beta. and
in the foreign T.sub.H epitope have been substituted or deleted,
and preferably have been substituted by leucin or isoleucine;
and/or
[0352] d) is a conjugate comprising a polyhydroxypolymer backbone
to which is separately coupled a polyamino acid as defined in a)
and/or a polyamino acid as defined in b) and/or a polyamino acid as
defined in c); and/or
[0353] e) is a conjugate comprising a polyhydroxypolymer backbone
to which is separately coupled 1) the foreign T.sub.H epitope and
2) a polyamino acid selected from the group consisting of a
subsequence as defined in a), a disrupted sequence of APP or
A.beta. as defined in b), and an APP or A.beta. derived amino acid
sequence that comprises 1 single methionine residue located in the
C-terminus, wherein other methionine residues in APP or A.beta. and
in the foreign T.sub.H epitope have been substituted or deleted,
and preferably have been substituted by leucin or isoleucine.
[0354] 2. The method according to paragraph 1, wherein a
substantial fraction of B-cell epitopes of APP or A.beta. are
preserved in the analogue and that
[0355] at least one first moiety is introduced which effects
targeting of the analogue to an antigen presenting cell (APC) or a
B-lymphocyte, and/or
[0356] at least one second moiety is introduced which stimulates
the immune system, and/or
[0357] at least one third moiety is introduced which optimizes
presentation of the analogue to the immune system.
[0358] 3. The method according to paragraph 2, wherein the first
and/or of the second and/or of the third moiety is/are attached as
side groups by covalent or non-covalent binding to suitable
chemical groups in the APP or A.beta. sequence.
[0359] 4. The method according to any one of the preceding
paragraphs, wherein the analogue comprises a fusion
polypeptide.
[0360] 5. The method according to any one of the preceding
paragraphs, wherein introduction of the amino acid substitution
and/or deletion and/or insertion and/or addition results in a
substantial preservation of the overall tertiary structure of APP
or A.beta..
[0361] 6. The method according to any one of the preceding
paragraphs, wherein the analogue includes duplication of at least
one B-cell epitope of APP or A.beta. and/or introduction of a
hapten.
[0362] 7. The method according to any one of the preceding
paragraphs, wherein the foreign T-cell epitope is immunodominant in
the animal.
[0363] 8. The method according to any one of the preceding
paragraphs, wherein the foreign T-cell epitope is promiscuous, such
as a foreign T-cell epitope which is selected from a natural
promiscuous T-cell epitope and an artificial MHC-II binding peptide
sequence.
[0364] 9. The method according to paragraph 8, wherein the natural
T-cell epitope is selected from a Tetanus toxoid epitope such as P2
or P30, a diphtheria toxoid epitope, an influenza virus
hemagluttinin epitope, and a P. falciparum CS epitope.
[0365] 10. The method according to any one of the preceding
paragraphs, wherein the analogue comprises B-cell epitopes which
are not exposed to the extracellular phase when present in a
cell-bound form of the precursor polypeptide A.beta..
[0366] 11. The method according to any one of the preceding
paragraphs, wherein the analogue lacks at least one B-cell epitope
which is exposed to the extracellular phase when present in a
cell-bound form of the precursor polypeptide.
[0367] 12. The method according to any one of the preceding
paragraphs, wherein the analogue comprises at most 9 consecutive
amino acids of SEQ ID NO: 2., such as at most 8, at most 7, at most
6, at most 5, at most 4, and at most 3 consecutive amino acids.
[0368] 13. The method according to paragraph 12, wherein the
analogue comprises at least one subsequence of SEQ ID NO: 2 so that
each such at least one subsequence of SEQ ID NO: 2 independently
consists of amino acid stretches selected from the group consisting
of 9 consecutive amino acids of SEQ ID NO: 2, 8 consecutive amino
acids of SEQ ID NO: 2, 7 consecutive amino acids of SEQ ID NO: 2, 6
consecutive amino acids of SEQ ID NO: 2, 5 consecutive amino acids
of SEQ ID NO: 2, 4 consecutive amino acids of SEQ ID NO: 2, and 3
consecutive amino acids of SEQ ID NO: 2.
[0369] 14. The method according to paragraph 22 or 23, wherein the
consecutive amino acids begin at an amino acid residue selected
from the group consisting of residue 672, 673, 674, 675, 676, 677,
678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690,
691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703,
704, 705, 706, 707, 708, 709, 710, 711, 712, 713, and 714.
[0370] 15. The method according to any one of the preceding
paragraphs, wherein presentation to the immune system is effected
by having at least two copies of an A.beta. derived fragment or the
analogue covalently of non-covalently linked to a carrier molecule
capable of effecting presentation of multiple copies of antigenic
determinants.
[0371] 16. The method according to any one of the preceding
paragraphs, variants d or e, wherein the polyamino acid and T.sub.H
epitope are attached to the polyhydroxypolymer by means of an amide
bond.
[0372] 17. The method according to any one of the preceding
paragraphs, variants d or e, wherein the the polyhydroxypolymer is
a polysaccharide.
[0373] 18. The method according to any one of the preceding
paragraphs, wherein the analogue has been formulated with an
adjuvant which facilitates breaking of autotolerance to
autoantigens.
[0374] 19. The method according to any one of the preceding
paragraphs, wherein an effective amount of the analogue is
administered to the animal via a route selected from the parenteral
route such as the intracutaneous, the subcutaneous, and the
intramuscular routes; the peritoneal route; the oral route; the
buccal route; the sublinqual route; the epidural route; the spinal
route; the anal route; and the intracranial route.
[0375] 20. The method according to paragraph 19, wherein the
effective amount is between 0.5 .mu.g and 2,000 .mu.g of the
analogue.
[0376] 21. The method according to any one of paragraphs 1-14,
variants a-c, wherein presentation of the analogue to the immune
system is effected by introducing nucleic acid(s) encoding the
analogue into the animal's cells and thereby obtaining in vivo
expression by the cells of the nucleic acid(s) introduced.
[0377] 22. The method according to paragraph 21, wherein the
nucleic acid(s) introduced is/are selected from naked DNA, DNA
formulated with charged or uncharged lipids, DNA formulated in
liposomes, DNA included in a viral vector, DNA formulated with a
transfection-facilitating protein or polypeptide, DNA formulated
with a targeting protein or polypeptide, DNA formulated with
Calcium precipitating agents, DNA coupled to an inert carrier
molecule, DNA encapsulated in chitin or chitosan, and DNA
formulated with an adjuvant.
[0378] 23. The method according to any one of paragraphs 19-22,
which includes at least one administration/introduction per year,
such as at least 2, at least 3, at least 4, at least 6, and at
least 12 administrations/introductions.
[0379] 24. A method for treating and/or preventing and/or
ameliorating Alzheimer's disease or other diseases and conditions
characterized by amyloid deposits, the method comprising
down-regulating APP or A.beta. according to the method of any one
of the preceding paragraphs to such an extent that the total amount
of amyloid is decreased or that the rate of amyloid formation is
reduced with clinical significance.
[0380] 25. An analogue of APP or A.beta. which is derived from an
animal APP or A.beta. wherein is introduced a modification which
has as a result that immunization of the animal with the analogue
induces production of antibodies against the animal's autologous
APP or A.beta., and wherein the analogue is as defined in any one
of paragraphs 1-17.
[0381] 26. An immunogenic composition comprising an immunogenically
effective amount of an analogue according to paragraph 25, the
composition further comprising a pharmaceutically and
immunologically acceptable carrier and/or vehicle and optionally an
adjuvant.
[0382] 27. A nucleic acid fragment which encodes an analogue
according to paragraph 25.
[0383] 28. A vector carrying the nucleic acid fragment according to
paragraph 27, such as a vector that is capable of autonomous
replication.
[0384] 29. The vector according to paragraph 28 which is selected
from the group consisting of a plasmid, a phage, a cosmid, a
mini-chromosome, and a virus.
[0385] 30. The vector according to paragraph 28 or 29, comprising,
in the 5'.fwdarw.3' direction and in operable linkage, a promoter
for driving expression of the nucleic acid fragment according to
paragraph 27, optionally a nucleic acid sequence encoding a leader
peptide enabling secretion of or integration into the membrane of
the polypeptide fragment, the nucleic acid fragment according to
paragraph 27, and optionally a terminator.
[0386] 31. The vector according to any one of paragraphs 28-30
which, when introduced into a host cell, is capable or incapable of
being integrated in the host cell genome.
[0387] 32. The vector according to paragraph 30 or 31, wherein the
promoter drives expression in a eukaryotic cell and/or in a
prokaryotic cell.
[0388] 33. A transformed cell carrying the vector of any one of
paragraphs 28-32, such as a transformed cell which is capable of
replicating the nucleic acid fragment according to paragraph
27.
[0389] 34. The transformed cell according to paragraph 33, which is
a microorganism selected from a bacterium, a yeast, a protozoan, or
a cell derived from a multicellular organism selected from a
fungus, an insect cell such as an S.sub.2 or an SF cell, a plant
cell, and a mammalian cell.
[0390] 35. The transformed cell according to paragraph 33 or 34,
which expresses the nucleic acid fragment according to paragraph
28, such as a transformed cell, which secretes or carries on its
surface, the analogue according to paragraph 25.
[0391] 36. The method according to any one of paragraphs 1-14,
variants a-c, wherein presentation to the immune system is effected
by administering a non-pathogenic microorganism or virus which is
carrying a nucleic acid fragment which encodes and expresses the
analogue.
[0392] 37. A composition for inducing production of antibodies
against amyloid, the composition comprising
[0393] a nucleic acid fragment according to paragraph 27 or a
vector according to any one of paragraphs 28-32, and
[0394] a pharmaceutically and immunologically acceptable carrier
and/or vehicle and/or adjuvant.
[0395] 38. A stable cell line which carries the vector according to
any one of paragraphs 28-32 and which expresses the nucleic acid
fragment according to paragraph 27, and which optionally secretes
or carries the analogue according to paragraph 25 on its
surface.
[0396] This application claims priority from U.S. Provisional
Patent Application No. 60/373,027 filed Apr. 16, 2002 and
60/337,543 filed Aug. 20, 2001. Priority is also claimed to Danish
Patent Application Numbers PA 2002 0058 filed Apr. 16, 2002 and PA
2001 01231 filed Aug. 20, 2001.
[0397] Reference is made to the U.S. Provisional Patent Application
No. 60/331,575 filed Nov. 11, 2001, No. 60/350,047 filed Jan. 17,
2002, No. 60/363,128 filed Mar. 11, 2002, and No. 60/382,991 filed
May 23, 2002, and to U.S. patent application Ser. Nos. 08/955,373
filed Oct. 21, 1997 and 10/080,101 filed Feb. 19, 2002.
[0398] Reference is also made to European Patent Number 0752886,
South Korean Patent Number 308444 and Australian Patent Number
707083, as well as to the following publications which relate to
the present invention:
[0399] Hertz, M., Juji, T., Tanaka, S. & Mouritsen, S. A
therapeutic RANKL vaccine induces neutralizing anti-RANKL
antibodies and prevents bone loss in ovariectomized mice. 23rd
Annual Meeting American Society of Bone and Mineral Research, 12-16
October 2001, Phoenix, Ariz., USA, Abstract 1043, (2001).
[0400] Hertz, M. et al. Active Vaccination Against IL-5 Bypasses
Immunological Tolerance and Ameliorates Experimental Asthma. J
Immunol 167, 3792-3799 (2001).
[0401] Hertz, M., Mouritsen, S.; Gautam, A. Emerging therapeutic
vaccines. Drug Discovery World Summer 2000, 49-53 (2001).
[0402] Dalum, I. et al. Therapeutic antibodies elicited by
immunization against TNF-alpha. Nat Biotechnol 17, 666-669
(1999).
[0403] Dalum, I. et al. Induction of cross-reactive antibodies
against a self protein by immunization with a modified self protein
containing a foreign T helper epitope. Mol Immunol 34, 1113-1120
(1997).
[0404] Each of the foregoing applications and patents, each
foregoing publication, and each document cited or referenced in
each of the foregoing applications and patents, including during
the prosecution of each of the foregoing applications and patents
("application and article cited documents"), and any manufacturer
's instructions or catalogues for any products cited or mentioned
in each of the foregoing applications and patents and articles and
in any of the application and article cited documents, are hereby
incorporated herein by reference. Furthermore, all documents cited
in this text, and all documents cited or referenced in documents
cited in this text, and any manufacturer's instructions or
catalogues for any products cited or mentioned in this text or in
any document hereby incorporated into this text, are hereby
incorporated herein by reference. Documents incorporated by
reference into this text or any teachings therein may be used in
the practice of this invention. Documents incorporated by reference
into this text are not admitted to be prior art. Furthermore,
authors or inventors on documents incorporated by reference into
this text are not to be considered to be "another" or "others" as
to the present inventive entity and vice versa, especially where
one or more authors or inventors on documents incorporated by
reference into this text are an inventor or inventors named in the
present inventive entity.
Sequence CWU 1
1
17 1 2313 DNA Homo sapiens CDS (1)..(2313) 1 atg ctg ccc ggt ttg
gca ctg ctc ctg ctg gcc gcc tgg acg gct cgg 48 Met Leu Pro Gly Leu
Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10 15 gcg ctg gag
gta ccc act gat ggt aat gct ggc ctg ctg gct gaa ccc 96 Ala Leu Glu
Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 20 25 30 cag
att gcc atg ttc tgt ggc aga ctg aac atg cac atg aat gtc cag 144 Gln
Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln 35 40
45 aat ggg aag tgg gat tca gat cca tca ggg acc aaa acc tgc att gat
192 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp
50 55 60 acc aag gaa ggc atc ctg cag tat tgc caa gaa gtc tac cct
gaa ctg 240 Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro
Glu Leu 65 70 75 80 cag atc acc aat gtg gta gaa gcc aac caa cca gtg
acc atc cag aac 288 Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val
Thr Ile Gln Asn 85 90 95 tgg tgc aag cgg ggc cgc aag cag tgc aag
acc cat ccc cac ttt gtg 336 Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys
Thr His Pro His Phe Val 100 105 110 att ccc tac cgc tgc tta gtt ggt
gag ttt gta agt gat gcc ctt ctc 384 Ile Pro Tyr Arg Cys Leu Val Gly
Glu Phe Val Ser Asp Ala Leu Leu 115 120 125 gtt cct gac aag tgc aaa
ttc tta cac cag gag agg atg gat gtt tgc 432 Val Pro Asp Lys Cys Lys
Phe Leu His Gln Glu Arg Met Asp Val Cys 130 135 140 gaa act cat ctt
cac tgg cac acc gtc gcc aaa gag aca tgc agt gag 480 Glu Thr His Leu
His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu 145 150 155 160 aag
agt acc aac ttg cat gac tac ggc atg ttg ctg ccc tgc gga att 528 Lys
Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile 165 170
175 gac aag ttc cga ggg gta gag ttt gtg tgt tgc cca ctg gct gaa gaa
576 Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu
180 185 190 agt gac aat gtg gat tct gct gat gcg gag gag gat gac tcg
gat gtc 624 Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser
Asp Val 195 200 205 tgg tgg ggc gga gca gac aca gac tat gca gat ggg
agt gaa gac aaa 672 Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly
Ser Glu Asp Lys 210 215 220 gta gta gaa gta gca gag gag gaa gaa gtg
gct gag gtg gaa gaa gaa 720 Val Val Glu Val Ala Glu Glu Glu Glu Val
Ala Glu Val Glu Glu Glu 225 230 235 240 gaa gcc gat gat gac gag gac
gat gag gat ggt gat gag gta gag gaa 768 Glu Ala Asp Asp Asp Glu Asp
Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255 gag gct gag gaa ccc
tac gaa gaa gcc aca gag aga acc acc agc att 816 Glu Ala Glu Glu Pro
Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile 260 265 270 gcc acc acc
acc acc acc acc aca gag tct gtg gaa gag gtg gtt cga 864 Ala Thr Thr
Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285 gag
gtg tgc tct gaa caa gcc gag acg ggg ccg tgc cga gca atg atc 912 Glu
Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala Met Ile 290 295
300 tcc cgc tgg tac ttt gat gtg act gaa ggg aag tgt gcc cca ttc ttt
960 Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe
305 310 315 320 tac ggc gga tgt ggc ggc aac cgg aac aac ttt gac aca
gaa gag tac 1008 Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp
Thr Glu Glu Tyr 325 330 335 tgc atg gcc gtg tgt ggc agc gcc atg tcc
caa agt tta ctc aag act 1056 Cys Met Ala Val Cys Gly Ser Ala Met
Ser Gln Ser Leu Leu Lys Thr 340 345 350 acc cag gaa cct ctt gcc cga
gat cct gtt aaa ctt cct aca aca gca 1104 Thr Gln Glu Pro Leu Ala
Arg Asp Pro Val Lys Leu Pro Thr Thr Ala 355 360 365 gcc agt acc cct
gat gcc gtt gac aag tat ctc gag aca cct ggg gat 1152 Ala Ser Thr
Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp 370 375 380 gag
aat gaa cat gcc cat ttc cag aaa gcc aaa gag agg ctt gag gcc 1200
Glu Asn Glu His Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala 385
390 395 400 aag cac cga gag aga atg tcc cag gtc atg aga gaa tgg gaa
gag gca 1248 Lys His Arg Glu Arg Met Ser Gln Val Met Arg Glu Trp
Glu Glu Ala 405 410 415 gaa cgt caa gca aag aac ttg cct aaa gct gat
aag aag gca gtt atc 1296 Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala
Asp Lys Lys Ala Val Ile 420 425 430 cag cat ttc cag gag aaa gtg gaa
tct ttg gaa cag gaa gca gcc aac 1344 Gln His Phe Gln Glu Lys Val
Glu Ser Leu Glu Gln Glu Ala Ala Asn 435 440 445 gag aga cag cag ctg
gtg gag aca cac atg gcc aga gtg gaa gcc atg 1392 Glu Arg Gln Gln
Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met 450 455 460 ctc aat
gac cgc cgc cgc ctg gcc ctg gag aac tac atc acc gct ctg 1440 Leu
Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu 465 470
475 480 cag gct gtt cct cct cgg cct cgt cac gtg ttc aat atg cta aag
aag 1488 Gln Ala Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu
Lys Lys 485 490 495 tat gtc cgc gca gaa cag aag gac aga cag cac acc
cta aag cat ttc 1536 Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His
Thr Leu Lys His Phe 500 505 510 gag cat gtg cgc atg gtg gat ccc aag
aaa gcc gct cag atc cgg tcc 1584 Glu His Val Arg Met Val Asp Pro
Lys Lys Ala Ala Gln Ile Arg Ser 515 520 525 cag gtt atg aca cac ctc
cgt gtg att tat gag cgc atg aat cag tct 1632 Gln Val Met Thr His
Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser 530 535 540 ctc tcc ctg
ctc tac aac gtg cct gca gtg gcc gag gag att cag gat 1680 Leu Ser
Leu Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile Gln Asp 545 550 555
560 gaa gtt gat gag ctg ctt cag aaa gag caa aac tat tca gat gac gtc
1728 Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn Tyr Ser Asp Asp
Val 565 570 575 ttg gcc aac atg att agt gaa cca agg atc agt tac gga
aac gat gct 1776 Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr
Gly Asn Asp Ala 580 585 590 ctc atg cca tct ttg acc gaa acg aaa acc
acc gtg gag ctc ctt ccc 1824 Leu Met Pro Ser Leu Thr Glu Thr Lys
Thr Thr Val Glu Leu Leu Pro 595 600 605 gtg aat gga gag ttc agc ctg
gac gat ctc cag ccg tgg cat tct ttt 1872 Val Asn Gly Glu Phe Ser
Leu Asp Asp Leu Gln Pro Trp His Ser Phe 610 615 620 ggg gct gac tct
gtg cca gcc aac aca gaa aac gaa gtt gag cct gtt 1920 Gly Ala Asp
Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val 625 630 635 640
gat gcc cgc cct gct gcc gac cga gga ctg acc act cga cca ggt tct
1968 Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly
Ser 645 650 655 ggg ttg aca aat atc aag acg gag gag atc tct gaa gtg
aag atg gat 2016 Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu
Val Lys Met Asp 660 665 670 gca gaa ttc cga cat gac tca gga tat gaa
gtt cat cat caa aaa ttg 2064 Ala Glu Phe Arg His Asp Ser Gly Tyr
Glu Val His His Gln Lys Leu 675 680 685 gtg ttc ttt gca gaa gat gtg
ggt tca aac aaa ggt gca atc att gga 2112 Val Phe Phe Ala Glu Asp
Val Gly Ser Asn Lys Gly Ala Ile Ile Gly 690 695 700 ctc atg gtg ggc
ggt gtt gtc ata gcg aca gtg atc gtc atc acc ttg 2160 Leu Met Val
Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu 705 710 715 720
gtg atg ctg aag aag aaa cag tac aca tcc att cat cat ggt gtg gtg
2208 Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val
Val 725 730 735 gag gtt gac gcc gct gtc acc cca gag gag cgc cac ctg
tcc aag atg 2256 Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His
Leu Ser Lys Met 740 745 750 cag cag aac ggc tac gaa aat cca acc tac
aag ttc ttt gag cag atg 2304 Gln Gln Asn Gly Tyr Glu Asn Pro Thr
Tyr Lys Phe Phe Glu Gln Met 755 760 765 cag aac tag 2313 Gln Asn
770 2 770 PRT Homo sapiens misc_feature (2098)..(2169) nucleotides
encoding transmembrane region 2 Met Leu Pro Gly Leu Ala Leu Leu Leu
Leu Ala Ala Trp Thr Ala Arg 1 5 10 15 Ala Leu Glu Val Pro Thr Asp
Gly Asn Ala Gly Leu Leu Ala Glu Pro 20 25 30 Gln Ile Ala Met Phe
Cys Gly Arg Leu Asn Met His Met Asn Val Gln 35 40 45 Asn Gly Lys
Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp 50 55 60 Thr
Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu 65 70
75 80 Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln
Asn 85 90 95 Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His Pro
His Phe Val 100 105 110 Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val
Ser Asp Ala Leu Leu 115 120 125 Val Pro Asp Lys Cys Lys Phe Leu His
Gln Glu Arg Met Asp Val Cys 130 135 140 Glu Thr His Leu His Trp His
Thr Val Ala Lys Glu Thr Cys Ser Glu 145 150 155 160 Lys Ser Thr Asn
Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile 165 170 175 Asp Lys
Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 180 185 190
Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 195
200 205 Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp
Lys 210 215 220 Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val
Glu Glu Glu 225 230 235 240 Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp
Gly Asp Glu Val Glu Glu 245 250 255 Glu Ala Glu Glu Pro Tyr Glu Glu
Ala Thr Glu Arg Thr Thr Ser Ile 260 265 270 Ala Thr Thr Thr Thr Thr
Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285 Glu Val Cys Ser
Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala Met Ile 290 295 300 Ser Arg
Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe 305 310 315
320 Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr
325 330 335 Cys Met Ala Val Cys Gly Ser Ala Met Ser Gln Ser Leu Leu
Lys Thr 340 345 350 Thr Gln Glu Pro Leu Ala Arg Asp Pro Val Lys Leu
Pro Thr Thr Ala 355 360 365 Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr
Leu Glu Thr Pro Gly Asp 370 375 380 Glu Asn Glu His Ala His Phe Gln
Lys Ala Lys Glu Arg Leu Glu Ala 385 390 395 400 Lys His Arg Glu Arg
Met Ser Gln Val Met Arg Glu Trp Glu Glu Ala 405 410 415 Glu Arg Gln
Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile 420 425 430 Gln
His Phe Gln Glu Lys Val Glu Ser Leu Glu Gln Glu Ala Ala Asn 435 440
445 Glu Arg Gln Gln Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met
450 455 460 Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr
Ala Leu 465 470 475 480 Gln Ala Val Pro Pro Arg Pro Arg His Val Phe
Asn Met Leu Lys Lys 485 490 495 Tyr Val Arg Ala Glu Gln Lys Asp Arg
Gln His Thr Leu Lys His Phe 500 505 510 Glu His Val Arg Met Val Asp
Pro Lys Lys Ala Ala Gln Ile Arg Ser 515 520 525 Gln Val Met Thr His
Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser 530 535 540 Leu Ser Leu
Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile Gln Asp 545 550 555 560
Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn Tyr Ser Asp Asp Val 565
570 575 Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp
Ala 580 585 590 Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu
Leu Leu Pro 595 600 605 Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln
Pro Trp His Ser Phe 610 615 620 Gly Ala Asp Ser Val Pro Ala Asn Thr
Glu Asn Glu Val Glu Pro Val 625 630 635 640 Asp Ala Arg Pro Ala Ala
Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser 645 650 655 Gly Leu Thr Asn
Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp 660 665 670 Ala Glu
Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu 675 680 685
Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly 690
695 700 Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr
Leu 705 710 715 720 Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His
His Gly Val Val 725 730 735 Glu Val Asp Ala Ala Val Thr Pro Glu Glu
Arg His Leu Ser Lys Met 740 745 750 Gln Gln Asn Gly Tyr Glu Asn Pro
Thr Tyr Lys Phe Phe Glu Gln Met 755 760 765 Gln Asn 770 3 45 DNA
Clostridium tetani CDS (1)..(45) DNA encoding P2 epitope 3 cag tac
atc aaa gct aac tcc aaa ttc atc ggt atc acc gag ctg 45 Gln Tyr Ile
Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu 1 5 10 15 4 15 PRT
Clostridium tetani 4 Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly
Ile Thr Glu Leu 1 5 10 15 5 63 DNA Clostridium tetani CDS (1)..(63)
DNA encoding P30 epitope 5 ttc aac aac ttc acc gta agc ttc tgg ctg
cgt gtt ccg aaa gtt agc 48 Phe Asn Asn Phe Thr Val Ser Phe Trp Leu
Arg Val Pro Lys Val Ser 1 5 10 15 gct agc cac ctg gaa 63 Ala Ser
His Leu Glu 20 6 21 PRT Clostridium tetani 6 Phe Asn Asn Phe Thr
Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser 1 5 10 15 Ala Ser His
Leu Glu 20 7 21 DNA Artificial Sequence Primer ME#105 used in
generating plasmid pAB3 7 caactcagct tcctttcggg c 21 8 21 DNA
Artificial Sequence Primer ME#178 used in generating plasmid pAB2 8
agatctcgat cccgcgaaat t 21 9 135 DNA Artificial Sequence Primer
ME#800 which encodes human abeta-43 fragment with E.coli optimized
codons 9 atggatgcag aattccgtca cgactccggt tacgaagttc accaccagaa
actggttttc 60 ttcgcagaag atgttggttc caacaaaggt gcaatcatcg
gtctgatggt tggcggtgtt 120 gttatcgcga cctag 135 10 31 DNA Artificial
Sequence Primer ME#801 used to generate human abeta-43 fragment
with E.coli optimized codons and appropriate restriction sites 10
gccggccatg gatgcagaat tccgtcacga c 31 11 39 DNA Artificial Sequence
Primer ME#802 used to generate human abeta-43 fragment with E.coli
optimized codons and appropriate restriction sites 11 gccggaagct
tctaggtcgc gataacaaca ccgccaacc 39 12 84 DNA Artificial Sequence
Primer ME#806, which contains sequence encoding P2 epitope 12
ccggcaagct tctacagctc ggtgataccg atgaatttgg agttagcttt gatgtactgg
60 gtcgcgataa caacaccgcc aacc 84 13 101 DNA Artificial Sequence
Primer ME#807 used in generating plasmid pAB3 13 gccggccatg
ggtttcaaca acttcaccgt tagcttctgg ctgcgtgttc cgaaagttag 60
cgcgagccac ctggaagatg cagaattccg tcacgactcc g 101 14 172 DNA
Artificial Sequence Primer ME#809 used in generating plsmid pAB4 14
gggccaagct tggatccggt cgcgataaca acaccgccaa ccatcagacc gatgattgca
60 cctttgttgg aaccaacatc ttctgcgaag aaaaccagtt tctggtggtg
aacttcgtaa 120 ccggagtcgt gacggaactc tgcatccagc tcggtgatac
cgatgaattt gg 172 15 30 DNA Artificial Sequence Primer ME#810 used
in generating pAB5 plasmid 15 ctggaagatg cagagttccg tcacgactcc 30
16 35 DNA Artificial Sequence Primer
ME#811 used in generating pAB5 plasmid 16 gcgccggatc cttcaacaac
ttcaccgtta gcttc 35 17 13 PRT Artificial Sequence HLA DR binding
sequence 17 Ala Lys Phe Val Ala Ala Trp Thr Leu Lys Ala Ala Ala 1 5
10
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