U.S. patent application number 13/838152 was filed with the patent office on 2013-09-05 for compounds for treating beta-amyloidoses.
The applicant listed for this patent is Andrea Dolischka, Christian Gieffers, Markus MANDLER, Frank Mattner, Oleksandr Otava. Invention is credited to Andrea Dolischka, Christian Gieffers, Markus MANDLER, Frank Mattner, Oleksandr Otava.
Application Number | 20130230545 13/838152 |
Document ID | / |
Family ID | 41401982 |
Filed Date | 2013-09-05 |
United States Patent
Application |
20130230545 |
Kind Code |
A1 |
MANDLER; Markus ; et
al. |
September 5, 2013 |
COMPOUNDS FOR TREATING BETA-AMYLOIDOSES
Abstract
The present invention relates to the use of mimotopes in the
treatment of diseases associated with .beta.-amyloid formation
and/or aggregation (.beta.-Amyloidoses) including Alzheimer's
disease, whereby said mimotopes are able to induce the in vivo
formation of antibodies directed to A.beta.1-40/42,
A.beta.pE3-40/42, A.beta.3-40/42 and A.beta.11-40/42.
Inventors: |
MANDLER; Markus; (Vienna,
AT) ; Gieffers; Christian; (Dossenheim, DE) ;
Mattner; Frank; (Vienna, AT) ; Dolischka; Andrea;
(Vienna, AT) ; Otava; Oleksandr; (Vienna,
AT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MANDLER; Markus
Gieffers; Christian
Mattner; Frank
Dolischka; Andrea
Otava; Oleksandr |
Vienna
Dossenheim
Vienna
Vienna
Vienna |
|
AT
DE
AT
AT
AT |
|
|
Family ID: |
41401982 |
Appl. No.: |
13/838152 |
Filed: |
March 15, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12997674 |
Dec 13, 2010 |
|
|
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PCT/AT2009/000236 |
Jun 12, 2009 |
|
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13838152 |
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Current U.S.
Class: |
424/185.1 ;
530/328; 530/329; 530/330 |
Current CPC
Class: |
A61P 37/04 20180101;
A61P 27/00 20180101; A61K 38/08 20130101; A61P 27/16 20180101; A61P
25/00 20180101; C07K 16/18 20130101; A61P 25/16 20180101; A61P
25/28 20180101; A61K 39/0007 20130101 |
Class at
Publication: |
424/185.1 ;
530/329; 530/328; 530/330 |
International
Class: |
A61K 39/00 20060101
A61K039/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 12, 2008 |
AT |
A 951/2008 |
Claims
1. A process for preventing, treating, or both preventing and
treating .beta.-amyloidoses, the process comprising administering
to an individual a medicament comprising a compound comprising an
amino acid sequence selected from the group consisting of SEFKHG(C)
(SEQ ID NO: 124), TLHEFRH(C) (SEQ ID NO: 151), ILFRHG(C) (SEQ ID
NO: 133), TSVFRH(C) (SEQ ID NO: 126), SQFRHY(C) (SEQ ID NO: 128),
LMFRHN(C) (SEQ ID NO: 129), SPNQFRH(C) (SEQ ID NO: 143), ELFKHHL(C)
(SEQ ID NO: 150), THTDFRH(C) (SEQ ID NO: 160), DEHPFRH(C) (SEQ ID
NO: 164), QSEFKHW(C) (SEQ ID NO: 172), ADHDFRH(C) (SEQ ID NO: 174),
YEFRHAQ(C) (SEQ ID NO: 182) and TEFRHKA(C) (SEQ ID NO: 187).
2. A process for preventing, treating, or both preventing and
treating .beta.-amyloidoses, the process comprising administering
to an individual a medicament comprising a compound comprising an
amino acid sequence
X.sub.1RX.sub.2DX.sub.3(X.sub.4).sub.n(X.sub.5).sub.m(X.sub.6).sub.o
(SEQ ID NO: 100) (Formula I), wherein X.sub.1 is isoleucine (I) or
valine (V), X.sub.2 is tryptophan (W) or tyrosine (Y), X.sub.3 is
threonine (T), valine (V), alanine (A), methionine (M), glutamine
(Q) or glycine (G), X.sub.4 is proline (P), alanine (A), tyrosine
(Y), serine (5), cysteine (C) or glycine (G), X.sub.5 is proline
(P), leucine (L), glycine (G) or cysteine (C), X.sub.6 is cysteine
(C), n, m and o are, independently, 0 or 1, wherein the compound
has a binding capacity to an antibody which is specific for an
epitope of an amyloid-beta-peptide (A.beta.) comprising an amino
acid sequence EFRHDSGY (SEQ ID NO: 102) or pEFRHDSGY (SEQ ID NO:
103).
3. The process of claim 2, wherein the compound comprises an amino
acid sequence selected from the group consisting of IRWDTP(C) (SEQ
ID NO: 106), VRWDVYP(C) (SEQ ID NO: 107), IRYDAPL(C) (SEQ ID NO:
108), IRYDMAG(C) (SEQ ID NO: 109), IRWDTSL(C) (SEQ ID NO: 110),
IRWDQP(C) (SEQ ID NO: 111), IRWDG(C) (SEQ ID NO: 112) and IRWDGG(C)
(SEQ ID NO: 113).
4. A process for preventing, treating, or both preventing and
treating .beta.-amyloidoses, the process comprising administering
to an individual a medicament comprising a compound comprising an
amino acid sequence
EX.sub.1WHX.sub.2X.sub.3(X.sub.4).sub.n(X.sub.5).sub.m (SEQ ID NO:
101) (Formula II), wherein X.sub.1 is valine (V), arginine (R) or
leucine (L), X.sub.2 is arginine (R) or glutamic acid (E), X.sub.3
is alanine (A), histidine (H), lysine (K), leucine (L), tyrosine
(Y) or glycine (G), X.sub.4 is proline (P), histidine (H),
phenylalanine (F) or glutamine (Q) or Cysteine X.sub.5 is cysteine
(C), n and m are, independently, 0 or 1, wherein the compound has a
binding capacity to an antibody which is specific for an epitope of
an amyloid-beta-peptide (A.beta.) comprising an amino acid sequence
EVHHQKL (SEQ ID NO: 104).
5. The process of claim 4, wherein the compound comprises an amino
acid sequence selected from the group consisting of EVWHRHQ(C) (SEQ
ID NO: 114), ERHEKH(C) (SEQ ID NO: 115), EVWHRLQ(C) (SEQ ID NO:
116), ELWHRYP(C) (SEQ ID NO: 117), ELWHRAF(C) (SEQ ID NO: 118),
ELWHRA(C) (SEQ ID NO: 119), EVWHRG(C) (SEQ ID NO: 120), EVWHRH(C)
(SEQ ID NO: 121) and ERHEK(C) (SEQ ID NO: 122).
6. A process for preventing, treating, or both preventing and
treating .beta.-amyloidoses, the process comprising administering
to an individual a medicament comprising a compound comprising an
amino acid sequence selected from the group consisting of QDFRHY(C)
(SEQ ID NO: 123), SEFKHG(C) (SEQ ID NO: 124), TSFRHG(C) (SEQ ID NO:
125), TSVFRH(C) (SEQ ID NO: 126), TPFRHT(C) (SEQ ID NO: 127),
SQFRHY(C) (SEQ ID NO: 128), LMFRHN(C) (SEQ ID NO: 129), SAFRHH(C)
(SEQ ID NO: 130), LPFRHG(C) (SEQ ID NO: 131), SHFRHG(C) (SEQ ID NO:
132), ILFRHG(C) (SEQ ID NO: 133), QFKHDL(C) (SEQ ID NO: 134),
NWFPHP(C) (SEQ ID NO: 135), EEFKYS(C) (SEQ ID NO: 136), NELRHST(C)
(SEQ ID NO: 137), GEMRHQP(C) (SEQ ID NO: 138), DTYFPRS(C) (SEQ ID
NO: 139), VELRHSR(C) (SEQ ID NO: 140), YSMRHDA(C) (SEQ ID NO: 141),
AANYFPR(C) (SEQ ID NO: 142), SPNQFRH(C) (SEQ ID NO: 143),
SSSFFPR(C) (SEQ ID NO: 144), EDWFFWH(C) (SEQ ID NO: 145),
SAGSFRH(C) (SEQ ID NO: 146), QVMRHHA(C) (SEQ ID NO: 147),
SEFSHSS(C) (SEQ ID NO: 148), QPNLFYH(C) (SEQ ID NO: 149),
ELFKHHL(C) (SEQ ID NO: 150), TLHEFRH(C) (SEQ ID NO: 151),
ATFRHSP(C) (SEQ ID NO: 152), APMYFPH(C) (SEQ ID NO: 153),
TYFSHSL(C) (SEQ ID NO: 154), HEPLFSH(C) (SEQ ID NO: 155),
SLMRHSS(C) (SEQ ID NO: 156), EFLRHTL(C) (SEQ ID NO: 157),
ATPLFRH(C) (SEQ ID NO: 158), QELKRYY(C) (SEQ ID NO: 159),
THTDFRH(C) (SEQ ID NO: 160), LHIPFRH(C) (SEQ ID NO: 161),
NELFKHF(C) (SEQ ID NO: 162), SQYFPRP(C) (SEQ ID NO: 163),
DEHPFRH(C) (SEQ ID NO: 164), MLPFRHG(C) (SEQ ID NO: 165),
SAMRHSL(C) (SEQ ID NO: 166), TPLMFWH(C) (SEQ ID NO: 167),
LQFKHST(C) (SEQ ID NO: 168), ATFRHST(C) (SEQ ID NO: 169),
TGLMFKH(C) (SEQ ID NO: 170), AEFSHWH(C) (SEQ ID NO: 171),
QSEFKHW(C) (SEQ ID NO: 172), AEFMHSV(C) (SEQ ID NO: 173),
ADHDFRH(C) (SEQ ID NO: 174), DGLLFKH(C) (SEQ ID NO: 175),
IGFRHDS(C) (SEQ ID NO: 176), SNSEFRR(C) (SEQ ID NO: 177),
SELRHST(C) (SEQ ID NO: 178), THMEFRR(C) (SEQ ID NO: 179),
EELRHSV(C) (SEQ ID NO: 180), QLFKHSP(C) (SEQ ID NO: 181),
YEFRHAQ(C) (SEQ ID NO: 182), SNFRHSV(C) (SEQ ID NO: 183),
APIQFRH(C) (SEQ ID NO: 184), AYFPHTS(C) (SEQ ID NO: 185),
NSSELRH(C) (SEQ ID NO: 186), TEFRHKA(C) (SEQ ID NO: 187),
TSTEMWH(C) (SEQ ID NO: 188), SQSYFKH(C) (SEQ ID NO: 189), (C)SEFKH
(SEQ ID NO: 190), SEFKH(C) (SEQ ID NO: 191), (C)HEFRH (SEQ ID NO:
192) and HEFRH(C) (SEQ ID NO: 193).
7. The process of claim 6, wherein the compound comprises an amino
acid sequence selected from the group consisting of QDFRHY(C) (SEQ
ID NO: 123), SEFKHG(C) (SEQ ID NO: 124), TSFRHG(C) (SEQ ID NO:
125), TSVFRH(C) (SEQ ID NO: 126), TPFRHT(C) (SEQ ID NO: 127),
SQFRHY(C) (SEQ ID NO: 128), LMFRHN(C) (SEQ ID NO: 129), SAFRHH(C)
(SEQ ID NO: 130), LPFRHG(C) (SEQ ID NO: 131), SHFRHG(C) (SEQ ID NO:
132), ILFRHG(C) (SEQ ID NO: 133), QFKHDL(C) (SEQ ID NO: 134),
NWFPHP(C) (SEQ ID NO: 135), EEFKYS(C) (SEQ ID NO: 136), SPNQFRH(C)
(SEQ ID NO: 143), ELFKHHL(C) (SEQ ID NO: 150), TLHEFRH(C) (SEQ ID
NO: 151), THTDFRH(C) (SEQ ID NO: 160), DEHPFRH(C) (SEQ ID NO: 164),
QSEFKHW(C) (SEQ ID NO: 172), ADHDFRH(C) (SEQ ID NO: 174),
DGLLFKH(C) (SEQ ID NO: 175), EELRHSV(C) (SEQ ID NO: 180),
YEFRHAQ(C) (SEQ ID NO: 182), TEFRHKA(C) (SEQ ID NO: 187), (C)SEFKH
(SEQ ID NO: 190), SEFKH(C) (SEQ ID NO: 191), (C)HEFRH (SEQ ID NO:
192) and HEFRH(C) (SEQ ID NO: 193).
8. The process of claim 6, wherein the compound is a polypeptide
comprising 7 to 20 amino acid residues.
9. The process of claim 6, wherein the compound is coupled to a
pharmaceutically acceptable carrier.
10. The process of claim 6, wherein the compound is formulated for
intravenous, subcutaneous, intradermal or intramuscular
administration.
11. The process of claim 6, wherein the compound is formulated with
an adjuvant.
12. The process of claim 6, wherein the medicament comprises 0.1 ng
to 10 mg of the compound, preferably 10 ng to 1 mg, in particular
100 ng to 10 .mu.g.
13. An isolated peptide comprising an amino acid sequence selected
from the group consisting of IRWDTP(C) (SEQ ID NO: 106), VRWDVYP(C)
(SEQ ID NO: 107), IRYDAPL(C) (SEQ ID NO: 108), IRYDMAG(C) (SEQ ID
NO: 109), IRWDTSL(C) (SEQ ID NO: 110), IRWDQP(C) (SEQ ID NO: 111),
IRWDG(C) (SEQ ID NO: 112), IRWDGG(C) (SEQ ID NO: 113), EVWHRHQ(C)
(SEQ ID NO: 114), ERHEKH(C) (SEQ ID NO: 115), EVWHRLQ(C) (SEQ ID
NO: 116), ELWHRYP(C) (SEQ ID NO: 117), ELWHRAF(C) (SEQ ID NO: 118),
ELWHRA(C) (SEQ ID NO: 119), EVWHRG(C) (SEQ ID NO: 120), EVWHRH(C)
(SEQ ID NO: 121), ERWHEK(C) (SEQ ID NO: 122), QDFRHY(C) (SEQ ID NO:
123), SEFKHG(C) (SEQ ID NO: 124), TSFRHG(C) (SEQ ID NO: 125),
TSVFRH(C) (SEQ ID NO: 126), TPFRHT(C) (SEQ ID NO: 127), SQFRHY(C)
(SEQ ID NO: 128), LMFRHN(C) (SEQ ID NO: 129), SAFRHH(C) (SEQ ID NO:
130), LPFRHG(C) (SEQ ID NO: 131), SHFRHG(C) (SEQ ID NO: 132),
ILFRHG(C) (SEQ ID NO: 133), QFKHDL(C) (SEQ ID NO: 134), NWFPHP(C)
(SEQ ID NO: 135), EEFKYS(C) (SEQ ID NO: 136), NELRHST(C) (SEQ ID
NO: 137), GEMRHQP(C) (SEQ ID NO: 138), DTYFPRS(C) (SEQ ID NO: 139),
VELRHSR(C) (SEQ ID NO: 140), YSMRHDA(C) (SEQ ID NO: 141),
AANYFPR(C) (SEQ ID NO: 142), SPNQFRH(C) (SEQ ID NO: 143),
SSSFFPR(C) (SEQ ID NO: 144), EDWFFWH(C) (SEQ ID NO: 145),
SAGSFRH(C) (SEQ ID NO: 146), QVMRHHA(C) (SEQ ID NO: 147),
SEFSHSS(C) (SEQ ID NO: 148), QPNLFYH(C) (SEQ ID NO: 149),
ELFKHHL(C) (SEQ ID NO: 150), TLHEFRH(C) (SEQ ID NO: 151),
ATFRHSP(C) (SEQ ID NO: 152), APMYFPH(C) (SEQ ID NO: 153),
TYFSHSL(C) (SEQ ID NO: 154), HEPLFSH(C) (SEQ ID NO: 155),
SLMRHSS(C) (SEQ ID NO: 156), EFLRHTL(C) (SEQ ID NO: 157),
ATPLFRH(C) (SEQ ID NO: 158), QELKRYY(C) (SEQ ID NO: 159),
THTDFRH(C) (SEQ ID NO: 160), LHIPFRH(C) (SEQ ID NO: 161),
NELFKHF(C) (SEQ ID NO: 162), SQYFPRP(C) (SEQ ID NO: 163),
DEHPFRH(C) (SEQ ID NO: 164), MLPFRHG(C) (SEQ ID NO: 165),
SAMRHSL(C) (SEQ ID NO: 166), TPLMFWH(C) (SEQ ID NO: 167),
LQFKHST(C) (SEQ ID NO: 168), ATFRHST(C) (SEQ ID NO: 169),
TGLMFKH(C) (SEQ ID NO: 170), AEFSHWH(C) (SEQ ID NO: 171),
QSEFKHW(C) (SEQ ID NO: 172), AEFMHSV(C) (SEQ ID NO: 173),
ADHDFRH(C) (SEQ ID NO: 174), DGLLFKH(C) (SEQ ID NO: 175),
IGFRHDS(C) (SEQ ID NO: 176), SNSEFRR(C) (SEQ ID NO: 177),
SELRHST(C) (SEQ ID NO: 178), THMEFRR(C) (SEQ ID NO: 179),
EELRHSV(C) (SEQ ID NO: 180), QLFKHSP(C) (SEQ ID NO: 181),
YEFRHAQ(C) (SEQ ID NO: 182), SNFRHSV(C) (SEQ ID NO: 183),
APIQFRH(C) (SEQ ID NO: 184), AYFPHTS(C) (SEQ ID NO: 185),
NSSELRH(C) (SEQ ID NO: 186), TEFRHKA(C) (SEQ ID NO: 187),
TSTEMWH(C) (SEQ ID NO: 188), SQSYFKH(C) (SEQ ID NO: 189), (C)SEFKH
(SEQ ID NO: 190), SEFKH(C) (SEQ ID NO: 191), (C)HEFRH (SEQ ID NO:
192) and HEFRH(C) (SEQ ID NO: 193).
14. A process for treating, ameliorating symptoms of, or both
treating and ameliorating symptoms of a synucleopathy, the process
comprising administering to an individual a medicament comprising
the peptide of claim 13.
15. The process of claim 14, wherein the synucleopathy is selected
from the group consisting of Parkinson's Disease, Dementia with
Lewy Bodies, multiple system atrophy and neurodegeneration with
brain iron accumulation.
16. A compound comprising the peptide of claim 13, coupled to a
pharmaceutically acceptable carrier.
17. A pharmaceutical formulation comprising the peptide of claim
13.
18. A vaccine comprising the peptide of claim 13.
19. The process of claim 9, wherein the pharmaceutically acceptable
carrier is KLH (Keyhole Limpet Hemocyanin).
20. The process of claim 11, wherein the adjuvant is aluminium
hydroxide.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. patent
application Ser. No. 12/997,674 filed Dec. 13, 2010 which is a
National Stage Entry of PCT/AT2009/000236 filed Jun. 12, 2009 and
claims priority of Austrian Patent Application No. A 951/2008 filed
Jun. 12, 2008, the entire contents of which are hereby incorporated
by reference.
[0002] The present invention relates to the use of mimotopes for
the prevention, treatment and diagnosis of diseases associated with
.beta.-amyloid formation and/or aggregation
(.beta.-Amyloidoses).
[0003] Various degenerative diseases are characterized by the
aberrant polymerization and accumulation of specific proteins.
These so called proteopathies include neurological disorders such
as Alzheimer's disease, Parkinson's disease and Huntington's
disease as well as diverse systemic disorders including the
amyloidoses. The present invention relates to the prevention,
treatment and diagnosis of proteopathies associated with
.beta.-amyloid proteins summarised under the term
.beta.-Amyloidoses. The most prominent form of .beta.-Amyloidoses
is Alzheimer's disease (AD). Other examples include but are not
limited to Dementia with Lewy bodies and Dementia in Down
syndrome.
[0004] AD is characterized by the abnormal accumulation of
extracellular amyloid plaques--closely associated with extensive
astrocytosis and microgliosis as well as dystrophic neurones and
neuronal loss. These amyloid plaques mainly consist of the
Amyloid-.beta. (A.beta.) peptides A.beta.40 and A.beta.42 derived
from the Amyloid Precursor Protein (APP), which is expressed on
various cell types in the nervous system. A.beta. peptides are
considered to be directly involved in the pathogenesis and
progression of AD.
[0005] APP is normally processed by two cleavage steps to form the
currently known forms of Abeta x-40/42/43. The first cleavage is
performed by the so called beta-site APP-cleaving enzymes 1 and 2
(BACE1 and BACE2); the second proteolytic step is performed by the
gamma-Secretase Complex (reviewed in De Strooper et al. J Cell Sci
113 (2000): 1857-1870).
[0006] BACE enzymes recognise two sites in the N-terminal portion
of the presumptive A.beta. peptide: the first interaction of BACE
with APP leads to a cut at the sequence DAEFR (SEQ ID NO: 105)
(pos. 1 in A.beta.) and formation of Abeta 1-X. Alternatively BACE
can also cut at future position 11 within A.beta. resulting in the
fragment II-X. Thus BACE mediated APP processing creates a variety
of different A.beta. species with full length Abeta 1-40/42 as
major contribuent. Gamma-Secretase activity leads to production of
3 main fragments: A.beta. 1-40/42/43. Once these peptides are
produced they are further processed by Amino-peptidases resulting
in their subsequent stepwise degradation. These further steps lead
to formation of other forms like for example A.beta.3-40/42
respectively. In humans on average 60-85% of amyloid plaque
material is formed by A.beta.40/42 derivatives which are
N-terminally truncated and frequently modified. The relative
amounts of N-terminally truncated A.beta. species are variable in
respect of A.beta. levels, mutations and BACE activity.
[0007] The most abundant truncated forms of A.beta. are:
A.beta.3-40/42 and A.beta.11-40/42. Both peptides contain an
N-terminal glutamate residue, which is frequently modified
enzymatically to pyroglutamate, resulting in the formation of
A.beta.3(pE)-40/42 and A.beta.11(pE)-40/42, respectively. Because
the amino terminus of the Abeta 3(pE) and 11(pE) peptides is
blocked by internal lactam, it is protected from the proteolytic
action of aminopeptidases other than pyroglutamate-specific ones
and can thus remain stable in tissues.
[0008] The most prominent form of N-terminally truncated modified
amyloid is formed by the peptide 3(pE)-40/42, which is thought to
constitute up to 50% of all truncated forms. This means that this
isoforms constitute 25-40% of all amyloid peptides in AD brains.
Another prominent form of truncated A.beta. peptides is
A.beta.11-40/42: Naslund et al. and Huse et al. could demonstrate
that there is a significant level of these truncated species
detectable in human brains of AD patients as well as in
infra-clinical patients for AD. Furthermore these peptides undergo
intramolecular dehydration and form stable (pE) forms with similar
consequences as described for 3(pE)-40/42.
[0009] It has been shown previously that truncated and modified
peptides are more stable in neural tissue than full length A.beta..
Additionally, N-terminally truncated forms of A.beta. are more
amyloidogenic than unmodified A.beta. peptides, thus enhancing the
rate of plaque formation, and also show neurotoxic activity when
applied to neurons in culture as well as in in vivo experiments.
Truncated forms of A.beta. can already be detected in diffuse
aggregations of A.beta. in early stages of AD and might be involved
in early plaque formation, acting as individual seeds in vivo.
[0010] There is compelling evidence that the occurrence of
N-terminally truncated A.beta. species is correlated with
increasing severity and early onset of neurodegeneration in
sporadic and familial Alzheimer disease as well as Down Syndrome
patients. The aggregatory effects in conjunction with the increased
stability of these peptides make them a potentially dangerous
player in AD development. Analysis in infraclinical patients
suffering from familial AD or Down-Syndrome have unequivocally
shown that A.beta. 3(pE)-42 can be detected during the earliest
stages of disease, also called the "seed" stages. At this time no
or only minor neurological symptoms can be detected although
plaques are starting to accumulate which bear the A.beta. 3(pE)-42
peptide species. Thus, data from such patients are implying a link
between early formation of truncated A.beta. species and disease
onset as well as progression.
[0011] In light of these findings it seems to be important to
decrease the amount of these peptide species in AD patients to
modify disease progression and reduce toxicity and accompanying
cognitive decline. An optimal AD-vaccine should thus elicit an
immune response which is able to target the most prominent forms of
A.beta. peptides present in the brain of AD patients.
[0012] Indeed, immunotherapeutic treatment using active and passive
immunisation strategies to target full length A.beta., led to
reduction of A.beta. plaques and had beneficial impact on disease
progression in animal models of AD. Passive vaccination experiments
in mouse models of AD clearly showed, that antibodies specifically
directed against the N- and C-termini of A.beta.40/42 are able to
reduce plaque burden in the brain and also improve cognitive
functions in treated animals as judged from behavioural analyses.
Similar observations have been made in active vaccination
experiments, using different approaches to induce immune responses
directed against A.beta.40/42 in mice. All of these approaches used
full length A.beta.40/42 or fragments containing the native
sequence of A.beta. and most reduced the amyloid burden in
transgenic mouse models. Importantly, these animals also showed
improved cognitive functions. Strikingly, Lernere et al. (Am J
Pathol 165 (2004): 283-297) could reproduce these results in
non-human primates which showed clear reduction of plaque
deposition and associated pathology in response to active
vaccination with full length A.beta.. However, the first phase IIa
clinical vaccination trial in AD patients using full length
A.beta.42 as antigen had to be discontinued due to severe
neuroinflammatory side effects including brain infiltration of
autoreactive T-cells. Nevertheless, studies investigating the
clinical effects in patients treated with AN-1792 revealed that
patients who developed an antibody response against A.beta.42 but
did not suffer from meningoencephalitis performed better in
cognitive tests than non-responding patients, indicating that
immunotherapy might be a very useful treatment approach in AD.
[0013] Most importantly, recent results obtained from autopsy cases
analysing patients which underwent AN1792 vaccination showed a
clearance of full length A.beta. species from the brain but a
persistence of N-terminally truncated forms of A.beta.. This
underscores the necessity of the invention of novel vaccines which
are targeting full length A.beta. as well as N-terminally truncated
and modified forms of this molecule.
[0014] Inducing an immune response against A.beta.40/42 peptides in
humans can interfere with cognitive decline in AD patients, but a
safe Alzheimer vaccine has to avoid the formation of autoreactive T
cells. Vaccination using native A.beta.40/42 peptides or fragments
thereof suffers from the intrinsic risk of inducing autoimmune
disease in patients, as the immune response cannot exclusively be
targeted to A.beta..
[0015] It is an object of the present invention to provide
compounds and medicaments which can be used to treat and/or prevent
.beta.-Amyloidoses including Alzheimer's disease. These compounds
should show no or a significantly reduced risk of inducing
autoimmune diseases when administered to an individual. According
to another object of the present invention said compound may be
able to induce the in vivo formation of antibodies in an individual
which are directed to truncated and/or stabilised forms of A.beta.,
which usually are the major components of amyloid deposits.
[0016] Therefore the present invention relates to the use of at
least one compound comprising the amino acid sequence
X.sub.1RX.sub.2DX.sub.3(X.sub.4).sub.n(X.sub.5).sub.m(X.sub.6).sub.o
(SEQ ID NO: 100) (Formula I),
wherein [0017] X.sub.1 is isoleucine (I) or valine (V), [0018]
X.sub.2 is tryptophan (W) or tyrosine (Y), [0019] X.sub.3 is
threonine (T), valine (V), alanine (A), methionine (M), glutamine
(Q) or glycine (G), [0020] X.sub.4 is proline (P), alanine (A),
tyrosine (Y), serine (S), cysteine (C) or glycine (G), [0021]
X.sub.5 is proline (P), leucine (L), glycine (G) or cysteine (C),
[0022] X.sub.6 is cysteine (C), [0023] n, m and o are,
independently, 0 or 1,
[0024] said compound having a binding capacity to an antibody which
is specific for an epitope of the amyloid-beta-peptide (A.beta.)
comprising the amino acid sequence EFRHDSGY (SEQ ID NO: 102) and/or
pEFRHDSGY (SEQ ID NO: 103)
[0025] for producing a medicament for preventing and/or treating
.beta.-amyloidoses.
[0026] It surprisingly turned out, that a compound comprising an
amino acid sequence of the formula I is able to induce the in vivo
formation of antibodies which are directed to the truncated A.beta.
forms A.beta.pE3-40/42 and A.beta.3-40/42. The antibodies formed
are able to bind to said A.beta. fragments resulting in the
disintegration of A.beta. plaques.
[0027] Formula I and II and all other peptidic molecules disclosed
herein mimic the natural occurring A.beta. peptides and variants
A.beta.1-40/42, A.beta.pE3-40/42, A.beta.3-40/42 and
A.beta.11-40/42, so that compounds comprising the amino acid
sequences disclosed herein are able to induce the formation of
respective antibodies.
[0028] The invention presented herein refers to antigens which do
not contain sequences of the native A.beta. peptide and mimic the
structure of neo-epitopes not detectable by mimotopes such as
described in WO 2004/062556. Such a mimotope-based AD vaccine would
therefore induce antibody responses exclusively reacting with the
pathological A.beta. molecules mentioned herein but not with
parental structures like APP. Furthermore, mimotopes do not contain
potential T-cell self-epitopes and avoid induction of detrimental
autoreactive T-cells.
[0029] ".beta.-Amyloidoses", as used herein, refers to various
degenerative diseases which are characterized by the aberrant
polymerization and accumulation of specific proteins so called
proteopathies. The present invention relates to the prevention,
treatment and diagnosis of proteopathies associated with
.beta.-amyloid proteins summarized under the term
.beta.-Amyloidoses. The most prominent form of .beta.-Amyloidoses
is Alzheimer's disease (AD). Other examples include but are not
limited to Dementia with Lewy bodies and Dementia in Down syndrome.
Further examples are Lewy body dementia, myositis, sporadic
inclusion body myositis, hereditary cerebral hemorrhage with
amyloidosis (dutch type), cerebral amyloid angiopathy, A.beta.
related angiitis.
[0030] According to a particularly preferred embodiment of the
present invention ".beta.-Amyloidoses" is Alzheimer's disease.
[0031] According to a preferred embodiment of the present invention
the compound comprises a peptide having an amino acid sequence
selected from the group consisting of IRWDTP(C) (SEQ ID NO: 106),
VRWDVYP(C) (SEQ ID NO: 107), IRYDAPL(C) (SEQ ID NO: 108),
IRYDMAG(C) (SEQ ID NO: 109), IRWDTSL(C) (SEQ ID NO: 110), IRWDQP(C)
(SEQ ID NO: 111), IRWDG(C) (SEQ ID NO: 112) and IRWDGG(C) (SEQ ID
NO: 113).
[0032] Particularly preferred compounds of the present invention
comprise or consist of the above identified amino acid sequences,
whereby the C-terminus of said peptide may or may not comprise a
cysteine residue (indicated by the use of brackets) so that the
compound obtained may be coupled, e.g., to a carrier molecule.
However, it is of course also possible to link to the N-terminus of
said peptide a cysteine residue.
[0033] According to a particularly preferred embodiment of the
present invention the amino acid sequence is IRWDTP(C) (SEQ ID NO:
106), VRWDVYP(C) (SEQ ID NO: 107), IRYDAPL(C) (SEQ ID NO: 108) or
IRYDMAG(C) (SEQ ID NO: 109).
[0034] Another aspect of the present invention relates to the use
of at least one compound comprising the amino acid sequence
EX.sub.1WHX.sub.2X.sub.3(X.sub.4).sub.n(X.sub.5).sub.m(SEQ ID NO:
101) (Formula II),
wherein [0035] X.sub.1 is valine (V), arginine (R) or leucine (L),
[0036] X.sub.2 is arginine (R) or glutamic acid (E), [0037] X.sub.3
is alanine (A), histidine (H), lysine (K), leucine (L), tyrosine
(Y) or glycine (G), [0038] X.sub.4 is proline (P), histidine (H),
phenylalanine (F), glutamine (Q) or cysteine (C) [0039] X.sub.5 is
cysteine (C), [0040] n and m are, independently, 0 or 1,
[0041] said compound having a binding capacity to an antibody which
is specific for an epitope of the amyloid-beta-peptide (A.beta.)
comprising the amino acid sequence EVHHQKL (SEQ ID NO: 104)
[0042] for producing a medicament for preventing and/or treating
.beta.-amyloidoses.
[0043] The administration of a compound comprising an amino acid
sequence of formula II provokes an immune response against the
truncated A.beta. form A.beta.11-40/42.
[0044] According to a preferred embodiment of the present invention
the compound comprises a peptide having an amino acid sequence
selected from the group consisting of EVWHRHQ(C) (SEQ ID NO: 114),
ERHEKH(C) (SEQ ID NO: 115), EVWHRLQ(C) (SEQ ID NO: 116), ELWHRYP(C)
(SEQ ID NO: 117), ELWHRAF(C) (SEQ ID NO: 118), ELWHRA(C) (SEQ ID
NO: 119), EVWHRG(C) (SEQ ID NO: 120), EVWHRH(C) (SEQ ID NO: 121)
and ERHEK(C) (SEQ ID NO: 122), preferably EVWHRHQ(C) (SEQ ID NO:
114), ERHEKH(C) (SEQ ID NO: 115), EVWHRLQ(C) (SEQ ID NO: 116),
ELWHRYP(C) (SEQ ID NO: 117) and ELWHRAF(C) (SEQ ID NO: 118).
[0045] Another aspect of the present invention relates to the use
of at least one compound comprising an amino acid sequence selected
from the group consisting of QDFRHY(C) (SEQ ID NO: 123), SEFKHG(C)
(SEQ ID NO: 124), TSFRHG(C) (SEQ ID NO: 125), TSVFRH(C) (SEQ ID NO:
126), TPFRHT(C) (SEQ ID NO: 127), SQFRHY(C) (SEQ ID NO: 128),
LMFRHN(C) (SEQ ID NO: 129), SAFRHH(C) (SEQ ID NO: 130), LPFRHG(C)
(SEQ ID NO: 131), SHFRHG(C) (SEQ ID NO: 132), ILFRHG(C) (SEQ ID NO:
133), QFKHDL(C) (SEQ ID NO: 134), NWFPHP(C) (SEQ ID NO: 135),
EEFKYS(C) (SEQ ID NO: 136), NELRHST(C) (SEQ ID NO: 137), GEMRHQP(C)
(SEQ ID NO: 138), DTYFPRS(C) (SEQ ID NO: 139), VELRHSR(C) (SEQ ID
NO: 140), YSMRHDA(C) (SEQ ID NO: 141), AANYFPR(C) (SEQ ID NO: 142),
SPNQFRH(C) (SEQ ID NO: 143), SSSFFPR(C) (SEQ ID NO: 144),
EDWFFWH(C) (SEQ ID NO: 145), SAGSFRH(C) (SEQ ID NO: 146),
QVMRHHA(C) (SEQ ID NO: 147), SEFSHSS(C) (SEQ ID NO: 148),
QPNLFYH(C) (SEQ ID NO: 149), ELFKHHL(C) (SEQ ID NO: 150),
TLHEFRH(C) (SEQ ID NO: 151), ATFRHSP(C) (SEQ ID NO: 152),
APMYFPH(C) (SEQ ID NO: 153), TYFSHSL(C) (SEQ ID NO: 154),
HEPLFSH(C) (SEQ ID NO: 155), SLMRHSS(C) (SEQ ID NO: 156),
EFLRHTL(C) (SEQ ID NO: 157), ATPLFRH(C) (SEQ ID NO: 158),
QELKRYY(C) (SEQ ID NO: 159), THTDFRH(C) (SEQ ID NO: 160),
LHIPFRH(C) (SEQ ID NO: 161), NELFKHF(C) (SEQ ID NO: 162),
SQYFPRP(C) (SEQ ID NO: 163), DEHPFRH(C) (SEQ ID NO: 164),
MLPFRHG(C) (SEQ ID NO: 165), SAMRHSL(C) (SEQ ID NO: 166),
TPLMFWH(C) (SEQ ID NO: 167), LQFKHST(C) (SEQ ID NO: 168),
ATFRHST(C) (SEQ ID NO: 169), TGLMFKH(C) (SEQ ID NO: 170),
AEFSHWH(C) (SEQ ID NO: 171), QSEFKHW(C) (SEQ ID NO: 172),
AEFMHSV(C) (SEQ ID NO: 173), ADHDFRH(C) (SEQ ID NO: 174),
DGLLFKH(C) (SEQ ID NO: 175), IGFRHDS(C) (SEQ ID NO: 176),
SNSEFRR(C) (SEQ ID NO: 177), SELRHST(C) (SEQ ID NO: 178),
THMEFRR(C) (SEQ ID NO: 179), EELRHSV(C) (SEQ ID NO: 180),
QLFKHSP(C) (SEQ ID NO: 181), YEFRHAQ(C) (SEQ ID NO: 182),
SNFRHSV(C) (SEQ ID NO: 183), APIQFRH(C) (SEQ ID NO: 184),
AYFPHTS(C) (SEQ ID NO: 185), NSSELRH(C) (SEQ ID NO: 186),
TEFRHKA(C) (SEQ ID NO: 187), TSTEMWH(C) (SEQ ID NO: 188),
SQSYFKH(C) (SEQ ID NO: 189), (C)SEFKH (SEQ ID NO: 190), SEFKH(C)
(SEQ ID NO: 191), (C)HEFRH (SEQ ID NO: 192) and HEFRH(C) (SEQ ID
NO: 193) for producing a medicament for preventing and/or treating
.beta.-amyloidoses.
[0046] Each of these compounds is able to induce the in vivo
formation of antibodies directed to A.beta.1-40/42,
A.beta.pE3-40/42 and A.beta.3-40/42. Therefore these compounds are
particularly well suited to treat and/or prevent
.beta.-amyloidoses, such as AD, because the administration of one
compound results in the formation of antibodies which are capable
to recognize the three major A.beta. forms A.beta.1-40/42,
A.beta.pE3-40/42 and A.beta.3-40/42.
[0047] According to a preferred embodiment of the present invention
the compound comprises or consists of a peptide having an amino
acid sequence selected from the group consisting of QDFRHY(C)_(SEQ
ID NO: 123), SEFKHG(C) (SEQ ID NO: 124), TSFRHG(C) (SEQ ID NO:
125), TSVFRH(C) (SEQ ID NO: 126), TPFRHT(C) (SEQ ID NO: 127),
SQFRHY(C) (SEQ ID NO: 128), LMFRHN(C) (SEQ ID NO: 129), SAFRHH(C)
(SEQ ID NO: 130), LPFRHG(C) (SEQ ID NO: 131), SHFRHG(C) (SEQ ID NO:
132), ILFRHG(C) (SEQ ID NO: 133), QFKHDL(C) (SEQ ID NO: 134),
NWFPHP(C) (SEQ ID NO: 135), EEFKYS(C) (SEQ ID NO: 136), SPNQFRH(C)
(SEQ ID NO: 143), TLHEFRH(C) (SEQ ID NO: 151), THTDFRH(C) (SEQ ID
NO: 160), DEHPFRH(C) (SEQ ID NO: 164), QSEFKHW(C) (SEQ ID NO: 172),
ADHDFRH(C) (SEQ ID NO: 174), DGLLFKH(C) (SEQ ID NO: 175),
EELRHSV(C) (SEQ ID NO: 180), TEFRHKA(C) (SEQ ID NO: 187), (C)SEFKH
(SEQ ID NO: 190), SEFKH(C) (SEQ ID NO: 191), (C)HEFRH (SEQ ID NO:
192) and HEFRH(C) (SEQ ID NO: 193) preferably SEFKHG(C) (SEQ ID NO:
124), TSVFRH(C) (SEQ ID NO: 126), SQFRHY(C) (SEQ ID NO: 128),
LMFRHN(C) (SEQ ID NO: 129), ILFRHG(C) (SEQ ID NO: 133), SPNQFRH(C)
(SEQ ID NO: 143), ELFKHHL(C) (SEQ ID NO: 150), TLHEFRH(C) (SEQ ID
NO: 151), THTDFRH(C) (SEQ ID NO: 160), DEHPFRH(C) (SEQ ID NO: 164),
QSEFKHW(C) (SEQ ID NO: 172), ADHDFRH(C) (SEQ ID NO: 174),
YEFRHAQ(C) (SEQ ID NO: 182), TEFRHKA(C) (SEQ ID NO: 187).
[0048] The amino acid sequences disclosed herein are considered to
be mimotopes of the epitopes of A.beta. comprising the amino acid
sequence EFRHDSGY (SEQ ID NO: 102), pEFRHDSGY (SEQ ID NO: 103) or
EVHHQKL (SEQ ID NO: 104). According to the present invention the
term "mimotope" refers to a molecule which has a conformation that
has a topology equivalent to the epitope of which it is a mimic.
The mimotope binds to the same antigen-binding region of an
antibody which binds immunospecifically to a desired antigen. The
mimotope will elicit an immunological response in a host that is
reactive to the antigen to which it is a mimic. The mimotope may
also act as a competitor for the epitope of which it is a mimic in
in vitro inhibition assays (e.g. ELISA inhibition assays) which
involve the epitope and an antibody binding to said epitope.
However, a mimotope of the present invention may not necessarily
prevent or compete with the binding of the epitope of which it is a
mimic in an in vitro inhibition assay although it is capable to
induce a specific immune response when administered to a
mammal.
[0049] As used herein, the term "epitope" refers to an immunogenic
region of an antigen which is recognized by a particular antibody
molecule. In general, an antigen will possess one or more epitopes,
each capable of binding an antibody that recognizes the particular
epitope.
[0050] The mimotopes of the present invention can be synthetically
produced by chemical synthesis methods which are well known in the
art, either as an isolated peptide or as a part of another peptide
or polypeptide. Alternatively, the peptide mimotope can be produced
in a microorganism which produces the peptide mimotope which is
then isolated and if desired, further purified. The peptide
mimotope can be produced in microorganisms such as bacteria, yeast
or fungi, in eukaryote cells such as a mammalian or an insect cell,
or in a recombinant virus vector such as adenovirus, poxvirus,
herpesvirus, Simliki forest virus, baculovirus, bacteriophage,
sindbis virus or sendai virus. Suitable bacteria for producing the
peptide mimotope include E. coli, B. subtilis or any other
bacterium that is capable of expressing peptides such as the
peptide mimotope. Suitable yeast types for expressing the peptide
mimotope include Saccharomyces cerevisiae, Schizosaccharomyces
pombe, Candida, Pichia pastoris or any other yeast capable of
expressing peptides. Corresponding methods are well known in the
art. Also methods for isolating and purifying recombinantly
produced peptides are well known in the art and include e.g. as gel
filtration, affinity chromatography, ion exchange chromatography
etc.
[0051] To facilitate isolation of the peptide mimotope, a fusion
polypeptide may be made wherein the peptide mimotope is
translationally fused (covalently linked) to a heterologous
polypeptide which enables isolation by affinity chromatography.
Typical heterologous polypeptides are His-Tag (e.g. His.sub.6 (SEQ
ID NO: 194); 6 histidine residues (SEQ ID NO: 194)), GST-Tag
(Glutathione-Stransferase) etc. The fusion polypeptide facilitates
not only the purification of the mimotopes but can also prevent the
mimotope polypeptide from being degraded during purification. If it
is desired to remove the heterologous polypeptide after
purification the fusion polypeptide may comprise a cleavage site at
the junction between the peptide mimotope and the heterologous
polypeptide. The cleavage site consists of an amino acid sequence
that is cleaved with an enzyme specific for the amino acid sequence
at the site (e.g. proteases).
[0052] The mimotopes of the present invention may also be modified
at or nearby their N- and/or C-termini so that at said positions a
cysteine residue is bound thereto. In a preferred embodiment
terminally positioned (located at the N- and C-termini of the
peptide) cysteine residues are used to cyclize the peptides through
a disulfide bond. The cysteine residue may also serve to bind to
said peptide/compound a further molecule (e.g. a carrier).
[0053] The mimotopes of the present invention may also be used in
various assays and kits, in particular in immunological assays and
kits. Therefore, it is particularly preferred that the mimotope may
be part of another peptide or polypeptide, particularly an enzyme
which is used as a reporter in immunological assays. Such reporter
enzymes include e.g. alkaline phosphatase or horseradish
peroxidase.
[0054] The mimotopes according to the present invention preferably
are antigenic polypeptides which in their amino acid sequence vary
from the amino acid sequence of A.beta. or of fragments of A.beta..
In this respect, the inventive mimotopes may not only comprise
amino acid substitutions of one or more naturally occurring amino
acid residues but also of one or more non-natural amino acids (i.e.
not from the 20 "classical" amino acids) or they may be completely
assembled of such non-natural amino acids. Moreover, the inventive
antigens which induce antibodies directed and binding to
A.beta.1-40/42, A.beta.pE3-40/42, A.beta.3-40/42 and
A.beta.11-40/42 may be assembled of D- or L-amino acids or of
combinations of DL-amino acids and, optionally, they may have been
changed by further modifications, ring closures or derivatizations.
Suitable antibody-inducing antigens may be provided from
commercially available peptide libraries. Preferably, these
peptides are at least 7 amino acids, and preferred lengths may be
up to 16, preferably up to 14 or 20 amino acids (e.g. 5 to 16 amino
acid residues). According to the invention, however, also longer
peptides may very well be employed as antibody-inducing antigens.
Furthermore the mimotopes of the present invention may also be part
of a polypeptide and consequently comprising at their N- and/or
C-terminus at least one further amino acid residue.
[0055] For preparing the mimotopes of the present invention (i.e.
the antibody-inducing antigens disclosed herein), of course also
phage libraries, peptide libraries are suitable, for instance
produced by means of combinatorial chemistry or obtained by means
of high throughput screening techniques for the most varying
structures (Display: A Laboratory Manual by Carlos F. Barbas
(Editor), et al.; Willats WG Phage display: practicalities and
prospects. Plant Mol. Biol. 2002 December; 50(6):837-54).
[0056] Furthermore, according to the invention also
anti-A.beta.1-40/42-, -A.beta.pE3-40/42-, -A.beta.3-40/42- and
-A.beta.11-40/42-antibody-inducing antigens based on nucleic acids
("aptamers") may be employed, and these, too, may be found with the
most varying (oligonucleotide) libraries (e.g. with 2-180 nucleic
acid residues) (e.g. Burgstaller et al., Curr. Opin. Drug Discov.
Dev. 5(5) (2002), 690-700; Famulok et al., Acc. Chem. Res. 33
(2000), 591-599; Mayer et al., PNA.beta. 98 (2001), 4961-4965,
etc.). In antibody-inducing antigens based on nucleic acids, the
nucleic acid backbone can be provided e.g. by the natural
phosphor-diester compounds, or also by phosphorotioates or
combinations or chemical variations (e.g. as PNA), wherein as
bases, according to the invention primarily U, T, A, C, G, H and mC
can be employed. The 2'-residues of the nucleotides which can be
used according to the present invention preferably are H, OH, F,
Cl, NH.sub.2, O-methyl, O-ethyl, O-propyl or O-butyl, wherein the
nucleic acids may also be differently modified, i.e. for instance
with protective groups, as they are commonly employed in
oligonucleotide synthesis. Thus, aptamer-based antibody-inducing
antigens are also preferred antibody-inducing antigens within the
scope of the present invention.
[0057] According to a preferred embodiment of the present invention
the compound is coupled to a pharmaceutically acceptable carrier,
preferably KLH (Keyhole Limpet Hemocyanin), tetanus toxoid,
albumin-binding protein, bovine serum albumin, a dendrimer (MAP;
Biol. Chem. 358: 581), peptide linkers (or flanking regions) as
well as the adjuvant substances described in Singh et al., Nat.
Biotech. 17 (1999), 1075-1081 (in particular those in Table 1 of
that document), and O'Hagan et al., Nature Reviews, Drug Discovery
2 (9) (2003), 727-735 (in particular the endogenous
immunopotentiating compounds and delivery systems described
therein), or mixtures thereof. The conjugation chemistry (e.g. via
heterobifunctional compounds such as GMBS and of course also others
as described in "Bioconjugate Techniques", Greg T. Hermanson) in
this context can be selected from reactions known to the skilled
man in the art. Moreover, the vaccine composition may be formulated
with an adjuvant, preferably a low soluble aluminium composition,
in particular aluminium hydroxide. Of course, also adjuvants like
MF59 aluminium phosphate, calcium phosphate, cytokines (e.g., IL-2,
IL-12, GM-CSF), saponins (e.g., QS21), MDP derivatives, CpG oligos,
LPS, MPL, polyphosphazenes, emulsions (e.g., Freund's, SAF),
liposomes, virosomes, iscoms, cochleates, PLG microparticles,
poloxamer particles, virus-like particles, heat-labile enterotoxin
(LT), cholera toxin (CT), mutant toxins (e.g., LTK63 and LTR72),
microparticles and/or polymerized liposomes may be used.
[0058] The compound of the present invention is preferably bound to
the carrier or adjuvant via a linker, which is selected from the
group consisting of NHS-poly (ethylene oxide) (PEO) (e.g.
NHS-PEO.sub.4-maleimide).
[0059] A vaccine which comprises the present compound (mimotope)
and the pharmaceutically acceptable carrier may be administered by
any suitable mode of application, e.g. i.d., i.v., i.p., i.m.,
intranasally, orally, subcutaneously, etc. and in any suitable
delivery device (O'Hagan et al., Nature Reviews, Drug Discovery 2
(9), (2003), 727-735). The compound of the present invention is
preferably formulated for intravenous, subcutaneous, intradermal or
intramuscular administration (see e.g. "Handbook of Pharmaceutical
Manufacturing Formulations", Sarfaraz Niazi, CRC Press Inc,
2004).
[0060] The medicament (vaccine) according to the present invention
contains the compound according to the invention in an amount of
from 0.1 ng to 10 mg, preferably 10 ng to 1 mg, in particular 100
ng to 100 .mu.g, or, alternatively, e.g. 100 fmol to 10 .mu.mol,
preferably 10 .mu.mol to 1 .mu.mol, in particular 100 .mu.mol to
100 nmol. Typically, the vaccine may also contain auxiliary
substances, e.g. buffers, stabilizers etc.
[0061] Another aspect of the present invention relates to the use
of a compound as defined above for treating and/or ameliorating
symptoms of synucleopathy.
[0062] It surprisingly turned out that the compounds of the present
invention can also be used to treat and ameliorate symptoms
associated with synucleopathies.
[0063] Amyloidoses and synucleopathies are associated with the
cerebral accumulation of .beta.-amyloid and .alpha.-synuclein,
respectively. Some patients show clinical and pathological features
of both diseases, raising the possibility of overlapping pathogenic
pathways. These patients are also classified as suffering from a
newly identified syndrome described as Dementia with Lewy Bodies or
Parkinson's disease with dementia (DLB/PDD). In a recent transgenic
animal model for DLB/PDD it has been shown that overexpression of
both, .alpha.-synuclein and Amyloid Precursor Protein (hAPP), in
mice leads to the development of cognitive and motor alterations
accompanied by loss of cholinergic neurons and reduction in
synaptic vesicles, formation of extensive amyloid plaques, and
haSYN-immunoreactive intraneuronal fibrillar inclusions. All of
these features are also found in the DLB/PDD syndrome. It has been
described recently that both molecules are potentially able to
interact and to form hybrid oligomers in vitro. It has also been
shown that overexpression of the APP can exacerbate some of the
pathologic effects of .alpha.-synuclein overexpression. In
contrast, .alpha.-synuclein is able to enhance secretion and
toxicity of amyloid beta peptides and could thus also increase the
effects of .beta.-amyloid supporting the notion of overlapping
pathogenic pathways in neurodegenerative processes.
[0064] In both proteopathies progressive accumulation of peptide
oligomers has been identified as one of the central toxic events
leading to the various alterations typical for either
synucleopathies or amyloidoses. Despite this mechanistic
similarity, it is hypothesized that .alpha.-synuclein and A.beta.
have distinct, as well as convergent, pathogenic effects on the
integrity and function of the brain. Synucleins are believed to
affect motoric function more severely than cognitive function,
whereas amyloid .beta. peptides are described to have opposite
effects. The reason for this discrepancy is currently unknown but
it precludes a clear description of the interdependencies and
effects of both molecules.
[0065] The treatment approach presented in the current invention is
describing an immunotherapy targeting A.beta. which will lead to
the removal of mainly extracellular amyloid. It is thus believed to
relieve the amyloid associated alterations ranging from plaque
deposition to neuronal death as well as to memory problems and
cognitive decline. The subcellular localization of synucleins
however indicates that these intracellular proteins are mainly
active at the synapse, especially confined to synaptic vesicles.
Interestingly, also synuclein accumulations, which are the unifying
pathologic hallmark of synucleopathies, are mainly detectable
intracellularly. Additionally, the pathogenic mechanism underlying
synucleopathies is believed to be attributable to intraneuronal
changes ranging from mitochondrial dysfunction, .alpha.-cumulation
of abnormally folded, ubiquitinated or phosphorylated proteins as
well as accumulation of alpha synuclein. These alterations are
consequently resulting in changes in synaptic functions, synaptic
failure, and loss of dopaminergic neurons and classical clinical
signs of synucleopathies. In contrast A.beta. is mainly detectable
extraneuronally and amyloid plaques as well as fibrils,
protofibrils and oligomers of beta amyloid can exert neurotoxic
functions when applied extracellularly or intracerebrally. Thus it
is a surprising finding to the expert that an approach mainly
targeting extracellular amyloid would reduce the symptoms of
synucleopathies like PD, which are affecting mainly intracellular
processes leading to the typical symptoms described below. It is
even more surprising as it is currently believed that the
overlapping effects of both molecules are caused by direct
interactions of the two proteins which should mainly occur
intracellularly. According to the present invention the term
"synucleinopathy" includes all neurodegenerative disorders
characterized by pathological synuclein aggregations. Several
neurodegenerative disorders including Parkinson's Disease (PD),
Lewy Body Disease (LBD), Diffuse Lewy Body Disease (DLBD), Dementia
with Lewy Bodies (DLB), Parkinsonism with Dementia (PDD), Multiple
System Atrophy (MSA) and Neurodegeneration with Brain Iron
Accumulation type I (NBIA Type I) are collectively grouped as
synucleinopathies.
[0066] "Symptoms of synucleopathy", as used herein, refers to those
symptoms of the synucleopathies, in particular Parkinson's disease,
which affect the motor and non-motor behaviour of a patient
suffering from said disease. "Motor symptoms" include resting
tremor, Bradykinesia, rigidity, postural instability, stooped
posture, dystonia, fatigue, impaired fine motor dexterity and motor
coordination, impaired gross motor coordination, poverty of
movement (decreased arm swing), akathisia, speech problems, such as
softness of voice or slurred speech caused by lack of muscle
control, loss of facial expression, or "masking", micrographia,
difficulty swallowing, sexual dysfunction, drooling etc.
"Non-motor" symptoms include pain, dementia or confusion, sleep
disturbances, constipation, skin problems, depression, fear or
anxiety, memory difficulties and slowed thinking, urinary problems,
fatigue and aching, loss of energy, compulsive behaviour, cramping
etc.
[0067] According to a preferred embodiment of the present invention
the synucleopathy is selected from the group of Parkinson's
Disease, Dementia with Lewy Bodies, multiple system atrophy and
neurodegeneration with brain iron accumulation. Particularly
preferred is Parkinson's disease.
[0068] Another aspect of the present invention relates to a peptide
having or consisting of an amino acid sequence selected from the
group consisting of IRWDTP(C) (SEQ ID NO: 106), VRWDVYP(C) (SEQ ID
NO: 107), IRYDAPL(C) (SEQ ID NO: 108), IRYDMAG(C) (SEQ ID NO: 109),
IRWDTSL(C) (SEQ ID NO: 110), IRWDQP(C) (SEQ ID NO: 111), IRWDG(C)
(SEQ ID NO: 112), IRWDGG(C) (SEQ ID NO: 113), EVWHRHQ(C) (SEQ ID
NO: 114), ERHEKH(C) (SEQ ID NO: 115), EVWHRLQ(C) (SEQ ID NO: 116),
ELWHRYP(C) (SEQ ID NO: 117), ELWHRAF(C) (SEQ ID NO: 118), ELWHRA(C)
(SEQ ID NO: 119), EVWHRG(C) (SEQ ID NO: 120), EVWHRH(C) (SEQ ID NO:
121), ERWHEK(C) (SEQ ID NO: 122), QDFRHY(C) (SEQ ID NO: 123),
SEFKHG(C) (SEQ ID NO: 124), TSFRHG(C) (SEQ ID NO: 125), TSVFRH(C)
(SEQ ID NO: 126), TPFRHT(C) (SEQ ID NO: 127), SQFRHY(C) (SEQ ID NO:
128), LMFRHN(C) (SEQ ID NO: 129), SAFRHH(C) (SEQ ID NO: 130),
LPFRHG(C) (SEQ ID NO: 131), SHFRHG(C) (SEQ ID NO: 132), ILFRHG(C)
(SEQ ID NO: 133), QFKHDL(C) (SEQ ID NO: 134), NWFPHP(C) (SEQ ID NO:
135), EEFKYS(C) (SEQ ID NO: 136), NELRHST(C) (SEQ ID NO: 137),
GEMRHQP(C) (SEQ ID NO: 138), DTYFPRS(C) (SEQ ID NO: 139),
VELRHSR(C) (SEQ ID NO: 140), YSMRHDA(C) (SEQ ID NO: 141),
AANYFPR(C) (SEQ ID NO: 142), SPNQFRH(C) (SEQ ID NO: 143),
SSSFFPR(C) (SEQ ID NO: 144), EDWFFWH(C) (SEQ ID NO: 145),
SAGSFRH(C) (SEQ ID NO: 146), QVMRHHA(C) (SEQ ID NO: 147),
SEFSHSS(C) (SEQ ID NO: 148), QPNLFYH(C) (SEQ ID NO: 149),
ELFKHHL(C) (SEQ ID NO: 150), TLHEFRH(C) (SEQ ID NO: 151),
ATFRHSP(C) (SEQ ID NO: 152), APMYFPH(C) (SEQ ID NO: 153),
TYFSHSL(C) (SEQ ID NO: 154), HEPLFSH(C) (SEQ ID NO: 155),
SLMRHSS(C) (SEQ ID NO: 156), EFLRHTL(C) (SEQ ID NO: 157),
ATPLFRH(C) (SEQ ID NO: 158), QELKRYY(C) (SEQ ID NO: 159),
THTDFRH(C) (SEQ ID NO: 160), LHIPFRH(C) (SEQ ID NO: 161),
NELFKHF(C) (SEQ ID NO: 162), SQYFPRP(C) (SEQ ID NO: 163),
DEHPFRH(C) (SEQ ID NO: 164), MLPFRHG(C) (SEQ ID NO: 165),
SAMRHSL(C) (SEQ ID NO: 166), TPLMFWH(C) (SEQ ID NO: 167),
LQFKHST(C) (SEQ ID NO: 168), ATFRHST(C) (SEQ ID NO: 169),
TGLMFKH(C) (SEQ ID NO: 170), AEFSHWH(C) (SEQ ID NO: 171),
QSEFKHW(C) (SEQ ID NO: 172), AEFMHSV(C) (SEQ ID NO: 173),
ADHDFRH(C) (SEQ ID NO: 174), DGLLFKH(C) (SEQ ID NO: 175),
IGFRHDS(C) (SEQ ID NO: 176), SNSEFRR(C) (SEQ ID NO: 177),
SELRHST(C) (SEQ ID NO: 178), THMEFRR(C) (SEQ ID NO: 179),
EELRHSV(C) (SEQ ID NO: 180), QLFKHSP(C) (SEQ ID NO: 181),
YEFRHAQ(C) (SEQ ID NO: 182), SNFRHSV(C) (SEQ ID NO: 183),
APIQFRH(C) (SEQ ID NO: 184), AYFPHTS(C) (SEQ ID NO: 185),
NSSELRH(C) (SEQ ID NO: 186), TEFRHKA(C) (SEQ ID NO: 187),
TSTEMWH(C) (SEQ ID NO: 188), SQSYFKH(C) (SEQ ID NO: 189), (C)SEFKH
(SEQ ID NO: 190), SEFKH(C) (SEQ ID NO: 191), (C)HEFRH (SEQ ID NO:
192) and HEFRH(C) (SEQ ID NO: 193). As indicated by the use of the
parenthesis the peptides of the present invention may or may not
comprise the cysteine residue at the C- or N-terminus. Consequently
the present invention encompasses also the following amino acid
sequences: IRWDTP (SEQ ID NO: 195), VRWDVYP (SEQ ID NO: 196),
IRYDAPL (SEQ ID NO: 197), IRYDMAG (SEQ ID NO: 198), IRWDTSL (SEQ ID
NO: 199), IRWDQP (SEQ ID NO: 200), IRWDG (SEQ ID NO: 201), IRWDGG
(SEQ ID NO: 202), EVWHRHQ (SEQ ID NO: 203), ERHEKH (SEQ ID NO:
204), EVWHRLQ (SEQ ID NO: 205), ELWHRYP (SEQ ID NO: 206), ELWHRAF
(SEQ ID NO: 207), ELWHRA (SEQ ID NO: 208), EVWHRG (SEQ ID NO: 209),
EVWHRH (SEQ ID NO: 210), ERHEK (SEQ ID NO: 211), QDFRHY (SEQ ID NO:
212), SEFKHG (SEQ ID NO: 213), TSFRHG (SEQ ID NO: 214), TSVFRH (SEQ
ID NO: 215), TPFRHT (SEQ ID NO: 216), SQFRHY (SEQ ID NO: 217),
LMFRHN (SEQ ID NO: 218), SAFRHH (SEQ ID NO: 219), LPFRHG (SEQ ID
NO: 220), SHFRHG (SEQ ID NO: 221), ILFRHG (SEQ ID NO: 222), QFKHDL
(SEQ ID NO: 223), NWFPHP (SEQ ID NO: 224), EEFKYS (SEQ ID NO: 225),
NELRHST (SEQ ID NO: 226), GEMRHQP (SEQ ID NO: 227), DTYFPRS (SEQ ID
NO: 228), VELRHSR (SEQ ID NO: 229), YSMRHDA (SEQ ID NO: 230),
AANYFPR (SEQ ID NO: 231), SPNQFRH (SEQ ID NO: 232), SSSFFPR (SEQ ID
NO: 233), EDWFFWH (SEQ ID NO: 234), SAGSFRH (SEQ ID NO: 235),
QVMRHHA (SEQ ID NO: 236), SEFSHSS (SEQ ID NO: 237), QPNLFYH (SEQ ID
NO: 238), ELFKHHL (SEQ ID NO: 239), TLHEFRH (SEQ ID NO: 240),
ATFRHSP (SEQ ID NO: 241), APMYFPH (SEQ ID NO: 242), TYFSHSL (SEQ ID
NO: 243), HEPLFSH (SEQ ID NO: 244), SLMRHSS (SEQ ID NO: 245),
EFLRHTL (SEQ ID NO: 246), ATPLFRH (SEQ ID NO: 247), QELKRYY (SEQ ID
NO: 248), THTDFRH (SEQ ID NO: 249), LHIPFRH (SEQ ID NO: 250),
NELFKHF (SEQ ID NO: 251), SQYFPRP (SEQ ID NO: 252), DEHPFRH (SEQ ID
NO: 253), MLPFRHG (SEQ ID NO: 254), SAMRHSL (SEQ ID NO: 255),
TPLMFWH (SEQ ID NO: 256), LQFKHST (SEQ ID NO: 257), ATFRHST (SEQ ID
NO: 258), TGLMFKH (SEQ ID NO: 259), AEFSHWH (SEQ ID NO: 260),
QSEFKHW (SEQ ID NO: 261), AEFMHSV (SEQ ID NO: 262), ADHDFRH (SEQ ID
NO: 263), DGLLFKH (SEQ ID NO: 264), IGFRHDS (SEQ ID NO: 265),
SNSEFRR (SEQ ID NO: 266), SELRHST (SEQ ID NO: 267), THMEFRR (SEQ ID
NO: 268), EELRHSV (SEQ ID NO: 269), QLFKHSP (SEQ ID NO: 270),
YEFRHAQ (SEQ ID NO: 271), SNFRHSV (SEQ ID NO: 272), APIQFRH (SEQ ID
NO: 273), AYFPHTS (SEQ ID NO: 274), NSSELRH (SEQ ID NO: 275),
TEFRHKA (SEQ ID NO: 276), TSTEMWH (SEQ ID NO: 277), SQSYFKH (SEQ ID
NO: 278), (C)SEFKH (SEQ ID NO: 190), SEFKH (SEQ ID NO: 279), HEFRH
(SEQ ID NO: 280) and HEFRH (SEQ ID NO: 280).
[0069] According to a preferred embodiment the peptide is coupled
to a pharmaceutically acceptable carrier, preferably KLH (Keyhole
Limpet Hemocyanin).
[0070] Yet another aspect of the present invention relates to a
pharmaceutical formulation, preferably a vaccine, comprising at
least one peptide according to the present invention. Said
pharmaceutical formulation may be employed to treat individuals
suffering from .beta.-Amyloidoses including Alzheimer's disease or
prevent the formation of A.beta.-plaques in an individual to impede
the formation of .beta.-Amyloidoses including Alzheimer's
disease.
[0071] The present invention is further illustrated by the
following figures and examples, however without being restricted
thereto.
[0072] FIG. 1 shows binding of monoclonal antibody MV-001 to
specific peptides and recombinant proteins;
[0073] FIG. 2 shows binding of monoclonal antibody MV-003 to
specific peptides and recombinant proteins;
[0074] FIG. 3 shows binding of monoclonal antibody MV-004 to
specific peptides and recombinant proteins;
[0075] FIGS. 4A, 4B and 4C show typical binding assays with
mimotopes for .beta.-amyloid and N-terminally truncated and/or
posttranslationally modified .beta.-amyloid fragments;
[0076] FIGS. 5A, 5B and 5C show typical inhibition assays with
mimotopes for .beta.-amyloid and N-terminally truncated and/or
posttranslationally modified .beta.-amyloid fragments;
[0077] FIGS. 6A, 6B and 6C show examples for in vivo
characterisations of the immune response elicited by mimotope
vaccination (injected peptide/irrelevant peptide);
[0078] FIGS. 7A, 7B and 7C show examples for in vivo
characterisation of the immune response elicited by mimotope
vaccination against Amyloid Beta fragments;
[0079] FIGS. 8A and 8B show examples for in vivo characterisation
of the immune response elicited by mimotope vaccination against
full length A.beta.40/42.
[0080] FIG. 9 shows areas occupied by amyloid plaques. Tg2576 were
injected 6 times with mimotope vaccines adjuvanted with aluminium
hydroxide (ALUM) by s.c. inoculation at monthly intervals. Control
mice received PBS-ALUM only. Area occupied by amyloid plaques shown
as percent of the control group. Gr1 . . . control group; Gr2 . . .
received p4381; Gr3 . . . received p4390; Gr4 . . . received
p4715
[0081] FIG. 10 shows areas occupied by amyloid plaques. Tg2576 were
injected 6 times with AFFITOPE vaccines adjuvanted with aluminium
hydroxide (ALUM) by s.c. inoculation at monthly intervals. Control
mice received PBS-ALUM only. Area occupied by amyloid plaques shown
as percent of the control group. Gr1 . . . control group; Gr2 . . .
received p4395.
EXAMPLES
Methods
[0082] The antibodies used for the mimotope identification
according to the present invention detect amino acid sequences
derived from human A.beta. but do not bind to full length human
APP. The sequences detected include EFRHDS (=original epitope aa3-8
of A.beta.), p(E)FRHDS (=original epitope of the modified aa3-8 of
A.beta.), EVHHQK (=original epitope aa11-16 of A.beta.). The
antibody may be a monoclonal or polyclonal antibody preparation or
any antibody part or derivative thereof, the only prerequisite is
that the antibody molecule specifically recognises at least one of
the epitopes mentioned above (derived from human A.beta.), but does
not bind to full length human APP.
[0083] The mimotopes are identified and further characterised with
such monoclonal antibodies and peptide libraries.
Example 1
Generation of Monoclonal Antibodies to Specifically Detect
.beta.-Amyloid and N-Terminally Truncated and/or
Posttranslationally Modified .beta.-Amyloid Fragments
Example 1a
Generation of Monoclonal Antibody MV-001
[0084] A monoclonal antibody derived from the fusion of experiment
Alz-5 was generated: In experiment Alz-5 C57/B16 mice were
immunized repeatedly with original A.beta. epitope DAEFRHDSGYC (SEQ
ID NO: 89) coupled to KLH (Keyhole Limpet Hemocyanin) and Alum
(Aluiminium Hydroxide) as adjuvant. p4371-peptide-specific,
antibody-producing hybridomas were detected by ELISA (p1253- and
p4371-peptide-coated ELISA plates). Human A.beta.40/42 (recombinant
protein) was used as positive control peptide: hybridomas
recognizing the recombinant protein immobilised on ELISA plates
were included because they are binding both peptide and full length
A.beta. specifically. P1454 (Human A.beta. 33-40) was used as
negative control peptide. Furthermore hybridomas were tested
against p4373. Only hybridomas with no or limited p4373 binding
were used for further antibody development.
[0085] The Hybridoma clone (MV-001 (internal name 824; IgG1) was
purified and analysed for specific detection of p1253, p4371,
p4373, p1454 and A.beta. respectively. MV-001 recognized the
injected epitope (p1253) as well as the specific epitope (p4371)
and full length A.beta. protein (recombinant protein; obtained from
Bachem AG, Bubendorf, Switzerland) in ELISA. It however did not
detect p1454 in ELISA. Furthermore, the MV-001 antibodies basically
failed to detect the peptide p4373 encoding the pyroglutamate
version of A.beta.3-10 (30 times lower titer than the original
epitopes).
Example 1b
Generation of Monoclonal Antibody MV-003
[0086] A monoclonal antibody derived from the fusion of experiment
Alz-16 was generated: In experiment Alz-16 BalbC mice were
immunized repeatedly with the epitope p(E)FRHDSC (SEQ ID NO: 92)
(p4373) coupled to KLH (Keyhole Limpet Hemocyanin) and Alum
(Aluiminium Hydroxide) as adjuvant. p4373-peptide-specific,
antibody-producing hybridomas were detected by ELISA
(p4373-peptide-coated ELISA plates). p1253, p1454 and A.beta.40/42
were used as negative control peptides. Furthermore, hybridomas
were tested against p4371. Only hybridomas with no or limited p4371
binding were used for further antibody development in order to
guarantee for pyroglutamate-specificity.
[0087] The Hybridoma clone (MV-003 (internal name D129; IgG1) was
purified and analysed for specific detection of p1253, p4371,
p4373, p1454 and A.beta. respectively. MV-003 recognized the
injected epitope (p4373) but failed to detect p1454, p1253 or full
length A.beta. protein (recombinant protein; obtained from Bachem
AG, Bubendorf, Switzerland) in ELISA. Furthermore, the MV-003
antibodies failed to detect the peptide p4371 encoding the normal
version of A.beta.3-10 (15 times lower titer than the original
epitope).
Example 1c
Generation of Monoclonal Antibody MV-004
[0088] A monoclonal antibody derived from the fusion of experiment
Alz-15 was generated: In experiment Alz-15 BalbC mice were
immunized repeatedly with the epitope EVHHQKC (SEQ ID NO: 91)
(p4372) coupled to KLH (Keyhole Limpet Hemocyanin) and Alum
(Aluiminium Hydroxide) as adjuvant. p4372-peptide-specific,
antibody-producing hybridomas were detected by ELISA
(p4372-peptide-coated ELISA plates). P4376, p4378, p1454 and
A.beta.40/42 were used as negative control peptides. Only
hybridomas with no or limited p4376 and p4378 binding were used for
further antibody development in order to guarantee for specificity
against the free N-Terminus at position aa11.
[0089] The Hybridoma clone (MV-004 (internal name B204; IgG1) was
purified and analysed for specific detection of p4372, p4376,
p4378, p1454 and A.beta. respectively. MV-004 recognized the
injected epitope (p4372) but failed to detect p1454, p4376 and
p4378 as well as full length A.beta. protein (recombinant protein;
obtained from Bachem AG, Bubendorf, Switzerland) in ELISA. The
failure to detect p4376, p4378 demonstrates specificity for the
free N-terminus at position aa11 in truncated A.beta..
Example 2
Phage Display, In Vitro Binding and Inhibition ELISA
[0090] Phage Display libraries used in this example were: Ph.D. 7:
New England BioLabs E8102L (linear 7mer library). Phage Display was
done according to manufacturer's protocol (www.neb.com).
[0091] After 2 or 3 subsequent rounds of panning, single phage
clones were picked and phage supernatants were subjected to ELISA
on plates coated with the antibody that was used for the panning
procedure. Phage clones that were positive in this ELISA (strong
signal for the target, but no signal for unspecific control) were
sequenced. From DNA sequences, peptide sequences were deduced.
These peptides were synthesized and characterised in binding and
inhibition ELISA. Additionally, some novel mimotopes were created
by combining sequence information from mimotopes identified in the
screen to support the identification of a consensus sequence for a
mimotope vaccination.
[0092] 1. In Vitro Binding Assay (ELISA)
[0093] Peptides derived from Phage Display as well as variants
thereof were coupled to BSA and bound to ELISA plates (1 .mu.M; as
indicated in the respective figures) and subsequently incubated
with the monoclonal antibody that was used for the screening
procedure to analyse binding capacity of identified peptides.
[0094] 2. In Vitro Inhibition Assay (ELISA)
[0095] Different amounts of peptides (concentrations ranging from
10 .mu.g to 0.08 .mu.g; serial dilutions), derived from Phage
Display were incubated with the monoclonal antibody that was used
for the screening procedure. Peptides diminishing subsequent
binding of the antibody to the original epitope coated on ELISA
plates were considered as inhibiting in this assay.
Example 3
In Vivo Testing of Mimotopes: Analysis of Immunogenicity and
Crossreactivity
[0096] 1. In Vivo Testing of Mimotopes
[0097] Inhibiting as well as non-inhibiting peptides were coupled
to KLH and injected into mice (wildtype C57/B16 mice; subcutaneous
injection into the flank) together with an appropriate adjuvant
(aluminium hydroxide). Animals were vaccinated 3-6 times in
biweekly intervals and sera were taken biweekly as well. Titers to
injected peptides, as well as to an irrelevant peptide were
determined with every serum. Furthermore, titers against the
recombinant human A.beta. protein, and against original peptides
were determined respectively. In general sera were analysed by
reaction against peptides coupled to Bovine Serum Albumin (BSA) and
recombinant full length proteins which were immobilised on ELISA
plates. Titers were determined using anti mouse IgG specific
antibodies. For detailed results see FIGS. 6, 7 and 8
respectively.
[0098] 2. Results
2.1. Identification of Specific Monoclonal Antibodies (mAB)
Directed Against N-Terminally Truncated and Modified Forms of
A.beta.:
[0099] FIG. 1 depicts the characterisation of the monoclonal
antibody MV-001 (internal name 824; IgG1) derived from experiment
Alz-5 demonstrating specificity for full length A.beta. and A.beta.
truncated at position E3.
[0100] FIG. 2 depicts the characterisation of the monoclonal
antibody MV-003 (internal name D129; IgG1) derived from experiment
Alz-16 demonstrating specificity for A.beta. truncated and
posttranslationally modified at position p(E)3.
[0101] FIG. 3 depicts the characterisation of the monoclonal
antibody MV-004 (internal name B204; IgG1) derived from experiment
Alz-15 demonstrating specificity for A.beta. truncated at position
E11.
2.2. Screening with Specific mABs Directed Against n-Terminally
Truncated and Modified Forms of A.beta.:
2.2.1. Phage Display Library pH.D. 7
[0102] 2.2.1.1. Screening with Monoclonal Antibody Directed Against
p4373
[0103] 8 Sequences were identified by screening PhD 7 phage display
libraries in this screen: Table 1A summarises the peptides
identified and their binding capacity as compared to the original
epitope.
2.2.1.2. Screening with Monoclonal Antibody Directed Against
p4372
[0104] 9 Sequences were identified by screening PhD 7 phage display
libraries in this screen: Table 1B summarises the peptides
identified and their binding capacity as compared to the original
epitope.
2.2.1.3. Screening with Monoclonal Antibody Directed Against
p4371
[0105] 71 Sequences were identified by screening PhD 7 and PhD12
phage display libraries in this screen: Table 1C summarises the
peptides identified and their binding capacity as compared to the
original epitope.
TABLE-US-00001 TABLE 1A mimotopes binding to the parental antibody
MV-003 Internal Peptide SEQ ID Binding number No. Sequence Capacity
p4395 1 IRWDTPC 2 p4396 2 VRWDVYPC 1 p4397 3 IRYDAPLC 1 p4399 4
IRYDMAGC 1 p4728 5 IRWDTSLC 3 p4756 6 IRWDQPC 3 p4792 7 IRWDGC 1
p4793 8 IRWDGGC 2 Legend to Table 1A: the binding capacity is coded
by the following binding code: 1: X describes the dilution factor
of the parental AB. OD halfmax binding code 1: X 0 no binding : 0 1
weak binding : <16000 2 medium binding : 16-60000 3 strong
binding : >60000
TABLE-US-00002 TABLE 1B mimotopes binding to the parental antibody
MV-004 Internal Peptide SEQ ID Binding number No. Sequence Capacity
p4417 9 EVWHRHQC 2 p4418 10 ERWHEKHC 3 p4419 11 EVWHRLQC 3 p4420 12
ELWHRYPC 2 p4665 13 ELWHRAFC 2 p4786 14 ELWHRAC 1 p4788 15 EVWHRGC
1 p4789 16 EVWHRHC 1 p4790 17 ERWHEKC 1 Legend to Table 1B: the
binding capacity is coded by the following binding code: 1: X
describes the dilution factor of the parental AB. OD halfmax
binding code 1: X 0 no binding : 0 1 weak binding : <24000 2
medium binding : 24-96000 3 strong binding : >96000
TABLE-US-00003 TABLE 1C mimotopes binding to the parental antibody
MV-001 Internal Peptide SEQ ID Binding number No. Sequence Capacity
p4380 18 QDFRHYC 2 p4381 19 SEFKHGC 3 p4382 20 TSFRHGC 2 p4383 21
TSVFRHC 3 p4384 22 TPFRHTC 2 p4385 23 SQFRHYC 2 p4386 24 LMFRHNC 3
p4387 25 SAFRHHC 2 p4388 26 LPFRHGC 2 p4389 27 SHFRHGC 2 p4390 28
ILFRHGC 3 p4391 29 QFKHDLC 2 p4392 30 NWFPHPC 1 p4393 31 EEFKYSC 2
p4701 32 NELRHSTC 3 p4702 33 GEMRHQPC 3 p4703 34 DTYFPRSC 2 p4704
35 VELRHSRC 2 p4705 36 YSMRHDAC 2 p4706 37 AANYFPRC 2 p4707 38
SPNQFRHC 3 p4708 39 SSSFFPRC 2 p4709 40 EDWFFWHC 1 p4710 41
SAGSFRHC 3 p4711 42 QVMRHHAC 2 p4712 43 SEFSHSSC 3 p4713 44
QPNLFYHC 1 p4714 45 ELFKHHLC 3 p4715 46 TLHEFRHC 3 p4716 47
ATFRHSPC 2 p4717 48 APMYFPHC 2 p4718 49 TYFSHSLC 2 p4719 50
HEPLFSHC 1 p4721 51 SLMRHSSC 2 p4722 52 EFLRHTLC 3 p4723 53
ATPLFRHC 3 p4724 54 QELKRYYC 1 p4725 55 THTDFRHC 3 p4726 56
LHIPFRHC 3 p4727 57 NELFKHFC 2 p4729 58 SQYFPRPC 2 p4730 59
DEHPFRHC 3 p4731 60 MLPFRHGC 2 p4732 61 SAMRHSLC 2 p4733 62
TPLMFWHC 1 p4734 63 LQFKHSTC 2 p4735 64 ATFRHSTC 2 p4736 65
TGLMFKHC 2 p4737 66 AEFSHWHC 2 p4738 67 QSEFKHWC 3 p4739 68
AEFMHSVC 2 p4740 69 ADHDFRHC 3 p4741 70 DGLLFKHC 3 p4742 71
IGFRHDSC 2 p4743 72 SNSEFRRC 3 p4744 73 SELRHSTC 3 p4745 74
THMEFRRC 3 p4746 75 EELRHSVC 3 p4747 76 QLFKHSPC 3 p4748 77
YEFRHAQC 3 p4749 78 SNFRHSVC 3 p4750 79 APIQFRHC 3 p4751 80
AYFPHTSC 2 p4752 81 NSSELRHC 3 p4753 82 TEFRHKAC 3 p4754 83
TSTEMWHC 1 p4755 84 SQSYFKHC 3 p4800 85 CSEFKH 3 p4801 86 SEFKHC 3
p4802 87 CHEFRH 3 p4803 88 HEFRHC 3 Legend to Table 1C: the binding
capacity is coded by the following binding code: 1: X describes the
dilution factor of the parental AB OD halfmax binding code 1: X 0
no binding : 0 1 weak binding : <4000 2 medium binding :
4000-20000 3 strong binding : >20000
2.3. In Vitro Characterisation of Mimotopes Identified in Screening
Phage Display Libraries with Monoclonal Antibodies Directed Against
n-Terminally Truncated and Modified Forms of A.beta.:
[0106] FIGS. 4 and 5 show representative examples for binding and
inhibition assays used to characterise mimotopes in vitro. Data
obtained are summarised in Tables 1 and 2 respectively.
[0107] MV-003 Mimotopes: From the 8 sequences presented 6 sequences
inhibit binding of the p(E)3-7A.beta. specific monoclonal antibody
in in vitro competition experiments: Additional 2 sequences were
identified that do not inhibit binding of monoclonal antibody in in
vitro competition experiments but still retain binding capacity to
the parental antibody (Table 2A).
[0108] MV-004 Mimotopes: All the 9 sequences presented inhibit
binding of the monoclonal antibody specifically binding the free
N-terminus of A.beta. truncated at position E11 in in vitro
competition experiments: (Table 2B).
[0109] MV-001 Mimotopes: From the 71 sequences presented 27
sequences inhibit binding of the monoclonal antibody specifically
directed against A.beta. truncated at position E3 in in vitro
competition experiments: Additional 44 sequences were identified
that do not inhibit binding of monoclonal antibody in in vitro
competition experiments but still retain binding capacity to the
parental antibody (Table 2C).
[0110] Table 2: mimotopes identified in this invention giving
positive results in inhibiting assays
TABLE-US-00004 TABLE 2A MV-003 Mimotopes Internal Peptide SEQ ID
Inhibition number No. Sequence Capacity p4395 1 IRWDTPC 1 p4397 3
IRYDAPLC 1 p4728 5 IRWDTSLC 2 p4756 6 IRWDQPC 1 p4792 7 IRWDGC 1
p4793 8 IRWDGGC 1 Legend to Table 2A: the inhibition capacity is
coded by the following code: Weak inhibition means more peptide is
required to lower AB binding than with the original epitope; strong
inhibition means similar peptide amounts are required for mimotope
and original epitope for lowering AB binding. Mimotopes are
compared to the original peptide as standard. OD at 10 ug peptide
used in the assay is used to calculate the competition capacity
compared to original peptide. competition code 0 no inhibition (OD
of 10 ug peptide above 12 times of original peptide) 1 Weaker than
original epitope (OD of 10 ug peptide below 12 times of original
peptide) 2 strong inhibition (as original epitope; OD of 10 ug
peptide below 5 times of original peptide)
TABLE-US-00005 TABLE 2B MV-004 Mimotopes Internal Peptide SEQ ID
Inhibition number No. Sequence Capacity p4417 9 EVWHRHQC 1 p4418 10
ERWHEKHC 2 p4419 11 EVWHRLQC 2 p4420 12 ELWHRYPC 1 p4665 13
ELWHRAFC 2 p4786 14 ELWHRAC 1 p4788 15 EVWHRGC 1 p4789 16 EVWHRHC 1
p4790 17 ERWHEKC 2 Legend to Table 2B: the inhibition capacity is
coded by the following code: Weak inhibition means more peptide is
required to lower AB binding than with the original epitope; strong
inhibition means similar peptide amounts are required for mimotope
and original epitope for lowering AB binding. Mimotopes are
compared to the original peptide as standard. OD at 10 ug peptide
used in the assay is used to calculate the competition capacity
compared to original peptide. competition code 0 no inhibition (OD
of 10 ug peptide above 5 times of original peptide) 1 Weaker than
original epitope (OD of 10 ug peptide below 5 times of original
peptide) 2 strong inhibition (as original epitope; OD of 10 ug
peptide below 2 times of original peptide)
TABLE-US-00006 TABLE 2C MV-001 Mimotopes Internal Peptide SEQ ID
Inhibition number No. Sequence Capacity p4380 18 QDFRHYC 1 p4381 19
SEFKHGC 1 p4382 20 TSFRHGC 1 p4383 21 TSVFRHC 1 p4384 22 TPFRHTC 1
p4385 23 SQFRHYC 1 p4386 24 LMFRHNC 1 p4387 25 SAFRHHC 1 p4388 26
LPFRHGC 1 p4389 27 SHFRHGC 1 p4390 28 ILFRHGC 1 p4391 29 QFKHDLC 1
p4392 30 NWFPHPC 1 p4393 31 EEFKYSC 1 p4707 38 SPNQFRHC 1 p4715 46
TLHEFRHC 2 p4725 55 THTDFRHC 1 p4730 59 DEHPFRHC 1 p4738 67
QSEFKHWC 1 p4740 69 ADHDFRHC 1 p4741 70 DGLLFKHC 1 p4746 75
EELRHSVC 1 p4753 82 TEFRHKAC 2 p4800 85 CSEFKH 2 p4801 86 SEFKHC 1
p4802 87 CHEFRH 2 p4803 88 HEFRHC 2 Legend to Table 2C: the
inhibition capacity is coded by the following code: Weak inhibition
means more peptide is required to lower AB binding than with the
original epitope; strong inhibition means similar peptide amounts
are required for mimotope and original epitope for lowering AB
binding. Mimotopes are compared to the original peptide as
standard. OD at 10 ug peptide used in the assay is used to
calculate the competition capacity compared to original peptide.
competition code 0 no inhibition (OD of 10 ug peptide above 3 times
of original peptide) 1 Weaker than original epitope (OD of 10 ug
peptide below 3 times of original peptide) 2 strong inhibition (as
original epitope; OD of 10 ug peptide below 2 times of original
peptide)
TABLE-US-00007 TABLE 3 Non-mimotope peptides Internal SEQ Peptide
ID number No. Sequence p1253 89 DAEFRHDSGYC p4371 90 EFRHDS-C p4372
91 EVHHQK-C p4373 92 p(E)FRHDS-C p4374 93 p(E)VHHQKLVFC p4376 94
GYEVHHQKC p4377 95 EVHHQKLVFC p4378 96 C-EVHHQKLVFF p1454 97
CGLMVGGVV A.beta.1-40 98 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL MVGGVV
A.beta.1-42 99 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL MVGGVVIA
sAPPalpha 100 alpha-Secretase induced cleavage product derived from
human APP (gi: 112927)
2.4. In Vivo Characterisation of Mimotopes Identified in Screening
Phage Display Libraries with a Monoclonal Antibody Directed Against
N-Terminally Truncated and Modified Forms of A.beta.:
[0111] Female C57/b16 mice, 5-6 mice per group, were subcutaneously
immunized with 30 .mu.g peptide coupled to KLH. Control groups were
administered original epitope-KLH conjugates respectively. As
adjuvant alum was used (always 1 mg per mouse). The peptides
administered were all able to bind to monoclonal antibodies
specifically although some of the peptides did not inhibit the
binding of the original epitope to its parental antibody in vitro
(in an in vitro inhibition assay). The in vitro ELISA assay to
determine the antibody titer was performed with sera of single mice
after each vaccination in a two week interval (see FIGS. 6 and 7
respectively). The wells of the ELISA plate were coated with
mimotope-BSA conjugate and an irrelevant peptide-BSA conjugate
(negative control). The positive control was performed by reaction
of the parental antibody with the respective mimotope-BSA
conjugate. The detection was performed with anti-mouse IgG.
Additionally, recombinant proteins were immobilised on ELISA plates
and sera reacted accordingly. FIGS. 6 to 8 show representative
examples for assays used to characterise mimotopes in vivo.
[0112] FIG. 6 shows examples for in vivo characterisations of the
immune response elicited by mimotope vaccination by analysing the
immune response against injected peptide and an irrelevant peptide,
containing an unrelated sequence. In all three examples shown, the
original epitopes and the mimotopes, elicit immune responses
against the injected peptides but fail to induce a relevant immune
response against an unrelated sequence (p1454).
[0113] As example for MV-003-mimotopes, original epitope p4373 and
the mimotopes p4395, p4396, p4397, and p4399 are depicted in FIG.
6A. All vaccines are mounting similar immune responses against
their respective mimotopes. Neither original epitope p4373-vaccine
treated nor the animals treated with mimotope p4395, p4396, p4397
or p4399-vaccines mount relevant titers against irrelevant peptide
p1454 (11.times.-25.times. less than injected peptides).
[0114] As example for MV-004-mimotopes original epitope p4372 and
the mimotopes p4417, p4418, p4419, and p4420 are depicted in FIG.
6B. All vaccines are mounting similar immune responses against
their respective mimotopes. Neither original epitope p4372-vaccine
treated nor the animals treated with mimotope p4417, p4418, p4419,
and p4420-vaccines mount relevant titers against irrelevant peptide
p1454 (20-80.times. less than injected peptides).
[0115] As example for MV-001-mimotopes original epitope p4371 and
the mimotopes p4381, p4382, and p4390 are depicted in FIG. 6C. All
vaccines are mounting similar immune responses against their
respective mimotopes. Neither original epitope p4371-vaccine
treated nor the animals treated with mimotope p4381, p4382, and
p4390-vaccines mount relevant titers against irrelevant peptide
p1454 (>10.times. less than injected peptides).
[0116] FIG. 7 shows examples for in vivo characterisations of the
immune response elicited by mimotope vaccination against the
respective original epitope of the parental antibody as well as
against peptides derived of other forms of truncated species of
A.beta..
[0117] As example for MV-003-mimotopes, original epitope p4373 and
the mimotopes p4395, p4396, p4397, and p4399 are depicted in FIG.
7A. 3/4 Mimotope vaccines indicated mount detectable immune
responses against the original epitope p4373. A similar phenomenon
can be detected analysing cross reactivity against the non-modified
form as displayed by p4371. The original epitope p4373-vaccine and
2/4 Mimotope vaccines mount relevant titers against p4371.
Surprisingly, the mimotopes selected by MV-003, which is
specifically binding to p4373 are also inducing a immune reaction
cross reacting with the unmodified form of the original
epitope.
[0118] As example for MV-004-mimotopes, original epitope p4372 and
the mimotopes p4417, p4418, p4419, and p4420 are depicted in FIG.
7B. 3/4 Mimotope vaccines shown mount detectable immune responses
against the original epitope p4372.
[0119] As example for MV-001-mimotopes, original epitope p4371 and
the mimotopes p4381, p4382, and p4390 are depicted in FIG. 7C. All
Mimotope vaccines depicted mount detectable immune responses
against the original epitope p4371. A similar phenomenon as
described for MV-003 derived mimotopes can be detected analysing
cross reactivity against the pyroglutamate-modified form as
displayed by p4373. The original epitope p4371-vaccine and all
Mimotope vaccines mount relevant titers against p4373.
Surprisingly, the mimotopes selected by MV-001, which is
specifically binding to p4371 are inducing a immune reaction cross
reacting better with the modified form of the original epitope than
the original epitope induced immune reaction or the parental
antibody. Thus these mimotopes might surprisingly be able to induce
but are not necessarily inducing a broader immune reaction than the
parental antibody and can be used for a more wide targeting of
forms of A.beta..
[0120] FIG. 8 shows examples for in vivo characterisations of the
immune response elicited by mimotope vaccination against full
length A.beta.. Surprisingly, the mimotopes selected by using
MV-001 and MV-003 induce a cross reaction not only with the
truncated or modified short epitopes used to create the antibodies
but also induce cross reactivity to full length, non modified forms
of A.beta. as good as the original sequence or even more
efficiently than p4371/p4373. For MV-002 original epitope as well
as for the mimotopes identified, no such cross reactivity can be
detected demonstrating a transfer of specificity of the antibody to
the free N-Terminus of unmodified A.beta.11-40/42. Thus the
mimotopes presented in this invention constitute optimised vaccine
candidates to target a broad spectrum of naturally occurring forms
of the A.beta. peptides as have been found in the brain of AD
patients. The forms include but are not limited to A.beta.1-40/42,
and N-terminally truncated forms like A.beta.3-40/42,
A.beta.(pE)3-40/42 and unmodified A.beta.11-40/42 respectively.
[0121] In Table 4 and 5 further examples of the immune response
elicited by mimotope vaccination against full length A.beta. by
using MV-001 and MV-003 derived mimotopes are described.
TABLE-US-00008 TABLE 4 In vivo characterisation of mimotopes:
MV-001 Internal Peptide SEQ ID Detection of number No.
A.beta./truncated/modified forms p4381 19 + p4383 21 + p4385 23 +
p4386 24 + p4390 28 + p4707 38 + p4714 45 + p4715 46 + p4725 55 +
p4730 59 + p4738 67 + p4740 69 + p4748 77 + p4753 82 +
[0122] All peptides listed in Table 4 mount specific immune
reactions against full length and/or truncated and modified forms
of A.beta. or fragments thereof.
TABLE-US-00009 TABLE 5 In vivo characterisation of mimotopes:
MV-003 Internal Peptide SEQ ID Detection of number No.
A.beta./truncated/modified forms p4395 1 + p4396 2 + p4397 3 +
p4399 4 +
[0123] All peptides listed in Table 5 mount specific immune
reactions against full length and/or truncated and modified forms
of A.beta. or fragments thereof.
2.5: In Vivo Characterisation of Mimotopes for the Efficacy to
Reduce AD Like Disease in Transgenic Animals
[0124] The Tg2576 AD mouse model was used to study the preclinical
efficacy of the mimotope vaccines. This transgenic line is
expressing human APP carrying the Swedish double mutation at aa
position 670/671 under the control of a hamster prion protein (PrP)
promoter which results in overexpression of the protein. It is
currently one of the most widely employed models in AD research.
The Tg2576 model recapitulates various hallmarks of AD pathology
including disease-specific amyloid plaque deposition and
astrocytosis. As all other AD model systems available to date, it
does not reflect all cardinal neuropathological features of AD.
[0125] To assess whether treatment with mimotopes is capable of
preventing cerebral A.beta. accumulation, Tg2576 mice were s.c.
injected 6 times at monthly intervals with peptide-KLH conjugates
adsorbed to ALUM (adjuvant: aluminium hydroxide) or PBS adsorbed to
ALUM (referred to as PBS or control) alone. Up to eight weeks after
the last immunization, animals were sacrificed, their brains
harvested and analyzed for their A.beta. load (AD-like pathology).
The mice were sacrificed under deep anaesthesia. Subsequently, the
brain was isolated, fixed in 4% PFA and dehydrated by graded
Ethanol series followed by incubation in Xylene and paraffin
embedding. Each paraffin-embedded brain was sectioned at 7 .mu.M
using a slicing microtome and sections were mounted on glass
slides.
[0126] As a method to assay AD-like pathology in Tg2576 animals, we
analyzed the relative area occupied by amyloid deposits in the
brain of treated animals. This analysis was performed using an
automated area recognition programme. To identify the plaques,
sections were stained with the monoclonal antibody (mAb) 3A5
(specific for A.beta.40/42). Mimotope treated animals were compared
to control animals. All animals have been sacrificed at an age of
13, 5-14 months. For this analysis 3 slides/animal covering the
cortex and hippocampus were selected, stained with mAb 3A5 and
subsequently documented using the Mirax-system (Zeiss). For the
calculation of the area occupied by amyloid plaques, we analysed up
to four individual sections per slide and sections carrying tissue
artefacts and aberrant staining intensities have been excluded
after inspection of the result pictures.
[0127] For the mimotopes derived from MV001 an area analysis using
three exemplary candidates was performed: Analysis was performed
following repeated vaccination using peptide-KLH conjugate
vaccines. The control group showed an average occupation of 0.35%
as compared to 0.11%, 0.14% and 0.22% for the mimotope treated
animals respectively. This corresponds to a reduction following
mimotope treatment of 67% in group 2, a 60% reduction in group 3
and a 36% reduction in group 4 (see FIG. 9).
[0128] A similar picture can be detected for the group of MV003
derived mimotopes. Here the example of p4395 is depicted. As
described for the MV001 derived mimotopes, an analysis of the area
occupied by amyloid plaques following peptide-conjugate vaccination
has been performed. The control group showed an average occupation
of 0.35% as compared to 0.21% for the mimotope treated animals
respectively. This corresponds to a reduction following mimotope
treatment of 38% in group 2 (see FIG. 10).
[0129] Thus, this set of data clearly indicates a beneficial effect
of mimotope vaccine treatment on AD like pathology in transgenic
animals.
Sequence CWU 1
1
28317PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 1Ile Arg Trp Asp Thr Pro Cys 1 5
28PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 2Val Arg Trp Asp Val Tyr Pro Cys 1 5
38PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 3Ile Arg Tyr Asp Ala Pro Leu Cys 1 5
48PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 4Ile Arg Tyr Asp Met Ala Gly Cys 1 5
58PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 5Ile Arg Trp Asp Thr Ser Leu Cys 1 5
67PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 6Ile Arg Trp Asp Gln Pro Cys 1 5
76PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 7Ile Arg Trp Asp Gly Cys 1 5
87PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 8Ile Arg Trp Asp Gly Gly Cys 1 5
98PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 9Glu Val Trp His Arg His Gln Cys 1 5
108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 10Glu Arg Trp His Glu Lys His Cys 1 5
118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 11Glu Val Trp His Arg Leu Gln Cys 1 5
128PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 12Glu Leu Trp His Arg Tyr Pro Cys 1 5
138PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 13Glu Leu Trp His Arg Ala Phe Cys 1 5
147PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 14Glu Leu Trp His Arg Ala Cys 1 5
157PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 15Glu Val Trp His Arg Gly Cys 1 5
167PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 16Glu Val Trp His Arg His Cys 1 5
177PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 17Glu Arg Trp His Glu Lys Cys 1 5
187PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 18Gln Asp Phe Arg His Tyr Cys 1 5
197PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 19Ser Glu Phe Lys His Gly Cys 1 5
207PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 20Thr Ser Phe Arg His Gly Cys 1 5
217PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 21Thr Ser Val Phe Arg His Cys 1 5
227PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 22Thr Pro Phe Arg His Thr Cys 1 5
237PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 23Ser Gln Phe Arg His Tyr Cys 1 5
247PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 24Leu Met Phe Arg His Asn Cys 1 5
257PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 25Ser Ala Phe Arg His His Cys 1 5
267PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 26Leu Pro Phe Arg His Gly Cys 1 5
277PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 27Ser His Phe Arg His Gly Cys 1 5
287PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 28Ile Leu Phe Arg His Gly Cys 1 5
297PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 29Gln Phe Lys His Asp Leu Cys 1 5
307PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 30Asn Trp Phe Pro His Pro Cys 1 5
317PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 31Glu Glu Phe Lys Tyr Ser Cys 1 5
328PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 32Asn Glu Leu Arg His Ser Thr Cys 1 5
338PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 33Gly Glu Met Arg His Gln Pro Cys 1 5
348PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 34Asp Thr Tyr Phe Pro Arg Ser Cys 1 5
358PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 35Val Glu Leu Arg His Ser Arg Cys 1 5
368PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 36Tyr Ser Met Arg His Asp Ala Cys 1 5
378PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 37Ala Ala Asn Tyr Phe Pro Arg Cys 1 5
388PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 38Ser Pro Asn Gln Phe Arg His Cys 1 5
398PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 39Ser Ser Ser Phe Phe Pro Arg Cys 1 5
408PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 40Glu Asp Trp Phe Phe Trp His Cys 1 5
418PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 41Ser Ala Gly Ser Phe Arg His Cys 1 5
428PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 42Gln Val Met Arg His His Ala Cys 1 5
438PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 43Ser Glu Phe Ser His Ser Ser Cys 1 5
448PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 44Gln Pro Asn Leu Phe Tyr His Cys 1 5
458PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 45Glu Leu Phe Lys His His Leu Cys 1 5
468PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 46Thr Leu His Glu Phe Arg His Cys 1 5
478PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 47Ala Thr Phe Arg His Ser Pro Cys 1 5
488PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 48Ala Pro Met Tyr Phe Pro His Cys 1 5
498PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 49Thr Tyr Phe Ser His Ser Leu Cys 1 5
508PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 50His Glu Pro Leu Phe Ser His Cys 1 5
518PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 51Ser Leu Met Arg His Ser Ser Cys 1 5
528PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 52Glu Phe Leu Arg His Thr Leu Cys 1 5
538PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 53Ala Thr Pro Leu Phe Arg His Cys 1 5
548PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 54Gln Glu Leu Lys Arg Tyr Tyr Cys 1 5
558PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 55Thr His Thr Asp Phe Arg His Cys 1 5
568PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 56Leu His Ile Pro Phe Arg His Cys 1 5
578PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 57Asn Glu Leu Phe Lys His Phe Cys 1 5
588PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 58Ser Gln Tyr Phe Pro Arg Pro Cys 1 5
598PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 59Asp Glu His Pro Phe Arg His Cys 1 5
608PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 60Met Leu Pro Phe Arg His Gly Cys 1 5
618PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 61Ser Ala Met Arg His Ser Leu Cys 1 5
628PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 62Thr Pro Leu Met Phe Trp His Cys 1 5
638PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 63Leu Gln Phe Lys His Ser Thr Cys 1 5
648PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 64Ala Thr Phe Arg His Ser Thr Cys 1 5
658PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 65Thr Gly Leu Met Phe Lys His Cys 1 5
668PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 66Ala Glu Phe Ser His Trp His Cys 1 5
678PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 67Gln Ser Glu Phe Lys His Trp Cys 1 5
688PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 68Ala Glu Phe Met His Ser Val Cys 1 5
698PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 69Ala Asp His Asp Phe Arg His Cys 1 5
708PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 70Asp Gly Leu Leu Phe Lys His Cys 1 5
718PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 71Ile Gly Phe Arg His Asp Ser Cys 1 5
728PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 72Ser Asn Ser Glu Phe Arg Arg Cys 1 5
738PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 73Ser Glu Leu Arg His Ser Thr Cys 1 5
748PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 74Thr His Met Glu Phe Arg Arg Cys 1 5
758PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 75Glu Glu Leu Arg His Ser Val Cys 1 5
768PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 76Gln Leu Phe Lys His Ser Pro Cys 1 5
778PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 77Tyr Glu Phe Arg His Ala Gln Cys 1 5
788PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 78Ser Asn Phe Arg His Ser Val Cys 1 5
798PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 79Ala Pro Ile Gln Phe Arg His Cys 1 5
808PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 80Ala Tyr Phe Pro His Thr Ser Cys 1 5
818PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 81Asn Ser Ser Glu Leu Arg His Cys 1 5
828PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 82Thr Glu Phe Arg His Lys Ala Cys 1 5
838PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 83Thr Ser Thr Glu Met Trp His Cys 1 5
848PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 84Ser Gln Ser Tyr Phe Lys His Cys 1 5
856PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 85Cys Ser Glu Phe Lys His 1 5
866PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 86Ser Glu Phe Lys His Cys 1 5
876PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 87Cys His Glu Phe Arg His 1 5
886PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 88His Glu Phe Arg His Cys 1 5
8911PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 89Asp Ala Glu Phe Arg His Asp Ser Gly
Tyr Cys 1 5 10 907PRTArtificial SequenceDescription of Artificial
Sequence Synthetic mimotope peptide 90Glu Phe Arg His Asp Ser Cys 1
5 917PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 91Glu Val His His Gln Lys Cys 1 5
927PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 92Glu Phe Arg His Asp Ser Cys 1 5
9310PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mimotope peptide 93Glu Val His His Gln Lys Leu Val Phe
Cys 1 5 10 949PRTArtificial SequenceDescription of Artificial
Sequence Synthetic mimotope peptide 94Gly Tyr Glu Val His His Gln
Lys Cys 1 5 9510PRTArtificial SequenceDescription of Artificial
Sequence Synthetic mimotope peptide 95Glu Val His His Gln Lys Leu
Val Phe Cys 1 5 10 9611PRTArtificial SequenceDescription of
Artificial Sequence Synthetic mimotope peptide 96Cys Glu Val His
His Gln Lys Leu Val Phe Phe 1 5 10 979PRTArtificial
SequenceDescription of Artificial Sequence Synthetic mimotope
peptide 97Cys Gly Leu Met Val Gly Gly Val Val 1 5 9840PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
98Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1
5 10 15 Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile
Ile 20 25 30 Gly Leu Met Val Gly Gly Val Val 35 40
9942PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 99Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu
Val His His Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp Val Gly
Ser Asn Lys Gly Ala Ile Ile 20 25 30 Gly Leu Met Val Gly Gly Val
Val Ile Ala 35 40 1008PRTArtificial SequenceDescription of
Artificial Sequence Synthetic mimotope peptide 100Xaa Arg Xaa Asp
Xaa Xaa Xaa Cys 1 5 1018PRTArtificial SequenceDescription of
Artificial Sequence Synthetic mimotope peptide 101Glu Xaa Trp His
Xaa Xaa Xaa Cys 1 5 1028PRTArtificial SequenceDescription of
Artificial Sequence Synthetic amyloid-beta fragment peptide 102Glu
Phe Arg His Asp Ser Gly Tyr 1 5 1038PRTArtificial
SequenceDescription of Artificial Sequence Synthetic amyloid-beta
fragment peptide 103Glu Phe Arg His Asp Ser Gly Tyr 1 5
1047PRTArtificial SequenceDescription of Artificial Sequence
Synthetic amyloid-beta fragment peptide 104Glu Val His His Gln Lys
Leu 1 5 1055PRTArtificial SequenceDescription of Artificial
Sequence Synthetic amyloid-beta fragment peptide 105Asp Ala Glu Phe
Arg 1 5 1067PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 106Ile Arg Trp Asp Thr Pro Cys 1 5
1078PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 107Val Arg Trp Asp Val Tyr Pro Cys 1 5
1088PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 108Ile Arg Tyr Asp Ala Pro Leu Cys 1 5
1098PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 109Ile Arg Tyr Asp Met Ala Gly Cys 1 5
1108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 110Ile Arg Trp Asp Thr Ser Leu Cys 1
5 1117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 111Ile Arg Trp Asp Gln Pro Cys 1 5
1126PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 112Ile Arg Trp Asp Gly Cys 1 5 1137PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 113Ile
Arg Trp Asp Gly Gly Cys 1 5 1148PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 114Glu Val Trp His Arg His
Gln Cys 1 5 1158PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 115Glu Arg Trp His Glu Lys His Cys 1 5
1168PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 116Glu Val Trp His Arg Leu Gln Cys 1 5
1178PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 117Glu Leu Trp His Arg Tyr Pro Cys 1 5
1188PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 118Glu Leu Trp His Arg Ala Phe Cys 1 5
1197PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 119Glu Leu Trp His Arg Ala Cys 1 5
1207PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 120Glu Val Trp His Arg Gly Cys 1 5
1217PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 121Glu Val Trp His Arg His Cys 1 5
1227PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 122Glu Arg Trp His Glu Lys Cys 1 5
1237PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 123Gln Asp Phe Arg His Tyr Cys 1 5
1247PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 124Ser Glu Phe Lys His Gly Cys 1 5
1257PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 125Thr Ser Phe Arg His Gly Cys 1 5
1267PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 126Thr Ser Val Phe Arg His Cys 1 5
1277PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 127Thr Pro Phe Arg His Thr Cys 1 5
1287PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 128Ser Gln Phe Arg His Tyr Cys 1 5
1297PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 129Leu Met Phe Arg His Asn Cys 1 5
1307PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 130Ser Ala Phe Arg His His Cys 1 5
1317PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 131Leu Pro Phe Arg His Gly Cys 1 5
1327PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 132Ser His Phe Arg His Gly Cys 1 5
1337PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 133Ile Leu Phe Arg His Gly Cys 1 5
1347PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 134Gln Phe Lys His Asp Leu Cys 1 5
1357PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 135Asn Trp Phe Pro His Pro Cys 1 5
1367PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 136Glu Glu Phe Lys Tyr Ser Cys 1 5
1378PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 137Asn Glu Leu Arg His Ser Thr Cys 1 5
1388PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 138Gly Glu Met Arg His Gln Pro Cys 1 5
1398PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 139Asp Thr Tyr Phe Pro Arg Ser Cys 1 5
1408PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 140Val Glu Leu Arg His Ser Arg Cys 1 5
1418PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 141Tyr Ser Met Arg His Asp Ala Cys 1 5
1428PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 142Ala Ala Asn Tyr Phe Pro Arg Cys 1 5
1438PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 143Ser Pro Asn Gln Phe Arg His Cys 1 5
1448PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 144Ser Ser Ser Phe Phe Pro Arg Cys 1 5
1458PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 145Glu Asp Trp Phe Phe Trp His Cys 1 5
1468PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 146Ser Ala Gly Ser Phe Arg His Cys 1 5
1478PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 147Gln Val Met Arg His His Ala Cys 1 5
1488PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 148Ser Glu Phe Ser His Ser Ser Cys 1 5
1498PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 149Gln Pro Asn Leu Phe Tyr His Cys 1 5
1508PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 150Glu Leu Phe Lys His His Leu Cys 1 5
1518PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 151Thr Leu His Glu Phe Arg His Cys 1 5
1528PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 152Ala Thr Phe Arg His Ser Pro Cys 1 5
1538PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 153Ala Pro Met Tyr Phe Pro His Cys 1 5
1548PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 154Thr Tyr Phe Ser His Ser Leu Cys 1 5
1558PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 155His Glu Pro Leu Phe Ser His Cys 1 5
1568PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 156Ser Leu Met Arg His Ser Ser Cys 1 5
1578PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 157Glu Phe Leu Arg His Thr Leu Cys 1 5
1588PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 158Ala Thr Pro Leu Phe Arg His Cys 1 5
1598PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 159Gln Glu Leu Lys Arg Tyr Tyr Cys 1 5
1608PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 160Thr His Thr Asp Phe Arg His Cys 1 5
1618PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 161Leu His Ile Pro Phe Arg His Cys 1 5
1628PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 162Asn Glu Leu Phe Lys His Phe Cys 1 5
1638PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 163Ser Gln Tyr Phe Pro Arg Pro Cys 1 5
1648PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 164Asp Glu His Pro Phe Arg His Cys 1 5
1658PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 165Met Leu Pro Phe Arg His Gly Cys 1 5
1668PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 166Ser Ala Met Arg His Ser Leu Cys 1 5
1678PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 167Thr Pro Leu Met Phe Trp His Cys 1 5
1688PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 168Leu Gln Phe Lys His Ser Thr Cys 1 5
1698PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 169Ala Thr Phe Arg His Ser Thr Cys 1 5
1708PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 170Thr Gly Leu Met Phe Lys His Cys 1 5
1718PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 171Ala Glu Phe Ser His Trp His Cys 1 5
1728PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 172Gln Ser Glu Phe Lys His Trp Cys 1 5
1738PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 173Ala Glu Phe Met His Ser Val Cys 1 5
1748PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 174Ala Asp His Asp Phe Arg His Cys 1 5
1758PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 175Asp Gly Leu Leu Phe Lys His Cys 1 5
1768PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 176Ile Gly Phe Arg His Asp Ser Cys 1 5
1778PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 177Ser Asn Ser Glu Phe Arg Arg Cys 1 5
1788PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 178Ser Glu Leu Arg His Ser Thr Cys 1 5
1798PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 179Thr His Met Glu Phe Arg Arg Cys 1 5
1808PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 180Glu Glu Leu Arg His Ser Val Cys 1 5
1818PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 181Gln Leu Phe Lys His Ser Pro Cys 1 5
1828PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 182Tyr Glu Phe Arg His Ala Gln Cys 1 5
1838PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 183Ser Asn Phe Arg His Ser Val Cys 1 5
1848PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 184Ala Pro Ile Gln Phe Arg His Cys 1 5
1858PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 185Ala Tyr Phe Pro His Thr Ser Cys 1 5
1868PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 186Asn Ser Ser Glu Leu Arg His Cys 1 5
1878PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 187Thr Glu Phe Arg His Lys Ala Cys 1 5
1888PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 188Thr Ser Thr Glu Met Trp His Cys 1 5
1898PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 189Ser Gln Ser Tyr Phe Lys His Cys 1 5
1906PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 190Cys Ser Glu Phe Lys His 1 5 1916PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 191Ser
Glu Phe Lys His Cys 1 5 1926PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 192Cys His Glu Phe Arg His 1
5 1936PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 193His Glu Phe Arg His Cys 1 5 1946PRTArtificial
SequenceDescription of Artificial Sequence Synthetic 6xHis tag
194His His His His His His 1 5 1956PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 195Ile
Arg Trp Asp Thr Pro 1 5 1967PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 196Val Arg Trp Asp Val Tyr
Pro 1 5 1977PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 197Ile Arg Tyr Asp Ala Pro Leu 1 5
1987PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 198Ile Arg Tyr Asp Met Ala Gly 1 5
1997PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 199Ile Arg Trp Asp Thr Ser Leu 1 5
2006PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 200Ile Arg Trp Asp Gln Pro 1 5 2015PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 201Ile
Arg Trp Asp Gly 1 5 2026PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 202Ile Arg Trp Asp Gly Gly 1
5 2037PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 203Glu Val Trp His Arg His Gln 1 5
2047PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 204Glu Arg Trp His Glu Lys His 1 5
2057PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 205Glu Val Trp His Arg Leu Gln 1 5
2067PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 206Glu Leu Trp His Arg Tyr Pro 1 5
2077PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 207Glu Leu Trp His Arg Ala Phe 1 5
2086PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 208Glu Leu Trp His Arg Ala 1 5 2096PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 209Glu
Val Trp His Arg Gly 1 5 2106PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 210Glu Val Trp His Arg His 1
5 2116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 211Glu Arg Trp His Glu Lys 1 5 2126PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 212Gln
Asp Phe Arg His Tyr 1 5 2136PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 213Ser Glu Phe Lys His Gly 1
5 2146PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 214Thr Ser Phe Arg His Gly 1 5 2156PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 215Thr
Ser Val Phe Arg His 1 5 2166PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 216Thr Pro Phe Arg His Thr 1
5 2176PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 217Ser Gln Phe Arg His Tyr 1 5 2186PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 218Leu
Met Phe Arg His Asn 1 5 2196PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 219Ser Ala Phe Arg His His 1
5 2206PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 220Leu Pro Phe Arg His Gly 1 5 2216PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 221Ser
His Phe Arg His Gly 1 5 2226PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 222Ile Leu Phe Arg His Gly 1
5 2236PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 223Gln Phe Lys His Asp Leu 1 5 2246PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 224Asn
Trp Phe Pro His Pro 1 5 2256PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 225Glu Glu Phe Lys Tyr Ser 1
5 2267PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 226Asn Glu Leu Arg His Ser Thr 1 5
2277PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 227Gly Glu Met Arg His Gln Pro 1 5
2287PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 228Asp Thr Tyr Phe Pro Arg Ser 1 5
2297PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 229Val Glu Leu Arg His Ser Arg 1 5
2307PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 230Tyr Ser Met Arg His Asp Ala 1 5
2317PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 231Ala Ala Asn Tyr Phe Pro Arg 1 5
2327PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 232Ser
Pro Asn Gln Phe Arg His 1 5 2337PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 233Ser Ser Ser Phe Phe Pro
Arg 1 5 2347PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 234Glu Asp Trp Phe Phe Trp His 1 5
2357PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 235Ser Ala Gly Ser Phe Arg His 1 5
2367PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 236Gln Val Met Arg His His Ala 1 5
2377PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 237Ser Glu Phe Ser His Ser Ser 1 5
2387PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 238Gln Pro Asn Leu Phe Tyr His 1 5
2397PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 239Glu Leu Phe Lys His His Leu 1 5
2407PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 240Thr Leu His Glu Phe Arg His 1 5
2417PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 241Ala Thr Phe Arg His Ser Pro 1 5
2427PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 242Ala Pro Met Tyr Phe Pro His 1 5
2437PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 243Thr Tyr Phe Ser His Ser Leu 1 5
2447PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 244His Glu Pro Leu Phe Ser His 1 5
2457PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 245Ser Leu Met Arg His Ser Ser 1 5
2467PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 246Glu Phe Leu Arg His Thr Leu 1 5
2477PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 247Ala Thr Pro Leu Phe Arg His 1 5
2487PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 248Gln Glu Leu Lys Arg Tyr Tyr 1 5
2497PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 249Thr His Thr Asp Phe Arg His 1 5
2507PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 250Leu His Ile Pro Phe Arg His 1 5
2517PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 251Asn Glu Leu Phe Lys His Phe 1 5
2527PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 252Ser Gln Tyr Phe Pro Arg Pro 1 5
2537PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 253Asp Glu His Pro Phe Arg His 1 5
2547PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 254Met Leu Pro Phe Arg His Gly 1 5
2557PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 255Ser Ala Met Arg His Ser Leu 1 5
2567PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 256Thr Pro Leu Met Phe Trp His 1 5
2577PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 257Leu Gln Phe Lys His Ser Thr 1 5
2587PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 258Ala Thr Phe Arg His Ser Thr 1 5
2597PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 259Thr Gly Leu Met Phe Lys His 1 5
2607PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 260Ala Glu Phe Ser His Trp His 1 5
2617PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 261Gln Ser Glu Phe Lys His Trp 1 5
2627PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 262Ala Glu Phe Met His Ser Val 1 5
2637PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 263Ala Asp His Asp Phe Arg His 1 5
2647PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 264Asp Gly Leu Leu Phe Lys His 1 5
2657PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 265Ile Gly Phe Arg His Asp Ser 1 5
2667PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 266Ser Asn Ser Glu Phe Arg Arg 1 5
2677PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 267Ser Glu Leu Arg His Ser Thr 1 5
2687PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 268Thr His Met Glu Phe Arg Arg 1 5
2697PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 269Glu Glu Leu Arg His Ser Val 1 5
2707PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 270Gln Leu Phe Lys His Ser Pro 1 5
2717PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 271Tyr Glu Phe Arg His Ala Gln 1 5
2727PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 272Ser Asn Phe Arg His Ser Val 1 5
2737PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 273Ala Pro Ile Gln Phe Arg His 1 5
2747PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 274Ala Tyr Phe Pro His Thr Ser 1 5
2757PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 275Asn Ser Ser Glu Leu Arg His 1 5
2767PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 276Thr Glu Phe Arg His Lys Ala 1 5
2777PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 277Thr Ser Thr Glu Met Trp His 1 5
2787PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 278Ser Gln Ser Tyr Phe Lys His 1 5
2795PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 279Ser Glu Phe Lys His 1 5 2805PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 280His
Glu Phe Arg His 1 5 2816PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 281Glu Phe Arg His Asp Ser 1
5 2826PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 282Glu Phe Arg His Asp Ser 1 5 2836PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 283Glu
Val His His Gln Lys 1 5
* * * * *