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Patent 2473987 Summary

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(12) Patent: (11) CA 2473987
(54) English Title: PEPTIDES ANTIBODIES DIRECTED THEREAGAINST AND METHODS USING SAME FOR DIAGNOSING AND TREATING AMYLOID-ASSOCIATED DISEASES
(54) French Title: PEPTIDES, ANTICORPS DIRIGES CONTRE LES MALADIES ASSOCIEES A L'AMYLOIDE ET PROCEDES UTILISANT CES PEPTIDES ET CES ANTICORPS EN VUE DE DIAGNOSTIQUER ET DE TRAITER CES MALADIES
Status: Deemed expired
Bibliographic Data
(51) International Patent Classification (IPC):
  • C12N 15/12 (2006.01)
  • A61K 38/06 (2006.01)
  • A61K 38/07 (2006.01)
  • A61K 39/395 (2006.01)
  • A61P 25/28 (2006.01)
  • C07K 5/08 (2006.01)
  • C07K 7/06 (2006.01)
  • C07K 14/47 (2006.01)
  • C07K 16/18 (2006.01)
  • C12N 15/11 (2006.01)
  • C12N 15/63 (2006.01)
  • A61K 38/00 (2006.01)
  • A61K 38/08 (2006.01)
(72) Inventors :
  • GAZIT, EHUD (Israel)
(73) Owners :
  • TEL AVIV UNIVERSITY FUTURE TECHNOLOGY DEVELOPMENT L.P. (Israel)
(71) Applicants :
  • TEL AVIV UNIVERSITY FUTURE TECHNOLOGY DEVELOPMENT L.P. (Israel)
(74) Agent: NORTON ROSE FULBRIGHT CANADA LLP/S.E.N.C.R.L., S.R.L.
(74) Associate agent:
(45) Issued: 2013-11-19
(86) PCT Filing Date: 2003-01-30
(87) Open to Public Inspection: 2003-08-07
Examination requested: 2007-11-07
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/IL2003/000079
(87) International Publication Number: WO2003/063760
(85) National Entry: 2004-07-21

(30) Application Priority Data:
Application No. Country/Territory Date
60/352,578 United States of America 2002-01-31
60/392,266 United States of America 2002-07-01
10/235,852 United States of America 2002-09-06
60/436,453 United States of America 2002-12-27

Abstracts

English Abstract




A peptide comprising at least 3 amino acid residues and less than 15 amino
acid residues, the peptide including an amino acid sequence as set forth in
SEQ ID NO: 7 as well as pharmaceutical compositions and kits including same,
and methods using same for diagnosing and treating amyloid associated diseases.


French Abstract

L'invention concerne un peptide comprenant au moins 3 et moins de 15 résidus d'acide aminé, ce peptide contenant une séquence d'acide aminé telle qu'elle parait dans SEQ ID NO:7, ainsi que des compositions pharmaceutiques et des nécessaires le contenant. L'invention concerne également des procédés utilisant ce peptide dans le diagnostic et le traitement de maladies associées à l'amyloïde.

Claims

Note: Claims are shown in the official language in which they were submitted.



74

WHAT IS CLAIMED IS:
1. Use of a peptide of 15 amino acids or less, the peptide including an
amino
acid sequence as set forth in SEQ ID NO: 7, wherein said peptide further
includes at
least one beta-breaker amino acid for the treatment or prevention of an
amyloid-
associated disease in an individual, wherein said amyloid-associated disease
is
selected from the group consisting of type II diabetes mellitus, SAA
amyloidosis,
hereditary Icelandic syndrome, multiple myeloma, and prion disease.
2. The use of claim 1, wherein said amino acid sequence further includes a
polar
uncharged amino acid selected from the group consisting of serine, threonine,
asparagine, glutamine and natural derivatives thereof.
3. The use of claim 1, wherein said amino acid sequence further includes at
least
one positively charged amino acid and at least one negatively charged amino
acid.
4. The use of claim 3, wherein said at least one positively charged amino
acid is
selected from the group consisting of lysine, arginine and natural and
synthetic
derivatives thereof.
5. The use of claim 3, wherein said at least one negatively charged amino
acid is
selected from the group consisting of aspartic acid, glutamic acid and natural
and
synthetic derivatives thereof.
6. The use of claim 1, wherein the peptide is as set forth in SEQ ID NO:
45.
7. The use of claim I, further comprising at least two serine residues at a
C-
terminus thereof.
8. The use of claim 1, wherein the peptide is a linear or cyclic peptide.
9. The use of claim 1, wherein said beta-beaker amino acid is proline.
10. A method of identifying compounds capable of disaggregation of amyloid


75

aggregates, the method comprising:
(a) contacting the compounds with amyloid aggregates which comprise a
labeled peptide of 15 amino acids or less, said peptide including an amino
acid
sequence as set forth in SEQ ID NO: 7, wherein said SEQ ID NO: 7 does not
comprise a beta breaker; and
(b) monitoring displacement of said labeled peptide by the compounds,
the compounds capable of displacing being useful in disaggregation of the
amyloid
aggregates.
11. The use of claim 1, wherein said priori disease is scrapie or bovine
spongiform
encephalopathy (BSE).
12. The use of claim 1, wherein said prion disease is human prion disease.
13. The use of claim 12, wherein said human prion disease is selected from
the
group consisting of kuru, Creutzfeldt-Jakob Disease (CJD), Gerstmann-
Streussler-
Sheinker Disease (GSS), and fatal familial insomnia (FFI).

Description

Note: Descriptions are shown in the official language in which they were submitted.


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PEPTIDES ANTIBODIES DIRECTED THEREAGAINST AND METHODS
USING SAME FOR DIAGNOSING AND TREATING AMYLOID-ASSOCIATED
DISEASES
FIELD AND BACKGROUND OF THE INVENTION
The present invention relates to peptides and antibodies directed thereagainst

which can be used to diagnose, prevent, and treat amyloid-associated diseases,
such
as Type II diabetes mellitus.
Amyloid material deposition (also referred to as amyloid plaque formation) is
a central feature of a variety of unrelated pathological conditions including
Alzheimer's disease, prion-related encephalopathies, type II diabetes
mellitus,
familial amyloidosis and light-chain amyloidosis.
Amyloid material is composed of a dense network of rigid, nonbranching
proteinaceous fibrils of indefinite length that are about 80 to 100 A in
diameter.
Amyloid fibrils contain a core structure of polypeptide chains arranged in
antiparallel
13-pleated sheets lying with their long axes perpendicular to the long axis of
the fibril
[Both et al. (1997) Nature 385:787-93; Glenner (1980) N. Eng. J. Med. 302:1283-

92].
Approximately twenty amyloid fibril proteins have been identified in-vivo
and correlated with specific diseases. These proteins share little or no amino
acid
sequence homology, however the core structure of the amyloid fibrils is
essentially
the same. This common core structure of amyloid fibrils and the presence of
common substances in amyloid deposits suggest that data characterizing a
particular
form of amyloid material may also be relevant to other forms of amyloid
material and
thus can be implemented in template design for the development of drugs
against
amyloid-associated diseases such as type II diabetes mellitus, Alzheimer's
dementia
or diseases and prion-related encephalopathies.
Furthermore, amyloid deposits do not appear to be inert in vivo, but rather
are
in a dynamic state of turnover and can even regress if the formation of
fibrils is
halted [Gillmore et al. (1997) Br. J. Haematol. 99:245-56].
Thus, therapies designed to inhibiting the production of amyloid polypeptides
or inhibiting amyloidosis may be useful for treating amyloid associated
diseases.

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Inhibition of the production of amyloid polypeptides - Direct inhibition of
the production of amyloid polypeptides may be accomplished, for example,
through
the use of antisense oligonucleotides such as against human islet amyloid
polypeptide
messenger RNA (mRNA). In vitro, the addition of antisense oligonucleotides or
the
expression of antisense complementary DNA against islet amyloid polypeptide
mRNA
increased the insulin mRNA and protein content of cells, demonstrating the
potential
effectiveness of this approach [Kulkarni et al. (1996) J. Endocrinol. 151:341-
8;
Novials et al. (1998) Pancreas 17:182-6]. However, no experimental results
demonstrating the in vivo effectiveness of such antisense molecules have been
demonstrated.
Inhibition of the formation of amyloid fibrils - Amyloid, including islet
amyloid, contains potential stabilizing or protective substances, such as
serum
amyloid P component, apolipoprotein E, and perlecan. Blocking their binding to

developing amyloid fibrils could inhibit amyloidogenesis [Kahn et al. (1999)
Diabetes 48:241-53], as could treatment with antibodies specific for certain
parts of
an amyloidogenic protein [Solomon et al. (1997) Proc. Natl. Acad. Sci. USA
94:4109-121.
The following summarizes current attempts to engineer drugs having the
capability of destabilizing amyloid structures.
Destabilizing compounds - Heparin sulfate has been identified as a
component of all amyloids and has also been implicated in the earliest stages
of
inflammation-associated amyloid induction. Kisilevsky and co-workers (Mature
Med. 1:143-148, 1995) described the use of low molecular weight anionic
sulfonate
or sulfate compounds that interfere with the interaction of heparin sulfate
with the
inflammation-associated amyloid precursor and the 13 peptide of Alzheimer's
disease
(AD). Heparin sulfate specifically influences the soluble amyloid precursor
(SAA2)
to adopt an increased 13-sheet structure characteristic of the protein-folding
pattern of
amyloids. These anionic sulfonate or sulfate compounds were shown to inhibit
heparin accelerated AP fibril formation and were able to disassemble preformed
fibrils in vitro, as monitored by electron micrography. Moreover, these
compounds
substantially arrested murine splenic inflammation-associated amyloid
progression in
vivo in acute and chronic models. However, the most potent compound [i.e.,
poly-

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(vinylsulfonate)] showed acute toxicity. Similar toxicity has been observed
with
another compound, IDOX (Anthracycline 4'-iodo-4'-deoxy-doxorubicin), which has

been observed to induce amyloid resorption in patients with immunoglobin light

chain amyloidosis (AL) [Merlini et al. (1995) Proc. Natl. Acad. Sci. USA].
Destabilizing antibodies - Anti-I3-amy1oid monoclonal antibodies have been
shown to be effective in disaggregating 13 -amyloid plaques and preventing 13-
amyloid
plaque formation in vitro (U.S. Pat. No. 5,688,561). However, no experimental
results demonstrating the in vivo effectiveness of such antibodies have been
demonstrated.
Destabilizing peptides - The finding that the addition of synthetic peptides
that disrupt the 13-pleated sheets ("13-sheet breakers") dissociated fibrils
and prevented
amyloidosis [Soto et al. (1998) Nat. Med. 4:822-6] is particularly promising
from a
clinical point of view. In brief, a penta-residue peptide inhibited amyloid
beta-
protein fibrillogenesis, disassembled preformed fibrils in vitro and prevents
neuronal
death induced by fibrils in cell culture. In addition, the beta-sheet breaker
peptide
significantly reduced amyloid beta-protein deposition in vivo and completely
blocked
the formation of amyloid fibrils in a rat brain model of amyloidosis.
Small molecules ¨ The potential use of small molecules which bind the
amyloid polypeptide, stabilizing the native fold of the protein has been
attempted in
the case of the transthyretin (TTR) protein [Peterson (1998) Proc. Natl. Acad.
Sci.
USA 95:12965-12960; Oza (1999) Bioorg. Med. Chem. Lett. 9:1-6]. Thus far, it
has
been demonstrated that molecules such as thyroxine and flufenamic acid are
capable
of preventing the conformation change, leading to amyloid formation. However,
the
use of the compounds in animal models has not been proved yet and might be
compromised due to the presence in blood or proteins, other than TTR, capable
of
binding these ligands.
Antioxidants - Another proposed therapy has been the intake of antioxidants
in order to avoid oxidative stress and maintain amyloid proteins in their
reduced state
(i.e., monomers and dimers). The use of sulfite was shown to lead to more
stable
monomers of the TTR both in vitro and in vivo [Altland (1999) Neurogenetics
2:183-
188]. However, a complete characterization of the antioxidant effect is still
not

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available and the interpretation of results concerning possible therapeutic
strategies
remains difficult.
While reducing the present invention to practice, the present inventors have
demonstrated that contrary to the teachings of U.S. Pat. No. 6,359,112 to
Kapurniotu,
peptide aggregation into amyloid fibrils is governed by aromatic interactions.
Such
findings enable to efficiently and accurately design peptides, which can be
used to
diagnose and treat amyloid-associated diseases.
SUMMARY OF THE INVENTION
According to one aspect of the present invention there is provided a peptide
comprising at least 3 amino acid residues and less than 15 amino acid
residues, the
peptide including an amino acid sequence as set forth in SEQ ID NO: 7.
According to further features in preferred embodiments of the invention
described below the amino acid sequence further includes a polar uncharged
amino
acid selected from the group consisting of serine, threonine, asparagine,
glutamine and
natural derivatives thereof.
According to still further features in the described preferred embodiments the

amino acid sequence further includes at least one positively charged amino
acid and at
least one negatively charged amino acid.
According to still further features in the described preferred embodiments the
at least one positively charged amino acid is selected from the group
consisting of
lysine, arginine and natural and synthetic derivatives thereof.
According to still further features in the described preferred embodiments the

at least one negatively charged amino acid is selected from the group
consisting of
aspartic acid, glutamic acid and natural and synthetic derivatives thereof.
According to still further features in the described preferred embodiments the

amino acid sequence is selected from the group consisting of SEQ ID NO: 4, 12-
19
and 27-45.
According to still further features in the described preferred embodiments the
peptide is selected from the group consisting of SEQ ID NOs. 4, 12-19 and 27-
45.
According to another aspect of the present invention there is provided a
peptide
comprising at least 3 amino acid residues and less than 15 amino acid
residues, the

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peptide including an amino acid sequence selected from the group consisting of
SEQ
ID NO: 4, 12-19 and 27-44 wherein the peptide is capable of self-aggregating
under
physiological conditions.
According to yet another aspect of the present invention there is provided a
5 peptide selected from the group consisting of SEQ ID NOs: 8, 10-11, 21-22
and 25.
According to still another aspect of the present invention there is provided a

peptide having an amino acid sequence selected from the group consisting of
SEQ ID
NOs: 4, 12-19 and 27-44.
According to an additional aspect of the present invention there is provided a
peptide having an amino acid sequence selected from the group consisting of
SEQ ID
NOs: 8,10-11 and 21-22.
According to yet an additional aspect of the present invention there is
provided a method of treating or preventing an amyloid-associated disease in
an
individual, the method comprising providing to the individual a
therapeutically
effective amount of a peptide having at least 3 amino acid residues and less
than 15
amino acid residues, the peptide including an amino acid sequence as set forth
in SEQ
ID NO: 7.
According to still an additional aspect of the present invention there is
provided
a method of treating or preventing an amyloid-associated disease in an
individual, the
method comprising providing to the individual therapeutically effective amount
of a
peptide having at least 3 amino acid residues and less than 15 amino acid
residues, the
peptide including an amino acid sequence selected from the group consisting of
SEQ
ID NOs: 4, 12-19 and 27-45.
According to still further features in the described preferred embodiments the
peptide is an active ingredient of a pharmaceutical composition which also
includes a
physiologically acceptable carrier.
According to a further aspect of the present invention there is provided a
method of treating or preventing an amyloid-associated disease in an
individual, the
method comprising providing to the individual a therapeutically effective
amount of a
peptide selected from the group consisting of SEQ ID NOs: 8, 10-11, 21-22 and
25,
wherein the peptide is an active ingredient of a pharmaceutical compositions
which
also includes a physiologically acceptable carrier.

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According to yet a further aspect of the present invention there is provided a

method of treating or preventing an amyloid-associated disease in an
individual, the
method comprising providing to the individual a therapeutically effective
amount of a
peptide selected from the group consisting of SEQ ID NOs: 4, 12-19 and 27-45.
According to still a further aspect of the present invention there is provided
a
method of treating or preventing an amyloid-associated disease in an
individual, the
method comprising providing to the individual therapeutically effective amount
of a
peptide having at least 3 amino acid residues and less than 15 amino acid
residues, the
peptide including an amino acid sequence selected from the group consisting of
SEQ
ID NOs: 8, 10-11, 21-22 and 25, wherein the peptide is an active ingredient of
a
pharmaceutical composition which also includes a physiologically acceptable
carrier.
According to still further features in the described preferred embodiments the

peptide is expressed from a nucleic acid construct.
According to still a further aspect of the present invention there is provided
a
pharmaceutical composition for treating or preventing an amyloid-associated
disease
comprising as an active ingredient a peptide having at least 3 amino acid
residues and
less than 15 amino acid residues, the peptide including an amino acid sequence
as set
forth in SEQ ID NO: 7 and a pharmaceutically acceptable carrier or diluent.
According to still a further aspect of the present invention there is provided
a
pharmaceutical composition for treating or preventing an amyloid-associated
disease
comprising as an active ingredient a peptide selected from the group
consisting of
SEQ ID NOs: 8, 10-11, 21-22 and 25 and a pharmaceutically acceptable carrier
or
diluent.
According to still a further aspect of the present invention there is provided
a
pharmaceutical composition for treating or preventing an amyloid-associated
disease
comprising as an active ingredient a peptide having at least 3 amino acid
residues and
less than 15 amino acid residues, the peptide including an amino acid sequence

selected from the group consisting of SEQ 1D NOs: 8, 10-11, 21-22 and 25 and a

pharmaceutically acceptable carrier or diluent.
According to still a further aspect of the present invention there is provided
a
pharmaceutical composition for treating or preventing an amyloid-associated
disease
comprising as an active ingredient a peptide having at least 3 amino acid
residues and

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less than 15 amino acid residues, the peptide including an amino acid sequence

selected from the group consisting of SEQ ID NOs: 4, 12-19 and 27-45 and a
pharmaceutically acceptable carrier or diluent.
According to still a further aspect of the present invention there is provided
a
pharmaceutical composition for treating or preventing an amyloid-associated
disease
comprising as an active ingredient a peptide selected from the group
consisting of SEQ
ID NOs: 4, 12-19 and 27-45 and a pharmaceutically acceptable carrier or
diluent.
According to still a further aspect of the present invention there is provided
a nucleic
acid construct comprising a polynucleotide segment encoding a peptide having
at least
3 amino acid residues and less than 15 amino acid residues, the peptide
including an
amino acid sequence as set forth in SEQ ID NO: 7.
According to still a further aspect of the present invention there is provided
A
nucleic acid construct comprising a polynucleotide segment encoding a peptide
selected from the group consisting of SEQ ID NOs: 8, 10-11, 21-22 and 25.
According to still a further aspect of the present invention there is provided
a
nucleic acid construct comprising a polynucleotide segment encoding a peptide
selected from the group consisting of SEQ ID NOs: 4, 12-19 and 27-45.
According to still a further aspect of the present invention there is provided
an
antibody or an antibody fragment comprising an antigen recognition region
capable of
binding a peptide including at least 3 amino acid residues and less than 15
amino acid
residues, the peptide including an amino acid sequence as set forth in SEQ ID
NO: 7.
According to still a further aspect of the present invention there is provided
a
pharmaceutical composition for treating or preventing an amyloid-associated
disease
comprising as an active ingredient an antibody or an antibody fragment having
an
antigen recognition region capable of binding a peptide including at least 3
amino acid
residues and less than 15 amino acid residues, the peptide including an amino
acid
sequence as set forth in SEQ ID NO: 7.
According to still a further aspect of the present invention there is provided
a
method of treating or preventing an amyloid-associated disease in an
individual, the
method comprising providing to the individual therapeutically effective amount
of an
antibody or an antibody fragment having an antigen recognition region capable
of

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binding a peptide including at least 3 amino acid residues and less than 15
amino acid
residues, the peptide including an amino acid sequence as set forth in SEQ NO:
7.
According to still further features in the described preferred embodiments the

peptide further comprising at least two serine residues at a C-terminus
thereof.
According to still further features in the described preferred embodiments the
peptide is a linear or cyclic peptide.
According to still further features in the described preferred embodiments the

peptide further includes at least one beta-breaker amino acid.
According to still further features in the described preferred embodiments the
beta-beaker amino acid is proline.
The present invention successfully addresses the shortcomings of the presently

known configurations by providing novel peptides, compositions and methods,
which
can be used to diagnose and treat amyloid associated diseases such as type II
Diabetes
mellitus.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention is herein described, by way of example only, with reference to
the accompanying drawings. With specific reference now to the drawings in
detail, it
is stressed that the particulars shown are by way of example and for purposes
of
illustrative discussion of the preferred embodiments of the present invention
only,
and are presented in the cause of providing what is believed to be the most
useful and
readily understood description of the principles and conceptual aspects of the

invention. In this regard, no attempt is made to show structural details of
the
invention in more detail than is necessary for a fundamental understanding of
the
invention, the description taken with the drawings making apparent to those
skilled in
the art how the several forms of the invention may be embodied in practice.
In the drawings:
FIG. 1 is a schematic illustration depicting the self-assembly ability and
hydrophobicity of a group of peptides from a number of amyloid proteins as
deduced
using Kyte and Dolittle scale. Note, that no correlation is observed between
hydrophobicity and the amyloidogenic potential of the analyzed peptides. The
only

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apparent indication for potential amyloid fibril formation in this group of
peptide is a
combination of aromatic nature and minimal length.
FIGs. 2a-c are schematic illustrations of amyloid binding with the inhibitory
aromatic reagents: Ro 47-1816/001 (Figure 2a), Thioflavin T (Figure 2b) and CR
dye
(Figure 2c).
FIGs. 3a-c are schematic illustrations of a primary sequence comparison
between human and rodent IAPP and the synthetic peptides of the present
invention.
Figure 3a is a sequence alignment of human and rodent IAPP. A block indicates
a
seven amino acid sub-sequence illustrating the major inconsistencies between
the
sequences. The "basic amyloidogenic unit" is presented by bold letters and
underlined. Figure 3b illustrates the chemical structure of the wild type IAPP
peptide
(SEQ ID NO: 1). Figure lc illustrates the primary sequences and SEQ ID NOs. of

the peptides derived from the basic amyloidogenic unit.
FIGs. 4a-b are graphs illustrating light absorbance at 405 nm as a function of
time during fibril formation thus reflecting the aggregation kinetics of IAPP-
derived
peptides. The following symbols are used: closed squares ¨ N1A, opened circles
-
G3A, closed circles ¨ wild type, opened triangles - L6A, opened squares ¨ I5A
and
closed triangles ¨ F2A.
FIG. 5 is a histogram depicting mean particle size of assembled IAPP peptide
and derivatives as measured by light scattering. Each column represents the
results
of 3-5 independent measurements.
FIGs. 6a-n are photomicrographs illustrating Congo Red binding to pre-
assembled IAPP peptides. Normal field and polarized field micrographs are
shown
respectively for each of the following aged peptide suspensions: NIA peptide
(Figures 6a-b), F2A peptide (Figures 6c-d), G3A peptide (Figures 6e-f), wild
type
peptide (Figures 6g-h), I5A peptide (Figures 6i-j) and L6A (Figures 6k-1).
FIGs. 7a-f are electron micrographs of "aged" IAPP peptide and derivatives.
N1A peptide (Figure 7a), F2A peptide (Figure 7b), G3A peptide (Figure 7c),
wild
type peptide (Figure 7d), I5A peptide (Figure 7e) and L6A (Figure 70. The
indicated
scale bar represents 100 nm.

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FIG. 8a is a nucleic acid sequence alignment of wild type hIAPP and a
corresponding sequence modified according to a bacterial codon usage. Modified

bases are underlined.
FIG. 8b is a schematic illustration of the pMALc2x-NN vector which is used
5 for cytoplasmic expression of the 48 lcDa MBP-IAPP protein. The V8 Ek
cleavage
site and the (His)6 tag are fused C-terminally to the malE tag vector
sequence. A
factor Xa cleavage site for removal of the MBP tag is indicated.
FIG. 9 is a protein gel GelCode Blue staining depicting bacterial expression
and purification of MBP and MBP-IAPP fusion protein. Bacterial cell extracts
were
10 generated and proteins were purified on an amylose resin column. Samples
including
25 lig protein were loaded in each of Lanes 1-3 whereas 5 ps protein were
loaded on
each of lanes 4-5. Proteins were resolved on a 12 % SDS-PAGE and visualized
with
GelCode Blue staining. A molecular weight marker is indicated on the left.
Lane 1 ¨
0.5 mM IPTG-induced soluble extract of MBP. Lane 2 ¨ 0.1 mM 1PTG-induced
soluble extract of MBP-IAPP. Lane 3 ¨ 0.5 mM IPTG-induced soluble extract of
MBP-IAPP. Lane 4 ¨ purified MBP. Lane 5 ¨ purified MBP-IAPP. An arrow
marks the MBP-IAPP.
FIGs. 10a-b are a dot-blot image (Figure 10a) and densitometric quantitation
thereof (Figure 10b) depicting putative amyloidogenic sequences in hIAPP.
FIG. 11 is a graphic illustration depicting light absorbance at 405 nm as a
function of time during fibril formation thus reflecting the aggregation
kinetics of
IAPP-derived peptides (SEQ ID NOs. 14-19). The following symbols are used:
closed squares ¨ FLVHSS, opened circles - FLVHS, closed diamonds ¨ NFLVHSS,
opened triangles - NFLVHSSNN, opened squares ¨ FLVH and closed triangles -
NFLVH.
FIGs. 12a-f are photomicrographs illustrating Congo Red binding to pre-
assembled IAPP peptides. Polarized field micrographs are shown for each of the

following one day aged peptide suspensions: NFLVHSSNN peptide (Figures 12a),
NFLVHSS (Figure 12b), FLVHSS (Figure 12c), NFLVH (Figure 12d), FLVHS
(Figure 12e) and FLVH (Figure 120.
FIGs. 13a-f are electron micrographs of "aged" TAPP peptides.
NFLVHSSNN peptide (Figures 13a), NFLVHSS (Figure 13b), FLVHSS (Figure

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13c), NFLVH (Figure 13d), FLVHS (Figure 13e) and FLVH (Figure 130. The
indicated scale bar represents 100 urn.
FIGs. 14a-f are graphs showing secondary structures in the insoluble TAPP
aggregates as determined by Fourier transformed infrared spectroscopy.
NFLVHSSNN peptide (Figures 14a), NFLVHSS (Figure 14b), FLVHSS (Figure
14c), NFLVH (Figure 14d), FLVHS (Figure 14e) and FLVH (Figure 140.
FIG. 15 is a chemical structure of a previously reported amyloidogenic
peptide fragment of Medin [Haggqvist (1999) P.roc. Natl. Acad. Sci. U S A
96:8669-
8674].
FIGs. 16a-b are graphs illustrating light absorbance at 405 nm as a function
of
time during fibril formation thus reflecting the aggregation kinetics of Medin-
derived
peptides. Figure 16a illustrates a short-term kinetic assay. Figure 16b
illustrates a
long-term kinetic assay.
FIGs. 17a-f are electron micrographs of "aged" Medin-derived peptides.
NFGSVQFA - Figures 17a, NFGSVQ - Figure 17b, NFGSV - Figure 17c, FGSVQ -
Figure 17d, GSVQ - Figure 17e and FGSV - Figure 17f. The indicated scale bar
represents 100 nm.
FIGs. 18a-f are photomicrographs illustrating Congo Red binding to pre-
assembled Medin-derived peptides. Polarized field micrographs are shown for
each
of the following aged peptide suspensions: NFGSVQFA - Figures 18a, NFGSVQ -
Figure 18b, NFGSV - Figure 18c, FGSVQ - Figure 18d, GSVQ - Figure 18e and
FGSV - Figure 18f.
FIGs. 19a-c depict the effect of an alanine mutation on the amyloidogenic
features of the hexapeptide amyloidogenic fragment of Medin. Figure 19a ¨ is a
graph illustrating light absorbance at 405 urn as a function of time during
fibril
formation thus reflecting the aggregation kinetics of Medin-derived alanine
mutant;
Figure 19b is an electron micrograph of "aged" Medin- derived alanine mutant,
The
scale bar represents 100 urn; Figure 19c ¨ is a photomicrograph illustrating
Congo
Red binding to pre-assembled Medin-derived peptide mutant.
FIGs. 20a-b are the amino acid sequence of human Calcitonin (Figure 20a)
and chemical structure of an amyloidogenic peptide fragment of human
Calcitonin
(Figure 20b). Underlined are residues 17 and 18 which are important to the

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12
oligomerization state and hormonal activity of Calcitonin [Kazantzis (2001)
Eur. J.
Biochem. 269:780-791].
FIGs. 21a-d are electron micrographs of "aged" Calcitonin-derived peptides.
DFNKF - Figure 21a, DFNK - Figure 21b, FNKF - Figure 21c and DFN - Figure 21d.
The indicated scale bar represents 100 nm.
FIGs. 22a-d are photomicrographs illustrating Congo Red binding to pre-
assembled Calcitonin-derived peptides. Polarized field micrographs are shown
for
each of the following aged peptide suspensions: DFNKF - Figure 22a, DFNK -
Figure 22b, FNKF - Figure 22c and DFN - Figure 22d.
FIG. 23 is a graphic illustration showing secondary structures in the
insoluble
Calcitonin aggregates as determined by Fourier transformed infrared
spectroscopy.
FIGs. 24a-c depict the effect an alanine mutation on the amyloidogenic
features of the pentapeptide amyloidogenic fragment of Calcitonin. Figure 24a
is an
electron micrograph of "aged" Calcitonin-derived alanine mutant. The scale bar
represents 100 urn; Figure 24b ¨ is a photomicrograph illustrating Congo Red
binding to pre-assembled Calcitonin-derived peptide mutant; Figure 24c is a
graph
showing secondary structures in the mutant peptide as determined by Fourier
transformed infrared spectroscopy.
FIG. 25 is an electron micrograph depicting self-assembly of "aged"
Lactotransferrin-derived peptide. The scale bar represents 100 mn.
FIG. 26 is an electron micrograph depicting self-assembly of "aged" Serum
amyloid A protein-derived peptide. The scale bar represents 100 urn.
FIG. 27 is an electron micrograph depicting self-assembly of "aged" BriL-
derived peptide. The scale bar represents 100 nm.
FIG. 28 is an electron micrograph depicting self-assembly of "aged" Gelsolin-
derived peptide. The scale bar represents 100 urn.
FIG. 29 is an electron micrograph depicting self-assembly of "aged" Serum
amyloid P-derived peptide. The scale bar represents 100 urn.
FIG. 30 is an electron micrograph depicting self-assembly of "aged"
hnmunoglobulin light chain-derived peptide. The scale bar represents 100 nm.
FIG. 31 is an electron micrograph depicting self-assembly of "aged" Cystatin
C-derived peptide. The scale bar represents 100 nm.

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FIG. 32 is an electron micrograph depicting self-assembly of "aged"
Transthyretin-derived peptide. The scale bar represents 100 nm.
FIG. 33 is an electron micrograph depicting self-assembly of "aged"
Lysozyme-derived peptide. The scale bar represents 100 nm.
FIG. 34 is an electron micrograph depicting self-assembly of "aged"
Fibrinogen-derived peptide. The scale bar represents 100 nm.
FIG. 35 is an electron micrograph depicting self-assembly of "aged" Insulin-
derived peptide. The scale bar represents 100 nm.
FIG. 36 is an electron micrograph depicting self-assembly of "aged"
Prolactin-derived peptide. The scale bar represents 100 nm.
FIG. 37 is an electron micrograph depicting self-assembly of "aged" Beta 2
microglobulin-derived peptide. The scale bar represents 100 nm.
FIG. 38 is a graphic representation of the effect of an inhibitory peptide on
IAPP self-assembly. Squares ¨ wild type (wt) IAPP peptide; triangles ¨ wt-IAPP
+
inhibitory peptide; circles ¨ no peptides.
FIG. 39 is a graphic illustration depicting light absorbance at 405 nm as a
function of time during fibril formation thus reflecting the aggregation
kinetics of
IAPP-derived peptides (SEQ ID NOs. 46-49).
FIG. 40 is a histogram representation illustrating turbidity of IAPP analogues
following seven day aging.
FIG. 41a-f are electron micrographs of "aged" IAPP analogues. NFGAILSS -
Figure 41a; NFGALLSS - Figure 41b; NIGALLSS - Figure 41c; NLGALLSS - Figure
41d; NVGALLSS - Figure 41e and NAGAILSS - Figure 41f. The indicated scale bar
represents 100 nm.
FIGs. 42a-c illustrate the binding of IAPP- NFGAILSS to analogues of the
minimal amyloidogenic sequence SNNXGALLSS (X = any natural amino acid but
cysteine). Figure 42a shows short exposure of the bound peptide-array. Figure
42b
shows long exposure of the bound peptide-array. Figure 42c shows quantitation
of
the short exposure (Figure 42a) using densitometry and arbitrary units.

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DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is of novel peptides antibodies directed thereagainst,
compositions including same and methods of utilizing each for diagnosing or
treating
amyloid associated diseases such as type II Diabetes mellitus.
The principles and operation of the present invention may be better
understood with reference to the drawings and accompanying descriptions.
Before explaining at least one embodiment of the invention in detail, it is to

be understood that the invention is not limited in its application to the
details of
construction and the arrangement of the components set forth in the following
description or illustrated in the drawings. The invention is capable of other
embodiments or of being practiced or carried out in various ways. Also, it is
to be
understood that the phraseology and terminology employed herein is for the
purpose
of description and should not be regarded as limiting.
Numerous therapeutic approaches for prevention of amyloid fibril formation
or disaggreagtion of amyloid material have been described in the prior art.
However,
current therapeutic approaches are limited by cytotoxicity, non-specificity
and
delivery barriers.
While reducing the present invention to practice and while searching for a
novel therapeutic modality to amyloid associated diseases, such as II diabetes
mellitus, the present inventor has identified a sequence characteristic of
amyloid
forming peptides which directs fibril formation. This finding suggests that
ordered
amyloidogenesis involves a specific pattern of molecular interactions rather
than the
previously described mechanism involving non-specific hydrophobic interactions

[Petkova (2002) Proc. Natl. Acad. Sci. U S A 99:16742-16747].
As is further illustrated hereinbelow and in the Examples section which
follows, the present inventor attributed a pivotal role for aromatic residues
in amyloid
formation. The involvement of aromatic residues in the process of amyloid
formation is in-line with the well-established role of it-such interactions in
molecular
recognition and self-assembly [Gillard et al (1997) Chem. Eur. J. 3: 1933-40;
Claessens and Stoddart, (1997) J. Phys. Org. Chem. 10: 254-72; Shetty et al
(1996) J.
Am. Chem. Soc. 118: 1019-27; McGuaghey et al (1998) it-stacking interactions:
Alive and well in proteins. J. Biol. Chem. 273, 15458-15463; Sun and Bernstein

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(1996) J. Phys. Chem. 100: 13348-66]. 7r-stacking interactions are non-bonded
interactions which are formed between planar aromatic rings. The steric
constrains
associated with the formation of those ordered stacking structures have a
fundamental role in self-assembly processes that lead to the formation of
5 supramolecular structures. Such 7r-stacking interactions, which are
probably entropy
driven, play a central role in many biological processes such as stabilization
of the
double-helix structure of DNA, core-packing and stabilization of the tertiary
structure
of proteins, host-guest interactions, and porphyrin aggregation in solution
[for further
review on the possible role of it-stacking interaction in the self-assembly of
amyloid
10 fibrils see Gazit (2002) FASEB J. 16:77-83].
Identification of an aromatic sequence which is sufficient for mediating
amyloid self-assembly enables for the first time, to generate highly efficient

diagnostic, prophylactic and therapeutic peptides which can be utilized to
treat or
diagnose diseases characterized by amyloid plaque formation.
15 Thus, according to one aspect of the present invention there is provided
a
peptide which includes the amino acid sequence set forth in SEQ ID NO: 7 and
is
capable of self aggregation into fibrils. As is further described hereinbelow,
peptides
possessing self aggregation capabilities and modificants thereof can be
utilized in
diagnostic and therapeutic applications.
The sequence set forth in SEQ 1D NO: 7 includes at least one aromatic amino
acid residue which, as is shown by the results presented in the Examples
section, is
pivotal to the formation of amyloid fibrils. It will be appreciated that
aromaticity
rather than hydrophobicity of the aromatic amino acid is the prevailing
chemical
feature in amyloid self-assembly as illustrated in Examples 36-39 of the
Examples
section.
The aromatic amino acid can be any naturally occurring or synthetic aromatic
residue including, but not limited to, phenylalanine, tyrosine, tryptophan,
phenylglycine, or modificants, precursors or functional aromatic portions
thereof.
Examples of aromatic residues which can be used by the present invention are
provided in Table 2 below.

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As is demonstrated by the results provided in the Examples section which
follows, the present invention facilitates the design of peptides exhibiting
varying
degrees of self-aggregation kinetics and aggregate structure.
As used herein, the phrase "self-aggregation" refers to the capability of a
peptide to form aggregates (e.g. fibrils) in an aqueous solution. The ability
of a
peptide to self-aggregate and the kinetics and type of such self-aggregation
determines
a use for the peptide in treating or diagnosing amyloid diseases.
Since aggregation kinetics and aggregate structures are largely determined by
the specific residue composition and possibly the length of the peptides
generated (see
Figure 1), the present invention encompasses both longer peptides (e.g., 10-50
amino
acids) which include the sequences set forth in SEQ ID NOs: 4, 8, 10-19, 21-
22, 25 or
27-45, or shorter peptides (2-10 amino acid residues) including any of these
sequences. Due to their self-aggregating nature these peptides can be used as
potent
diagnostic reagents.
In order to enhance the rate of amyloid formation, the peptides of the present
invention preferably further include at least one polar and uncharged amino
acid
including but not limited to serine, threonine, asparagine, glutamine or
natural or
synthetic derivatives thereof (see Table 2).
Additionally, the peptides of the present invention may further include at
least
one pair of positively charged (e.g., lysine and arginine) and negatively
charged (e.g.,
aspartic acid and glutamic acid) amino acids (e.g., SEQ 1D NOs. 27-29). Such
amino
acid composition may be preferable, since as shown in Examples 21 of the
Examples
section, it is likely that electrostatic interactions between opposing charges
may direct
the formation of ordered antiparallel structure.
Since the present inventors have identified the sequence characteristics
governing fibril formation, the teachings of the present invention also enable
design of
peptides which would not aggregate into fibrils and be capable of either
preventing or
reducing fibril formation or disrupting preformed fibrils and thus can be used
as a
therapeutic agents.
For example, a peptide encompassed by SEQ ID NO: 9, 10, 11, 17, 19, 25 or
30 can be utilized for therapy since as is shown in the Examples section which

follows, such a peptide displays no aggregation (SEQ ID NO: 9) or slow
aggregation

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kinetics as compared to the wild type peptide (SEQ ID NOs: 9 and 10). It is
conceivable that since amyloid formation is a very slow process, these peptide

sequences will completely inhibit or significantly delay amyloidosis under
physiological conditions.
The term "peptide" as used herein encompasses native peptides (either
degradation products, synthetically synthesized peptides or recombinant
peptides) and
peptidomimetics (typically, synthetically synthesized peptides), as well as
peptoids
and semipeptoids which are peptide analogs, which may have, for example,
modifications rendering the peptides more stable while in a body or more
capable of
penetrating into cells. Such modifications include, but are not limited to N
terminus
modification, C terminus modification, peptide bond modification, including,
but not
limited to, CH2-NH, CH2-S, 0=C-NH,
CH2-0, CH2-CH2, S=C-NH,
CHH or CF=CH, backbone modifications, and residue modification. Methods for
preparing peptidomimetic compounds are well known in the art and are
specified, for
example, in Quantitative Drug Design, C.A. Ramsden Gd., Chapter 17.2, F.
Choplin
Pergamon Press (1992),
Further details in this respect are provided hereinunder.
Peptide bonds (-CO-NH-) within the peptide may be substituted, for example,
by N-methylated bonds (-N(CH3)-00-), ester bonds (-C(R)H-C-0-0-C(R)-N-),
ketomethylen bonds (-CO-CH2-), a-aza bonds (-NH-N(R)-009, wherein R is any
allcyl, e.g., methyl, carba bonds (-CH2-NH-), hydroxyethylene bonds (-CH(OH)-
CH2-
), thioamide bonds (-CS-NH-), olefinic double bonds (-CHH-), retro amide bonds

(-NH-CO-), peptide derivatives (-N(R)-CH2-00-), wherein R is the "normal" side

chain, naturally presented on the carbon atom.
These modifications can occur at any of the bonds along the peptide chain and
even at several (2-3) at the same time.
Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted for
synthetic non-natural acid such as Phenylglycine, TIC, naphthylelanine (Nol),
ring-
methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.
In addition to the above, the peptides of the present invention may also
include
one or more modified amino acids or one or more non-amino acid monomers (e.g.
fatty acids, complex carbohydrates etc).

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As used herein in the specification and in the claims section below the term
"amino acid" or "amino acids" is understood to include the 20 naturally
occurring
amino acids; those amino acids often modified post-translationally in vivo,
including,
for example, hydroxyproline, phosphoserine and phosphothreonine; and other
unusual
amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine,
isodesmosine, nor-valine, nor-leucine and omithine. Furthermore, the term
"amino
acid" includes both D- and L-amino acids.
Tables 1 and 2 below list naturally occurring amino acids (Table 1) and non-
conventional or modified amino acids (Table 2) which can be used with the
present
invention.
Table 1
Amino Acid Three-Letter Abbreviation One-letter Symbol
alanine Ala A
Arginine Arg
Asparagine Asn
Aspartic acid Asp
Cysteine Cys
Glutanaine Gin
Glutamic Acid Glu
glycine Gly
Histidine His
isoleucine lie
leucine Leu
Lysine Lys
Methionine Met
phenylalanine Phe
Proline Pro
Serine Ser
Threonine Thr
tryptophan Trp
tyrosine Tyr
Valine Val V
Any amino acid as above Xaa X
Table 2
Non-conventional amino acid Code Non-conventional amino acid Code
a-aminobutyric acid Abu L-N-methylalanine Nmala
a-amino-a-methylbutyrate Mgabu L-N-methylarginine Nmarg
arninocyclopropane- Cpro L-N-methylasparagine Nmasn
carboxylate L-N-methylaspartic acid Nmasp
aminoisobutyric acid Aib L-N-methylcysteine Nmcys
arninonorbornyl- Norb L-N-methylglutamine Nmgin
carboxylate L-N-methylglutamic acid Nmglu
cyclohexylalanine Chexa L-N-methylhistidine Nmhis
cyclopentylalanine Cpen L-N-methylisolleucine Nmile
D-alanine Dal L-N-methylleucine Nmleu
D-arginine Darg L-N-methyllysine Nmlys

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D-aspartic acid Dasp L-N-methylmethionine Nmmet
D-cysteine Dcys L-N-methylnorleucine Nninle
D-glutatnine Dgln L-N-methylnorvaline Nmnva
D-glutamic acid Dglu L-N-methylomithine Nmom
D-histidine Dhis L-N-methylphenylalanine Nmphe
D-isoleucine Dile L-N-methylproline Nmpro
D-leucine Dleu L-N-methylserine Nmser
D-lysine Dlys L-N-methylthreonine Nmthr
D-methionine Dmet L-N-methyltryptophan Nmtrp
D-omithine -Dorn L-N-methyltyrosine Nmtyr
D-phenylalanine Dphe L-N-methylvaline Nmval
D-proline Dpro .L-N-methylethylglycine Nmetg
D-serine Dser L-N-methyl-t-butylglycine Nmtbug
D-threonine Dthr L-norleucine Nle
D-tryptophan Dtrp L-norvaline Nva
D-tyrosine Dtyr a-methyl-arninoisobutyrate Maib
D-valine Dval a-methyl-y-aminobutyrate Mgabu
¨
D-a-methylalanine Dmala a-methylcyclohexylalanine Mchexa
D-a-methylarginine Dmarg a-methylcyclopentylalanine Mcpen
D-a-methylasparagine Dmasn a-methyl-a-napthylalanine Manap
D-a-methylaspartate Dmasp a- methylpenicillamine Mpen
D-a-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu
D-a-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg
D-a-methylhistidine Dmhis N-(3-aminopropyl)glycine Nom
D-a-methylisoleucine Dmile N- amino-a-methylbutyrate Nmaabu
D-a-methylleucine Dmleu a-napthylalanine Anap
D-a-methyllysine Dmlys N-benzylglycine Nphe
D-a-methylmethionine . Dmmet N-(2-carbamylethyl)glycine Ngln
D-a-methylomithine Dmorn N-(carbamyhnethyl)glycine Nasn
D-a-methylphenylalanine Dmphe N-(2-
carboxyethyl)glycine Nglu
D-a-methylproline Dmpro N-(carboxymethyl)glycine Nasp
D-a-methylserine Dmser N-cyclobutylglycine Ncbut
D-a-methylthreonine Dmthr N-cycloheptylglycine Nchep
D-a-methyltryptophan Dmtrp N-cyclohexylglycine Nchex
D-a-methyltyrosine Dmty N-cyclodecylglycine Ncdec
D-a-methylvaline Dmval N-cyclododeclglycine Ncdod
D-a-methylalnine Dnmala ,N-cyclooctylglycine Ncoct
s
D-a-methylarginine Dnrnarg N-cyclopropylglycine Ncpro
D-a-methylasparagine Dnmasn N-cycloundecylglycine Ncund
D-a-methylasparatate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm
D-a-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine Nbhe
D-N-methylleucine Drunleu N-(3-indolylyethyl) glycine Nhtrp
_.
D-N-methyllysine Dnmlys N-methyl-7-amirtobutyrate Nmgabu
N-methylcyclohexylalanine ,Nmchexa D-N-
methylmethionine Drunmet
D-N-methylornithine Dnmom N-methylcyclopentylalanine Nmcpen
N-methylglycine Nala D-N-methylphenylalanine Dnmphe
N-methylaminoisobutyrate Nmaib D-N-
methylproline Dnmpro
N-(1-methylpropyl)glycine , Nile D-N-
methylserine Dnmser
N-(2-methylpropyl)glycine Nile D-N-
methylserine Drunser

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N-(2-methylpropyl)glycine Nleu D-N-methylthreonine
Dnmthr
D-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine Nva
D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nrnanap
D-N-methylvaline Dnmval N-methylpenicillamine Nmpen
y-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr
L-t-butylglycine Thug N-(thiomethyl)glycine Ncys
L-ethylglycine Etg penicillarnine Pen
L-homophenylalanine Hphe L-a-methylalanine Mala
L-a-methylarginine Marg L-a-methylasparagine Masn
L-a-methylaspartate Masp L-a-methyl-t-butylglycine Mtbug
L-a-methylcysteine Mcys L-methylethylglycine Metg
L-a-methylglutamine Mgln L-a-methylglutamate Mglu
L-a-methylhistidine Mhis L-a-methylhomo phenylalanine Mhphe
L-a-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet
D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg
D-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine Nthr
D-N-methylhistidine Dnmhis N-(hydroxyethyl)glycine Nser
D-N-methylisoleucine Dnmile N-(Unidazolylethyl)glycine , Nhis
D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp
D-N-methyllysine Dmnlys N-methyl-y-aminobutyrate Nmgabu
N-methylcyclohexylalanine Nmchexa D-N-methylmethionine
Dmrunet
D-N-methylomithine Dnmom N-methylcyclopentylalanine Nmcpen
N-methylglycine Nala D-N-methylphenylalanine Drunphe
N-methylaminoisobutyrate Nmaib D-N-methylproline
Drurpro
N-(1-methylpropyl)glycine Nile D-N-methylserine
Dnmser
N-(2-methylpropyl)glycine Nleu D-N-methylthreonine
Dnmthr
D-N-methyltryptophan ,Dnmtrp N-(1-methylethyl)glycine Nval
D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap
D-N-methylvaline Dnmval N-methylpenicillamine Nmpen
y-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr
L-t-butylglycine Thug N-(thiomethyl)glycine Ncys
L-ethylglycine Etg penicillamine Pen
L-homophenylalanine Hphe L-a-methylalanine Mala
L-a-methylarginine Marg L-a-methylasparagine Masn
L-a-methylaspartate Masp L-a-methyl-t-butylglycine Mtbug
L-a-methylcysteine Mcys L-methylethylglycine Metg
L-a-methylglutamine Mgln L-a-methylglutamate Mglu
L-a-methylhistidine Mhis L-a-methylhomophenylalanine Mhphe
L-a-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet
L-a-methylleucine Mleu L-a-methyllysine Mlys
L-a-methylmethionine Mmet L-a-methylnorleucine Mnle
L-a-methylnorvaline Mnva L-a-methylomithine Mom
L-a-methylphenylalanine Mphe , L-a-methylproline
Mpro
L-a-methylserine Crms L-a-methylthreonine Mthr
-
L-a-methylvaline Mtrp L-a-methyltyrosine Mtyr
N-(N-(2,2-diphenylethyl) N-(N-(3,3-diphenylpropyl)
carbamylmethyl-glycine Nnblun carbamylmethyl(1)glycine Nnbhe
1-carboxy-1-(2,2-diphenyl Nmbc
ethylamino)cyclopropane

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Since the present peptides are preferably utilized in therapeutics or
diagnostics
which require the peptides to be in soluble form, the peptides of the present
invention
preferably include one or more non-natural or natural polar amino acids,
including but
not limited to serine and threonine which are capable of increasing peptide
solubility
due to their hydroxyl-containing side chain.
For therapeutic application, the peptides of the present invention preferably
further include at least one beta-sheet breaker amino acid residue such as
proline (e.g.,
SEQ ED NO. 45, see background section) which is characterized by a limited phi
angle
of about -60 to +25 rather than the typical beta sheet phi angle of about -120
to -140
degrees, thereby disrupting the beta sheet structure of the amyloid fibril.
The peptides of the present invention are preferably utilized in a linear
form,
although it will be appreciated that in cases where cyclization does not
severely
interfere with peptide characteristics, cyclic forms of the peptide can also
be utilized.
Cyclic peptides can either be synthesized in a cyclic form or configured so as
to assume a cyclic form under desired conditions (e.g., physiological
conditions).
Thus, the present invention provides conclusive data as to the identity of the

structural determinant of amyloid peptides, which directs fibril assembly.
As such, the present invention enables design of a range of peptide sequences,

which can be utilized for prevention/treatment or diagnosis of amyloidosis.
It will be appreciated that the present inventor could identify the consensus
aromatic sequence of the present invention (SEQ ID NO: 7) in numerous amyloid
related proteins (see Examples 6-35 of the Examples section). Thus, the
present
invention enables accurate identification of amyloidogenic fragments in
essentially
all amyloidogenic proteins.
Furthermore, the fact that small aromatic molecules, such as Ro 47-1816/001
[Kuner et al. (2000) J. Biol. Chem. 275:1673-8, see Figure 2a] and 3-p-toluoy1-
244'-
(3-diethylaminopropoxy)-phnyl]-benzofuran [Twyman (1999) Tetrahedron Letters
40:9383-9384] have been demonstrated effective in inhibiting the
polymerization of
the beta polypeptide of Alzheimer's disease [Findeis et al. (2000) Biochem.
Biophys.
Acta 1503:76-84], while amyloid specific dyes such as Congo-Red (Figure 2b)
and
thioflavin T (Figure 2c), which contain aromatic elements are generic amyloid

CA 02473987 2011-02-02
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formation inhibitors, substantiate the recognition motif of the present
invention as
sufficient for amyloid self-assembly.
The availability of the peptides of the present invention allows for the
generation of antibodies directed thereagainst, which may be used to
dissociate or
prevent the formation of amyloid plaques
The term "antibody" refers to intact antibody molecules as well as functional
fragments thereof, such as Fab, F(ab1)2, and Fv that are capable of binding to

macrophages. These functional antibody fragments are defined as follows: (i)
Fab,
the fragment which contains a monovalent antigen-binding fragment of an
antibody
molecule, can be produced by digestion of whole antibody with the enzyme
papain to
yield an intact light chain and a portion of one heavy chain; (ii) Fab', the
fragment of
an antibody molecule that can be obtained by treating whole antibody with
pepsin,
followed by reduction, to yield an intact light chain and a portion of the
heavy chain;
two Fab' fragments are obtained per antibody molecule; (iii) (FaV)2, the
fragment of
the antibody that can be obtained by treating whole antibody with the enzyme
pepsin
without subsequent reduction; F(abi)2 is a dimer of two Fab' fragments held
together
by two disulfide bonds; (iv) Fv, defined as a genetically engineered fragment
containing the variable region of the light chain and the variable region of
the heavy
chain expressed as two chains; and (v) Single chain antibody ("SCA"), a
genetically
engineered molecule containing the variable region of the light chain and the
variable
region of the heavy chain, linked by a suitable polypeptide linker as a
genetically
fused single chain molecule.
Methods of making these fragments are known in the art. (See for example,
Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory,
New York, 1988).
Methods of generating antibodies (i.e., monoclonal and polyclonal) are well
known in the art. Antibodies may be generated via any one of several methods
known
in the art, which methods can employ induction of in vivo production of
antibody
molecules, screening irrununoglobulin libraries or panels of highly specific
binding
reagents as disclosed [Orlandi D.R. et al. (1989) Proc. Natl. Acad. Sci.
86:3833-3837,
Winter G. et al. (1991) Nature 349:293-299] or generation of monoclonal
antibody
molecules by continuous cell lines in culture. These include but are not
limited to, the

CA 02473987 2011-02-02
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hybridoma technique, the human B-cell hybridoma technique, and the Epstein-Bar-

Virus (EBV)-hybridoma technique [Kohler G., et at. (1975) Nature 256:495-497,
Kozbor D., et at. (1985) J. Inununol. Methods 81:31-42, Cote R.J. et at.
(1983) Proc.
Natl. Acad. Sci. 80:2026-2030, Cole S.P. et al. (1984) Mol. Cell. Biol. 62:109-
1201
Antibody fragments according to the present invention can be prepared by
proteolytic hydrolysis of the antibody or by expression in E. coli or
mammalian cells
(e.g. Chinese hamster ovary cell culture or other protein expression systems)
of DNA
encoding the fragment.
Antibody fragments can be obtained by pepsin or papain digestion of whole
antibodies by conventional methods. For example, antibody fragments can be
produced by enzymatic cleavage of antibodies with pepsin to provide a 5S
fragment
denoted F(ab1)2. This fragment can be further cleaved using a thiol reducing
agent, and
optionally a blocking group for the sulfhydryl groups resulting from cleavage
of
disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively,
an
enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an
Fc
fragment directly. These methods are described, for example, by Goldenberg,
U.S.
Pat. Nos. 4,036,945 and 4,331,647, and references contained therein. See also
Porter, R.
R., Biochem. J., 73: 119-126, 1959. Other methods of cleaving antibodies, such
as
separation heavy chains to form monovalent light-heavy chain fragments,
further
cleavage of fragments, or other enzymatic, chemical, or genetic techniques may
also be
used, so long as the fragments bind to the antigen that is recognized by the
intact
antibody.
Fv fragments comprise an association of VH and VL chains. This association
may be noncovalent, as described in Inbar et al., Proc. Nat'l Acad. Sci. USA
69:2659-
62, 1972. Alternatively, the variable chains can be linked by an
intermolecular
disulfide bond or cross-linked by chemicals such as glutaraldehyde.
Preferably, the Fv
fragments comprise VH and VL chains connected by a peptide linker. These
single-
chain antigen binding proteins (sFv) are prepared by constructing a structural
gene
comprising DNA sequences encoding the VH and VL domains connected by an
oligonucleotide. The structural gene is inserted into an expression vector,
which is
subsequently introduced into a host cell such as E. coli. The recombinant host
cells
synthesize a single polypeptide chain with a linker peptide bridging the two V

CA 02473987 2011-02-02
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domains. Methods for producing sFvs are described, for example, by Whitlow and
Filpula, Methods, 2: 97-105, 1991; Bird et al., Science 242:423-426, 1988;
Pack et al.,
Bio/Technology 11:1271-77, 1993; and Ladner et al., U.S. Pat. No. 4,946,778.
Another form of an antibody fragment is a peptide coding for a single
complementarity-determining region (CDR). CDR peptides ("minimal recognition
units") can be obtained by constructing genes encoding the CDR of an antibody
of
interest. Such genes are prepared, for example, by using the polymerase chain
reaction
to synthesize the variable region from RNA of antibody-producing cells. See,
for
example, Larrick and Fry, Methods, 2: 106-10, 1991.
For human applications, the antibodies of the present invention are preferably

humanized. Humanized forms of non-human (e.g., murine) antibodies are chimeric

molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such

as Fv, Fab, Fab', F(ab)2 or other antigen-binding subsequences of
antibodies)
which contain minimal sequence derived from non-human immunoglobulin.
Humanized antibodies include human immunoglobulins (recipient antibody) in
which residues form a complementary determining region (CDR) of the recipient
are
replaced by residues from a CDR of a non-human species (donor antibody) such
as
mouse, rat or rabbit having the desired specificity, affinity and capacity. In
some
instances, Fv framework residues of the human immunoglobulin are replaced by
corresponding non-human residues. Humanized antibodies may also comprise
residues which are found neither in the recipient antibody nor in the imported
CDR
or framework sequences. In general, the humanized antibody will comprise
substantially all of at least one, and typically two, variable domains, in
which all or
substantially all of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are those of a
human
immunoglobulin consensus sequence. The humanized antibody optimally also will
include at least a portion of an immunoglobulin constant region (Fe),
typically that of
a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et
al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-
596
(1992)].

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Methods for humanizing non-human antibodies are well known in the art.
Generally, a humanized antibody has one or more amino acid residues introduced

into it from a source which is non-human. These non-human amino acid residues
are
often referred to as import residues, which are typically taken from an import
variable
5 domain. Humanization can be essentially performed following the method of
Winter
and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al.,
Nature
332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)1, by
substituting rodent CDRs or CDR sequences for the corresponding sequences of a

human antibody. Accordingly, such humanized antibodies are chimeric antibodies
10 (U.S. Pat. No. 4,816,567), wherein substantially less than an intact
human variable
domain has been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human antibodies in
which
some CDR residues and possibly some FR residues are substituted by residues
from
analogous sites in rodent antibodies.
15 Human antibodies can also be produced using various techniques known in
the art, including phage display libraries [Hoogenboom and Winter, J. Mol.
Biol.,
227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)]. The techniques
of Cole
et al. and Boerner et al. are also available for the preparation of human
monoclonal
antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R.
Liss, p.
20 77 (1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)1
Similarly, human
can be made by introducing of human inununoglobulin loci into transgenic
animals,
e.g., mice in which the endogenous immunoglobulin genes have been partially or

completely inactivated. Upon challenge, human antibody production is observed,

which closely resembles that seen in humans in all respects, including gene
25 rearrangement, assembly, and antibody repertoire. This approach is
described, for
example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;
5,633,425;
5,661,016, and in the following scientific publications: Marks et al.,
Bio/Technology
10, 779-783 (1992); Lonberg et al., Nature 368 856-859 (1994); Morrison,
Nature
368 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);
Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern.
Rev.
Immunol. 13 65-93 (1995).

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As is mentioned hereinabove, one specific use for the peptides of the present
invention is prevention or treatment of diseases associated with amyloid
plaque
formation.
Thus, according to yet another aspect of the present invention, there is
provided a method of treating an amyloid-associated disease in an individual.
Preferred individual subjects according to the present invention are mammals
such as
canines, felines, ovines, porcines, equines, bovines, humans and the like.
The term "treating" refers to reducing or preventing amyloid plaque formation,

or substantially decreasing plaque occurrence in the affected tissue. The
phrase
"amyloid plaque" refers to fibrillar amyloid as well as aggregated but not
fibrillar
amyloid, hereinafter "protofibrillar amyloid", which may be pathogenic as
well. For
example, an aggregated but not necessarily fibrillar form of IAPP was found to
be
toxic in culture. As shown by Anaguiano and co-workers [(2002) Biochemistry
41:11338-43] protofibrillar IAPP, like protofibrillar a-synucelin, which is
implicated
in Parkinson's disease pathogenesis, permeabilized synthetic vesicles by a
pore-like
mechanism. The formation of the of the IAPP amyloid pore was temporally
correlated to the formation of early IAPP oligomers and disappearance thereof
to the
appearance of amyloid fibrils. These results suggest that protofibrillar IAPP
may be
critical to type II diabetes mellitus as other protofibrillar proteins are
critical to the
development of Alzheimer's and Parkinson's diseases.
Amyloid-associated diseases treated according to the present invention
include, but are not limited to, type II diabetes mellitus, Alzheimer's
disease (AD),
early onset Alzheimer's disease, late onset Alzheimer's disease,
presymptomatic
Alzheimer's disease, Perkinson's disease, SAA amyloidosis, hereditary
Icelandic
syndrome, multiple myeloma, medullary carcinoma, aortic medical carcinoma,
Insulin injection amyloidosis, prion-systematic amyloidosis, choronic
inflammation
amyloidosis, Huntington's disease, senile systemic amyloidosis, pituitary
gland
amyloidosis, Hereditary renal amyloidosis, familial British dementia, Finnish
hereditary amyloidosis, familial non-neuropathic amyloidosis [Gazit (2002)
Curr.
Med. Chem. 9:1667-1675] and prion diseases including scrapie of sheep and
goats
and bovine spongiform encephalopathy (BSE) of cattle [Wilesmith and Wells
(1991)
Curr Top Microbiol Immunol 172: 21-38] and human prion diseases including (i)

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kuru, (ii) Creutzfeldt-Jakob Disease (Cm), (iii) Gerstmann-Streussler-Sheinker

Disease (GSS), and (iv) fatal familial insomnia (FFI) [Gajdusek (1977) Science
197:
943-960; Medori, Tritschler et al. (1992) N Engl J Med 326: 444-449].
The method includes providing to the individual a therapeutically effective
amount of the peptide of the present invention. The peptide can be provided
using
any one of a variety of delivery methods. Delivery methods and suitable
formulations
are described hereinbelow with respect to pharmaceutical compositions.
It will be appreciated that when utilized for treatment of amyloid diseases,
the
peptide of the present invention includes an amino acid sequence suitable for
preventing fibril formation, reducing fibril formation, or disaggregating
formed
aggregates by competitive destabilization of the preformed aggregate. For
example,
SEQ ID NO: 45 can be utilized for treatment of amyloid diseases, particularly
type II
diabetes mellitus since as shown in Example 35 of the Examples section which
follows, such a sequence interferes with TAPP self-assembly as demonstrated by
the
decreased ability of the amyloidogenic peptide to bind thioflavin T in the
presence of
an inhibitory peptide.
Alternatively, the peptides set forth in SEQ ID NOs: 10 or 11 can be used as
potent inhibitors of type II diabetes since as shown in the Examples section
which
follows, substitution of either leucine or isoleucine in the peptide elicits
very slow
kinetics of aggregation. Since amyloid formation in vivo is a very slow
process, it is
conceivable that under physiological conditions no fibrilization will occur
upon the
substitution of isoleucine or leucine to alanine in the context of the full
length IAPP.
Alternatively, self-aggregating peptides such as those set forth in SEQ ID
NOs.
17, 19 and 28-30, can be used as potent inhibitors of amyloid fibrilization,
since such
peptides can form heteromolecular complexes which are not as ordered as the
homomolecular assemblies formed by amyloid fragments.
It will be appreciated that since one of the main obstacles in using short
peptide fragments in therapy is their proteolytic degradation by
stereospecific cellular
proteases, the peptides of the present invention are preferably synthesized
from D-
isomers of natural amino acids [i.e., inverso peptide analogues, Tjemberg
(1997) J.
Biol. Chem. 272:12601-5, Gazit (2002) Curr. Med. Chem. 9:1667-1675].

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Additionally, the peptides of the present invention include retro, inverso and

retro-inverso analogues thereof. It will be appreciated that complete or
extended
partial retro-inverso analogues of hormones have generally been found to
retain or
enhance biological activity. Retro-inversion has also found application in the
area of
rational design of enzyme inhibitors (see U.S. Pat. No. 6,261,569).
As used herein a "retro peptide" refers to peptides which are made up of L-
amino acid residues which are assembled in opposite direction to the native
peptide
sequence.
RetTo-inverso modification of naturally occurring polypeptides involves the
synthetic assembly of amino acids with a-carbon stereochemistry opposite to
that of
the corresponding L-amino acids, i.e., D- or D-allo-amino acids in inverse
order to the
native peptide sequence. A rerto inverso analogue, thus, has reversed termini
and
reversed direction of peptide bonds, while essentially maintaining the
topology of the
side chains as in the native peptide sequence.
Additionally, since one of the main issues in amyloid fibril formation is the
transition of the amyloid polypeptide from the native form to stacked (3-
strand
structure, inhibitory peptides preferably include N-methylated amino acids
which
constrain peptide-backbone due to steric effects [Kapurniotu (2002) 315:339-
350].
For example, aminoisobutyric acid (Aib or methyl alanine) is known to
stabilize an a-
helical structure in short natural peptides. Furthermore, the N-methylation
also affects
the intermolecular NH to CO H-bonding ability, thus suppressing the formation
of
multiplayer 13-strands, which are stabilized by H-bonding interactions.
It will be further appreciated that addition of organic groups such as a
cholyl
groups to the N-terminal or C-terminal of the peptides of the present
invention is
preferred since it was shown to improve potency and bioavailability (e.g.,
crossing the
blood brain barrier in the case of neurodegenerative diseases) of therapeutic
peptides
[Findeis (1999) Biochemistry 38:6791-6800]. Furthermore, introducing a charged

amino acid to the recognition motif, may result in electrostatic repulsion
which
inhibits further growth of the amyloid fibrils [Lowe (2001) J. Mol. Biol.
40:7882-
7889].
As mentioned hereinabove, the antibodies of the present invention may also be
used to treat amyloid-associated diseases.

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The peptides and/or antibodies of the present invention can be provided to an
individual per se, or as part of a pharmaceutical composition where it is
mixed with a
pharmaceutically acceptable carrier.
As used herein a "pharmaceutical composition" refers to a preparation of one
or more of the active ingredients described herein with other chemical
components
such as physiologically suitable carriers and excipients. The purpose of a
pharmaceutical composition is to facilitate administration of a compound to an

organism.
Herein the term "active ingredient" refers to the peptide or antibody
preparation, which is accountable for the biological effect.
Hereinafter, the phrases "physiologically acceptable carrier" and
"pharmaceutically acceptable carrier" which may be interchangeably used refer
to a
carrier or a diluent that does not cause significant irritation to an organism
and does
not abrogate the biological activity and properties of the administered
compound. An
adjuvant is included under these phrases. One of the ingredients included in
the
pharmaceutically acceptable carrier can be for example polyethylene glycol
(PEG), a
biocompatible polymer with a wide range of solubility in both organic and
aqueous
media (Mutter et al. (1979).
Herein the term "excipient" refers to an inert substance added to a
pharmaceutical composition to further facilitate administration of an active
ingredient.
Examples, without limitation, of excipients include calcium carbonate, calcium
phosphate, various sugars and types of starch, cellulose derivatives, gelatin,
vegetable
oils and polyethylene glycols.
Techniques for formulation and administration of drugs may be found in
"Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest

edition.
Suitable routes of administration may, for example, include oral, rectal,
transmucosal, especially transnasal, intestinal or parenteral delivery,
including
intramuscular, subcutaneous and intramedullary injections as well as
intrathecal,
direct intraventricular, intravenous, inrtaperitoneal, intranasal, or
intraocular
injections.

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Alternately, one may administer a preparation in a local rather than systemic
manner, for example, via injection of the preparation directly into a specific
region of
a patient's body.
Pharmaceutical compositions of the present invention may be manufactured by
5 processes well known in the art, e.g., by means of conventional mixing,
dissolving,
granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping
or
lyophilizing processes.
Pharmaceutical compositions for use in accordance with the present invention
may be formulated in conventional manner using one or more physiologically
10 acceptable carriers comprising excipients and auxiliaries, which
facilitate processing
of the active ingredients into preparations which, can be used
pharmaceutically.
Proper formulation is dependent upon the route of administration chosen.
For injection, the active ingredients of the invention may be formulated in
aqueous solutions, preferably in physiologically compatible buffers such as
Hank's
15 solution, Ringer's
solution, or physiological salt buffer. For transmucosal
administration, penetrants appropriate to the barrier to be permeated are used
in the
formulation. Such penetrants are generally known in the art.
For oral administration, the compounds can be formulated readily by
combining the active compounds with pharmaceutically acceptable carriers well
20 known in the art. Such carriers enable the compounds of the invention to
be
formulated as tablets, pills, dragees, capsules, liquids, gels, syrups,
slurries,
suspensions, and the like, for oral ingestion by a patient. Pharmacological
preparations for oral use can be made using a solid excipient, optionally
grinding the
resulting mixture, and processing the mixture of granules, after adding
suitable
25 auxiliaries if desired, to obtain tablets or dragee cores. Suitable
excipients are, in
particular, fillers such as sugars, including lactose, sucrose, mannitol, or
sorbitol;
cellulose preparations such as, for example, maize starch, wheat starch, rice
starch,
potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-
cellulose, sodium carbomethylcellulose; and/or physiologically acceptable
polymers
30 such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents
may be added,
such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such
as sodium alginate.

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Dragee cores are provided with suitable coatings. For this purpose,
concentrated sugar solutions may be used which may optionally contain gum
arabic,
talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium
dioxide,
lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs
or
pigments may be added to the tablets or dragee coatings for identification or
to
characterize different combinations of active compound doses.
Pharmaceutical compositions, which can be used orally, include push-fit
capsules made of gelatin as well as soft, sealed capsules made of gelatin and
a
plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain
the active
ingredients in admixture with filler such as lactose, binders such as
starches,
lubricants such as talc or magnesium stearate and, optionally, stabilizers. In
soft
capsules, the active ingredients may be dissolved or suspended in suitable
liquids,
such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In
addition,
stabilizers may be added. All formulations for oral administration should be
in
dosages suitable for the chosen route of administration.
For buccal administration, the compositions may take the form of tablets or
lozenges formulated in conventional manner.
For administration by nasal inhalation, the active ingredients for use
according
to the present invention are conveniently delivered in the form of an aerosol
spray
presentation from a pressurized pack or a nebulizer with the use of a suitable
propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-
tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the
dosage
unit may be determined by providing a valve to deliver a metered amount.
Capsules
and cartridges of, e.g., gelatin for use in a dispenser may be formulated
containing a
powder mix of the compound and a suitable powder base such as lactose or
starch.
The preparations described herein may be formulated for parenteral
administration, e.g., by bolus injection or continuous infusion. Formulations
for
injection may be presented in unit dosage form, e.g., in ampoules or in
multidose
containers with optionally, an added preservative. The compositions may be
suspensions, solutions or emulsions in oily or aqueous vehicles, and may
contain
formulatory agents such as suspending, stabilizing and/or dispersing agents.

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Pharmaceutical compositions for parenteral administration include aqueous
solutions of the active preparation in water-soluble form. Additionally,
suspensions
of the active ingredients may be prepared as appropriate oily or water based
injection
suspensions. Suitable lipophilic solvents or vehicles include fatty oils such
as sesame
oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or
liposomes.
Aqueous injection suspensions may contain substances, which increase the
viscosity
of the suspension, such as sodium carboxymethyl cellulose, sorbitol or
dextran.
Optionally, the suspension may also contain suitable stabilizers or agents
which
increase the solubility of the active ingredients to allow for the preparation
of highly
concentrated solutions.
Alternatively, the active ingredient may be in powder form for constitution
with a suitable vehicle, e.g., sterile, pyrogen-free water based solution,
before use.
The preparation of the present invention may also be formulated in rectal
compositions such as suppositories or retention enemas, using, e.g.,
conventional
suppository bases such as cocoa butter or other glycerides.
Pharmaceutical compositions suitable for use in context of the present
invention include compositions wherein the active ingredients are contained in
an
amount effective to achieve the intended purpose. More specifically, a
therapeutically
effective amount means an amount of active ingredients effective to prevent,
alleviate
or ameliorate symptoms of disease or prolong the survival of the subject being
treated.
Determination of a therapeutically effective amount is well within the
capability of those skilled in the art.
For any preparation used in the methods of the invention, the therapeutically
effective amount or dose can be estimated initially from in vitro assays. For
example,
a dose can be formulated in animal models and such information can be used to
more
accurately determine useful doses in humans.
Toxicity and therapeutic efficacy of the active ingredients described herein
can
be determined by standard pharmaceutical procedures in vitro, in cell cultures
or
experimental animals. The data obtained from these in vitro and cell culture
assays
and animal studies can be used in formulating a range of dosage for use in
human.
The dosage may vary depending upon the dosage form employed and the route of
administration utilized. The exact formulation, route of administration and
dosage

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33
can be chosen by the individual physician in view of the patient's condition.
[See e.g.,
Fingl, et al., (1975) "The Pharmacological Basis of Therapeutics", Ch. 1 p.1].
Depending on the severity and responsiveness of the condition to be treated,
dosing can be of a single or a plurality of administrations, with course of
treatment
lasting from several days to several weeks or until cure is effected or
diminution of the
disease state is achieved.
The amount of a composition to be administered will, of course, be dependent
on the subject being treated, the severity of the affliction, the manner of
administration, the judgment of the prescribing physician, etc.
Compositions including the preparation of the present invention formulated in
a compatible pharmaceutical carrier may also be prepared, placed in an
appropriate
container, and labeled for treatment of an indicated condition.
Compositions of the present invention may, if desired, be presented in a pack
or dispenser device, such as an FDA approved kit, which may contain one or
more
unit dosage forms containing the active ingredient. The pack may, for example,
comprise metal or plastic foil, such as a blister pack. The pack or dispenser
device
may be accompanied by instructions for administration. The pack or dispenser
may
also be accommodated by a notice associated with the container in a form
prescribed
by a governmental agency regulating the manufacture, use or sale of
pharmaceuticals,
which notice is reflective of approval by the agency of the form of the
compositions or
human or veterinary administration. Such notice, for example, may be of
labeling
approved by the U.S. Food and Drug Administration for prescription drugs or of
an
approved product insert.
It will be appreciated that the peptides or antibodies of the present
invention
can also be expressed from a nucleic acid construct administered to the
individual
employing any suitable mode of administration, described hereinabove (i.e., in-
vivo
gene therapy). Alternatively, the nucleic acid construct is introduced into a
suitable
cell via an appropriate gene delivery vehicle/method (transfection,
transduction,
homologous recombination, etc.) and an expression system as needed and then
the
modified cells are expanded in culture and returned to the individual (i.e.,
ex-vivo
gene therapy).
To enable cellular expression of the peptides or antibodies of the present

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34
invention, the nucleic acid construct of the present invention further
includes at least
one cis acting regulatory element. As used herein, the phrase "cis acting
regulatory
element" refers to a polynucleotide sequence, preferably a promoter, which
binds a
trans acting regulator and regulates the transcription of a coding sequence
located
downstream thereto.
Any available promoter can be used by the present methodology. In a
preferred embodiment of the present invention, the promoter utilized by the
nucleic
acid construct of the present invention is active in the specific cell
population
transformed. Examples of cell type-specific and/or tissue-specific promoters
include
promoters such as albumin that is liver specific [Pinkert et al., (1987) Genes
Dev.
1:268-277], lymphoid specific promoters [Calame et al., (1988) Adv. Immunol.
43:235-275]; in particular promoters of T-cell receptors [Winoto et al.,
(1989) EMBO
J. 8:729-733] and immunoglobulins; [Banerji et al. (1983) Cell 33729-740],
neuron-
specific promoters such as the neurofilament promoter [Byrne et al. (1989)
Proc. Natl.
Acad. Sci. USA 86:5473-5477], pancreas-specific promoters [Edlunch et al.
(1985)
Science 230:912-916] or mammary gland-specific promoters such as the milk whey

promoter (U.S. Pat. No. 4,873,316 and European Application Publication No.
264,166). The nucleic acid construct of the present invention can further
include an
enhancer, which can be adjacent or distant to the promoter sequence and can
function
in up regulating the transcription therefrom.
The constructs of the present methodology preferably further include an
appropriate selectable marker and/or an origin of replication. Preferably, the

construct utilized is a shuttle vector, which can propagate both in E. coli
(wherein the
construct comprises an appropriate selectable marker and origin of
replication) and be
compatible for propagation in cells, or integration in a gene and a tissue of
choice.
The construct according to the present invention can be, for example, a
plasmid, a
bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome.
Currently preferred in vivo nucleic acid transfer techniques include
transfection with viral or non-viral constructs, such as adenovirus,
lentivirus, Herpes
simplex I virus, or adeno-associated virus (AAV) and lipid-based systems.
Useful
lipids for lipid-mediated transfer of the gene are, for example, DOTMA, DOPE,
and
DC-Chol [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)]. The
most

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preferred constructs for use in gene therapy are viruses, most preferably
adenoviruses,
AAV, lentiviruses, or retroviruses. A viral construct such as a retroviral
construct
includes at least one transcriptional promoter/enhancer or locus-defining
element(s),
or other elements that control gene expression by other means such as
alternate
5 splicing, nuclear RNA export, or post-translational modification of
messenger. Such
vector constructs also include a packaging signal, long terminal repeats
(LTRs) or
portions thereof, and positive and negative strand primer binding sites
appropriate to
the virus used, unless it is already present in the viral construct. In
addition, such a
construct typically includes a signal sequence for secretion of the peptide or
antibody
10 from a host cell in which it is placed. Preferably the signal sequence
for this purpose
is a mammalian signal sequence. Optionally, the construct may also include a
signal
that directs polyadenylation, as well as one or more restriction sites and a
translation
termination sequence. By way of example, such constructs will typically
include a 5'
LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA
15 synthesis, and a 3' LTR or a portion thereof. Other vectors can be used
that are non-
viral, such as cationic lipids, polylysine, and dendrimers.
Because of the self-aggregating nature of the peptides of the present
invention
it is conceivable that such peptides can also be used as potent detectors of
amyloid
fibrils/plaques in biological samples. This is of a special significance to
amyloid-
20 associated diseases such as Alzheimer's disease wherein unequivocal
diagnosis can
only be made after postmortem examination of brain tissues for the hallmark
neurofibrillary tangles (NFT) and neuritic plaques.
Thus, according to yet another aspect of the present invention there is
provided a method of detecting a presence or an absence of an amyloid fibril
in a
25 biological sample.
The method is effected by incubating the biological sample with a peptide of
the present invention capable of co-aggregating with the amyloid fibril and
detecting
= the peptide, to thereby detect the presence or the absence of amyloid
fibril in the
biological sample. A variety of peptide reagents, which are capable of
recognizing
30 conformational ensembles are known in the art some of which are reviewed
in
Bursavich (2002) J. Med. Chem. 45(3): 541-58 and in Baltzer Chem Rev.
101(10):3153-63.

CA 02473987 2011-02-02
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The biological sample utilized for detection can be any body sample such as
blood (serum or plasma), sputum, ascites fluids, pleural effusions, urine,
biopsy
specimens, isolated cells and/or cell membrane preparation. Methods of
obtaining
tissue biopsies and body fluids from mammals are well known in the art.
The peptide of the present invention is contacted with the biological sample
under conditions suitable for aggregate formation (i.e., buffer, temperature,
incubation
time etc.); suitable conditions are described in Example 2 of the Examples
section.
Measures are taken not to allow pre-aggregation of peptides prior to
incubation with
the biological sample. To this end freshly prepared peptide stocks are
preferably
used.
Protein complexes within a biological sample can be detected via any one of
several methods known in the art, which methods can employ biochemical and/or
optical detection schemes.
To facilitate complex detection, the peptides of the present invention are
highlighted preferably by a tag or an antibody. It will be appreciated that
highlighting
can be effected prior to, concomitant with or following aggregate formation,
depending on the highlighting method. As used herein the term "tag" refers to
a
molecule, which exhibits a quantifiable activity or characteristic. A tag can
be a
fluorescent molecule including chemical fluorescers such as fluorescein or
polypeptide fluorescers such as the green fluorescent protein (OF?) or related
proteins
In such case, the tag can be quantified via its fluorescence,
which is generated upon the application of a suitable excitatory light.
Alternatively, a
tag can be an epitope tag, a fairly unique polypeptide sequence to which a
specific
antibody can bind without substantially cross reacting with other cellular
epitopes.
Such epitope tags include a Myc tag, a Flag tag, a His tag, a leucine tag, an
IgG tag, a
streptavidin tag and the like.
Alternatively, aggregate detection can be effected by the antibodies of the
present invention.
Thus, this aspect of the present invention provides a method of assaying or
screening biological samples, such as body tissue or fluid suspected of
including an
amyloid fibril.

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It will be appreciated that such a detection method can also be utilized in an

assay for uncovering potential drugs useful in prevention or disaggregation of
amyloid
deposits. For example, the present invention may be used for high throughput
screening of test compounds. Typically, the co-aggregating peptides of the
present
invention are radiolabeled, to reduce assay volume. A competition assay is
then
effected by monitoring displacement of the label by a test compound [Han
(1996) J.
Am. Chem. Soc. 118:4506-7 and Esler (1996) Chem. 271:8545-8].
It will be appreciated that the peptides of the present invention may also be
used as potent detectors of amyloid deposits in-vivo. A designed peptide
capable of
binding amyloid deposits, labeled non-radioactively or with a radio-isotope,
as is well
known in the art can be administered to an individual to diagnose the onset or

presence of amyloid-related disease, discussed hereinabove. The binding of
such a
labeled peptide after administration to amyloid or amyloid-like deposits can
be
detected by in vivo imaging techniques known in the art.
The peptides of the present invention can be included in a diagnostic or
therapeutic kit. For example, peptide sets of specific disease related
proteins or
antibodies directed thereagainst can be packaged in a one or more containers
with
appropriate buffers and preservatives and used for diagnosis or for directing
therapeutic treatment.
Thus, the peptides can be each mixed in a single container or placed in
individual containers. Preferably, the containers include a label. Suitable
containers
include, for example, bottles, vials, syringes, and test tubes. The containers
may be
formed from a variety of materials such as glass or plastic.
In addition, other additives such as stabilizers, buffers, blockers and the
like
may also be added.
The peptides of such kits can also be attached to a solid support, such as
beads, array substrate (e.g., chips) and the like and used for diagnostic
purposes.
Peptides included in kits or immobilized to substrates may be conjugated to a
detectable label such as described hereinabove.
The kit can also include instructions for determining if the tested subject is
suffering from, or is at risk of developing, a condition, disorder, or disease
associated
with amyloid polypeptide of interest.

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Additional objects, advantages, and novel features of the present invention
will become apparent to one ordinarily skilled in the art upon examination of
the
following examples, which are not intended to be limiting. Additionally, each
of the
various embodiments and aspects of the present invention as delineated
hereinabove
and as claimed in the claims section below finds experimental support in the
following examples.
EXAMPLES
Reference is now made to the following examples, which together with the
above descriptions, illustrate the invention in a non limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures
utilized in the present invention include molecular, biochemical,
microbiological and
recombinant DNA techniques. Such techniques are thoroughly explained in the
literature. See, for example, "Molecular Cloning: A laboratory Manual"
Sambrook et
al., (1989); "Current Protocols in Molecular Biology" Volumes
Ausubel, R. M.,
ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John
Wiley and
Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular
Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant
DNA", Scientific American Books, New York; Birren et al. (eds) "Genome
Analysis:
A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press,
New
York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828;
4,683,202;
4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook",
Volumes I-111 Cellis, J. E., ed. (1994); "Current Protocols in Immunology"
Volumes
Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical
Immunology"
(8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds),

"Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York
(1980); available immunoassays are extensively described in the patent and
scientific
literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752;
3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;
3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;
"Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid
Hybridization"

CA 02473987 2011-02-02
39
Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation"
Hames,
B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I.,
ed.
(1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide
to
Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317,
Academic Press; "PCR Protocols: A Guide To Methods And Applications",
Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein
Purification and Characterization - A Laboratory Course Manual" CSHL Press
(1996). Other general references are provided throughout this document. The
procedures
therein are believed to be well known in the art and are provided for the
convenience of
the reader.
EXAMPLE 1
Alanine scan of the hIAPP basic amylodogenic unit ¨ rational and peptide
synthesis
Pancreatic amyloid is found in more than 95 % of type II diabetes patients.
Pancreatic amyloid is formed by the aggregation of a 37 amino acid long islet
amyloid polypeptide (LAPP, GenBank Accession No. gi:4557655), the cytotoxicity

thereof being directly associated with the development of the disease. IAPP
amyloid
formation follows a nucleation-dependent polymerization process, which
proceeds
through conformational transition of soluble IAPP into aggregated 13-sheets.
Recently it has been shown that a hexapeptide (22-27) (NFGA1L, SEQ ID NO: 111)

of IAPP, also termed as the "basic amyloidogenic unit" is sufficient for the
formation
of I3-sheet-containing amyloid fibrils [Konstantinos et al. (2000) J. Mol.
Biol.
295:1055-1071].
To gain further insight into the specific role of the residues that compose
"the
"basic amyloidogenic unit", a systematic alanine scan was performed. Amino-
acids
were replaced with alanine in order to specifically change the molecular
interface of
the peptides, without significantly changing their hydrophobicity or tendency
to form
(3-sheet structures. alanine-scan was preformed in the context of the block
that is
unique to human IAPP (Figure 3a). This block includes two serine residues that

follow the NFGAIL motif in the full-length polypeptide. These eight amino-acid

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peptide sequences were used since the shorter peptides are hydrophobic and as
s such
less soluble. Figure 3b shows a schematic representation of the chemical
structure of
the wild-type peptide while Figure 3c indicates the amino-acid substitutions
in the
different mutant peptides that were generated.
5 Methods and Reagents - Peptide synthesis was performed by PeptidoGenic
Research & Co. Inc (Livermore, CA USA). The sequence identity of the peptides
was confirmed by ion spray mass-spectrometry using a Perkin Elmer Sciex API I
spectrometer. The purity of the peptides was confirmed by reverse phase high-
pressure liquid chromatography (RP-HPLC) on a C18 column, using a linear
gradient
10 of 10 to 70% acetonitrile in water and 0.1% trifluoroacetic acid (TFA).
EXAMPLE 2
Kinetics of aggregation of IAPP peptide fragment and mutant derivatives as
monitored by turbidity measurements
15 To study self-assembly of the IAPP peptide derived fragments,
aggregation
and insolubilization kinetics were monitored using turbidity measurements at
405
Kinetic aggregation assay ¨ Fresh peptide stock solutions were prepared by
dissolving lyophilized form of the peptides in DMSO, a disaggregating solvent,
at a
20 concentration of 100 mM. To avoid any pre-aggregation, fresh stock
solutions were
prepared prior to each and every experiment. Peptide stock solutions were
diluted
into assay buffer and plated in 96-well plates as follows: 2 IA of peptides
stock
solutions were added to 98 pi of 10 mM Tris pH 7.2, resulting in a 2 mM final
concentration of the peptide in the presence of 2% DMSO. Turbidity data was
25 measured at 405 nm. A buffer solution including 2 % DMSO was used as a
blank.
Turbidity was measured at room temperature over several time points.
Results - As shown in Figure 4a, wild-type peptide fragment (SEQ ID NO: 1)
showed an aggregation kinetic profile that was very similar to those
previously
reported for non-seeded hIAPP hexapeptide [Tenidis et al. (2000) J. Mol. Biol
30 295:1055-71]. Such a profile is strongly indicative of a nucleation-
dependent
polymerization mechanism [Jarrett and Lansbury (1992) Biochemistry 31:6865-
70].
Following a lag-time of 20 minutes, wild type peptide self-assembled into
insoluble

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41
fibrils. Peptide G3A (SEQ ID NO: 4) showed essentially the same profile as
that of
wild type peptide. The N1A peptide (SEQ ID NO: 2) mediated higher kinetics of
aggregation, albeit with different kinetic profile as compared to that of wild-
type
peptide. Interestingly, the aggregation of NIA seemed to be less nucleation-
dependent. Substitution of the isoleucine or leucine to alanine (peptides I5A,
SEQ
ID NO: 5 and L6A, SEQ ID NO: 6 respectively) reduced the kinetics of
aggregation
but did not abolish it completely. Substitution of the phenylalanine residue
to alanine
(peptide F2A, SEQ ID NO:3) led to a total loss of peptide ability to
aggregate. The
F2A peptide was completely soluble in the aqueous assay buffer.
Altogether, kinetic aggregation studies of the amyloidogenic fragments
suggested a major role to the phenylalanine residue in the process of amyloid
formation by the IAPP active fragment.
EXAMPLE 3
Measurement of aggregate mean particle size
While the turbidity assay provided an important estimate regarding the
aggregation potential and kinetics of the various peptides, it did not provide

information about the size of the actual aggregates formed. It will be
appreciated that
although the apparent hydrodynamic diameter of amyloid structures varies due
to
irregularity of the amyloid structure, it may still provide a clear indication
about the
order of magnitude of the structure formed and present a quantitative
criterion for
comparing the structures formed by the various peptides.
Therefore, the average size of the aggregates, formed by the various peptides,

was determined using dynamic light scattering (DLS) experiments.
Method - Freshly prepared peptide stock solutions at a concentration of 10
mM were diluted in 10 mM Tris buffer pH 7.2 and further filtrated through a
0.2 pm
filter to a final concentration of 100 1AM peptide and 1% DMSO. Particle size
measurement was conducted with a laser-powered ALV-NIBS/RPPS non-invasive
backscattering instrument. Autocorrelation data was fitted using the ALV-
NIBS/HPPS software to derive average apparent hydrodynamic diameters.
Results - The average apparent hydrodynamic diameters of the structures that
were formed by the various peptides are presented in Figure 5.

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Altogether, the apparent hydrodynamic diameter of the structures formed by
the various peptides seemed to be consistent with the results obtained by the
turbidity
assay. As with the turbidity assay, the wild-type peptide and G3A peptide
formed
particles of very similar hydrodynamic diameters. Smaller structures were
observed
with the derivative peptides: N1A, I5A and L6A. Thus, in accordance with the
turbidity assay, the DLS experiments clearly illustrate that no large
particles were
formed by the F2A peptide under the indicated experimental conditions.
EXAMPLE 4
Examination of amyloidogenic performance of wild type peptide and
derivatives through Congo Red (CR) binding assay
Congo red (CR) staining combined with polarization microscopy was utilized
to test amyloidogenicity of the peptides of the present invention. Amyloid
fibrils in
general, and fibrilar IAPP in particular, bind CR and exhibit gold/green
birefringence
under polarized light [Cooper (1974) Lab. Invest. 31:232-8; Lansbury (1992)
Biochemistry 31:6865-701.
Method and reagents ¨ Peptide solutions incubated in a 10 mM Tris buffer
(pH 7) for four days were dried on a glass microscope slide. Staining was
effected by
the addition of 1 mM CR in 10 mM Tris buffer pH 7.2 followed by a 1 minute
incubation. To remove excess CR, slides were rinsed with double-distilled
water and
dried. Saturated CR solutions solubilized in 80% ethanol (v/v) were used for
poorly
aggregating peptides. In such cases, staining was effected without rinsing.
Birefringence was determined using a WILD Makroskop m420 (x70) equipped with
a polarizing stage.
Results - Wild type, N1A and G3A peptides bound CR and exhibited the
characteristic green/gold birefringence (see Figures 6g, 6a and 6e for normal
field and
Figures 6h, 6b and 6f for polarized light microscopy, respectively). Peptides
I5A and
L6A, bound CR and exhibited rare but characteristic birefringence (Figures 6i
and 6k
for normal field and Figures 6j and 61 for polarized light, respectively).
Peptide F2A
(NAG/tit) showed no capability of binding CR (Figure 6c for normal field and
Figure 6d for polarized light). Dried buffer solution stained with CR was used
as a
negative control (see Figures 6m and 6n for normal and polarized light,
respectively).

CA 02473987 2011-02-02
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Interestingly, no significant difference in binding was observed for the
negative
control and the F2A peptide.
To substantiate the inability of F2A peptide to form fibrils, a peptide
solution
incubated for 14 days was used in the binding assay. Although some degree of
aggregation was visually observed following two weeks of peptide "aging", CR
staining showed no amyloid structure (results not shown). Under the same
conditions
wild-type peptide incubation resulted in significant CR birefringence.
EXAMPLE 5
Mtrastructu. ral analysis of the fibrillogenic peptide and mutants
The fibrillogenic potential of the various peptides was assessed by electron
microscopy analysis.
Method ¨ Peptide solutions (2 mM peptide in 10 mM Tris buffer pH 7.2),
were incubated overnight at room temperature. Fibrils formation was assessed
using
10 pl sample placed on 200-mesh copper grids, covered with carbon-stabilized
formvar film (SPI Supplies, West Chester PA). Following 20-30 seconds of
incubation, excess fluid was removed and the grids were negatively stained
with 2%
TM
uranyl acetate in water. Samples were viewed in a JEOL 1200EX electron
microscope
operating at 80 kV.
Results ¨ To further characterize the structures formed by the various
peptides,
negative staining electron microscopy analysis was effected. In accordance
with
previous results, filamentous structures were observed for all peptides
(Figures la-f)
but F2A which generated amorphous fibrils (Figure 7b). Frequency of appearance
of
fibrils formed by the I5A and L6A peptides (Figures 7e and 7f, respectively)
was
lower in comparison to that of wild type (Figure 7d), NIA, and G3A peptides
(Figures
7a and 7c, respectively). Although the EM fields shown for peptides F2A, I5A
and
L6A, were rarely observed, the results presented by these images support the
quantitative results presented in the previous sections and thus provide
qualitative
analysis of fibril morphology.
The tangled net-like structures that were observed for the wild-type, NIA, and
G3A peptides could be explained by the fast kinetics of formation of these
fibrils (see
Example 2). More distinct structures and longer fibrils, albeit less frequent,
were

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44
observed with peptides I5A and L6A. These longer fibrils may be a result of a
slower
kinetics, which allow for a more ordered fibril organization.
Taken together, the qualitative results of the electron microscopy and CR
analyses strongly suggest that the phenylalanine residue in the hexaamyloid
peptide is
crucial for its amyloidogenic potential.
EXAMPLE 6
Mapping recognition domains in the hIAPP basic amyloidogenic unit ¨
rational and MBP-IAPP fusion protein synthesis
To systematically map and compare potential recognition domains, the ability
of hIAPP (GenBank Accession No. gi:4557655) to interact with an array of 28
membrane-spotted overlapping peptides that span the entire sequence of hIAPP
(i.e.,
hIAPPI-to, hIAPP2_11...., hIAPP28-37) was addressed [Mazor (2002) J. Mol.
Biol.
322:1013-24].
Materials and Experimental Procedures
Bacterial strains - E. coli strain TG-1 (Amersham Pharmacia, Sweden) was
used for molecular cloning and plasmid propagation. The bacterial strain
BL21(DE3)
(Novagen, USA) was used for protein overexpression.
Engineering synthetic IAPP and MBP-IAPP fusion proteins ¨ A synthetic
DNA sequence of human IAPP modified to include a bacterial codon usage (SEQ ID
NO: 58) was generated by annealing 8 overlapping primers (SEQ ID NOs. 50-57).
PCR was effected through 30 cycles of 1 minute at 95 C, one minute at 55 C,
and
one minute at 72 C. The annealing product was ligated and amplified using
primers
IAPP1 (SEQ ID NO: 50) and IAPP8 (SEQ ID NO: 57). An MBP-IAPP (MBP
GenBank Accession No. gi:2654021) fusion sequence was then constructed using
the
IAPP synthetic template, which was amplified using primer YAR2 (SEQ ID NO: 60)

and primer YAR1 (SEQ ID NO. 59), thereby introducing a V8 Ek cleavage site and
a
(His)6 tag at the N-terminus of IAPP. The two primers included a Not I and an
Nco I
cloning sites, respectively. The resultant PCR product was digested with Nco I
and
Not I and ligated into the pMALc2x-NN expression vector. The pMALc2x-NN
expression vector was constructed by cloning the polylinker site of pMALc-NNI9
into
pMALc2x (New England Biolabs, USA) [BACH (2001) J. Mol. Biol. 312:79-93].

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Protein expression and purification ¨ E. coli BL21 cells transformed with
expression plasmid pMALc2x-IAPP encoding MBP-IAPP under the strong Ptac
promoter were grown in 200 ml of LB medium supplemented with 100 1.1g/m1
ampicillin and 1% (WN) glucose. Once reaching an optical density of A600 =
0.8,
5 protein expression was induced with 0.1 or 0.5 mM IPTG at 30 C for 3
hours (h).
Cell extracts were prepared in 20 mM Tric-HC1 (pH 7.4), 1 mM EDTA, 200
mM NaC1 and a protease inhibitors cocktail (Sigma) using a freeze-thaw
followed by
a brief sonication as previously described [Gazit (1999) J. Biol. Chem.
274:2652-
2657]. Protein extracts were clarified by centrifugation at 20,000 g and
stored at 4 C.
10 MBP-IAPP fusion protein was purified by passing the extract over an
amylose resin
column (New England Biolabs, USA) and recovered by elution with 20 mM maltose
in the same buffer. Purified MBP-IAPP was stored at 4 C. Protein
concentration
was determined using the Pierce Coomassie plus reagent (Pierce, USA) with BSA
as a
standard. MBP and MBP-IAPP protein fractions were analyzed on SDS/12 %
15 polyaciylamide gels, which were stained with GelCode Blue (Pierce, USA).
To study whether the disulfide bond in the MBP-IAPP are oxidized, purified
MBP and MBP-IAPP proteins were reacted with 5 equivalents of N-iodoacetyl-N'-
(8-
sulfo- 1 -naphthyl) ethylenediamine (IAEDANS) (Sigma, Rehovot, Israel) for
overnight at room temperature in the dark. Free dye was separated from labeled
20 protein by gel filtration chromatography on a QuickSpin G-25 Sephadex
column.
MBP and MBP-IAPP fluorescence was then determined. Only small fluorescence
labeling was detected (on average less than 0.1 probe molecules per protein
molecules) and there was no significant difference between the labeling of MBP
and
MBP-IAPP, which suggested that the disulfide bridge in the expressed IAPP
25 molecules was predominantly oxidized.
Results
Expression and purification of recombinant MBP-IAPP - Since previous
attempts to express the intact hIAPP in bacteria were unsuccessful, the
protein was
expressed as an MBP fusion, which protected MAPP from undesirable aggregation
30 during expression [Bach (2001) J. Mol. Biol. 312:79-93]. Synthesis of
the fusion
protein was effected using a bacterial codon usage as shown in Figure 8a. The
resulting fusion sequence was cloned into pMALc2x-NN as shown in Figure 8b and

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46
introduced into E. coli BL21(DE3). Growth conditions, cell extract preparation
and
protein purification were effected as described hereinabove. IPTG induction
resulted
in the accumulation of high levels of MBP-IAPP in the soluble fraction with
less then
5% of the MBP-IAPP fusion protein was found in the insoluble fraction of the
cell
extract (data not shown). Aliquots from typical purification steps of MBP and
MBP-
IAPP are shown in Figure 9. As shown, the 48 kDa MBP-IAPP accumulated to 25%
of the total soluble protein as calculated by densitometric scanning of
GelCode Blue-
stained SDS/Polyacrylamide gels. When induced at 30 C in a shake flask (A600 =

2.0), MBP-IAPP accumulated as soluble protein in the cytoplasm at a level of
about
150 mg/1 of cell culture. Despite losses during purification, MBP-IAPP was
purified
to near-homogeneity at a yield of 80 mg/1 of cells. For future application and

convenient homogeneity purification of IAPP, in addition to the factor Xa
cleavage
site for removal of the MBP tag, an additional His-Tag was also included
(Figure 8b).
The His-Tag could be removed by Ek V8 cleavage at the N-terminal Lys residue
of
the IAPP sequence, resulting in the release of wild type TAPP.
EXAMPLE 7
Identification of molecular recognition sequences in the hL4PP polypeptide
IAPP peptide array construction - Decamers corresponding to consecutive
overlapping sequences of hIAPP1_37 SEQ ID NOs. 61-88) were synthesizes on a
cellulose membrane matrix using the SPOT technique (Jerini AG, Berlin,
Germany).
The peptides were covalently bound to a Whatman 50 cellulose support (Whatman,

Maidstone, England) via the C-terminal amino-acids. N-terminal acetylation was

used for peptide scanning because of higher stability to peptide degradation,
and
better representation of the native recognition motif.
Peptides Synthesis - Peptide synthesis was effected using solid-phase
synthesis
methods performed by Peptron, Inc. (Taejeon, Korea). Correct identity of the
peptides
was confirmed by ion spray mass-spectrometry using a HP 1100 series LC/MSD
[Hewlett-Packard Company, Palo Alto, CA]. The purity of the peptides was
confirmed by reverse phase high-pressure liquid chromatography (RP-HPLC) on a
C18
column, using a 30 minute linear gradient of 0 to 100% acetonitrile in water
and 0.1%
trifluoroacetic acid (TFA) at flow rate of 1 ml/min.

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Binding studies ¨ The cellulose peptide array was initially blocked with 5 %
(V/V) non fat milk in Tris buffered saline (TBS, 20 mM Tris pH 7.5, 150 mM
NaC1).
Thereafter, cellulose membrane was incubated in the presence of 10 pg/m1 MBP-
IAPPI.37 at 4 C for 12 h in the same blocking buffer. The cellulose membrane
was
-rm
then washed repeatedly with 0.05 % Tween 20 in TBS. MBP-IAPP1.37 bound to the
cellulose membrane was detected with an anti MBP monoclonal antibody (Sigma,
Israel). HRP-conjugated goat anti mouse antibodies (Jackson Laboratories, USA)

were used as a secondary antibody. Immunoblots were developed using the
Renaissance western blot Chemiluminesce,nce Reagent (NEN, USA) adcording to
io Manufacturer's instructions and signal was quantified using densitometry.
Regeneration of the cellulose membrane for reuse was carried out by sequential

washing with Regeneration buffer I including 62.5 mM Tris, 2% SDS, 100 Mm 2-
mercaptoethanol, pH 6.7, and Regeneration buffer 11 including 8 M urea, 1%
SDS,
0.1% 2-mercaptoethanol. Efficiency of the washing steps was monitored by
contacting the membrane with the chemiluminescence reagent, as described.
Results
Identification of binding sequences in the IAPP polypeptide - To identify
structural motifs in the IAPP molecule that mediates the intermolecular
recognition
between laIAPP molecules, 28 possible overlapping decamers corresponding to
amino acids 1-10 up to 28-37 of the hIAPP1.37 molecule were synthesized on a
cellulose membrane matrix. Cellulose membrane-bound peptides were incubated
with MBP-11LAPP 1_37 overnight Following washing of the cellulose membrane in
a
high-salt buffer, inununoblots on the cellulose membrane were analyzed and
binding
was quantified by densitometry (Figure 10b). It will be appreciated that the
measured
binding is semiquantitative, since peptide coupling efficiency during
synthesis can
vary.
As shown in Figures 10a-b, a number of peptide segments exhibited binding to
MBP-IAPP; An amino acid sequence localized to the center of the LAPP
polypeptide
hIAPP7-16 to hIAPP12-20 displayed the most prominent binding to MBP-MAPPI.
37.. Another binding region was identified at the C-terminal part of IAPP
(hIAPP19-28
to hIAPP21.30), although binding in this case was considerably less prominent;
A third
binding spot was located to the N-terminal part of IAPP (hIAPP2_11), however,
no

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typical distribution around a central motif was evident in this case,
suggesting that this
result may be false. Even after overexposure of the blot (data not shown), no
binding
near-ihis peptide (to either MAPPi_io or hIAPP3_12) was detected. Furthermore
given
the close proximity of the 2-11 region to the disulfide bridge may not allow
the
process of fibrillization under physiological conditions.
To rule out involvement of MBP itself in binding the arrayed peptides, the
peptide coupled cellulose membrane was incubated with MBP alone and analyzed
by
immunobloting. No binding was identified after development of the membrane
(not
shown).
These results identified in addition to the previously defined binding motif
of
hIAPP [i.e., basic amyloidogenic unit, MAPP20-29, Westermark (1990) Proc.
Natl.
Acad. Sci. 13:5036-40; Tenidis (2000) J. Mol. Biol. 295:1055-1071; Azriel and
Gazit
(2001) J. Biol. Chem. 276:34156-34161], a major central domain of molecular
recognition within hIAPP. The profile of the binding distribution of the
peptide array
(Figure 10b), suggests that NFVLH (SEQ ID No. 17) may serve as the core
recognition motif
EXAMPLE 8
Characterization of aggregation kinetics of hIAPP peptide fragments as
monitored by turbidity measurements
Binding analysis of the recombinant MBP-hIAPP fusion protein to the hIAPP
peptide array (Example 7), identified a putative self-assembly domain within
the
central part of the hIAPP protein.
In order to identify the minimal structural motif that is capable of forming
amyloid fibrils, a series of peptides encompassed within the putative self-
assembly
domain were tested for aggregation as monitored using turbidity measurements
at
405 nm.
Table 3 below, illustrates the examined peptides.
Table 3
HIAPP peptide fragments SEQ ID NO: Peptide sequence
(hIAPP coordinates)
14-22 14 NFLVHSSNN
14-20 15 NFLVHSS
15-20 16 FLVHSS

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14-18 17 NFLVH
15-19 18 FLVHS
15-18 19 FLVH
Materials and Experimental Procedures
Kinetic Aggregation Assay ¨ freshly prepared peptide stock solutions were
generated by dissolving the lyophilized form of the peptides in dimethyl
sulfoxide
(DMSO) at a concentration of 100 mg/ml. To avoid any pre-aggregation, fresh
stock
solutions were prepared for each experiment. Peptide stock solutions were
diluted
into the assay buffer in enzyme-linked immunosorbent assay (ELISA) plate wells
as
follows: 8 !IL of peptide stock solutions were added to 92 pi, of 10 mM Tris,
pH 7.2
(hence the final concentration of the peptide was 8 mg/ml in the presence of
8%
DMSO). Turbidity data were collected at 405 urn. Buffer solution containing
the
same amount of DMSO as the tested samples was used as blank, which was
subtracted from the results. Turbidity was measured continuously at room
temperature using THERMOmax ELISA plate reader (Molecular Devices, Sunnyvale
CA).
Results
Turbidity assay was performed in-order to determine the ability of the various

peptides (Table 3) to aggregate in an aqueous medium. Fresh stock solutions of
the
different peptide fragments were made in DMSO, and then diluted into a Tris
buffer
solution and turbidity, as a hallmark of protein aggregation, was monitored
for two
hours. As shown in Figure 11, the peptides NFLVHSS, FLVHSS and FLVHS
exhibited high turbidity. It will be appreciated that the lag-time, as was
previously
reported for amyloid formation by the NFGAIL short peptide [Tenidis (2000)
Supra],
is very short or lacking at all and thus could not be detected under these
experimental
conditions, however the aggregation kinetic profiles were similar to those
obtained for
the hexapeptide hIAPP22_27 (NFGAIL). On the other hand, the peptide NFLVHSSNN
exhibited very low turbidity, while NFLVH and FLVH have shown almost no
turbidity at all. Even after significantly longer incubation no significant
turbidity was
observed with the latter two peptides. The lack of amyloid fibrils formation
may be
due to electrostatic repulsion of the partially charged histidine residues.

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EXAMPLE 9
Examination of hL4PP peptide amyloidogenic through Congo Red (CR)
binding assay
Congo red (CR) staining combined with polarization microscopy was utilized
5 to test amyloidogenicity of the peptides of the present invention.
Amyloid fibrils
bind CR and exhibit gold/green birefringence under polarized light [Puchtler
(1966)
J. Histochem. Cytochem. 10:355-364].
Materials and Experimental Procedures
Congo Red Staining and Birefringence ¨ A 10 1.11., suspension of 8 mg/ml
10 peptide solution in 10 mM Tris buffer, pH 7.2 aged for at least one day
was allowed
to dry overnight on a glass microscope slide. Staining was performed by the
addition
of a 10 I.LL suspension of saturated Congo Red (CR) and NaCl in 80% ethanol
(v/v)
solution as previously described [Puchtler (1966) Supra]. The solution was
filtered
via 0.45 pm filter. The slide was then dried for few hours. Birefringence was
15 determined with a SZX-12 Stereoscope (Olympus, Hamburg, Germany)
equipped
with cross polarizers.
Results
Congo Red Staining and Birefringence- In order to determine any possible
amyloidal nature of the aggregates formed at the turbidity assay (see Example
8), a
20 CR birefringence assay was performed. Peptide fragments were tested for
amyloidogenecity by staining with CR and examination under a light microscope
equipped with cross-polarizers. Consistent with the kinetic assay results, and
as
shown in Figures 120b-c and 12e, the peptides NFLVHSS, FLVHSS and FLVHS
exhibited a typical birefringence. On the other hand, peptides NFLVHSSNN,
NFLVH
25 and FLVH exhibited very weak birefringence or no birefringence at all
(Figures 12a,
12d and 120. Peptide NFLVHSSNN exhibited a weaker characteristic birefringence

(Figure 12a). T he peptide NFLVH exhibited a powerful smear of birefringence
at the
edges of the sample (Figure 12d). The peptide FLVH exhibited no birefringence
(Figure 120. In order to test whether the FLVH peptide did not form amyloid
fibrils
30 due to a long lag-time, a sample of five days aged peptide solution was
examined.
The same peptide was also tested in aqueous solution and at very high
concentrations
(10 mg/ml), however no Birefringence was detected in all cases indicating the
peptide

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did not form amyloid (data not shown).
EXAMPLE 10
Ultrastructural analysis of the fibrillogenic hIAPP peptides
The fibrillogenic potential of the various peptides was assessed by electron
microscopy analysis.
Materials and Experimental Procedures
Transmission Electron Microscopy ¨ A 10 I, sample of 8 mg/ml peptide
solution in 10 inM Tris buffer, pH 7.2 aged for at least one day was placed on
400-
mesh copper grids (SPI supplies, West Chester PA) covered by carbon-stabilized
Formvar film. Following 1 minute, excess fluid was removed, and the grid was
then
negatively stained with 2% uranyl acetate in water for another two minutes.
Samples
were viewed in a JEOL 1200EX electron microscope operating at 80 kV.
Results
To further characterize the structures formed by the various peptides,
negative
staining electron microscopy analysis was effected. In accordance with
previous
results, all peptide fragments exhibited fibrillar structures except the FLVH
peptide in
which only amorphous aggregates were found (Figures 13a-f). NFLVHSSNN peptide
exhibited long thin coiling filaments similar to those formed by the full-
length peptide
as described above (Figure 13a). Peptides NFLVHSS, FLVHSS, FLVHS exhibited
large broad ribbon-like fibrils as described for the NFGAIL fragment [Tenidis
(2000)
Supra., Figures 13c-e, respectively]. The fibrils formed by NFLVH peptide were
thin
and short and could be considered as protofilaments rather than filaments.
Their
appearance was at much lower frequency, and the EM picture does not represent
the
general fields but rather rare events (Figure 13d). As shown in Figure 13f,
the FLVH
peptide mediated the formation of amorphous aggregates.
EXAMPLE 11
Secondary structure analysis of hL4PP peptide fragments
Fourier transform infrared spectroscopy (FT-IR) was effected to determine the
secondary structure of the hIAPP amyloidogenic peptide fibrils and the non-
fibrillar
peptides.

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Materials and Experimental Procedures
Fourier Transform Infrared Spectroscopy - Infrared spectra were recorded
using a Nicolet Nexus 470 FT-IR spectrometer with a DTGS detector. Samples of
aged peptide solutions, taken from turbidity assay, were suspended on a CaF2
widows (Sigma)-plate and dried by vacuum. The peptide deposits were
resuspended
with double-distilled water and subsequently dried to form thin films. The
resuspension procedure was repeated twice to ensure maximal hydrogen to
deuterium
exchange. The measurements were taken using a 4 cm' resolution and 2000 scans
averaging. The transmittance minima values were determined by the OMMC
to analysis program (Nicolet).
Results
FT-IR studies ¨ As shown in Figure 14a-f, all the fibrillar peptides exhibited

FT-1R spectra with a well-defined minimum bands typical for f3-sheet structure
around
1620-1640 cm-I. On the other hand the spectrum of the tetrapeptide FLVH that
has no
appearance for fibrils according to the other methods, is typical for a random
coil
structure. The NFLVHSSNN peptide spectrum exhibited a transmittance minimum at

1621 cm' indicating a large 13 -sheet content, as well as minima at 1640 cm-1
and 1665
suggesting presence of non- 13 structures. Another minor minimum was observed
at
1688 cm-1 indicative for anti-parallel 13-sheet (Figure 14a). The NFLVHSS
peptide
spectrum exhibited major minimum band at 1929 cm' 1675 cm-I. this spectrum is
classical for an anti-parallel 13 -sheet structure (Figure 14b). A similar
spectrum was
observed for the peptide FLVHS with a major minimum at 1625 cm-1 and a minor
minimum at 1676 cm-I (Figure 14e). The spectrum of FLVHSS peptide showed also
a
major minimum at 1626 cm-1. The spectrum had also some minor minima around
1637-1676 cm-1 but those were shaped more like noise than signal (Figure 14c).
The
spectrum of NFLVH peptide showed a minimum at 1636 cm-I which was also
indicative of 13-sheet, however, in comparison with the other spectra, this
band was
shifted which could indicate presence of non-13 structures, as well as
observed minima
at 1654 cm-1 and 1669 cm-1 (Figure 14d). By contrast, the FLVH peptide
spectrum
exhibited no minimum at 1620-1640 cm-I, but showed multiple minima around 1646-

1675 cm-I typical to random coil structure (Figure 140.
To study whether the FLVH tetrapeptide could not form amyloid fibrils at all

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or the undetectable fibrils formation was a result of a slow kinetics, a
solution of the
peptide at the same experimental conditions was incubated for two months and
the
existence of fibrils was tested. However, no evidence for amyloid fibril
formation
was detected using EM microscopy, CR staining, or FT-IR spectroscopy. These
results may suggest that tetrapeptides are incapable of forming fibrils due to
energetic
consideration. That is, the energetic contribution of the stacking of a strand
composed
of three peptide bonds is lower than the entropic cost of oligomerization.
Taken together, the ultrastructural observations are consistent with the
findings
as determined by the turbidity and Congo red birefringence assays. All
together the
experimental data identified a novel pentapeptide element within the MAPP
peptide,
the FLVHS peptides, which has strong amyloid forming capability.
Interestingly, an
NFLVH peptide found in the same central domain of the hIAPP polypeptide was
found to be amyloidogenic however, the ability thereof to form fibrils was
somehow
inferior.
EXAMPLE 12
Identification of the minimal amyloidogenic peptide fragment of Medin
Background
Medin (GenBank Accession No. gi:5174557) is the main constitute of aortic
medial amyloid deposits [Haggqvist (1999) Proc. Natl. Acad. Sci. USA. 96:8674-
8669]. Previous studies found aortic medial amyloid in 97% of the subjects
above
the age of 50 [Mucchiano (1992) Am. J. Pathol. 140:811-877]. However, the
pathological role of those amyloid deposits is still unknown. It was suggested
that
these amyloid play a role in the diminished elasticity of aortic vessels that
is related
to old age [Mucchiano (1992) Supra; Haggqvist (1999) Supra]. While the study
clearly identified a tryptic peptide NFGSVQFV as the medin amyloidogenic
peptide,
the minimal sequence of the peptide that is still amyloidogenic and the
molecular
determinants that mediate the amyloid formation process were not determined.
Such
information is critical for true understanding of the fibrillization process
in the
specific case of Medin but also as a paradigm for the process of amyloid
fibrils
formation in general.

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The minimal active fragment of Medin was determined using functional and
structural analyses of truncated analogues derived from the published
octapeptide
[llaggqvist (1999) Supra].
Materials and Experimental procedures
Peptide synthesis is described in Example 7.
Table 4 below illustrates the studied peptides.
Table 4
Peptide sequence SEQ ID NO:
NH2-NFGSVQVF-COOH 20
NH2-NFGSVQ -COOH 21
NH2-NFGSV -COOH 22
NH2- FGSVQ -COOH 23
NH2- GSVQ -COOH 24
NH2- FGSV -COOH 25
NH2-NAGSVQ -COOH 26
Results
In order to get further insights into the structural elements of Medin that
retain the molecular information needed to mediate a process of molecular
recognition and self-assembly, the ability of short peptide fragments and
analogues of
Medin to form amyloid fibrils in vitro was studied. Figure 15a shows a
schematic
representation of the chemical structure of the largest peptide fragment
studied.
EXAMPLE 13
Kinetics of aggregation of Medin-derived peptide fragments
Turbidity assay was effected as described in Example 8.
In order to get first insights regarding the aggregation potential of the
various
Medin derived peptides, turbidity assay was performed. Freshly made stocks of
the
amyloidogenic octapeptide and truncated analogues thereof were prepared in
DMSO.
The peptides were than diluted to aqueous solution and the turbidity was
monitored
by following the absorbance at 405 nm as a function of time. As shown in
Figure
16a, the NFGSV pentapeptide exhibited the highest degree of aggregation within
minutes of incubation. Physical examination of the solution indicated that the
peptide formed a gel structure. The kinetics of aggregation of the NFGSVQV
octapeptide was too fast to be measured since turbidity was already observed
immediately with the dilution into aqueous solution (Figures 16a-b). Similar
fast

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kinetics were also observed with the GSVQ tetrapeptide. The truncated NFGSVQ,
FGSVQ, and FGSV peptides showed a gradual increase in turbidity over ¨30
minutes
(Figure 16b) which was followed by a slight decrease, which could be explained
by
sedimentation of large aggregates. Altogether, such kinetics and turbidity
values were
5 similar to those previously observed with amyloidogenic peptides of
similar size
(Azriel and Gazit, 2001).
EXAMPLE 14
Ultrastructural analysis of Medin-derived peptide fragments
10 Electron microscopy analysis was effected as described in Example 10.
The fibrillization potential of Medin-derived peptide fragments was effected
by electron microscopy (EM) using negative staining. Stock solutions of the
peptide
fragments were suspended and aged for 4 days. Fibrillar structures were
clearly seen
in solutions that contained both the NFGSVQFA octapeptide (Figure 17a) and the
15 truncated NFGSVQ (Figure 17b). In both cases the structures were similar
to those
observed with much longer polypeptides, such the IAPP and the 0-amyloid (AP)
polypeptides. The shorter gel-forming NFGSV pentapeptide did not form a
typical
amyloid structure but a network of fibrous structures (Figure 17c). It should
be noted
that fibrous networks were recently observed upon the gelation of the
glutathione
20 peptide [Lyon and Atkins, (2001) J. Am. Chem. Soc. 123:4408-4413]. No
typical
fibrils could be detected in solutions that contained the FGSVQ pentapeptide,
the
GSVQ tetrapeptide, or the FGSV tetrapeptide in spite of extensive search.
While in
the case of the FGSVQ peptide (Figure 17d) somewhat fibrillar and ordered
structure
could be seen, although significantly different than those formed by typical
25 amyloidogenic peptide), in the case of the GSVQ and the FGSV peptides,
no fibrillar
structures could be found (Figures 17e and 17f, respectively).
EXAMPLE 15
Examination of amyloidogenic performance of Medin-derived peptides
30 through Congo Red (CR) binding assay
CR staining was effected as described in Example 9.

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A CR staining was effected to determine whether the structures formed by the
various Medin-derived peptides show a typical birefringence. As shown in
Figure
18b, the NFGSVQ hexapeptide bound CR and exhibited a characteristic bright and

strong green-gold birefringence. The NFGSVQFV octapeptide also exhibited
significant birefringence (Figure 18a), although less typical than that
observed with
the hexapeptide. The gel-forming NFGSV peptide deposits exhibited very low
degree of birefringence (Figure 18c). The FGSVQ and FGSV peptide showed no
birefringence upon staining with CR (Figures 18d and 18f, respectively). There
was
clearly no significant difference between those two peptides and a negative
control
(i.e., buffer solution with no peptide) Interestingly, unexpected high level
of
birefringence was observed with the GSVQ tetrapeptide (Figure 18e), while the
morphology of the structures formed therefrom (Figure 18e) was clearly
different
from that of amyloid fibrils, indicating that these structures may have a
significant
degree of order that is reflected in strong birefringence.
EXAMPLE 16
The effect of phenylalanine substitution on the self-assembly of Medin
T elucidate a possible role for the phenylalanine residue in the process of
amyloid fibrils formation by the minimal amyloid-forming hexapeptide, the
phenylalanine amino acids was replaced with an alanine. The alanine-
substituted
peptide was prepared and examined in the same way as described for the various

fragments of Medin. As shown in Figure 19a, a significantly lower turbidity
was
observed with the alanine-substituted peptide as compared to the wild-type
hexapeptide. When aged solution of the NAGSVQ peptide was visualized by EM, no
clear fibrillar structures could be detected (Figure 19b). This is in complete
contrast to
the high abundance fibrillar structures seen with the wild-type peptide
(Figure 17b).
Furthermore, the structures that were visualized did not show any degree of
order as
observed with the NFGSV and FGSVQ peptides as described above, Figures 17c-d,
but were very similar to the completely non-fibrillar structures as were
observed with
the FGSV tetrapeptide (Figure 17e). Interestingly, some degree of
birefringence could
still be detected (Figure 19c) with the alanine-substituted peptide (as was
observed
with the GSVQ peptide, Figure 18e). These results raise further doubts
regarding the

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use of CR staining as a sole indicator of amyloid formation [Khurana (2001) J.
Biol.
Chem. 276:22715-22721].
Altogether these results show that the truncated fragment of Medin which is
capable of forming amyloid fibrils is the hexapeptide NFGSVQ (SEQ ID NO: 21),
although a shorter pentapeptide fragment, NFGSV (SEQ ID NO: 22), exhibited a
network of fibrous structures which were not typical of amyloids. The amyloid
forming NFGSVQ hexapeptide is noticeably similar to the minimal amyloidogenic
fragment of the islet amyloid polypeptide (IAPP, see Examples 1-5). Taken
together,
the results are consistent with the assumed role of stacking interactions in
the self-
assembly processes that lead to the formation of amyloid fibrils and the
suggested
correlation between amyloid fibrils and 0-helix structures.
EXAMPLE 17
Identification of the minimal amyloidogenic peptide fragment of human
Calcitonin
Human Calcitonin (hCT, GenBank Accession No. gi:179880) is a 32 amino
acid long polypeptide hormone that is being produced by the C-cells of the
thyroid
and is involve in calcium homeostasis [Austin and Health (1981) N. Engl. J.
Med.
304:269-278; Copp (1970) Annu. Rev. Physiol. 32:61-86; Zaidi (2002) Bone
30:655-
663]. Amyloid fibrils composed of hCT were found to be associated with
medullary
carcinoma of the thyroid [Kedar (1976) Isr. J. Sci. 12:1137; Berger (1988)
Arch. A.
Pathol. Mat. Histopathol. 412:543-551; Arvinte (1993) J. Biol. Chem. 268:6415-
6422]. Interestingly, synthetic hCT was found to form amyloid fibrils in vitro
with
similar morphology to the deposits found in the thyroid [Kedar (1976) Supra;
Berger
(1988) Supra; Arvinte (1993) Supra; Benvenga (1994) J. Endocrinol. Invest.
17:119-
122; Bauer (1994) Biochemistry 33:12276-12282; Kanaori (1995) Biochemistry
34:12138-43; Kamihara (2000) Protein Sci. 9:867-877]. The in vitro process of
amyloid formation is affected by the pH of the medium [23]. Electron
microscopy
experiments have revealed that the fibrils formed by hCT are approximately 80A
in
diameter and up to several micrometers in length. The fibrils are often
associated with
one another and in vitro amyloid formation is affected by the pH of the medium

[Kamihara (2000) Supra.].

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Calcitonin has been used as a drug for various diseases including Paget's
disease and osteoporosis. However, the tendency of hCT to associate and form
amyloid fibrils in aqueous solutions at physiological pH is a significant
limit for its
efficient use as a drug [Austin (1981) Supra; Copp (1970) Supra; Zaidi (2002)
Supra].
Salmon CT [Zaidi (2002) Supra], the clinically used alternative to hCT, causes
immunogenic reaction in treated patients due to low sequence homology.
Therefore,
understanding the mechanism of amyloid formation by hCT and controlling this
process is highly important not only in the context of amyloid formation
mechanism
but also as a step toward improved therapeutic use of Calcitonin.
Circular dichroism (CD) studies have shown that in water monomeric hCT has
little ordered secondary structure at room temperature [Arvinte (1993) Supra].

However, studies of hCT fibrils using circular dichroism, fluorescence, and
infrared
spectroscopy revealed that fibrillated hCT molecules have both a-helical and
I3-sheet
secondary structure components [Bauer (1994) Supra]. NMR spectroscopy studies
have shown that in various structure promoting solvents like TFE/H20, hCT
adopts an
amphiphilic a-helical conformation, predominantly in the residue range 8-22
[Meadows (1991) Biochemistry 30:1247-1254; Motta (1991) Biochemistry 30:10444-
10450]. In DMSO/1120, a short double-stranded antiparallel [3-sheet form in
the
central region made by residues 16-21 [Mona (1991) Biochemistry 30:2364-71].
Based on this structural data and the proposed role of aromatic residues in
the
process of amyloid formation, the present inventor has identified a short
peptide
fragment, which is sufficient for mediating Calcitonin self-assembly [Reches
(2002) J.
Biol. Chem. 277:35475-80].
The studied peptides ¨ Based on the previously reported susceptibility of
amyloid formation to acidic pH [Kanaori (1995) Supra], it was suggested that
negatively-charged amino-acids, which undergo protonation at low pH, may play
a
key role in the process of amyloid formation. The only negatively-charged
amino-acid
in hCT is Asp15 (Figure 20a). Furthermore, a critical role for residues Lys18
and Phel9
in the oligomerization state and bioactivity of hCT was recently shown
[Kazantzis
(269) Eur. J. Biochem. 269:780-91]. Together with the occurrence of two
phenylalanine residues in the region focused the structural analysis of the
amyloidogenic determinants in hCT to amino acids 15-19. Figure 20b shows a

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schematic representation of the chemical structure of the longest peptide and
Table 5
below, indicates the various peptide fragments that were used in the study.
Table 5
Amino acid coordinates on Peptide sequence SEQ ID NO:
hCT
15-19 NH2-DFNKF-COOH 27
16-19 NH2- FNKF-COOH 28
15-18 NH2-DFNK -COOH 29
15-17 NH2-DFN -COOH 30
F>A 15-19 NH2-DANKF-COOH 31
EXAMPLE 18
Ultrastructural analysis of Calcitonin-derived peptide fragments
Electron microscopy analysis was effected as described in Example 10.
The fibrillization potential of Calcitonin-derived peptide fragments was
effected by electron microscopy (EM) using negative staining. Stock solutions
of the
peptide fragments were suspended in 0.02M NaC1, 0.01M Tris pH 7.2, aged for 2
days
and negatively stained. Fibrillar structures, similar to those formed by the
full-length
polypeptide [Arvinte (1993) Supra; Benvenga (1994) Supra; Bauer (1994) Supra;
Kanaori (1995) Supra; Kamihara (2000) Supra], were clearly seen with high
frequency in solutions that contained the DFNKF pentapeptide (Figure 21a). The
shorter DFNK tetrapeptide also formed fibrillar structures (Figure 21b).
However, the
structures formed were less ordered as compared to those formed by the DFNKF
pentapeptide. The amount of fibrillar structures formed by DFNK was also lower
as
compared to the DFNKF peptapeptide. No clear fibrils could be detected using
- solutions that contained the FNKF tetrapeptide and the DFN tripeptide, in
spite of
extensive search. In the case of the FNKF tetrapeptide only amorphous
aggregates
could be found (Figure 21c). The DFN tripeptide formed more ordered structures

(Figure 21d) that resembled the structure formed by gel-forming tripeptide
[Lyon
(2001) Supra]. To study whether the FNKF tetrapeptide and the DFN tripeptide
peptide cannot form fibrils whatsoever or the observation is a result of slow
kinetics, a
solution of the peptides at the same experimental conditions was incubated for
two
weeks. Also in this case no clear fibrillar structures could be detected (data
not
shown).

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EXAMPLE 19
Examination of amyloidogenic performance of Calcitonin-derived peptides
through Congo Red (CR) binding assay
CR staining was effected as described in Example 9.
5 A CR staining was effected to determine whether the structures formed by
the
various hCT-derived peptides show a typical birefringence. As shown in Figures

22a-d, all the studies peptides showed some degree of birefringence. However,
the
green birefringence, which was observed with the DFNKF-pentapeptide was clear
and strong (Figure 22a). The level of birefringence that was observed with the
other
10 peptides was lower but significant since no birefringence could be
detected using
control solutions which did not contain the peptides. The lower level of
birefringence of the DFNK tetrapeptide (Figure 22b) was consistent with the
lower
extent of fibrillization as observed using EM (Figure 21b). It will be
appreciated,
though, that the birefringence observed with the FNKF tetrapeptide and the DFN
15 tripeptide might represent some degree of ordered structures [Lyon
(2001) Supra].
EXAMPLE 20
Secondary structure of the aggregated hCT-derived peptides
FT-IR spectroscopy was effected as described in Example 11.
20 Amyloid deposits are characteristic of fibrils rich with (3-pleated
sheet
structures. To get a quantitative information regarding the secondary
structures that
were formed by the various peptide fragments FT-IR spectroscopy was used. Aged

peptide solutions were dried on CaF2 plates forming thin films as described in

Example 11. As shown in Figure 23, the DFNKF pentapeptide exhibited a double
25 minima (at 1639 cm-1 and 1669 cm-1) an amide I FT-1R spectrum that is
consistent
with anti-parallel (3-sheet structure and is remarkably similar to the
spectrum of the
amyloid-forming hexapeptide fragment of the islet amyloid polypeptide [Tenidis

(2000) Supra]. The amide I spectrum observed with the DFNK tetrapeptide
(Figure
23) was less typical of a 13-sheet structure. While it exhibited a minimum at
1666
30 cm-1 that may reflect an anti-parallel (3-sheet it lacked the typical
minimum around
1620-1640 cm-1 that is typically observed with 0-sheet structures. The FNKF
tetrapeptide exhibited a FT-IR spectrum that is typical of a non-ordered
structure

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61
(Figure 23) and is similar to spectra of the short non-amyloidogenic fragments
of the
islet amyloid polypeptide [Tenidis (2000) Supra]. The DFN tripeptide exhibited
a
double minima (at 1642 cm-1 and 1673 cm-I, Figure 23) amide I FT-IR spectrum
that
is consistent with a mixture of 13-sheet and random structures. This may
further
indicate that the structures observed by EM visualization may represent some
degree
of ordered structure composed of predominantly f3-sheet structural elements.
EXAMPLE 21
The effect of phenylalanine substitution on the self-assembly of Calcitonin-
derived peptides
In order to get insight into a possible role for the phenylalanine residues in
the
process of Calcitonin self-assembly, the phenylalanine amino acids were
replaced
with alanine in the context of the pentapeptide (SEQ ID NO: 31). When aged
solution
of the DANKF pentapeptide was visualized by EM, no clear fibrillar structures
could
be detected (Figure 24a). Structures that were visualized exhibited some
degree of
order (as compared to the amorphous aggregates seen with the FNKF
tetrapeptide),
however, no green-gold birefringence could be observed (Figure 24b). The FT-IR

spectrum of the DANICA pentapeptide was similar to that of the FNKF
tetrapeptide
and other short non-amyloidogenic peptide, typical of non-ordered structures
[Tenidis
(2000) Supra]. Taken together, the effect of the phenylalanine to alanine
substitution
is very similar to the effect of such a change in the context of a short
amyloid-forming
fragment of the islet amyloid polypeptide [Azriel (2001) Supra].
Altogether, the ability of an hCT-derived pentapeptide (SEQ ID NO: 27) to
form well-ordered amyloid fibrils was demonstrated. The typical fibrillar
structure as
seen by electron microscopy visualization (Figure 21a), the very strong green
birefringence upon staining with CR (Figure 22a), and the typical anti-
parallel n-sheet
structure (Figure 23a), all indicate that the DFNKF pentapeptide is a very
potent
amyloid forming agent. Other pentapeptides capable of self-assembling were
shown
in hereinabove. Yet, in terms of the degree of birefringence and electron
microscopy
morphology, the hCT fragment seems to be the pentapeptide with the highest
amyloidogenic potential similar to the potent amyloidogenic fragment of the (3-

amyloid (An) polypeptide, KLVFFAE [Balbach (2000) Biochemistry 39:13748-59].

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It is possible that electrostatic interactions between the opposing charges on
the lysine
and asp artic acids direct the formation of ordered antiparallel structure.
Interestingly,
the DFNK polypeptide exhibited a significantly lower amyloidogenic potential
as
compared to the DFNKF peptide. It is possible that a pentapeptide is a lower
limit for
potent amyloid former. This is consistent with recent results that demonstrate
that two
pentapeptides of IAPP, NFLVH and FLVHS, can form amyloid fibrils, but their
common denominator, the tetrapeptide FLVH, could not form such fibrils (see
Examples 1-5).
EXAMPLE 22
Identification of an amyloidogenic peptide from Lactotransferrin
Amyloid fibril formation by lactotransferrin (GenBank Accession No.
gi:24895280) is associated familial subepithelial corneal amyloid formation
[Sacchettini and Kelly (2002) Nat Rev Drug Discov 1:267-75]. Based on the
proposed role of aromatic residues in amyloid self-assembly, the amyloidogenic
features of a Lactotransferrin-derived peptide, LFNQTG (SEQ 11) NO: 32) were
studied.
Materials and Experimental Procedures ¨ Described in Examples 7 and 10.
Results ¨ To characterize the ability of the Lactotransferrin-derived peptide
to
form fibrilar suprarnolecular ultrastructures, negative staining electron
microscopy
analysis was effected. As shown in Figure 25, under mild conditions,
filamentous
structures were observed for the selected peptide, suggesting that LFNQTG of
Lactotransferrin is important for the polypeptide self-assembly. These results
further
substantiate the ability of the present invention to predict amyloidogenic
peptide
sequences.
EXAMPLE 23
Identification of an amyloidogenic peptide from Serum amyloid A protein
Fragments of Serum amyloid A proteins (GenBank Accession No. gi:134167)
were found in amyloid-state in cases of Chronic inflammation amyloidosis
(Westermark et al. (1992) Biochem. Biophys. Res. Commun. 182: 27-33). Based on

the proposed role of aromatic residues in amyloid self-assembly, the
amyloidogenic

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features of a Serum amyloid A protein-derived peptide, SFFSFL (SEQ lD NO: 33)
were studied.
Materials and Experimental Procedures ¨ Described in Examples 7 and 10.
Results ¨ To characterize the ability of the Serum amyloid A protein-derived
peptide to form fibrilar supramolecular ultrastructures, negative staining
electron
microscopy analysis was effected. As shown in Figure 26, under mild
conditions,
filamentous structures were observed for the selected peptide, suggesting that
SFFSFL
of serum amyloid A protein is important for the polypeptide self-assembly.
These
results further substantiate the ability of the present invention to predict
amyloidogenic
peptide sequences.
EXAMPLE 24
Identification of an amyloidogenic peptide from BriL
The human BRI gene is located on chromosome 13. The amyloid fibrils of the
BriL gene product (GenBank Accession No. gi:12643343) are associated with
neuronal dysfunction and dementia (Vidal et al (1999) Nature 399, 776-781).
Based
on the proposed role of aromatic residues in amyloid self-assembly, the
amyloidogenic
features of a BriL-derived peptide, FENKF (SEQ ID NO: 34) were studied.
Materials and Experimental Procedures ¨ Described in Examples 7 and 10.
Results ¨ To characterize the ability of the BriL-derived peptide to form
fibrilar
supramolecular ultrastructures, negative staining electron microscopy analysis
was
effected. As shown in Figure 27, under mild conditions, filamentous structures
were
observed for the selected peptide, suggesting that FENKF of BriL is important
for the
polypeptide self-assembly. These results further substantiate the ability of
the present
invention to predict amyloidogenic peptide sequences.
EXAMPLE 25
Identification of an amyloidogenic peptide from Gelsolin
Fragments of Gelsolin proteins (GenBank Accession No. gi:4504165) were
found in amyloid-state in cases of Finnish hereditary amyloidosis [Maury and
Nurmiaho-Lassila (1992) Biochem. Biophys. Res. Commun. 183: 227-311. Based on

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the proposed role of aromatic residues in amyloid self-assembly, the
amyloidogenic
features of a Gelsolin-derived peptide, SFNNG (SEQ ID NO: 35) were studied.
Materials and Experimental Procedures ¨ Described in Examples 7 and 10.
Results ¨ To characterize the ability of the Gelsolin-derived peptide to form
fibrilar supramolecular ultrastructures, negative staining electron microscopy
analysis
was effected. As shown in Figure 28, under mild conditions, filamentous
structures
were observed for the selected peptide, suggesting that SFNNG of BriL is
important
for the polypeptide self-assembly. These results further substantiate the
ability of the
present invention to predict amyloidogenic peptide sequences.
EXAMPLE 26
Identification of an amyloidogenic peptide from Serum amyloid P
Amyloid fibril formation by beta-amyloid is promoted by interaction with
serum amyloid-P (GenBank Accession No. gi:2144884). Based on the proposed role
of aromatic residues in amyloid self-assembly, the amyloidogenic features of a
Serum
amyloid P-derived peptide, LQNFTL (SEQ ID NO: 36) were studied.
Materials and Experimental Procedures ¨ Described in Examples 7 and 10.
Results ¨ To characterize the ability of the Serum amyloid P-derived peptide
to
form fibrilar supramolecular ultrastructures, negative staining electron
microscopy
analysis was effected. As shown in Figure 29, under mild conditions,
filamentous
structures were observed for the selected peptide, suggesting that LQNFTL of
Serum
amyloid P is important for the polypeptide self-assembly. These results
further
substantiate the ability of the present invention to predict amyloidogenic
peptide
sequences.
EXAMPLE 27
Identification of an amyloidogenic peptide from Immunoglobulin light
chain
Amyloid fibrils formation by Immunoglobulin light chain (GenBank Accession
No. gi:625508) is associated with primary systemic amyloidosis [Sacchettini
and
Kelly (2002) Nat Rev Drug Discov 1:267-75]. Based on the proposed role of
aromatic

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residues in amyloid self-assembly, the amyloidogenic features of an
hnmunoglobulin
light chain-derived peptide, TLIFGG (SEQ ID NO: 37) were studied.
Materials and Experimental Procedures ¨ Described in Examples 7 and 10.
Results ¨ To characterize the ability of the immunoglobulins light chain-
5 derived peptide to form fibrilar supramolecular ultrastructures, negative
staining
electron microscopy analysis was effected. As shown in Figure 30, under mild
conditions, filamentous structures were observed for the selected peptide,
suggesting
that TLIFGG of the immunoglobulin light chain is important for the polypeptide
self-
assembly. These results further substantiate the ability of the present
invention to
10 predict amyloidogenic peptide sequences.
EXAMPLE 28
Identification of an amyloidogenic peptide from Cystatin C
Amyloid fibril formation by Cystatin C (GenBank Accession No. gi:4490944)
15 is associated with hereditary cerebral amyloid angiopathy [Sacchettini
and Kelly
(2002) Nat Rev Drug Discov 1:267-751. Based on the proposed role of aromatic
residues in amyloid self-assembly, the amyloidogenic features of a Cystatin C-
derived
peptide, RALDFA (SEQ ID NO: 38) were studied.
Materials and Experimental Procedures ¨ Described in Examples 7 and 10.
20 Results ¨ To characterize the ability of the Cystatin C-derived peptide
to form
fibrilar supramolecular ultrastructures, negative staining electron microscopy
analysis
was effected. As shown in Figure 31, under mild conditions, filamentous
structures
were observed for the selected peptide, suggesting that RALDFA of the Cystatin
C is
important for the polypeptide self-assembly. These results further
substantiate the
25 ability of the present invention to predict amyloidogenic peptide
sequences.
EXAMPLE 29
Identification of an amyloidogenic peptide from Transthyretin
Amyloid fibril formation by Transthyretin (GenBank Accession No. gi:72095)
30 is associated with familial amyloid polyneuropathy (Sacchettini and
Kelly (2002) Nat
Rev Drug Discov 1:267-75). Based on the proposed role of aromatic residues in

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amyloid self-assembly, the amyloidogenic features of an Transthyretin-derived
peptide, GLVFVS (SEQ ID NO: 39) were studied.
Materials and Experimental Procedures ¨ Described in Examples 7 and 10.
Results ¨ To characterize the ability of the Transthyretin-derived peptide to
form fibrilar supramolecular ultrastructures, negative staining electron
microscopy
analysis was effected. As shown in Figure 32, under mild conditions,
filamentous
structures were observed for the selected peptide, suggesting that GLVFVS of
Transthyretin is important for the polypeptide self-assembly. These results
further
substantiate the ability of the present invention to predict amyloidogenic
peptide
sequences.
EXAMPLE 30
Identification of an amyloidogenic peptide from Lysozyme
Amyloid fibril formation by Lysozyme (GenBan.k Accession No. gi:299033) is
associated with familial non-neuropathic amyloidosis [Sacchettini and Kelly
(2002)
Nat Rev Drug Discov 1:267-75]. Based on the proposed role of aromatic residues
in
amyloid self-assembly, the amyloidogenic features of a Lysozyme-derived
peptide,
GTFQIN (SEQ ID NO: 40) were studied.
Materials and Experimental Procedures ¨ Described in Examples 7 and 10.
Results ¨ To characterize the ability of the Lysozyme-derived peptide to form
fibrilar supramolecular ultrastructures, negative staining electron microscopy
analysis
was effected. As shown in Figure 33, under mild conditions, filamentous
structures
were observed for the selected peptide, suggesting that GTFQIN of Lysozyme is
important for the polypeptide self-assembly. These results further
substantiate the
ability of the present invention to predict amyloidogenic peptide sequences.
EXAMPLE 31
Identification of an amyloidogenic peptide from Fibrinogen
Amyloid fibril formation by Fibrinogen (GenBank Accession No. gi:11761629)
is associated with hereditary renal amyloidosis (Sacchettini and Kelly (2002)
Nat Rev
Drug Discov 1:267-75). Based on the proposed role of aromatic residues in
amyloid

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self-assembly, the amyloidogenic features of a Fibrinogen-derived peptide,
SGIFTN
(SEQ ID NO: 41) were studied.
Materials and Experimental Procedures ¨ Described in Examples 7 and 10.
Results ¨ To characterize the ability of the Fibrinogen -derived peptide to
form fibrilar supramolecular ultrastructures, negative staining electron
microscopy
analysis was effected. As shown in Figure 34, under mild conditions,
filamentous
structures were observed for the selected peptide, suggesting that SGIFTN of
Fibrinogen is important for the polypeptide self-assembly. These results
further
substantiate the ability of the present invention to predict amyloidogenic
peptide
sequences.
EXAMPLE 32
Identification of an amyloidogenic peptide from Insulin
Amyloid fibril formation by Insulin (GenBank Accession No. gi:229122) is
associated with injection-localized amyloidosis [Sacchettini and Kelly (2002)
Nat Rev
Drug Discov 1:267-75]. Based on the proposed role of aromatic residues in
arnyloid
self-assembly, the amyloidogenic features of an insulin-derived peptide, ERGFF
(SEQ
ID NO: 42) were studied.
Materials and Experimental Procedures ¨ Described in Examples 7 and 10.
Results ¨ To characterize the ability of the Insulin-derived peptide to form
fibrilar supramolecular ultrastructures, negative staining electron microscopy
analysis
was effected. As shown in Figure 35, under mild conditions, filamentous
structures
were observed for the selected peptide, suggesting that ERGFF of insulin is
important for the polypeptide self-assembly. These results further
substantiate the
ability of the present invention to predict amyloidogenic peptide sequences.
EXAMPLE 33
Identification of an amyloidogenic peptide from prolactin
Amyloid fibrils formation by prolactin (GenBank Accession No. gi:4506105)
is associated with pituitary-gland amyloidosis (Sacchettini and Kelly (2002)
Nat Rev
Drug Discov 1:267-75). Based on the proposed role of aromatic residues in
amyloid

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self-assembly, the amyloidogenic features of a prolactin-derived peptide,
RDFLDR
(SEQ ID NO: 43) were studied.
Materials and Experimental Procedures ¨ Described in Examples 7 and 10.
Results ¨ To characterize the ability of the prolactin-derived peptide to form
fibrilar supramolecular ultrastructures, negative staining electron microscopy
analysis
was effected. As shown in Figure 36, under mild conditions, filamentous
structures
were observed for the selected peptide, suggesting that RDFLDR of prolactin is

important for the polypeptide self-assembly. These results further
substantiate the
ability of the present invention to predict amyloidogenic peptide sequences.
EXAMPLE 34
Identification of an amyloidogenic peptide from Beta-2-microglobulin
Amyloid fibrils formation by beta-2-microtublin (GenBank Accession No.
gi:70065) is associated haemodialysis-related amyloidosis (Sacchettini and
Kelly
(2002) Nat Rev Drug Discov 1:267-75). Based on the proposed role of aromatic
residues in amyloid self-assembly, the amyloidogenic features of a beta-2-
microtublin
-derived peptide, SNFLN (SEQ ID NO: 44) were studied.
Materials and Experimental Procedures ¨ Described in Examples 7 and 10.
Results ¨ To characterize the ability of the beta-2-microtublin-derived
peptide
to form fibrilar supramolecular ultrastructures, negative staining electron
microscopy
analysis was effected. As shown in Figure 37, under mild conditions,
filamentous
structures were observed for the selected peptide, suggesting that SNFLN of
beta-2-
microtublin is important for the polypeptide self-assembly. These results
further
substantiate the ability of the present invention to predict amyloidogenic
peptide
sequences.
EXAMPLE 35
Inhibition of amyloid formation an amyloidogenic peptide identified according
to
the teachings of the present invention
The ability of amyloidogenic peptides of IAPP, identified according to the
teachings of the present invention to inhibit amyloid formation by the full-
length
polypeptide was tested by the addition of beta-breaker proline residues to the

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recognition sequence as set forth in the peptide sequence NFLVHPP (SEQ ID NO:
45).
The degree of amyloid fibrils formation with and without the inhibitor was
assessed using thioflavin T (ThT) as molecular indicator. The degree of
fluorescence
of the ThT dye is directly correlated with the amount of amyloid fibrils in
the solution
[LeVine H 3rd. (1993) Protein Sci. 2:404-410. IAPP solutions (41.1M hIAPPin 10

mM Tris buffer pH 7.2), were incubated in the presence or absence of 401.1M of
the
modified peptide (i.e., NFLVHPP) at room temperature. Fibril formation was
determined by a ten fold dilution of the solutions into a solution that
contained 3 i.tM
thioflavin T (ThT) in 50 mM sodium phosphate pH 6.0 and determination of
fluorescence at 480 nm with excitation at 450 nm using a LS50B
spectroflurimeter
(Perkin Elinor, Wellesley, MA). As a control 10 mM Tris buffer pH 7.2 were
diluted
into the ThT solution and fluorescence was determined as described.
Result ¨ As shown in Figure 38, while the IAPP alone showed high levels of
ThT fluorescence as expected for amyloidogenic protein, there was a
significant
increase in fluorescence in the presence of the inhibitory peptide. Thus,
these results
validate the NFLVH sequence as the amyloidogenic determinant in the IAPP
polypeptide.
EXAMPLE 36
Signcance of hydrophobic residues in amyloid assembly
The significance of an aromatic residue in the basic amyloidogenic unit of
IAPP has been demonstrated in Examples 1-5. As described, substitution of a
phenylalanine to an alanine abolished the ability of an amyloidogenic fragment
(NAGAIL, SEQ ID NO: 9) to form amyloid fibrils in vitro. Based on this
observation, the remarkable occurrence of aromatic residues in other short
amyloid
related sequences (Examples 12-35), and the well-known role of 7c-stacking in
processes of self-assembly in chemistry and biochemistry, it was suggested
that
stacking of aromatic residues may play a role in the process of amyloid
fibrils
formation [Gazit (2002) FASEB J. 16:77-83].
The study was further extended to indicate whether the phenylalanine residue
is critical due to aromaticity thereof, or rather due to its hydrophobic
nature. The

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effect of phenylalanine substitution with hydrophobic residues on the self
assembly of
the basic amyloidogenic unit of IAPP (i.e., NFGAIL peptide) was addressed.
The list of peptides used in the study and designation thereof is presented in

Table 6, below.
5 Table 6
Amino acid substitution and Peptide sequence SEQ ID NO:
coordinates on hIAPP
WT 22-29 NH2-NFGAILSS-COOH 46
F>I 22-29 NH2-NIGAILSS-COOH 47
F>L 22-29 NH2-NLGAILSS-COOH 48
F>V 22-29 NH2-NVGAILSS-COOH 49
F>A 22-29 NH2-NAGAILSS-COOH 89
It will be appreciated that while these hydrophobic amino acids are similar or

even slightly more hydrophobic than phenylalanine [Wolfenden (1981)
Biochemistry
20:849-855; Kyte (1982) J. Mol. Biol. 157:105-132; Radzicka (1988) ], they are
not
10 aromatic. Furthermore, valine and isoleucine, are considered to be very
strong P-sheet
formers [Chou (1974) Biochemistry 13:211-222; Chou (1978) Annu. Rev. Biochem.
47:251-276], which is assumed to be important to the formation of P-sheet rich

amyloid fibrils.
15 EXAMPLE 37
Characterization of the aggregation kinetics of hydrophobically modified
hIAPP peptide fragments as monitored by turbidity measurements
Experimental Procedures ¨ Effected as described in Example 8.
Results
20 To get insight into the aggregation potential of the hydrophobically-
modified
IAPP-derived peptide analogues, turbidity assay was performed. Freshly made
stock
solutions of the wild-type peptide and the various peptide mutants were made
in
DMSO. The peptides were then diluted to a buffer solution and the turbidity
was
monitored by following the absorbance at 405 nm as a function of time. As
shown in
25 Figure 39, significant increase in turbidity was observed for the wild-
type
NFGAILSS octapeptide within minutes following dilution thereof into the
aqueous
solution. The shape of the aggregation curve resembled that of a saturation
curve,
with a rapid increase in turbidity in the first hour, followed by a much
slower increase

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in turbidity over the entire incubation time monitored. This probably reflects
a rapid
aggregation process, with the number of free building blocks as the rate
limiting
factor. In contrast, none of the analogue peptides revealed any significant
aggregative behavior and the turbidity of all the hydrophobic analogues as
well as the
alanine-substituted analogues remained very low for at least 24 hours (Figure
39).
To determine whether the non-aggregative behavior of the hydrophobic
analogues is a result of extremely slow kinetics, peptide analogue solutions
were
incubated for 1 week in the same experimental conditions and endpoint
turbidity
values were determined. As shown in Figure 30, some low degree of turbidity
was
observed with the NIGAILSS, and lower extent for the NLGAILSS, NAGAILSS, and
NVGAILSS peptides in decreasing order of turbidity. However, even for the
NIGAILSS, the degree of turbidity was significantly lower as compared to the
wild-
type NFGAILSS protein (Figure 40). Moreover, there was no correlation between
aggregation potential and hydrophobicity or 13-sheet forming tendency, since
the
lower degree of aggregation was observed with the substitution to the highly
hydrophobic and 13-sheet former, valine. The slight decrease in the endpoint
turbidity
value of the NFGAILSS wild-type peptide, as compared to the values obtained
after
24 hours incubation, could reflect the formation of very large aggregates that
adhere
to the cuvette surface.
EXAMPLE 38
Ultrastructural analysis of hydrophobically modified hIAPP peptide fragments
Electron microscopy analysis was effected as described in Example 10.
An ultrastructural visualization of any possible structures formed by the
various analogous peptides was effected following five days of incubation.
This
structural analysis represents the most sensitive method since various
aggregates
were visualized individually. For that aim, the occurrence and characteristics
of the
formed structures were studied by electron microscopy using negative staining,
with
the same of peptide solution which were incubated in the aggregation assay
(Example
32). As expected, well-ordered fibrils were observed with the wild-type
peptide
NFGAILSS peptide fragment (Figures 41a-b). Some amorphous aggregates could be
also seen with the modified fragments (Figures 41c-f). However, those
structures

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72
were significantly less abundant on the microscope grid. Larger aggregative
structures were observed with the more hydrophobic substitutions as compared
to the
alanine analogues. Yet, unlike the ordered fibrillar structures that were seen
with the
NFGA1LSS peptide, as mentioned above, these aggregates were quite rare and did
not have ordered structures (Figures 41c-f). Those irregular and sporadic
structures
are consistent with some degree of non-specific aggregation as expected after
long
incubation of rather hydrophobic molecules.
EXAMPLE 39
Determination of the specific function of phenylalanine in the L4PP self
assembly
To determine the specific role of the phenylalanine residue in IAPP-self
assembly, a membrane-based binding assay was preformed in order to
systematically
explore the molecular determinants that facilitate the ability of the full-
length hIAPP
to recognize the "basic amyloidogenic unit". To this end, the ability of MBP-
IAPP
(see Example 6) to interact with an array of peptides in which the
phenylalanine
position was systematically altered (SEQ ID NOs. 91-110), was addressed.
Materials and Experimental Procedures ¨ see Examples 6-7.
Results
A peptide array corresponding to the SNNXGAILSS motif (SEQ NO: 90),
where X is any natural amino-acid but cysteine was constructed. As shown in
Figure
42a, binding of MBP-IAPP was clearly observed to peptides which contained the
aromatic tryptophan and phenylalanine residues at the X position (Figure 42a).

Interestingly, binding was also observed upon substitution of phenylalanine
with
basic amino acids such as arginine and lysine. In contrast, no binding was
observed
with any of the hydrophobic substitutions of the position, even after long
exposure of
the membrane (Figure 42b).
The short exposure binding was assessed using densitometry (Figure 42c). It
will be appreciated though, that the measured binding should be interpreted as

semiquantitative since the coupling efficiency during synthesis and therefore
the
amount of peptide per spot may vary. In this case, however, the marked
difference in
binding between the various peptide variants was very clear.

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Taken together, all these observations substantiate the role of aromatic
residues in the acceleration of amyloid formation processes.
Although the invention has been described in conjunction with specific
embodiments thereof, it is evident that many alternatives, modifications and
variations will be apparent to those skilled in the art. Accordingly, it is
intended to
embrace all such alternatives, modifications and variations that fall within
the spirit
and broad scope of the appended claims. In addition, citation or
identification of any
reference in this application shall not be construed as an admission that such
reference is
available as prior art to the present invention.

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SEQUENCE LISTING
<110> Tel Aviv University Future Technology Development L.P.
<120> PEPTIDES ANTIBODIES DIRECTED THEREAGAINST AND METHODS USING SAME
FOR DIAGNOSING AND TREATING AMYLOID-ASSOCIATED DISEASES
<130> 7723-168CA CC/gc
<140> 2,473,987
<141> 2003-01-30
<150> 60/352,578
<151> 2002-01-31
<150> 60/392,266
<151> 2002-07-01
<150> 10/235,852
<151> 2002-09-06
<150> 60/436,453
<151> 2002-12-27
<160> 111
<170> PatentIn version 3.1
<210> 1
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Synthetic peptide
<400> 1
Asn Phe Gly Ala Ile Leu Ser Ser
1 5
<210> 2 '
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Synthetic peptide
<400> 2
Ala Phe Gly Ala Ile Leu Ser Ser
1 5

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Title Date
Forecasted Issue Date 2013-11-19
(86) PCT Filing Date 2003-01-30
(87) PCT Publication Date 2003-08-07
(85) National Entry 2004-07-21
Examination Requested 2007-11-07
(45) Issued 2013-11-19
Deemed Expired 2018-01-30

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Application Fee $400.00 2004-07-21
Maintenance Fee - Application - New Act 2 2005-01-31 $100.00 2004-07-21
Registration of a document - section 124 $100.00 2004-12-01
Maintenance Fee - Application - New Act 3 2006-01-30 $100.00 2006-01-10
Maintenance Fee - Application - New Act 4 2007-01-30 $100.00 2006-12-20
Request for Examination $800.00 2007-11-07
Maintenance Fee - Application - New Act 5 2008-01-30 $200.00 2007-12-20
Maintenance Fee - Application - New Act 6 2009-01-30 $200.00 2008-12-22
Maintenance Fee - Application - New Act 7 2010-02-01 $200.00 2009-11-30
Maintenance Fee - Application - New Act 8 2011-01-31 $200.00 2010-10-25
Maintenance Fee - Application - New Act 9 2012-01-30 $200.00 2011-12-01
Maintenance Fee - Application - New Act 10 2013-01-30 $250.00 2012-12-19
Final Fee $468.00 2013-09-09
Maintenance Fee - Patent - New Act 11 2014-01-30 $250.00 2013-12-03
Maintenance Fee - Patent - New Act 12 2015-01-30 $250.00 2015-01-19
Maintenance Fee - Patent - New Act 13 2016-02-01 $250.00 2016-01-18
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
TEL AVIV UNIVERSITY FUTURE TECHNOLOGY DEVELOPMENT L.P.
Past Owners on Record
GAZIT, EHUD
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
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Document
Description 
Date
(yyyy-mm-dd) 
Number of pages   Size of Image (KB) 
Description 2004-07-21 93 4,100
Abstract 2004-07-21 1 52
Claims 2004-07-21 18 549
Drawings 2004-07-21 26 713
Cover Page 2004-10-04 1 34
Description 2004-12-02 102 4,201
Description 2011-02-02 74 3,865
Claims 2011-02-02 2 60
Claims 2011-12-23 2 56
Claims 2012-12-06 2 54
Cover Page 2013-10-16 1 38
Assignment 2004-07-21 4 115
PCT 2004-07-21 2 104
PCT 2004-07-21 1 50
Correspondence 2004-09-30 1 29
Assignment 2004-12-01 3 109
Prosecution-Amendment 2004-12-02 32 393
PCT 2004-07-22 7 256
Prosecution-Amendment 2009-08-21 6 161
Prosecution-Amendment 2007-11-07 1 42
Prosecution-Amendment 2010-08-03 4 209
Prosecution-Amendment 2011-02-02 27 1,188
Prosecution-Amendment 2011-06-30 3 170
Prosecution-Amendment 2011-12-23 7 373
Prosecution-Amendment 2012-06-11 3 182
Prosecution-Amendment 2012-12-06 5 260
Correspondence 2013-09-09 2 72

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