U.S. patent application number 17/382466 was filed with the patent office on 2021-11-18 for alzheimer abeta peptide binding polypeptide.
The applicant listed for this patent is Amylonix AB. Invention is credited to Torleif Hard, Hanna Lindberg, John Lofblom, Stefan Stahl.
Application Number | 20210355180 17/382466 |
Document ID | / |
Family ID | 1000005742182 |
Filed Date | 2021-11-18 |
United States Patent
Application |
20210355180 |
Kind Code |
A1 |
Stahl; Stefan ; et
al. |
November 18, 2021 |
ALZHEIMER ABETA PEPTIDE BINDING POLYPEPTIDE
Abstract
The present disclosure relates to a class of engineered
polypeptides having a binding affinity for amyloid .beta. (A.beta.)
peptides (in the following referred to as A.beta.), comprising the
amino acid sequence EX.sub.2X.sub.3YX.sub.5X.sub.6NLX.sub.9A
X.sub.11QLCAX.sub.16IX.sub.18X.sub.19X.sub.20 ED (SEQ ID NO:632).
The present disclosure also relates to the use of such A.beta.
peptide binding polypeptides as therapeutic, prognostic and/or
diagnostic agents.
Inventors: |
Stahl; Stefan; (Stockholm,
SE) ; Lofblom; John; (Tullinge, SE) ;
Lindberg; Hanna; (Stockholm, SE) ; Hard; Torleif;
(Uppsala, SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Amylonix AB |
Johanneshov |
|
SE |
|
|
Family ID: |
1000005742182 |
Appl. No.: |
17/382466 |
Filed: |
July 22, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16715798 |
Dec 16, 2019 |
11098096 |
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17382466 |
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15548208 |
Aug 2, 2017 |
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PCT/EP2016/053665 |
Feb 22, 2016 |
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16715798 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/4711 20130101;
C07K 2317/92 20130101; A61K 38/00 20130101 |
International
Class: |
C07K 14/47 20060101
C07K014/47 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 20, 2015 |
EP |
15156018.2 |
Sep 16, 2015 |
EP |
15185461.9 |
Claims
1. An A.beta. peptide binding polypeptide which comprises a first
moiety comprising a first A.beta. peptide binding motif BM1, a
second moiety comprising a second A.beta. peptide binding motif
BM2), and a linker, wherein each one of said binding motifs BM1 and
BM2 consists of the amino acid sequence selected from
TABLE-US-00035 i) (SEQ ID NO: 632)
EX.sub.2X.sub.3YX.sub.5X.sub.6NLX.sub.9A
X.sub.11QLCAX.sub.16IX.sub.18X.sub.19X.sub.20 ED
wherein, independently from each other, X.sub.2 is selected from I,
M, Q, R, T and Y; X.sub.3 is selected from H and V; X.sub.5 is
selected from F, I and L; X.sub.6 is selected from P and T; X.sub.9
is selected from N and T; X.sub.11 is selected from D and H;
X.sub.16 is selected from F and I; X.sub.18 is selected from N, Q
and R; X.sub.19 is selected from K and 5; and X.sub.20 is selected
from F, I, L and R; and ii) an amino acid sequence which has at
least 95% identity to the full-length sequence defined in i). and
wherein the amino acid sequence of the BM1 and the amino acid
sequence of the BM2 are the same or different
2. The A.beta. peptide binding polypeptide according to claim 1,
wherein at least one of the first and second moieties comprises a
binding module amino acid sequence, Bmod, selected from
TABLE-US-00036 iii) (SEQ ID NO: 562) [BM]-DXaSQXbXcXdLLXe
EAKKLXfXgXhQA PXi
wherein BM is the BM1 or the BM2 as defined in claim 1, and,
independently of each other, Xa is selected from P, Q, R and 5; Xb
is selected from N, Q, R and 5; Xc is selected from A and 5; Xd is
selected from K, N and E; Xe is selected from A, S and C; Xf is
selected from E, N and 5; Xg is selected from D, E and 5; Xh is
selected from A and 5; and Xi is selected from no amino acid, A and
K, and iv) an amino acid sequence which has at least 97% identity
to the full-length sequence defined in iii).
3. The A.beta. peptide binding polypeptide according to claim 2,
wherein sequence iii) consists of the amino acid sequence
TABLE-US-00037 (SEQ ID NO: 563) [BM]-DXaSQXbAXdLLA EAKKLNDAQA
PXi
wherein BM is the BM1 or the BM2, and, independently of each other,
Xa is selected from P, Q, R and 5; Xb is selected from N, Q, R and
5; Xd is selected from K and N; and Xi is selected from no amino
acid, A and K.
4. The A.beta. peptide binding polypeptide according to claim 1,
wherein sequence i) of the BM1 is amino acid residues 4-25 in any
one of SEQ ID NO:1-106 and 118-553, and sequence i) of the BM2 is
amino acid residues 64-85 in any one of SEQ ID NO:1-17, 19-106 and
118-553 or amino acid residues 69-90 in SEQ ID NO:18.
5. The A.beta. peptide binding polypeptide according to claim 4,
wherein sequence i) of the BM1 is amino acid residues 4-25 in SEQ
ID NO:95 and sequence i) of the BM2 is amino acid residues 64-85 in
SEQ ID NO:95.
6. The A.beta. peptide binding polypeptide according to claim 2,
wherein sequence iii) of the Bmod of the first moiety is amino acid
residues 4-47 in any one of SEQ ID NO:1-106 and 118-553, and
sequence iii) of the Bmod of the second moiety is amino acid
residues 64-107 in any one of SEQ ID NO:1-17, 19-106 and 118-553 or
amino acid residues 69-112 in SEQ ID NO:18.
7. The A.beta. peptide binding polypeptide according to claim 6,
wherein sequence iii) of the Bmod of the first moiety is amino acid
residues 4-47 in SEQ ID NO:95, and sequence iii) of the Bmod of the
second moiety is amino acid residues 64-107 in SEQ ID NO:95.
8. An A.beta. peptide binding polypeptide comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1-106 and
118-553.
9. The A.beta. peptide binding polypeptide according to claim 1,
which is capable of binding to A.beta. peptide such that the
K.sub.D value of the interaction is at most 1.times.10.sup.-7
M.
10. A fusion protein or conjugate, comprising a first part
consisting of the A.beta. peptide binding polypeptide according to
claim 1; and a second part consisting of a polypeptide having a
biological activity.
11. The fusion protein or conjugate according to claim 10, wherein
said biological activity is a binding activity.
12. The fusion protein or conjugate according to claim 10, wherein
the second part is selected from the group consisting of antibodies
and antigen binding fragments thereof.
13. A composition comprising the A.beta. peptide binding
polypeptide according to claim 1 and at least one pharmaceutically
acceptable excipient or carrier.
14. The A.beta. binding polypeptide of claim 8, comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:1, 5,
9, 13, 14, 16, 18, 20, 21, 25, 26, 28, 33, 35, 37, 40, 42, 48, 49,
50, 51, 53, 54, 55, 59, 60, 61, 62, 64, 65, 70, 72, 75, 78, 84, 89,
90, 95, 96, 97, 100, 104 and 118-159.
15. The A.beta. binding polypeptide of claim 8, comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:18,
28, 35, 49, 50, 51, 55, 59, 64, 65, 70, 72, 78, 84, 95 and
118-159.
16. The A.beta. binding polypeptide of claim 8, comprising an amino
acid sequence selected from the group consisting of SEQ ID NO 18,
70 and 95.
17. The A.beta. binding polypeptide of claim 8, comprising an amino
acid sequence consisting of SEQ ID NO:95.
18. The fusion protein or conjugate according to claim 11, wherein
said binding activity is binding to a target selected from the
group consisting of Tau, apolipoprotein E (ApoE), beta-secretase 1
(BACE1), gamma-secretase, transferrin receptor (TfR), complement
factor Cis, presenilin 1, presenilin 2, nicastrin, alpha-synuclein
and Bri.
19. The composition of claim 13, further comprising at least one
active agent, said active agent selected from the group consisting
of inhibitors of neurotransmitter degradation, neurotransmitters,
acetylcholinesterase inhibitors, NMDA receptor antagonists, TNF
inhibitors, antihistamines, anti-viral agents, alpha-secretase
activators, inhibitors of beta- or gamma-secretase, inhibitors of
.alpha.-synuclein aggregation, inhibitors of tau aggregation,
calcium channel blockers, compounds effective against oxidative
stress, anti-apoptotic compounds, metal chelators, pirenzepin and
metabolites, attractants for A.beta. clearing/depleting cellular
components, inhibitors of N-terminal truncated A.beta. including
pyroglutamated A.beta.3-42, anti-inflammatory molecules, clozapine,
ziprasidone, risperidone, aripiprazole, olanzapine, tacrine,
rivastigmine, donepezil, galantamine, amyloid or tau modifying
drugs, vitamin B12, cysteine, acetylcholine precursor, lecithin,
choline, Ginkgo biloba, acetyl-L-carnitine, idebenone,
propentofylline, xanthine derivatives, and combinations
thereof.
20. A method for the treatment of an A.beta. peptide associated
condition, comprising administering to a subject in need thereof,
an effective amount of the A.beta. peptide binding polypeptide
according to claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/715,798 filed Dec. 16, 2019 which is a
continuation of U.S. patent application Ser. No. 15/548,208 filed
Aug. 2, 2017, which is a U.S. National Stage Application of
International Patent Application Serial No. PCT/EP2016/053665 filed
Feb. 22, 2016 which claims priority to European Patent Application
Serial No. 15185461.9, filed Sep. 16, 2015 and European Patent
Application No. 15156018.2, filed Feb. 20, 2015. All of which are
incorporated by reference in their entireties herein.
FIELD OF THE INVENTION
[0002] The present disclosure relates to a class of engineered
polypeptides having a binding affinity for amyloid .beta. (A.beta.)
peptide (in the following referred to as A.beta.). The present
disclosure also relates to the use of such A.beta. peptide binding
polypeptides as therapeutic, prognostic and/or diagnostic
agents.
BACKGROUND
[0003] Many different diseases, such as Alzheimer's disease, type
II diabetes, primary and secondary systemic amyloidosis, and
familial amyloid polyneuropathy 1, have been recognized as
belonging to the growing family of amyloid diseases. All amyloid
diseases have in common the presence of extracellular protein
aggregates that may or may not be fibrillar.
[0004] Alzheimer's disease (AD) is the leading cause of dementia
worldwide, with approximately 35 million people affected. Current
treatments for Alzheimer's disease provide only modest symptomatic
relief, and there is a great need for therapies that slow the
course of the disease and prevent or delay the disease in
susceptible individuals. Despite tremendous research efforts during
the last decades, there is currently no treatment that
significantly alters the course of the disease or efficiently stops
its development (Citron (2010) Nat Rev Drug Discov 9(5):387-398).
The pathological hallmarks of AD include large extracellular
amyloid plaques and intracellular neurofibrillary tangles in the
brains of affected individuals (Citron (2010), supra). The main
constituents of the amyloid plaques are the 40 and 42 amino acid
amyloid beta (A.beta.) peptides. It has been hypothesized that the
aggregation process of these peptides into oligomers, protofibrils
and fibrils play a pivotal role in the neuropathology of AD.
Consequently, therapeutic strategies targeting the production and
aggregation, as well as clearance of A.beta. from the brain, are
under investigation.
[0005] One promising approach for therapy is based on
administration of A.beta.-specific agents that bind directly to
A.beta. aggregates in the brain or to free A.beta. peptides in the
plasma (peripheral sink mechanism) (Citron (2010), supra).
Conventional antibodies have demonstrated the great potential of
immunotherapy, but have also been associated with severe side
effects, such as Fc-mediated pro-inflammatory immune responses.
Additionally, antibodies are large molecules and their size may
have negative effect on in vivo biodistribution, including uptake
into the brain. Therefore, alternative approaches using engineered
antibody alternatives and alternative scaffold proteins without
effector functions have been suggested to provide safer and more
effective therapies, and smaller scaffold proteins are presumed to
transfer across the blood-brain barrier faster than large
antibodies.
[0006] Several molecules with affinity for specific targets and
different non-antibody based scaffolds have been described in the
art. In particular, molecules related to protein Z, derived from
domain B of staphylococcal protein A (SPA) (Nilsson B et al (1987)
Protein Engineering 1:107-133), have been selected from libraries
of randomized protein Z molecules using different interaction
targets (see eg WO95/19374; WO00/63243; Nord K et al (1995) Prot
Eng 8:601-608; Nord K et al (1997) Nature Biotechnology
15:772-777).
[0007] A protein Z variant denoted Z.sub.A.beta.3, targeting
monomeric A.beta. with a 17 nM affinity (Gronwall et al (2007) J
Biotechnol 128(1):162-183; Hoyer et al (2008) Proc Natl Acad Sci
USA 105(13):5099-5104) has been described. Structural analysis has
shown that Z.sub.A.beta.3 binds the A.beta. peptide as a
disulfide-linked homodimer (due to an internal cysteine), and that
both the disulfide-linked dimeric Z variant and the A.beta. peptide
fold upon binding. The A.beta. peptide folds into a 8-hairpin
structure, allowing the first .alpha.-helices in both Z variant
units of the homodimer to adopt .beta.-sheet structures with
unstructured N-termini (Hoyer and Hard (2008) J. Mol. Biol.
378(2):398-411; Hoyer et al (2008), supra). In a recent in vivo
study using an A.beta. transgenic fruit fly model of AD,
Z.sub.A.beta.3 was shown to efficiently inhibit formation of toxic
amyloidogenic oligomers and plaques, thereby abolishing the
neurotoxic effects and restoring the life span of the flies (Hoyer
et al (2008), supra; Luheshi et al (2010) PLoS Biol
8(3):e1000334).
[0008] It has been demonstrated that there is a correlation between
the affinity of peripherally administered A.beta. specific agents
and the efflux of A.beta. from the brain to the plasma. Since
levels of A.beta. peptides in the blood are generally low, it is
important to use capturing agents of high affinity for such
applications.
[0009] Thus, there is a continued need for agents with a high
affinity for A.beta., which can be used as reagents in various
assays and processes where such an affinity is needed, such as
treatment, diagnosis and prognosis of AD.
SUMMARY OF THE INVENTION
[0010] It is an object of the present disclosure to provide new
A.beta. peptide binding agents, which could for example be used for
therapeutic, prognostic and diagnostic applications.
[0011] It is an object of the present disclosure to provide a
molecule allowing for efficient therapy, targeting various forms of
A.beta. peptide related pathologies while alleviating the
abovementioned and other drawbacks of current therapies.
[0012] It is furthermore an object of the present disclosure to
provide a molecule suitable for prognostic and diagnostic
applications.
[0013] These, and other objects which are evident to the skilled
person from the present disclosure, are met by the different
aspects of the invention as claimed in the appended claims and as
generally disclosed herein.
[0014] Thus, in a first aspect of the disclosure, there is provided
an A.beta. peptide binding polypeptide, which comprises [0015] a
first moiety comprising a first A.beta. peptide binding motif BM1,
[0016] a second moiety comprising a second A.beta. peptide binding
motif BM2, which motifs may be the same or different, and [0017] a
linker,
[0018] wherein each one of said binding motifs BM1 and BM2 consists
of an amino acid sequence selected from
TABLE-US-00001 i) (SEQ ID NO: 632)
EX.sub.2X.sub.3YX.sub.5X.sub.6NLX.sub.9A
X.sub.11QLCAX.sub.16IX.sub.18X.sub.19X.sub.20 ED
wherein, independently from each other,
[0019] X.sub.2 is selected from I, M, Q, R, T and Y;
[0020] X.sub.3 is selected from H and V;
[0021] X.sub.5 is selected from F, I and L;
[0022] X.sub.6 is selected from P and T;
[0023] X.sub.9 is selected from N and T;
[0024] X.sub.11 is selected from D and H;
[0025] X.sub.16 is selected from F and I;
[0026] X.sub.18 is selected from N, Q and R;
[0027] X.sub.19 is selected from K and 5; and
[0028] X.sub.20 is selected from F, I, L and R;
[0029] and [0030] ii) an amino acid sequence which has at least 95%
identity to the sequence defined in i).
[0031] The above definition of a class of sequence related, A.beta.
peptide binding polypeptides is based on a statistical analysis of
a number of random polypeptide variants of a truncated dimer
((Z.sub.A.beta.3A12).sub.2; SEQ ID NO:113) of the first generation
A.beta. peptide binding polypeptide Z.sub.A.beta.3. As detailed in
the background section, this first generation A.beta. peptide
binding polypeptide was originally identified based on its
interaction with A.beta. peptide in selection experiments, and its
binding surface has now been modified to create the variants
defined herein. The identified A.beta. peptide binding motifs (BM1
and BM2) correspond to the target binding region of the parent
scaffold, which constitutes a .beta. sheet and two .alpha. helices
when bound to A.beta. peptide.
[0032] As the skilled person will realize, the function of any
polypeptide, such as the A.beta. peptide binding capacity of the
polypeptide of the present disclosure, is dependent on the tertiary
structure of the polypeptide. It is therefore possible to make
minor changes to the sequence of amino acids in a polypeptide
without affecting the function thereof. Thus, the disclosure
encompasses modified variants of the A.beta. peptide binding
polypeptide, which have retained A.beta. peptide binding
characteristics. In this way, encompassed by the present disclosure
is an A.beta. peptide binding polypeptide comprising an amino acid
sequence with 95% or greater identity to a polypeptide as defined
in i). For example, it is possible that an amino acid residue
belonging to a certain functional group of amino acid residues
(e.g. hydrophobic, hydrophilic, polar etc) could be exchanged for
another amino acid residue from the same functional group.
[0033] In some embodiments, such changes may be made in any
position of the sequence of the A.beta. peptide binding polypeptide
as disclosed herein. In other embodiments, such changes may be made
only in the non-variable positions, also denoted scaffold amino
acid residues. In such cases, changes are not allowed in the
variable positions, i.e. positions denoted by an "X" in sequence
i).
[0034] The term "% identity", as used throughout the specification,
may for example be calculated as follows. The query sequence is
aligned to the target sequence using the CLUSTAL W algorithm
(Thompson et al (1994) Nucleic Acids Research, 22:4673-4680). A
comparison is made over the window corresponding to the shortest of
the aligned sequences. The shortest of the aligned sequences may in
some instances be the target sequence. In other instances, the
shortest of the aligned sequences may be the query sequence. The
amino acid residues at each position are compared and the
percentage of positions in the query sequence that have identical
correspondences in the target sequence is reported as %
identity.
[0035] In one embodiment of the first aspect, there is provided an
A.beta. peptide binding polypeptide, wherein, in sequence i) of
said binding motifs,
[0036] X.sub.2 is further selected from I, Q, and R;
[0037] X.sub.5 is further selected from F and L;
[0038] X.sub.16 is F; and
[0039] X.sub.20 is further selected from I and L.
[0040] In one embodiment of the first aspect, there is provided an
A.beta. peptide binding polypeptide as defined above, wherein
sequence i) in BM1 consists of the amino acid sequence
TABLE-US-00002 (SEQ ID NO: 558)
EX.sub.2X.sub.3YX.sub.5X.sub.6NLX.sub.9A
X.sub.11QLCAFIX.sub.18X.sub.19L ED
wherein, independently from each other,
[0041] X.sub.2 is selected from I, M, Q, R and Y;
[0042] X.sub.3 is selected from H and V;
[0043] X.sub.5 is selected from F, I and L;
[0044] X.sub.5 is selected from P and T;
[0045] X.sub.9 is selected from N and T;
[0046] X.sub.11 is selected from D and H;
[0047] X.sub.18 is selected from N, Q and R; and
[0048] X.sub.19 is selected from K and S.
[0049] In one embodiment, in sequence i) in BM1, X.sub.2 is further
selected from I and R; and X.sub.5 is further selected from F and
L.
[0050] In another embodiment, in sequence i) in BM1, X.sub.2 is
further selected from I, Q, R and Y; and X.sub.5 is further
selected from F and L.
[0051] In yet another embodiment, in sequence i) in BM1, X.sub.2 is
further selected from I and R; and X.sub.18 is further selected
from Q and R.
[0052] In still another embodiment, in sequence i) in BM1, X.sub.2
is further selected from I, M and R.
[0053] In a further embodiment, in sequence i) in BM1, X.sub.2 is
further selected from I and R; X.sub.3 is V; and X.sub.5 is further
selected from F and L.
[0054] In one embodiment, sequence i) in BM1 consists of the amino
acid sequence
TABLE-US-00003 SEQ ID NO: 559 EX.sub.2VYX.sub.5X.sub.6NLNA
X.sub.11QLCAFIX.sub.18SL ED
wherein, independently from each other,
[0055] X.sub.2 is selected from I, M and R;
[0056] X.sub.5 is selected from F, I and L;
[0057] X.sub.6 is selected from P and T;
[0058] X.sub.11 is selected from D and H; and
[0059] X.sub.18 is selected from N, Q and R.
[0060] In one embodiment of the first aspect, there is provided an
A.beta. peptide binding polypeptide as defined above, wherein
sequence i) in BM2 consists of the amino acid sequence
TABLE-US-00004 (SEQ ID NO: 560) EX.sub.2VYX.sub.5PNLX.sub.9A
X.sub.11QLCAX.sub.16IX.sub.18X.sub.19X.sub.20 ED
wherein, independently from each other,
[0061] X.sub.2 is selected from I, M, Q, R and T;
[0062] X.sub.5 is selected from F and L;
[0063] X.sub.9 is selected from N and T;
[0064] X.sub.11 is selected from D and H;
[0065] X.sub.16 is selected from F and I;
[0066] X.sub.18 is selected from N, Q and R;
[0067] X.sub.19 is selected from K and 5; and
[0068] X.sub.20 is selected from F, I, L and R.
[0069] In one embodiment, in sequence i) in BM2, X.sub.2 is further
selected from I, Q and R; X.sub.16 is F; and X.sub.20 is further
selected from I and L.
[0070] In another embodiment, in sequence i) in BM2, X.sub.2 is
further selected from R and T; and X.sub.5 is L.
[0071] In yet another embodiment, in sequence i) in BM2, X.sub.2 is
R; X.sub.5 is L; X.sub.16 is F; and X.sub.20 is further selected
from I and L.
[0072] In still another embodiment, in sequence i) in BM2, X.sub.2
is further selected from I, M, Q and R; X.sub.16 is F; and X.sub.20
is L.
[0073] In a further embodiment, in sequence i) in BM2, X.sub.2 is
further selected from I, Q and R; X.sub.16 is F; and X.sub.20 is
L.
[0074] In one embodiment, sequence i) in BM2 consists of the amino
acid sequence
TABLE-US-00005 (SEQ ID NO: 561) EX.sub.2VYX.sub.5X.sub.6NLNA
X.sub.11QLCAFIX.sub.18SX.sub.20 ED
wherein, independently from each other,
[0075] X.sub.2 is selected from I, M and R;
[0076] X.sub.5 is selected from F, I and L;
[0077] X.sub.6 is selected from P and T;
[0078] X.sub.11 is selected from D and H;
[0079] X.sub.18 is selected from N, Q and R; and
[0080] X.sub.20 is selected from I and L.
[0081] In additional embodiments of any one of BM1 and BM2 as
defined above, X.sub.6 may instead be selected from P, T, R and
Y.
[0082] As used herein, the designations "first binding motif" or
"BM1" and "second binding motif" or "BM2", as well as the
designations "first moiety" and "second moiety" are made for
clarity reasons to distinguish between the A.beta. peptide binding
motifs and moieties. These designations are not intended to refer
to the actual order of the different domains in the polypeptide.
Thus, for example, said BM2 or second moiety may without
restriction appear N-terminally or C-terminally of said BM1 or
first moiety in the A.beta. peptide binding polypeptide.
[0083] Herein, "X.sub.n" and "X.sub.m" are used to indicate amino
acid residues in positions n and m in the sequence i) as defined
above, wherein n and m are integers which indicate the position of
an amino acid residue within said sequence as counted from the
N-terminal end of said sequence. For example, X.sub.3 and X.sub.6
indicate the amino acid residue in positions three and six,
respectively, from the N-terminal end of sequence i).
[0084] In the embodiments of the first aspect, polypeptides are
provided wherein X.sub.n in sequence i) is independently selected
from a group of possible residues according to Table 1. The skilled
person will appreciate that X.sub.n may be selected from any one of
the listed groups of possible residues and that this selection is
independent from the selection of amino acid residues in X.sub.m,
wherein n.noteq.m. Thus, any of the listed possible residues in
position X.sub.n in Table 1 may be independently combined with any
of the listed possible residues any other variable position in
Table 1.
[0085] The skilled person will appreciate that Table 1 is to be
read as follows: In one embodiment according to the first aspect,
there is provided a polypeptide wherein amino acid residue
"X.sub.n" in sequence i) is selected from "Possible residues".
Thus, Table 1 discloses several specific and individualized
embodiments of the first aspect of the present disclosure. For
example, in one embodiment according to the first aspect, there is
provided a polypeptide wherein X.sub.2 in sequence i) is selected
from I, M, Q, R and Y, and in another embodiment according to the
first aspect, there is provided a polypeptide wherein X.sub.2 in
sequence i) is selected from M, Q, R and T. For avoidance of doubt,
the listed embodiments may be freely combined in yet other
embodiments. For example, one such combined embodiment is a
polypeptide in which X.sub.2 is selected from I, M, Q, R and Y,
while X.sub.6 is T, and X.sub.18 is selected from Q and R.
TABLE-US-00006 TABLE 1 X.sub.n Possible residues X.sub.2 I, M, Q,
R, Y X.sub.2 M, Q, R, T X.sub.2 I, Q, R, Y X.sub.2 I, M, R X.sub.2
T, R X.sub.2 I, M, Q, R X.sub.2 I, R, Y X.sub.2 I, Q, R X.sub.2 I,
R X.sub.2 R X.sub.2 I X.sub.3 V X.sub.3 H X.sub.5 F, L X.sub.5 F, I
X.sub.5 I X.sub.5 L X.sub.5 F X.sub.6 P X.sub.6 T X.sub.9 N X.sub.9
T X.sub.11 D X.sub.11 H X.sub.16 F X.sub.16 I X.sub.18 Q, R
X.sub.18 N, Q X.sub.18 N, R X.sub.18 Q X.sub.18 N X.sub.18 R
X.sub.19 K X.sub.19 S X.sub.20 I, L X.sub.20 L
[0086] In one embodiment of the first aspect, there is provided an
A.beta. peptide binding polypeptide, wherein at least one of said
first and second moieties comprises the amino acid sequence
X.sub.AGX.sub.B-[BM]
Wherein BM is BM1 or BM2 as defined herein, and, independently of
each other,
[0087] X.sub.A is selected from A and 5; and
[0088] X.sub.B is selected from G and R.
[0089] In another embodiment, there is provided an A.beta. peptide
binding polypeptide, wherein at least one of said first and second
moieties comprises a binding module amino acid sequence, Bmod,
selected from
TABLE-US-00007 iii) (SEQ ID NO: 562)
[BM]-DX.sub.aSQX.sub.bX.sub.cX.sub.dLLX.sub.e
EAKKLX.sub.fX.sub.gX.sub.hQA PX.sub.i
wherein BM is BM1 or BM2 as defined above, and, independently of
each other,
[0090] X.sub.a is selected from P, Q, R and 5;
[0091] X.sub.b is selected from N, Q, R and 5;
[0092] X.sub.c is selected from A and 5;
[0093] X.sub.d is selected from K, N and E;
[0094] X.sub.e is selected from A, S and C;
[0095] X.sub.f is selected from E, N and 5;
[0096] X.sub.g is selected from D, E and 5;
[0097] X.sub.h is selected from A and 5; and
[0098] X.sub.i is selected from no amino acid, A and K,
and [0099] iv) an amino acid sequence which has at least 97%
identity to a sequence defined in iii).
[0100] In one embodiment, sequence iii) of Bmod consists of the
amino acid sequence
TABLE-US-00008 (SEQ ID NO: 563) [BM]DX.sub.aSQX.sub.bAX.sub.dLLA
EAKKLNDAQA PX.sub.i
wherein [BM] is [BM1] or [BM2] as defined above, and
[0101] X.sub.a is selected from P, Q, R and 5;
[0102] X.sub.b is selected from N, Q, R and 5;
[0103] X.sub.d is selected from K and N; and
[0104] X.sub.i is selected from no amino acid, A and K.
[0105] In one embodiment, sequence iii) consists of an amino acid
sequence selected from
TABLE-US-00009 (SEQ ID NO: 564) [BM]-DPSQSANLLAEAKKLNDAQAP; (SEQ ID
NO: 565) [BM]-DPSQQANLLAEAKKLNDAQAP; (SEQ ID NO: 566)
[BM]-DSSQSANLLAEAKKLNDAQAP; (SEQ ID NO: 567)
[BM]-DPSQQAKLLAEAKKLNDAQAP; (SEQ ID NO: 568)
[BM]-DPSQNANLLAEAKKLNDAQAP; (SEQ ID NO: 569)
[BM]-DRSQQANLLAEAKKLNDAQAP; (SEQ ID NO: 570)
[BM]-DQSQRANLLAEAKKLNDAQAP; (SEQ ID NO: 571)
[BM]-DQSQQANLLAEAKKLNDAQAP; (SEQ ID NO: 572)
[BM]-DRSQSANLLAEAKKLNDAQAP; (SEQ ID NO: 573)
[BM]-DRSQRANLLAEAKKLNDAQAP; and (SEQ ID NO: 574)
[BM]-DRSQNANLLAEAKKLNDAQAP.
[0106] In a more specific embodiment, said first moiety comprises a
Bmod in which sequence iii) is an amino acid sequence selected
from
TABLE-US-00010 (SEQ ID NO: 575) [BM1]-DPSQSANLLAEAKKLNDAQAPA; (SEQ
ID NO: 576) [BM1]-DPSQQANLLAEAKKLNDAQAPA; (SEQ ID NO: 577)
[BM1]-DSSQSANLLAEAKKLNDAQAPA; (SEQ ID NO: 578)
[BM1]-DPSQRANLLAEAKKLNDAQAPA; (SEQ ID NO: 579)
[BM1]-DPSQQAKLLAEAKKLNDAQAPA; (SEQ ID NO: 580)
[BM1]-DPSQNANLLAEAKKLNDAQAPA; and (SEQ ID NO: 581)
[BM1]-DRSQNANLLAEAKKLNDAQAPA.
[0107] In another specific embodiment, said second moiety comprises
a Bmod in which sequence iii) is an amino acid sequence selected
from
TABLE-US-00011 (SEQ ID NO: 582) [BM2]-DPSQQANLLAEAKKLNDAQAPK; (SEQ
ID NO: 583) [BM2]-DPSQSANLLAEAKKLNDAQAPK; (SEQ ID NO: 584)
[BM2]-DRSQQANLLAEAKKLNDAQAPK; (SEQ ID NO: 585)
[BM2]-DQSQRANLLAEAKKLNDAQAPK; (SEQ ID NO: 586)
[BM2]-DQSQQANLLAEAKKLNDAQAPK; (SEQ ID NO: 587)
[BM2]-DPSQRANLLAEAKKLNDAQAPK; (SEQ ID NO: 588)
[BM2]-DRSQSANLLAEAKKLNDAQAPK; (SEQ ID NO: 589)
[BM2]-DRSQRANLLAEAKKLNDAQAPK; (SEQ ID NO: 590)
[BM2]-DSSQSANLLAEAKKLNDAQAPK; and (SEQ ID NO: 591)
[BM2]-DPSQNANLLAEAKKLNDAQAPK.
[0108] The first aspect of the disclosure also provides an A.beta.
peptide binding polypeptide as described above, wherein at least
one of said first and second moieties comprises the amino acid
sequence
X.sub.AGX.sub.B-[BMod]
wherein Bmod is as defined above, and, independently of each
other,
[0109] X.sub.A is selected from A and 5; and
[0110] X.sub.B is selected from G and R.
[0111] In one embodiment, at least one of said first and second
moieties comprises an amino acid sequence selected from
TABLE-US-00012 (SEQ ID NO: 592) AGG-[BM]-DPSQSANLLAEAKKLNDAQAP;
(SEQ ID NO: 593) AGG-[BM]-DPSQQANLLAEAKKLNDAQAP; (SEQ ID NO: 594)
AGG-[BM]-DSSQSANLLAEAKKLNDAQAP; (SEQ ID NO: 595)
AGG-[BM]-DPSQRANLLAEAKKLNDAQAP; (SEQ ID NO: 596)
AGR-[BM]-DPSQSANLLAEAKKLNDAQAP; (SEQ ID NO: 597)
AGG-[BM]-DPSQQAKLLAEAKKLNDAQAP; (SEQ ID NO: 598)
AGG-[BM]-DPSQNANLLAEAKKLNDAQAP; (SEQ ID NO: 599)
AGR-[BM]-DPSQQANLLAEAKKLNDAQAP; (SEQ ID NO: 600)
AGR-[BM]-DRSQQANLLAEAKKLNDAQAP; (SEQ ID NO: 601)
AGR-[BM]-DQSQRANLLAEAKKLNDAQAP; (SEQ ID NO: 602)
AGR-[BM]-DQSQQANLLAEAKKLNDAQAP; (SEQ ID NO: 603)
AGR-[BM]-DPSQRANLLAEAKKLNDAQAP; (SEQ ID NO: 604)
AGR-[BM]-DRSQSANLLAEAKKLNDAQAP; (SEQ ID NO: 605)
SGG-[BM]-DPSQSANLLAEAKKLNDAQAP; (SEQ ID NO: 606)
AGR-[BM]-DRSQRANLLAEAKKLNDAQAP; (SEQ ID NO: 607)
AGR-[BM]-DPSQNANLLAEAKKLNDAQAP; (SEQ ID NO: 608)
AGR-[BM]-DSSQSANLLAEAKKLNDAQAP; and (SEQ ID NO: 609)
AGR-[BM]-DRSQNANLLAEAKKLNDAQAP.
[0112] In one embodiment, said first moiety comprises an amino acid
sequence selected from
TABLE-US-00013 (SEQ ID NO: 610) AGG-[BM1]-DPSQSANLLAEAKKLNDAQAPA;
(SEQ ID NO: 611) AGG-[BM1]-DPSQQANLLAEAKKLNDAQAPA; (SEQ ID NO: 612)
AGG-[BM1]-DSSQSANLLAEAKKLNDAQAPA; (SEQ ID NO: 613)
AGG-[BM1]-DPSQRANLLAEAKKLNDAQAPA; (SEQ ID NO: 614)
AGR-[BM1]-DPSQSANLLAEAKKLNDAQAPA; (SEQ ID NO: 615)
AGG-[BM1]-DPSQQAKLLAEAKKLNDAQAPA; and (SEQ ID NO: 616)
AGG-[BM1]-DPSQNANLLAEAKKLNDAQAPA; (SEQ ID NO: 617)
AGR-[BM1]-DPSQNANLLAEAKKLNDAQAP; (SEQ ID NO: 618)
AGR-[BM1]-DSSQSANLLAEAKKLNDAQAP; and (SEQ ID NO: 619)
AGR-[BM1]-DRSQNANLLAEAKKLNDAQAP.
[0113] In one embodiment, said second moiety comprises an amino
acid sequence selected from
TABLE-US-00014 (SEQ ID NO: 620) AGR-[BM2]-DPSQQANLLAEAKKLNDAQAPK;
(SEQ ID NO: 621) AGR-[BM2]-DPSQSANLLAEAKKLNDAQAPK; (SEQ ID NO: 622)
AGR-[BM2]-DRSQQANLLAEAKKLNDAQAPK; (SEQ ID NO: 623)
AGR-[BM2]-DQSQRANLLAEAKKLNDAQAPK; (SEQ ID NO: 624)
AGR-[BM2]-DQSQQANLLAEAKKLNDAQAPK; (SEQ ID NO: 625)
AGR-[BM2]-DPSQRANLLAEAKKLNDAQAPK; (SEQ ID NO: 626)
AGR-[BM2]-DRSQSANLLAEAKKLNDAQAPK; (SEQ ID NO: 627)
AGG-[BM2]-DPSQSANLLAEAKKLNDAQAPK; (SEQ ID NO: 628)
SGG-[BM2]-DPSQSANLLAEAKKLNDAQAPK; (SEQ ID NO: 629)
AGR-[BM2]-DRSQRANLLAEAKKLNDAQAPK; (SEQ ID NO: 630)
AGG-[BM2]-DSSQSANLLAEAKKLNDAQAPK; and (SEQ ID NO: 631)
AGG-[BM2]-DPSQNANLLAEAKKLNDAQAPK.
[0114] Similarly to the discussion above in relation to sequence
i), "X.sub.y" and "X.sub.z" are used herein to indicate amino acid
residues in positions y and z in the sequences defined above,
wherein y and z represent letters A, B and a-i which indicate the
position of variable amino acid residues within said sequence
counted from the N-terminal end of said sequence. For example,
X.sub.a and X.sub.b indicate the first and second variable amino
acid residue, respectively, counted from the N-terminal end of
sequence iii), but do not have to be in positions one and two in
the amino acid sequence.
[0115] In embodiments according to the first aspect, there are
provided polypeptides wherein the variable amino acid residues
X.sub.y are independently selected from a group of possible
residues according to Table 2. The skilled person will appreciate
that X.sub.y may be selected from any one of the listed groups of
possible residues and that this selection is independent from the
selection of amino acids in X.sub.z, wherein y.noteq.z. Thus, any
of the listed possible residues in position X.sub.y in Table 2 may
be independently combined with any of the listed possible residues
in any other variable position in Table 2.
[0116] The skilled person will appreciate that Table 2 is to be
read as follows: In one embodiment according to the first aspect,
there is provided a polypeptide wherein amino acid residue
"X.sub.y" is selected from "Possible residues". Thus, Table 2
discloses several specific and individualized embodiments of the
first aspect of the present disclosure.
TABLE-US-00015 TABLE 2 X.sub.y Possible residues X.sub.A A X.sub.A
S X.sub.B G X.sub.B R X.sub.a P, Q, R X.sub.a P, R, S X.sub.a P, R
X.sub.a P, Q X.sub.a P, S X.sub.a P X.sub.a R X.sub.a S X.sub.a Q
X.sub.b Q, R, S X.sub.b N, R, S X.sub.b R, S X.sub.b R, Q X.sub.b
Q, S X.sub.b N, S X.sub.b N X.sub.b Q X.sub.b R X.sub.b S X.sub.b A
X.sub.b S X.sub.d N X.sub.d K X.sub.d E X.sub.e A X.sub.e S X.sub.e
C X.sub.f E X.sub.f N X.sub.f S X.sub.g D X.sub.g E X.sub.g S
X.sub.h A X.sub.h S X.sub.i A X.sub.i K X.sub.i no amino acid
[0117] Additionally, Table 3 lists possible combinations of 2, 3 or
6 of X.sub.A-B and X.sub.a-i. The skilled person will appreciate
that these combinations may be independently combined with any of
the listed possible residues in any other variable position in
Table 2.
TABLE-US-00016 TABLE 3 X.sub.y Possible residues residues
X.sub.A,X.sub.B A,G; A,R X.sub.A,X.sub.B A,G X.sub.A,X.sub.B A,R
X.sub.f,X.sub.g E,E; E,S; S,D; S,E; S,S; N,D X.sub.f,X.sub.g E,S
X.sub.f,X.sub.g S,E X.sub.f,X.sub.g S,D X.sub.f,X.sub.g N,D
X.sub.a,X.sub.b,X.sub.d P,Q,N; P,S,N; P,Q,K; Q,R,N; S,S,N
X.sub.a,X.sub.b,X.sub.d P,Q,N; P,S,N; Q,R,N X.sub.a,X.sub.b,X.sub.d
P,Q,N; P,S,N X.sub.a,X.sub.b,X.sub.d P,S,N X.sub.a,X.sub.b,X.sub.d
P,Q,N X.sub.a,X.sub.b,X.sub.d Q,R,N X.sub.a,X.sub.b,X.sub.d P,Q,K
X.sub.a,X.sub.b,X.sub.d S,S,N
X.sub.A,X.sub.B,X.sub.a,X.sub.b,X.sub.d,X.sub.i A,G,P,Q,N,A,
A,G,P,S,N,A, A,R,P,S,N,A, A,G,P,Q,K,A, A,R,Q,R,N,K, A,R,P,S,N,K,
ARPQNK, A,G,P,S,N,K, A,R,S,S,N,A, A,G,S,S,N,K
X.sub.A,X.sub.B,X.sub.a,X.sub.b,X.sub.d,X.sub.i A,G,P,Q,N,A,
A,R,P,S,N,A, A,G,P,S,N,A, A,R,Q,R,N,K, A,G,P,S,N,K
X.sub.A,X.sub.B,X.sub.a,X.sub.b,X.sub.d,X.sub.i A,G,P,Q,N,A,
A,R,Q,P,K,K X.sub.A,X.sub.B,X.sub.a,X.sub.b,X.sub.d,X.sub.i
A,G,P,Q,N,A X.sub.A,X.sub.B,X.sub.a,X.sub.b,X.sub.d,X.sub.i
A,G,P,S,N,A X.sub.A,X.sub.B,X.sub.a,X.sub.b,X.sub.d,X.sub.i
A,R,P,S,N,A X.sub.A,X.sub.B,X.sub.a,X.sub.b,X.sub.d,X.sub.i
A,G,P,Q,K,A X.sub.A,X.sub.B,X.sub.a,X.sub.b,X.sub.d,X.sub.i
A,R,Q,R,N,K X.sub.A,X.sub.B,X.sub.a,X.sub.b,X.sub.d,X.sub.i
A,R,P,S,N,K X.sub.A,X.sub.B,X.sub.a,X.sub.b,X.sub.d,X.sub.i
A,R,P,Q, N, K X.sub.A,X.sub.B,X.sub.a,X.sub.b,X.sub.d,X.sub.i
A,G,P,S,N,K X.sub.c,X.sub.d,X.sub.e,X.sub.f,X.sub.g,X.sub.h
A,N,A,N,D,A X.sub.c,X.sub.d,X.sub.e,X.sub.f,X.sub.g,X.sub.h
A,N,A,S,E,A X.sub.c,X.sub.d,X.sub.e,X.sub.f,X.sub.g,X.sub.h
A,N,A,S,D,A X.sub.c,X.sub.d,X.sub.e,X.sub.f,X.sub.g,X.sub.h
S,E,S,N,D,S X.sub.c,X.sub.d,X.sub.e,X.sub.f,X.sub.g,X.sub.h
S,E,S,S,E,S X.sub.c,X.sub.d,X.sub.e,X.sub.f,X.sub.g,X.sub.h
S,E,S,S,D,S X.sub.c,X.sub.d,X.sub.e,X.sub.f,X.sub.g,X.sub.h
S,E,C,N,D,S X.sub.c,X.sub.d,X.sub.e,X.sub.f,X.sub.g,X.sub.h
S,E,C,S,E,S X.sub.c,X.sub.d,X.sub.e,X.sub.f,X.sub.g,X.sub.h
S,E,C,S,D,S
[0118] As discussed above, polypeptides comprising minor changes as
compared to the above amino acid sequences, which changes do not
largely affect the tertiary structure or function of the
polypeptide, are also within the scope of the present disclosure.
Thus, sequence iv) has at least 97% identity to a sequence defined
by iii).
[0119] In one embodiment of the A.beta. peptide binding polypeptide
according to the first aspect of the disclosure, the definitions
above are subject to the proviso that at least one of the first and
second moiety of the polypeptide does not comprise an amino acid
sequence selected from
TABLE-US-00017 (SEQ ID NO: 554) VDNKFNKEGK GAPGEIHYLP NLNADQLCAF
IRSLEDDPSQ SANLLAEAKK LNDAQAPK and (SEQ ID NO: 555) VDNKFNKEIE
VATGEIVYLP NLNADQLCAF INSLEDDPSQ SANLLAEAKK LNDAQAPK.
[0120] As stated above, the A.beta. peptide binding polypeptide
according to the first aspect comprises a linker. In one
embodiment, said linker is arranged between said first moiety and
said second moiety as defined herein.
[0121] The skilled person is aware of different kinds of linkers
with different properties, such as flexible amino acid linkers,
rigid amino acid linkers and cleavable amino acid linkers. Linkers
have been used to for example increase stability or improve folding
of fusion proteins, to increase expression, improve biological
activity, enable targeting and alter pharmacokinetics of fusion
proteins.
[0122] Flexible linkers are often used when the joined domains
require a certain degree of movement or interaction, and may be
particularly useful in some embodiments of the A.beta. peptide
binding polypeptide as defined herein. Such linkers are generally
composed of small, non-polar (for example G) or polar (for example
S or T) amino acids. Some flexible linkers primarily consist of
stretches of G and S residues, for example (GGGGS (SEQ ID
NO:648)).sub.p and (SSSSG(SEQ ID NO:649)).sub.p. Adjusting the copy
number "p" allows for optimization of the linker in order to
achieve appropriate separation between the functional moieties or
to maintain necessary inter-moiety interaction. Apart from G and S
linkers, other flexible linkers are known in the art, such as G and
S linkers containing additional amino acid residues, such as T, A,
K and E, to maintain flexibility, as well as polar amino acid
residues to improve solubility.
[0123] In one embodiment of the first aspect disclosed herein, the
linker is a flexible linker comprising at least one amino acid
residue selected from the group consisting of glycine (G), serine
(S) and threonine (T). In one embodiment, said linker has a general
formula selected from (G.sub.nS.sub.m).sub.p and
(S.sub.nG.sub.m).sub.p, wherein, independently, n=1-7, m=0-7,
n+m.ltoreq. 8 and p=1-10. In one embodiment, n=1-5. In one
embodiment, m=0-5. In one embodiment, p=1-5. In one embodiment,
said linker is selected from the group consisting of S.sub.4G (SEQ
ID NO:649), (S.sub.4G).sub.3 (SEQ ID NO:634), (S.sub.4G).sub.4 (SEQ
ID NO:650) and (S.sub.4G).sub.8 (SEQ ID NO:651). In one embodiment,
said linker is selected from the group consisting of S.sub.4G (SEQ
ID NO:649), (S.sub.4G).sub.2 (SEQ ID NO:633), (S.sub.4G).sub.3 (SEQ
ID NO:634) and (S.sub.4G).sub.4 (SEQ ID NO:650). In a more specific
embodiment, n=4, m=1 and p=2-3. In one embodiment, said linker is
selected from the group consisting of (S.sub.4G).sub.2 (SEQ ID
NO:633) and (S.sub.4G).sub.3 (SEQ ID NO:634). In one embodiment,
said linker is (S.sub.4G).sub.2 (SEQ ID NO:633). In one embodiment,
said linker is (S.sub.4G).sub.3 (SEQ ID NO:634).
[0124] In another embodiment of the first aspect of the present
disclosure, there is provided an A.beta. peptide binding
polypeptide as defined herein, in which said linker comprises an
amino acid sequence selected from the group consisting of
VEVDNKFNKEMAS, VDNKFNKEMAS, VEVDNKFNKE, VDNKFNKE, AEAKYAKE,
ADNNFNK, ADNKFNK, ADAQQNNFNK, AQHDE, VDNKFNK, AEAKYAK, VDAKYAK and
ADAKYAK. In one embodiment, said sequence is selected from
VEVDNKFNKEMAS and VDNKFNKEMAS. In one embodiment, said sequence is
selected from VEVDNKFNKE and VDNKFNKE. In one embodiment, said
sequence is VEVDNKFNKEMAS. In another embodiment, said sequence is
VEVDNKFNKE.
[0125] The skilled person will appreciate that the properties of
the linker will generally not be changed by the addition of a small
number of amino acid residues to its N- or C-terminus. Thus, in a
further embodiment, said linker further comprises 1-5 additional
amino acid residues at the N- or C-terminal end of said linker,
such as 1-4 additional amino acid residues, such as 1-3 additional
amino acid residues, such as 3 additional amino acid residues. In
one embodiment, said additional amino acid residues are selected
from the group consisting of AS, MAS and RAS. In one embodiment,
said additional amino acid residues are RAS. In one embodiment,
said additional amino acid residues are MAS.
[0126] The skilled person will appreciate that the presence of a
linker arranged in any of the contexts mentioned above does not
exclude the presence of additional linkers in the same or any other
context. Thus, in one embodiment, the A.beta. peptide binding
polypeptide as defined herein further comprises at least one
additional linker, such as a linker selected from the group
consisting of flexible amino acid linkers, rigid amino acid linkers
and cleavable amino acid linkers. In one embodiment, said linker is
arranged between the A.beta. peptide binding polypeptide as
disclosed herein and a second or further part consisting of a
polypeptide having a desired biological activity. Further below is
a general discussion of alternatives for such a second part.
[0127] The skilled person will appreciate that the A.beta. peptide
binding polypeptides as disclosed herein may comprise any first
moiety in combination with any second moiety. Thus, the presence of
one particular first moiety as disclosed herein does neither limit
the choice of second moiety nor the choice of linker as disclosed
herein, and vice versa. The skilled person will appreciate that
first moiety, second moiety and linker as disclosed herein may be
independently combined.
[0128] As described in detail in the experimental section to
follow, the selection of A.beta. peptide binding polypeptides led
to the identification of a number of individual A.beta. peptide
binding motifs (BM1 and BM2). These motifs constitute individual
embodiments of sequence i) according to this aspect. The A.beta.
peptide binding motifs BM1 and BM2 of the A.beta. peptide
correspond to amino acid residues 4-25 and 64-85, respectively, in
the sequences with SEQ ID NO:1-17, 19-106 and 118-553, and to amino
acid residues 4-25 and 69-90, respectively, in SEQ ID NO:18, all as
presented in FIG. 1A-X. Hence, in one embodiment of the A.beta.
peptide binding polypeptide as defined herein, sequence i) of BM1
corresponds to amino acid residues 4-25 in any one of SEQ ID
NO:1-106 and 118-553, while sequence i) of BM2 corresponds to amino
acid residues 64-85 in any one of SEQ ID NO:1-17, 19-106 and
118-553 or to amino acid residues 69-90 in SEQ ID NO:18. In another
embodiment, sequence i) of BM1 corresponds to amino acid residues
4-25 in any one of SEQ ID NO:1-106, while sequence i) of BM2
corresponds to amino acid residues 64-85 in any one of SEQ ID
NO:1-17 and 19-106 or to amino acid residues 69-90 in SEQ ID NO:18.
In another embodiment, sequence i) of BM1 corresponds to amino acid
residues 4-25 in any one of SEQ ID NO:1, 5, 9, 13, 14, 16, 18, 20,
21, 25, 26, 28, 33, 35, 37, 40, 42, 48, 49, 50, 51, 53, 54, 55, 59,
60, 61, 62, 64, 65, 70, 72, 75, 78, 84, 89, 90, 95, 96, 97, 100,
104 and 118-159, while sequence i) of BM2 corresponds to amino acid
residues 64-85 in any one of SEQ ID NO:1, 5, 9, 13, 14, 16, 20, 21,
25, 26, 28, 33, 35, 37, 40, 42, 48, 49, 50, 51, 53, 54, 55, 59, 60,
61, 62, 64, 65, 70, 72, 75, 78, 84, 89, 90, 95, 96, 97, 100, 104
and 118-159 or to amino acid residues 69-90 in SEQ ID NO:18. In
another embodiment, sequence i) of BM1 corresponds to amino acid
residues 4-25 in any one of SEQ ID NO:1, 5, 9, 13, 14, 16, 18, 20,
21, 25, 26, 28, 33, 35, 37, 40, 42, 48, 49, 50, 53, 54, 59, 60, 61,
62, 70, 75, 78, 84, 89, 90, 95, 96, 97, 100 and 104, while sequence
i) of BM2 corresponds to amino acid residues 64-85 in any one of
SEQ ID NO:1, 5, 9, 13, 14, 16, 20, 21, 25, 26, 28, 33, 35, 37, 40,
42, 48, 49, 50, 53, 54, 59, 60, 61, 62, 70, 75, 78, 84, 89, 90, 95,
96, 97, 100 and 104 or to amino acid residues 69-90 in SEQ ID
NO:18. In another embodiment, sequence i) of BM1 corresponds to
amino acid residues 4-25 in any one of SEQ ID NO:18, 28, 35, 49,
50, 51, 55, 59, 64, 65, 70, 72, 78, 84, 95 and 118-159, while
sequence i) of BM2 corresponds to amino acid residues 64-85 in any
one of SEQ ID NO:28, 35, 49, 50, 51, 55, 59, 64, 65, 70, 72, 78,
84, 95 and 118-159 or to amino acid residues 69-90 in SEQ ID NO:18.
In another embodiment, sequence i) of BM1 corresponds to amino acid
residues 4-25 in any one of SEQ ID NO:18, 51, 55, 59, 64, 70, 78,
95 and 118-142, while sequence i) of BM2 corresponds to amino acid
residues 64-85 in any one of SEQ ID NO:51, 55, 59, 64, 70, 78, 95
and 118-142 or to amino acid residues 69-90 in SEQ ID NO:18. In
another embodiment, sequence i) of BM1 corresponds to amino acid
residues 4-25 in any one of SEQ ID NO:18, 55, 70, 95, 124-127,
130-133, 139 and 140, while sequence i) of BM2 corresponds to amino
acid residues 64-85 in any one of SEQ ID NO:55, 70, 95, 124-127,
130-133, 139 and 140 or to amino acid residues 69-90 in SEQ ID
NO:18. In another embodiment, sequence i) of BM1 corresponds to
amino acid residues 4-25 in any one of SEQ ID NO:18, 55, 70, 95,
124, 125, 132, 133, 139 and 140, while sequence i) of BM2
corresponds to amino acid residues 64-85 in any one of SEQ ID
NO:55, 70, 95, 124, 125, 132, 133, 139 and 140 or to amino acid
residues 69-90 in SEQ ID NO:18. In another embodiment, sequence i)
of BM1 corresponds to amino acid residues 4-25 in any one of SEQ ID
NO:18, 28, 35, 49, 50, 70, 84 and 95, while sequence i) of BM2
corresponds to amino acid residues 64-85 in any one of SEQ ID
NO:28, 35, 49, 50, 70, 84 and 95 or to amino acid residues 69-90 in
SEQ ID NO:18. In one embodiment, sequence i) of BM1 corresponds to
amino acid residues 4-25 in any one of SEQ ID NO:18, 70 and 95,
while sequence i) of BM2 corresponds to amino acid residues 64-85
in any one of SEQ ID NO:70 and 95 or amino acid residues 69-90 in
SEQ ID NO:18. In one particular embodiment, sequence i) of BM1
corresponds to amino acid residues 4-25 in SEQ ID NO:95 and
sequence i) of BM2 corresponds to amino acid residues 64-85 in SEQ
ID NO:95.
[0129] In another embodiment, there is provided an A.beta. peptide
binding polypeptide as disclosed herein, wherein the first moiety
comprises amino acid residues 1-25 in any one of SEQ ID NO:1-106
and 118-553 and the second moiety comprises amino acid residues
61-85 in any one of SEQ ID NO:1-17, 19-106 and 118-553 or amino
acid residues 66-90 in SEQ ID NO:18. In another embodiment, there
is provided an A.beta. peptide binding polypeptide as disclosed
herein, wherein the first moiety comprises amino acid residues 1-25
in any one of SEQ ID NO:1-106 and the second moiety comprises amino
acid residues 61-85 in any one of SEQ ID NO:1-17 and 19-106 or
amino acid residues 66-90 in SEQ ID NO:18. In another embodiment,
the first moiety comprises amino acid residues 1-25 in any one of
SEQ ID NO:1, 5, 9, 13, 14, 16, 18, 20, 21, 25, 26, 28, 33, 35, 37,
40, 42, 48, 49, 50, 51, 53, 54, 55, 59, 60, 61, 62, 64, 65, 70, 72,
75, 78, 84, 89, 90, 95, 96, 97, 100, 104 and 118-159 and the second
moiety comprises amino acid residues 61-85 in any one of SEQ ID
NO:1, 5, 9, 13, 14, 16, 18, 20, 21, 25, 26, 28, 33, 35, 37, 40, 42,
48, 49, 50, 51, 53, 54, 55, 59, 60, 61, 62, 64, 65, 70, 72, 75, 78,
84, 89, 90, 95, 96, 97, 100, 104 and 118-159 or amino acid residues
66-90 in SEQ ID NO:18. In another embodiment, the first moiety
comprises amino acid residues 1-25 in any one of SEQ ID NO:1, 5, 9,
13, 14, 16, 18, 20, 21, 25, 26, 28, 33, 35, 37, 40, 42, 48, 49, 50,
53, 54, 59, 60, 61, 62, 70, 75, 78, 84, 89, 90, 95, 96, 97, 100 and
104 and the second moiety comprises amino acid residues 61-85 in
any one of SEQ ID NO:1, 5, 9, 13, 14, 16, 20, 21, 25, 26, 28, 33,
35, 37, 40, 42, 48, 49, 50, 53, 54, 59, 60, 61, 62, 70, 75, 78, 84,
89, 90, 95, 96, 97, 100 and 104 or amino acid residues 66-90 in SEQ
ID NO:18. In another embodiment, the first moiety comprises amino
acid residues 1-25 in any one of SEQ ID NO:18, 28, 35, 49, 50, 51,
55, 59, 64, 65, 70, 72, 78, 84, 95 and 118-159 and the second
moiety comprises amino acid residues 61-85 in any one of SEQ ID
NO:28, 35, 49, 50, 51, 55, 59, 64, 65, 70, 72, 78, 84, 95 and
118-159 or amino acid residues 66-90 in SEQ ID NO:18. In another
embodiment, the first moiety comprises amino acid residues 1-25 in
any one of SEQ ID NO:18, 51, 55, 59, 64, 70, 78, 95 and 118-142 and
the second moiety comprises amino acid residues 61-85 in any one of
SEQ ID NO:51, 55, 59, 64, 70, 78, 95 and 118-142 or amino acid
residues 66-90 in SEQ ID NO:18. In another embodiment, the first
moiety comprises amino acid residues 1-25 in any one of SEQ ID
NO:18, 55, 70, 95, 124-127, 130-133, 139 and 140 and the second
moiety comprises amino acid residues 61-85 in any one of SEQ ID
NO:55, 70, 95, 124-127, 130-133, 139 and 140 or amino acid residues
66-90 in SEQ ID NO:18. In another embodiment, the first moiety
comprises amino acid residues 1-25 in any one of SEQ ID NO:18, 55,
70, 95, 124, 125, 132, 133, 139 and 140 and the second moiety
comprises amino acid residues 61-85 in any one of SEQ ID NO:55, 70,
95, 124, 125, 132, 133, 139 and 140 or amino acid residues 66-90 in
SEQ ID NO:18. In yet another embodiment, the first moiety comprises
amino acid residues 1-25 in any one of SEQ ID NO:18, 28, 35, 49,
50, 70, 84 and 95 and the second moiety comprises amino acid
residues 61-85 in any one of SEQ ID NO:28, 35, 49, 50, 70, 84 and
95 or amino acid residues 66-90 in SEQ ID NO:18. In one embodiment,
the first moiety comprises amino acid residues 1-25 in any one of
SEQ ID NO:18, 70 and 95 and the second moiety comprises amino acid
residues 61-85 in any one of SEQ ID NO:70 and 95 or amino acid
residues 66-90 in SEQ ID NO:18. In one particular embodiment, the
first moiety comprises amino acid residues 1-25 in SEQ ID NO:95 and
the second moiety comprises amino acid residues 61-85 in SEQ ID
NO:95.
[0130] In another embodiment, there is provided an A.beta. peptide
binding polypeptide as disclosed herein having a binding module,
Bmod, as defined above, wherein sequence iii) in Bmod of the first
moiety corresponds to amino acid residues 4-47 in any one of SEQ ID
NO:1-106 and 118-553, while sequence iii) in Bmod of the second
moiety comprises amino acid residues 64-107 in any one of SEQ ID
NO:1-17, 19-106 and 118-553 or amino acid residues 69-112 in SEQ ID
NO:18. In another embodiment, sequence iii) in Bmod of the first
moiety corresponds to amino acid residues 4-47 in any one of SEQ ID
NO:1-106, while sequence iii) in Bmod of the second moiety
comprises amino acid residues 64-107 in any one of SEQ ID NO:1-17
and 19-106 or amino acid residues 69-112 in SEQ ID NO:18. In
another embodiment, sequence iii) in Bmod of the first moiety
corresponds to amino acid residues 4-47 in any one of SEQ ID NO:1,
5, 9, 13, 14, 16, 18, 20, 21, 25, 26, 28, 33, 35, 37, 40, 42, 48,
49, 50, 51, 53, 54, 55, 59, 60, 61, 62, 64, 65, 70, 72, 75, 78, 84,
89, 90, 95, 96, 97, 100, 104 and 118-159, while sequence iii) in
Bmod of the second moiety comprises amino acid residues 64-107 in
any one of SEQ ID NO:1, 5, 9, 13, 14, 16, 20, 21, 25, 26, 28, 33,
35, 37, 40, 42, 48, 49, 50, 51, 53, 54, 55, 59, 60, 61, 62, 64, 65,
70, 72, 75, 78, 84, 89, 90, 95, 96, 97, 100, 104 and 118-159 or
amino acid residues 69-112 in SEQ ID NO:18. In another embodiment,
sequence iii) in Bmod of the first moiety corresponds to amino acid
residues 4-47 in any one of SEQ ID NO:1, 5, 9, 13, 14, 16, 18, 20,
21, 25, 26, 28, 33, 35, 37, 40, 42, 48, 49, 50, 53, 54, 59, 60, 61,
62, 70, 75, 78, 84, 89, 90, 95, 96, 97, 100 and 104, while sequence
iii) in Bmod of the second moiety comprises amino acid residues
64-107 in any one of SEQ ID NO:1, 5, 9, 13, 14, 16, 20, 21, 25, 26,
28, 33, 35, 37, 40, 42, 48, 49, 50, 53, 54, 59, 60, 61, 62, 70, 75,
78, 84, 89, 90, 95, 96, 97, 100 and 104 or amino acid residues
69-112 in SEQ ID NO:18. In another embodiment, sequence iii) in
Bmod of the first moiety corresponds to amino acid residues 4-47 in
any one of SEQ ID NO:18, 28, 35, 49, 50, 51, 55, 59, 64, 65, 70,
72, 78, 84, 95 and 118-159, while sequence iii) in Bmod of the
second moiety comprises amino acid residues 64-107 in any one of
SEQ ID NO:28, 35, 49, 50, 51, 55, 59, 64, 65, 70, 72, 78, 84, 95
and 118-159 or amino acid residues 69-112 in SEQ ID NO:18. In
another embodiment, sequence iii) in Bmod of the first moiety
corresponds to amino acid residues 4-47 in any one of SEQ ID NO:18,
51, 55, 59, 64, 70, 78, 95 and 118-142, while sequence iii) in Bmod
of the second moiety comprises amino acid residues 64-107 in any
one of SEQ ID NO:51, 55, 59, 64, 70, 78, 95 and 118-142 or amino
acid residues 69-112 in SEQ ID NO:18. In another embodiment,
sequence iii) in Bmod of the first moiety corresponds to amino acid
residues 4-47 in any one of SEQ ID NO:18, 55, 70, 95, 124-127,
130-133, 139 and 140, while sequence iii) in Bmod of the second
moiety comprises amino acid residues 64-107 in any one of SEQ ID
NO:55, 70, 95, 124-127, 130-133, 139 and 140 or amino acid residues
69-112 in SEQ ID NO:18. In another embodiment, sequence iii) in
Bmod of the first moiety corresponds to amino acid residues 4-47 in
any one of SEQ ID NO:18, 55, 70, 95, 124, 125, 132, 133, 139 and
140, while sequence iii) in Bmod of the second moiety comprises
amino acid residues 64-107 in any one of SEQ ID NO:55, 70, 95, 124,
125, 132, 133, 139 and 140 or amino acid residues 69-112 in SEQ ID
NO:18. In yet another embodiment, sequence iii) in Bmod of the
first moiety corresponds to amino acid residues 4-47 in any one of
SEQ ID NO:18, 28, 35, 49, 50, 70, 84 and 95, while sequence iii) in
Bmod of the second moiety comprises amino acid residues 64-107 in
any one of SEQ ID NO:28, 35, 49, 50, 70, 84 and 95 or amino acid
residues 69-112 in SEQ ID NO:18. In one embodiment, sequence iii)
in Bmod of the first moiety corresponds to amino acid residues 4-47
in any one of SEQ ID NO:18, 70 and 95, while sequence iii) in Bmod
of the second moiety comprises amino acid residues 64-107 in any
one of SEQ ID NO:70 and 95 or amino acid residues 69-112 in SEQ ID
NO:18. In one particular embodiment, sequence iii) in Bmod of the
first moiety corresponds to amino acid residues 4-47 in SEQ ID
NO:95 and sequence iii) in Bmod of the second moiety comprises
amino acid residues 64-107 in SEQ ID NO:95.
[0131] As discussed above, the skilled person will appreciate that
the 22 amino acid residue binding motifs, BM, as well as the
various stretches of 25, 44 or 47 amino acid residues as defined
herein and all comprising a BM, may all be combined independently.
For example, the BM or a 25 amino acid residue stretch comprising a
BM, may be grafted into another amino acid environment than that of
the original polypeptide, which new environment substantially
retains the three-dimensional structure of the original
environment. Thus, in another embodiment of the A.beta. peptide
binding polypeptide as disclosed herein, there is provided a
A.beta. peptide binding polypeptide wherein the first moiety
comprises an amino acid sequence corresponding to amino acid
residues 4-25, 1-25, 4-47 or 1-47 from any one of SEQ ID NO:1-106
and 118-553; and the second moiety comprises an amino acid sequence
corresponding to amino acid residues 64-85, 61-85, 64-107 or 61-107
from any one of SEQ ID NO:1-17, 19-106 and 118-553 or to amino acid
residues 69-90, 66-90, 69-112 or 66-112 from SEQ ID NO:18, and
wherein both amino acid sequences comprised in each of the first
and second moieties are from the same SEQ ID NO. Also, in another
embodiment of the A.beta. peptide binding polypeptide as disclosed
herein, there is provided a A.beta. peptide binding polypeptide
wherein the first moiety comprises an amino acid sequence
corresponding to amino acid residues 4-25, 1-25, 4-47 or 1-47 from
any one of SEQ ID NO:1-106; and the second moiety comprises an
amino acid sequence corresponding to amino acid residues 64-85,
61-85, 64-107 or 61-107 from any one of SEQ ID NO:1-17 and 19 to
106; or to amino acid residues 69-90, 66-90, 69-112 or 66-112 from
SEQ ID NO:18, and wherein both amino acid sequences comprised in
each of the first and second moieties are from the same SEQ ID
NO.
[0132] In another embodiment according to this aspect of the
disclosure, there is provided an A.beta. peptide binding
polypeptide, which comprises an amino acid sequence selected from
the group consisting of SEQ ID NO:1-106 and 118-553; such as from
the group consisting of SEQ ID NO:1-106; such as from the group
consisting of SEQ ID NO:1, 5, 9, 13, 14, 16, 18, 20, 21, 25, 26,
28, 33, 35, 37, 40, 42, 48, 49, 50, 51, 53, 54, 55, 59, 60, 61, 62,
64, 65, 70, 72, 75, 78, 84, 89, 90, 95, 96, 97, 100, 104 and
118-159; such as from the group consisting of SEQ ID NO:1, 5, 9,
13, 14, 16, 18, 20, 21, 25, 26, 28, 33, 35, 37, 40, 42, 48, 49, 50,
53, 54, 59, 60, 61, 62, 70, 75, 78, 84, 89, 90, 95, 96, 97, 100 and
104; such as from the group consisting of SEQ ID NO:18, 28, 35, 49,
50, 51, 55, 59, 64, 65, 70, 72, 78, 84, 95 and 118-159; such as
from the group consisting of SEQ ID NO:18, 51, 55, 59, 64, 70, 78,
95 and 118-142; such as from the group consisting of SEQ ID NO:18,
55, 70, 95, 124-127, 130-133, 139 and 140; such as from the group
consisting of SEQ ID NO:18, 55, 70, 95, 124, 125, 132, 133, 139 and
140; such as from the group consisting of SEQ ID NO:18, 28, 35, 49,
50, 70, 84 and 95; such as from the group consisting of SEQ ID NO
18, 70 and 95. In one embodiment, the A.beta. peptide binding
polypeptide comprises SEQ ID NO:95.
[0133] "Amyloid .beta.", "A.beta." or ".beta.-amyloid" as used
herein refer to amyloid .beta. proteins and peptides, amyloid
.beta. precursor protein (APP), as well as modifications, fragments
and functional equivalents thereof. In particular, by "amyloid
.beta. peptide" or "A.beta. peptide" is meant any fragment produced
by proteolytic cleavage of APP, especially fragments which are
involved in or associated with the amyloid pathologies. Such
fragments include, but are not limited to, A.beta..sub.1-38,
A.beta..sub.1-39, A.beta..sub.1-40, A.beta..sub.1-41,
A.beta..sub.1-42 and A.beta..sub.1-43.
[0134] The terms "A.beta. peptide binding" and "binding affinity
for A.beta. peptide" as used in this specification refer to a
property of a polypeptide which may be tested for example by ELISA
or by use of surface plasmon resonance (SPR) technology.
[0135] For example as described in the examples below, A.beta.
peptide binding affinity may be tested in an experiment in which
samples of the polypeptide are captured on antibody coated ELISA
plates and biotinylated A.beta. peptide is added, followed by
streptavidin conjugated HRP. TMB substrate is added and the
absorbance at 450 nm is measured using a multi-well plate reader,
such as Victor.sup.3 (Perkin Elmer). The skilled person may then
interpret the results obtained by such experiments to establish at
least a qualitative measure of the binding affinity of the
polypeptide for A.beta. peptide. If a quantitative measure is
desired, for example to determine the EC50 value (the half maximal
effective concentration) for the interaction, ELISA may also be
used. The response of the polypeptides against a dilution series of
biotinylated A.beta. peptide is measured using ELISA as described
above. The skilled person may then interpret the results obtained
by such experiments and EC50 values may be calculated from the
results using for example GraphPad Prism 5 and non-linear
regression.
[0136] A.beta. peptide binding affinity may also be tested in an
experiment in which A.beta. peptide, or a fragment thereof, is
immobilized on a sensor chip of a surface plasmon resonance (SPR)
instrument, and the sample containing the polypeptide to be tested
is passed over the chip. Alternatively, the polypeptide to be
tested is immobilized on a sensor chip of the instrument, and a
sample containing A.beta. peptide, or a fragment thereof, is passed
over the chip. The skilled person may then interpret the results
obtained by such experiments to establish at least a qualitative
measure of the binding affinity of the polypeptide for A.beta.
peptide. If a quantitative measure is desired, for example to
determine a K.sub.D value for the interaction, surface plasmon
resonance methods may also be used. Binding values may for example
be defined in a Biacore (GE Healthcare) or ProteOn XPR 36 (Bio-Rad)
instrument. A.beta. peptide is suitably immobilized on a sensor
chip of the instrument, and samples of the polypeptide whose
affinity is to be determined are prepared by serial dilution and
injected in random order. K.sub.D values may then be calculated
from the results using for example the 1:1 Langmuir binding model
of the BIAevaluation 4.1 software, or other suitable software,
provided by the instrument manufacturer.
[0137] In one embodiment, the A.beta. peptide binding polypeptide
is capable of binding to A.beta. peptide such that the EC50 value
of the interaction is at most 1.times.10.sup.-7 M, such as at most
1.times.10.sup.-8 M, such as at most 1.times.10.sup.-9 M, such as
at most 1.times.10.sup.-10 M.
[0138] In one embodiment, the A.beta. peptide binding polypeptide
is capable of binding to A.beta. peptide such that the K.sub.D
value of the interaction is at most 1.times.10.sup.-7 M, such as at
most 1.times.10.sup.-8 M, such as at most 1.times.10.sup.-9 M, such
as at most 1.times.10.sup.-10 M.
[0139] The skilled person will understand that various
modifications and/or additions can be made to an A.beta. peptide
binding polypeptide according to the first aspect disclosed herein
in order to tailor the polypeptide to a specific application
without departing from the scope of the present disclosure.
[0140] For example, in one embodiment there is provided a fragment
of an A.beta. peptide binding polypeptide as disclosed herein,
which fragment retains binding affinity for an A.beta. peptide.
[0141] An A.beta. peptide binding polypeptide encompassed by the
present disclosure may comprise a number of additional amino acid
residues. For example, in one embodiment, there is provided an
A.beta. peptide binding polypeptide as described herein, which
polypeptide has been extended by and/or comprises at least one
additional amino acid at at least one of the N terminus and the C
terminus. Such a polypeptide should be understood as a polypeptide
having one or more additional amino acid residues at the very first
and/or the very last position in the polypeptide chain. Thus, an
A.beta. peptide binding polypeptide may comprise any suitable
number of additional amino acid residues, for example at least one
additional amino acid residue. Suitably, an A.beta. peptide binding
polypeptide with additional amino acids will retain binding
affinity for an A.beta. peptide.
[0142] Each additional amino acid residue may individually or
collectively be added in order to, for example, improve or simplify
production, purification, stabilization in vivo or in vitro,
coupling or detection of the polypeptide. Such additional amino
acid residues may for example comprise one or more amino acid
residues added for the purpose of chemical coupling. One example of
this is the addition of a cysteine residue. Additional amino acid
residues may also provide a "tag" for purification or detection of
the polypeptide, such as a Hiss tag, a (HisGlu).sub.3 tag ("HEHEHE"
tag) or a "myc" (c-myc) tag or a "FLAG" tag for interaction with
antibodies specific to the tag or immobilized metal affinity
chromatography (IMAC) in the case of the Hiss-tag.
[0143] The further amino acids as discussed above may be coupled to
the A.beta. peptide binding polypeptide by means of chemical
conjugation (using known organic chemistry methods) or by any other
means, such as expression of the A.beta. peptide binding
polypeptide as a fusion protein or joined in any other fashion,
either directly or via a linker, for example an amino acid linker
as discussed above.
[0144] Fusion proteins, or conjugates, in which an A.beta. peptide
binding polypeptide described herein constitutes a first domain
(herein referred to as a first part), and which also comprises
second and optionally further domains (herein referred to as second
and further parts) are also contemplated and fall within the ambit
of the present disclosure. The second and optional further parts of
the fusion polypeptide or conjugate suitably have a desired
biological activity.
[0145] For example, such a desired biological activity could be
binding to the same target, A.beta. peptide, as the first part. In
one embodiment, this second and optional further A.beta. peptide
binding part could comprise one or more additional polypeptides as
disclosed in the first aspect above, making the fusion protein or
conjugate a multimer (such as a dimer, trimer, tetramer etc) of
A.beta. peptide binding polypeptides as disclosed herein. The amino
acid sequences of the individual, "monomer", polypeptide chains may
be the same or different. Also contemplated are constructs in which
the first part is as disclosed in the first aspect, whereas the
second and optional further parts are not.
[0146] Thus, in a second aspect of the present disclosure, there is
provided a fusion protein or a conjugate, comprising a first part
consisting of an A.beta. peptide binding polypeptide according to
the first aspect, and a second part consisting of a polypeptide
having a desired biological activity. In one embodiment, said
fusion protein or conjugate may additionally comprise further
parts, comprising desired biological activities that can be either
the same or different from the biological activity of the first
and/or second part. For the sake of clarity, the discussion to
follow relating to said second part is equally relevant for any
further parts which may be present in the fusion or conjugate as
defined herein.
[0147] Non-limiting examples of a desired biological activity
comprise a therapeutic activity, a binding activity and an
enzymatic activity.
[0148] In one embodiment, the second part having a desired
biological activity is a therapeutically active polypeptide.
[0149] Non-limiting examples of therapeutically active polypeptides
are biomolecules, such as molecules selected from the group
consisting of human endogenous enzymes, hormones, growth factors,
chemokines, cytokines and lymphokines.
[0150] Non-limiting examples of binding activities are binding
activities which increase the in vivo half-life of the fusion
protein or conjugate. In one particular embodiment, said binding
activity which increases the in vivo half-life of the fusion
protein or conjugate is an albumin binding activity. In one
embodiment, said albumin binding activity is provided by the
albumin binding domain of streptococcal protein G or a derivative
thereof, for example comprising the amino acid sequence SEQ ID
NO:107 or a derivative thereof. In another particular embodiment,
said binding activity which increases the in vivo half-life of the
fusion protein or conjugate is an FcRn binding activity. FcRn
binding may for example be provided by an antibody or a fragment
thereof having FcRn affinity (reviewed in Roopenian and Akilesh
(2007), Nat Rev Immunol 7:715-725), or by a Z variant with
engineered affinity for FcRn (Seijsing et al (2014), Proc Natl Acad
Sci USA 111:17110-5).
[0151] A binding activity as discussed above may be a binding
activity that acts to block a biological activity or a binding
activity that acts to stimulate a biological activity. Specific,
contemplated targets for binding of a second or optional further
part are selected from the group consisting of Tau, apolipoprotein
E (ApoE), beta-secretase 1 (BACE1), gamma-secretase, transferrin
receptor (TfR), complement factor C1s, presenilin 1, presenilin 2,
nicastrin, alpha-synuclein and Bri.
[0152] In one particular embodiment, the second part of said fusion
protein or conjugate may comprise an antibody or an antigen binding
fragment thereof. As is well known, antibodies are immunoglobulin
molecules capable of specific binding to a target (an antigen),
such as a carbohydrate, polynucleotide, lipid, polypeptide or
other, through at least one antigen recognition site located in the
variable region of the immunoglobulin molecule. As used herein, the
term "antibody or an antigen binding fragment thereof" encompasses
not only full-length or intact polyclonal or monoclonal antibodies,
but also antigen-binding fragments thereof, such as Fab, Fab',
F(ab').sub.2, Fab.sub.3, Fv and variants thereof, fusion proteins
comprising one or more antibody portions, humanized antibodies,
chimeric antibodies, minibodies, diabodies, triabodies,
tetrabodies, linear antibodies, single chain antibodies,
multispecific antibodies (e.g., bispecific antibodies) and any
other modified configuration of the immunoglobulin molecule that
comprises an antigen recognition site of the required specificity,
including glycosylation variants of antibodies, amino acid sequence
variants of antibodies and covalently modified antibodies. Further
examples of modified antibodies and antigen binding fragments
thereof include nanobodies, AlbudAbs, DARTs (dual affinity
re-targeting), BiTEs (bispecific T-cell engager), TandAbs (tandem
diabodies), DAFs (dual acting Fab), two-in-one antibodies, SMIPs
(small modular immunopharmaceuticals), FynomAbs (fynomers fused to
antibodies), DVD-Igs (dual variable domain immunoglobulin),
CovX-bodies (peptide modified antibodies), duobodies and triomAbs.
This listing of variants of antibodies and antigen binding
fragments thereof is not to be seen as limiting, and the skilled
person is aware of other suitable variants.
[0153] A full-length antibody comprises two heavy chains and two
light chains. Each heavy chain contains a heavy chain variable
region (V.sub.H) and first, second and third constant regions
(C.sub.H1, C.sub.H2 and C.sub.H3). Each light chain contains a
light chain variable region (V.sub.L) and a light chain constant
region (CL). Depending on the amino acid sequence of the constant
domain of its heavy chains, antibodies are assigned to different
classes. There are six major classes of antibodies: IgA, IgD, IgE,
IgG, IgM and IgY, and several of these may be further divided into
subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
The term "full-length antibody" as used herein refers to an
antibody of any class, such as IgD, IgE, IgG, IgA, IgM or IgY (or
any sub-class thereof). The subunit structures and
three-dimensional configurations of different classes of antibodies
are well known.
[0154] An "antigen binding fragment" is a portion or region of an
antibody molecule, or a derivative thereof, that retains all or a
significant part of the antigen binding of the corresponding
full-length antibody. An antigen binding fragment may comprise the
heavy chain variable region (V.sub.H), the light chain variable
region (V.sub.L), or both. Each of the V.sub.H and V.sub.L
typically contains three complementarity determining regions CDR1,
CDR2 and CDR3. The three CDRs in V.sub.H or V.sub.L are flanked by
framework regions (FR1, FR2, FR3 and FR4). As briefly listed above,
examples of antigen binding fragments include, but are not limited
to: (1) a Fab fragment, which is a monovalent fragment having a
V.sub.L-CL chain and a V.sub.H-C.sub.H1 chain; (2) a Fab' fragment,
which is a Fab fragment with the heavy chain hinge region, (3) a
F(ab')2 fragment, which is a dimer of Fab' fragments joined by the
heavy chain hinge region, for example linked by a disulfide bridge
at the hinge region; (4) an Fc fragment; (5) an Fv fragment, which
is the minimum antibody fragment having the V.sub.L and V.sub.H
domains of a single arm of an antibody; (6) a single chain Fv
(scFv) fragment, which is a single polypeptide chain in which the
V.sub.H and V.sub.L domains of an scFv are linked by a peptide
linker; (7) an (scFv).sub.2, which comprises two V.sub.H domains
and two V.sub.L domains, which are associated through the two
V.sub.H domains via disulfide bridges and (8) domain antibodies,
which can be antibody single variable domain (V.sub.H or V.sub.L)
polypeptides that specifically bind antigens.
[0155] Antigen binding fragments can be prepared via routine
methods. For example, F(ab').sub.2 fragments can be produced by
pepsin digestion of a full-length antibody molecule, and Fab
fragments can be generated by reducing the disulfide bridges of
F(ab').sub.2 fragments. Alternatively, fragments can be prepared
via recombinant technology by expressing the heavy and light chain
fragments in suitable host cells (e.g., E. coli, yeast, mammalian,
plant or insect cells) and having them assembled to form the
desired antigen-binding fragments either in vivo or in vitro. A
single-chain antibody can be prepared via recombinant technology by
linking a nucleotide sequence coding for a heavy chain variable
region and a nucleotide sequence coding for a light chain variable
region. For example, a flexible linker may be incorporated between
the two variable regions. The skilled person is aware of methods
for the preparation of both full-length antibodies and antigen
binding fragments thereof.
[0156] Thus, in one embodiment there is provided a fusion protein
or conjugate as defined herein, wherein said at least one antibody
or antigen binding fragment thereof is selected from the group
consisting of full-length antibodies, Fab fragments, Fab'
fragments, F(ab').sub.2 fragments, Fc fragments, Fv fragments,
single chain Fv fragments, (scFv).sub.2 and domain antibodies. In
one embodiment, said at least one antibody or antigen binding
fragment thereof is selected from full-length antibodies, Fab
fragments and scFv fragments. In one particular embodiment, said at
least one antibody or antigen binding fragment thereof is a
full-length antibody.
[0157] In one embodiment, the antibody or antigen binding fragment
thereof is selected from the group consisting of monoclonal
antibodies, human antibodies, humanized antibodies, chimeric
antibodies, and antigen-binding fragments thereof.
[0158] The term "monoclonal antibodies" as used herein refers to
antibodies having monovalent affinity, meaning that each antibody
molecule in a sample of the monoclonal antibody binds to the same
epitope on the antigen, whereas the term "polyclonal antibodies" as
used herein refers to a collection of antibodies that react against
a specific antigen, but in which collection there may be different
antibody molecules for example identifying different epitopes on
the antigen. Polyclonal antibodies are typically produced by
inoculation of a suitable mammal and are purified from the mammal's
serum. Monoclonal antibodies are made by identical immune cells
that are clones of a unique parent cell (for example a hybridoma
cell line). The term "human antibody" as used herein refers to
antibodies having variable and constant regions corresponding
substantially to, or derived from, antibodies obtained from human
subjects. The term "chimeric antibodies" as used herein, refers to
recombinant or genetically engineered antibodies, such as for
example mouse monoclonal antibodies, which contain polypeptides or
domains from a different species, for example human, introduced to
reduce the antibodies' immunogenicity. The term "humanized
antibodies" refers to antibodies from non-human species whose
protein sequences have been modified to increase their similarity
to antibody variants produced naturally in humans, in order to
reduce immunogenicity.
[0159] In one embodiment, there is provided a fusion protein or a
conjugate as disclosed herein, wherein antibody or antigen binding
fragment thereof has affinity for a target selected from the group
consisting of Tau, apolipoprotein E (ApoE), beta-secretase 1
(BACE1; Atwal et al (2011), Sci Transl Med 3:84ra43),
gamma-secretase, transferrin receptor (TfR; Yu et al, (2014) Sci
Transl Med 6:261ra154), complement factor C1s, presenilin 1,
presenilin 2, nicastrin, alpha-synuclein and Bri.
[0160] The blood-brain barrier (BBB) represents a major obstacle to
the delivery of drugs to the central nervous system (CNS). The BBB
is a highly selective permeability barrier that separates the
circulating blood from the brain extracellular fluid in the central
nervous system and one of its important functions is to block
circulating substances from entering or exiting the CNS, which
makes drug delivery to the brain challenging. It may therefore be
beneficial to target the fusion protein or conjugate to the brain
by fusion to a second part which promotes the transport across the
BBB. The major mechanisms for delivery of substances into the CNS
are transmembrane diffusion and saturable transport. Most CNS
therapeutics are small, lipid soluble molecules that are likely to
rely upon transmembrane diffusion to cross the BBB. Although
peptides, and even some small proteins, have a measurable
transmembrane diffusion, saturable transporters are believed to be
the most effective mechanism for delivering these molecules into
the CNS. Saturable transporters typically deliver 10 to 100 times
more of their main ligand to the CNS than would occur with
transmembrane diffusion. It may also be of interest to adapt the
fusion protein or conjugate such that it can exit the CNS after
having bound to the A.beta. target, so that it may for example
remove or sequester A.beta. peptide more efficiently.
[0161] Thus, in one embodiment, there is provided a fusion protein
or conjugate as disclosed herein, wherein the second part mediates
transport of the fusion protein or conjugate to the cerebrospinal
fluid and/or brain. In one embodiment, said second part is selected
from the group consisting of transferrin, ghrelin, insulin and
leptin; for example selected from transferrin and ghrelin.
[0162] In another embodiment, there is provided a fusion protein or
conjugate as disclosed, wherein the second part mediates transport
away from the cerebrospinal fluid and/or brain. In one such
embodiment, said second part is selected from the group consisting
of anti-FcRn antibodies (Roopenian and Akilesh (2007), supra) and
FcRn-binding Z variants (Seijsing et al (2014), supra) as disclosed
in the context of half-life extension above.
[0163] The disclosure furthermore encompasses polypeptides in which
the A.beta. peptide binding polypeptide according to the first
aspect and as comprised in a fusion protein or conjugate according
to the second aspect further comprises a label, such as a label
selected from the group consisting of fluorescent dyes and metals,
chromophoric dyes, chemiluminescent compounds and bioluminescent
proteins, enzymes, radionuclides and radioactive particles. In
other embodiments, the labeled A.beta. peptide binding polypeptide
is present as a moiety in a fusion protein or conjugate also
comprising a second and optionally further parts having a desired
biological activity. The label may in some instances be coupled
only to the A.beta. peptide binding polypeptide, and in some
instances both to the A.beta. peptide binding polypeptide and to
the second part of the fusion protein or conjugate. Furthermore, it
is also possible that the label may be coupled to a second part,
and not to the A.beta. peptide binding part. Hence, in yet another
embodiment, there is provided a A.beta. peptide binding polypeptide
comprising a second part, wherein said label is coupled to the
second part only. Such labels may for example be used for detection
of the polypeptide in vivo or in vitro and be useful in diagnosis
and therapy monitoring. The skilled person is aware of
radionuclides which may be useful in for example positron emission
tomography to detect A.beta. and plaques thereof.
[0164] In one embodiment, said A.beta. peptide binding polypeptide,
fusion protein or conjugate as described herein comprises a
chelating environment provided by a polyaminopolycarboxylate
chelator conjugated to the A.beta. peptide binding polypeptide via
a thiol group of a cysteine residue or an amine group of a lysine
residue.
[0165] In embodiments where the A.beta. peptide binding
polypeptide, fusion protein or conjugate is radiolabeled, such a
radiolabeled polypeptide may comprise a radionuclide. A majority of
radionuclides have a metallic nature and metals are typically
incapable of forming stable covalent bonds with elements presented
in proteins and peptides. For this reason, labeling of proteins and
peptides with radioactive metals is performed with the use of
chelators, i.e. multidentate ligands, which form non-covalent
compounds, called chelates, with the metal ions. In an embodiment
of the A.beta. peptide binding polypeptide, fusion protein or
conjugate, the incorporation of a radionuclide is enabled through
the provision of a chelating environment, through which the
radionuclide may be coordinated, chelated or complexed to the
polypeptide.
[0166] One example of a chelator is the polyaminopolycarboxylate
type of chelator. Two classes of such polyaminopolycarboxylate
chelators can be distinguished: macrocyclic and acyclic
chelators.
[0167] In one embodiment, the A.beta. peptide binding polypeptide,
fusion protein or conjugate comprises a chelating environment
provided by a polyaminopolycarboxylate chelator conjugated to the
A.beta. peptide binding polypeptide via a thiol group of a cysteine
residue or an epsilon amine group of a lysine residue.
[0168] The most commonly used macrocyclic chelators for
radioisotopes of indium, gallium, yttrium, bismuth, radioactinides
and radiolanthanides are different derivatives of DOTA
(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). In one
embodiment, a chelating environment of the A.beta. peptide binding
polypeptide, fusion protein or conjugate is provided by DOTA or a
derivative thereof. More specifically, in one embodiment, the
chelating polypeptides encompassed by the present disclosure are
obtained by reacting the DOTA derivative
1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic
acid-10-maleimidoethylacetamide (maleimidomonoamide-DOTA) with said
polypeptide.
[0169] Additionally, 1,4,7-triazacyclononane-1,4,7-triacetic acid
(NOTA) and derivatives thereof may be used as chelators. Hence, in
one embodiment, there is provided an A.beta. binding polypeptide,
fusion protein or conjugate, wherein the polyaminopolycarboxylate
chelator is 1,4,7-triazacyclononane-1,4,7-triacetic acid or a
derivative thereof.
[0170] The most commonly used acyclic polyaminopolycarboxylate
chelators are different derivatives of DTPA
(diethylenetriamine-pentaacetic acid). Hence, polypeptides having a
chelating environment provided by diethylenetriaminepentaacetic
acid or derivatives thereof are also encompassed by the present
disclosure.
[0171] In a third aspect of the present disclosure, there is
provided a polynucleotide encoding an A.beta. peptide binding
polypeptide or a fusion protein as described herein; an expression
vector comprising said polynucleotide; and a host cell comprising
said expression vector.
[0172] Also encompassed by this disclosure is a method of producing
a polypeptide or fusion protein as described above, comprising
culturing said host cell under conditions permissive of expression
of said polypeptide from its expression vector, and isolating the
polypeptide.
[0173] The A.beta. peptide binding polypeptide of the present
disclosure may alternatively be produced by non-biological peptide
synthesis using amino acids and/or amino acid derivatives having
protected reactive side-chains, the non-biological peptide
synthesis comprising [0174] step-wise coupling of the amino acids
and/or the amino acid derivatives to form a polypeptide according
to the first aspect having protected reactive side-chains, [0175]
removal of the protecting groups from the reactive side-chains of
the polypeptide, and [0176] folding of the polypeptide in aqueous
solution.
[0177] In another aspect, there is provided a composition
comprising an A.beta. peptide binding polypeptide, fusion protein
or conjugate as described herein and at least one pharmaceutically
acceptable excipient or carrier. In one embodiment, said
composition further comprises at least one additional active agent,
such as at least two additional active agents, such as at least
three additional active agents. Non-limiting examples of additional
active agents that may prove useful in such a composition are
agents selected from the group consisting of inhibitors of
neurotransmitter degradation, neurotransmitters,
acetylcholinesterase inhibitors, NMDA receptor antagonists, TNF
inhibitors, antihistamines, anti-viral agents, alpha-secretase
activators, inhibitors of beta- or gamma-secretase, inhibitors of
.alpha.-synuclein aggregation, inhibitors of tau aggregation,
calcium channel blockers, compounds effective against oxidative
stress, anti-apoptotic compounds, metal chelators, inhibitors of
DNA repair such as pirenzepin and metabolites, attractants for
A.beta. clearing/depleting cellular components, inhibitors of
N-terminal truncated A.beta. including pyroglutamated
A.beta..sub.3-42, anti-inflammatory molecules, atypical
antipsychotics such as clozapine, ziprasidone, risperidone,
aripiprazole and olanzapine, cholinesterase inhibitors (ChEIs) such
as tacrine, rivastigmine, donepezil and galantamine, M1 agonists
and other drugs including any amyloid or tau modifying drug and
nutritive supplements such as vitamin B12, cysteine, acetylcholine
precursor, lecithin, choline, Ginkgo biloba, acetyl-L-carnitine,
idebenone, propentofylline, and xanthine derivatives. In one
embodiment, the additional active agent(s) is/are selected from the
group consisting of acetylcholinesterase inhibitors, NMDA receptor
antagonists, TNF inhibitors, antihistamines and anti-viral
agents.
[0178] The skilled person will appreciate that said A.beta. peptide
binding polypeptide, fusion protein or conjugate, or a composition
comprising an A.beta. peptide binding polypeptide, fusion protein
or conjugate as described herein may be administered to a subject
using standard administration techniques, such as including oral,
intravenous, intraperitoneal, subcutaneous, intrathecal (e.g. via
the Ommaya reservoir), pulmonary, transdermal, intramuscular,
intranasal, buccal, sublingual or suppository administration. Thus,
in one embodiment, there is provided a composition or an A.beta.
peptide binding polypeptide, fusion protein, conjugate as disclosed
herein for administration via a route selected from the group
consisting of oral, intravenous, intraperitoneal, subcutaneous,
intrathecal, pulmonary, transdermal, intramuscular, intranasal,
buccal, sublingual or suppository administration, such as selected
from the group consisting of oral, intrathecal, intravenous and
intranasal administration, in particular intranasal
administration.
[0179] A.beta. peptide may also serve as a marker for diagnosis and
prognosis of A.beta. peptide associated disorders, such as
amyloidosis, which refers to a group of diseases and disorders
associated with amyloid plaque formation including secondary
amyloidosis and age-related amyloidosis including, but not limited
to, neurological disorders or conditions characterized by a loss of
cognitive memory capacity such as, for example, Alzheimer's disease
(AD), mild cognitive impairment (MCI), Lewybody dementia, Down's
syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch
type), the Guam Parkinson-dementia complex; as well as other
diseases which are based on or associated with amyloid-like
proteins such as cerebral amyloid angiopathy, primary and secondary
systemic amyloidosis, familial amyloid polyneuropathy 1,
progressive supranuclear palsy, multiple sclerosis, Creutzfeld
Jacob disease, Parkinson's disease, HIV-related dementia, ALS
(amyotropic lateral sclerosis), inclusion-body myositis (IBM), type
II diabetes, and senile cardiac amyloidosis; and various eye
diseases including glaucoma, macular degeneration, drusen-related
optic neuropathy, and cataract due to beta-amyloid deposition.
[0180] The A.beta. peptide binding polypeptide according to the
present disclosure may be useful as a therapeutic, diagnostic or
prognostic agent. Hence, in another aspect of the present
disclosure, there is provided an A.beta. peptide binding
polypeptide, fusion protein, conjugate or composition as described
herein for use as a medicament, a diagnostic agent or a prognostic
agent.
[0181] It is envisioned that the administration of an A.beta.
peptide binding polypeptide, fusion protein, conjugate or
composition as described herein to a subject afflicted with a
disease characterized by an over-representation of A.beta., or by
A.beta. aggregates or A.beta. deposits, would likely lower the
A.beta. toxicity and promote A.beta. degradation in the subject
(Luheshi et al (2010) PLOS Biology, March, Vol 8, Issue 3, el
000334). This, in turn, would also reduce the amount of insoluble
A.beta. in the body, because of the shift in equilibrium between
soluble and aggregated forms of the amyloid beta peptide. Lowering
the amounts of, and/or sequestering, A.beta. in this fashion serves
to reduce, and perhaps eliminate, the very source of the disease
itself.
[0182] Thus, in one embodiment, there is provided an A.beta.
peptide binding polypeptide, fusion protein, conjugate or
composition as described herein, for use as a medicament to reduce
the amount of free A.beta. peptide in blood. In one embodiment,
there is provided an A.beta. peptide binding polypeptide, fusion
protein, conjugate or composition as described herein, for use as a
medicament to slow down, stop or reverse the A.beta.
monomer-aggregate equilibrium. In one embodiment, there is provided
an A.beta. peptide binding polypeptide, fusion protein, conjugate
or composition as described herein, for use as a medicament to
prevent or reverse A.beta. aggregate formation. The skilled person
will appreciate that the three above mentioned modes of function of
an A.beta. peptide binding polypeptide, fusion protein, conjugate
or composition as disclosed herein, are not mutually exclusive.
Instead, an A.beta. peptide binding polypeptide, fusion protein,
conjugate or composition as disclosed herein may exert any one, two
or all three effects at the same time, or at different time points,
for example depending on the effective concentration of said
A.beta. peptide binding polypeptide, fusion protein, conjugate or
composition in the patient treated as well as on the stage of the
disease.
[0183] In one embodiment, there is provided an A.beta. peptide
binding polypeptide, fusion protein, conjugate or composition for
use in the treatment, diagnosis or prognosis of a A.beta. peptide
associated condition selected from the group consisting of
amyloidosis, which refers to a group of diseases and disorders
associated with amyloid plaque formation including secondary
amyloidosis and age-related amyloidosis including, but not limited
to, neurological disorders or conditions characterized by a loss of
cognitive memory capacity such as, for example, Alzheimer's disease
(AD), mild cognitive impairment (MCI), Lewybody dementia, Down's
syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch
type), the Guam Parkinson-dementia complex; as well as other
diseases which are based on or associated with amyloid-like
proteins such as cerebral amyloid angiopathy, primary and secondary
systemic amyloidosis, familial amyloid polyneuropathy 1,
progressive supranuclear palsy, multiple sclerosis, Creutzfeld
Jacob disease, Parkinson's disease, HIV-related dementia, ALS
(amyotropic lateral sclerosis), inclusion-body myositis (IBM), type
II diabetes, and senile cardiac amyloidosis; and various eye
diseases including glaucoma, macular degeneration, drusen-related
optic neuropathy, and cataract due to beta-amyloid deposition. In
one embodiment, the A.beta. peptide associated condition is
selected from the group consisting of dementia, cognitive
impairment, Alzheimer's disease, cerebral amyloid angiopathy,
Down's syndrome, type II diabetes, primary and secondary systemic
amyloidosis, familial amyloid polyneuropathy 1, glaucoma and
age-related macular degeneration. In one particular embodiment,
said condition is Alzheimer's disease.
[0184] In a related aspect, there is provided a method of detecting
A.beta. peptide, comprising providing a sample suspected to contain
A.beta. peptide, contacting said sample with an A.beta. peptide
binding polypeptide, fusion protein, conjugate or composition as
described herein, and detecting the binding of the A.beta. peptide
binding polypeptide, fusion protein, conjugate or composition to
indicate the presence of A.beta. peptide in the sample. In one
embodiment, said method further comprises an intermediate washing
step for removing non-bound polypeptide, fusion protein, conjugate
or composition, after contacting the sample.
[0185] In one embodiment, said method is a diagnostic or prognostic
method, for determining the presence of A.beta. peptide in a
subject, the method comprising the steps: [0186] contacting the
subject, or a sample isolated from the subject, with an A.beta.
peptide binding polypeptide, fusion protein, conjugate or
composition as described herein, and [0187] obtaining a value
corresponding to the amount of the A.beta. peptide binding
polypeptide, fusion protein, conjugate or composition that has
bound in said subject or to said sample.
[0188] In one embodiment, said method further comprises an
intermediate washing step for removing non-bound polypeptide,
fusion protein, conjugate or composition, after contacting the
subject or sample and before obtaining a value.
[0189] In one embodiment, said method further comprises a step of
comparing said value to a reference. Said reference may be scored
by a numerical value, a threshold or a visual indicator, for
example based on a color reaction. The skilled person will
appreciate that different ways of comparison to a reference are
known in the art and may be suitable for use.
[0190] In one embodiment of such a method, said subject is a
mammalian subject, such as a human subject.
[0191] In one embodiment of such a method, said sample is a
biological fluid sample, such as sample selected form the group
consisting of a whole blood sample, a plasma sample and a serum
sample.
[0192] In another embodiment of such a method, said sample is a
tissue sample.
[0193] In one embodiment, said method is performed in vivo.
[0194] In one embodiment, said method is performed in vitro.
[0195] In a related aspect, there is provided a method of treatment
of an A.beta. peptide associated condition, comprising
administering to a subject in need thereof an effective amount of
an A.beta. peptide binding polypeptide, fusion protein, conjugate
or composition as described herein. In a more specific embodiment
of said method, the A.beta. peptide binding polypeptide, fusion
protein, conjugate or composition as described herein reduces the
amount of free A.beta. peptide in blood; slows down, stops or
reverses the A.beta. monomer-aggregate equilibrium; prevents or
reverses A.beta. aggregate formation; or a combination thereof.
[0196] In one embodiment, said A.beta. peptide associated condition
is selected from the group consisting of amyloidosis, which refers
to a group of diseases and disorders associated with amyloid plaque
formation including secondary amyloidosis and age-related
amyloidosis including, but not limited to, neurological disorders
or conditions characterized by a loss of cognitive memory capacity
such as, for example, Alzheimer's disease (AD), mild cognitive
impairment (MCI), Lewybody dementia, Down's syndrome, hereditary
cerebral hemorrhage with amyloidosis (Dutch type), the Guam
Parkinson-dementia complex; as well as other diseases which are
based on or associated with amyloid-like proteins such as cerebral
amyloid angiopathy, primary and secondary systemic amyloidosis,
familial amyloid polyneuropathy 1, progressive supranuclear palsy,
multiple sclerosis, Creutzfeld Jacob disease, Parkinson's disease,
HIV-related dementia, ALS (amyotropic lateral sclerosis),
inclusion-body myositis (IBM), type II diabetes, and senile cardiac
amyloidosis; and various eye diseases including glaucoma, macular
degeneration, drusen-related optic neuropathy, and cataract due to
beta-amyloid deposition. In one embodiment, the A.beta. peptide
associated condition is selected from the group consisting of
dementia, cognitive impairment, Alzheimer's disease, cerebral
amyloid angiopathy, Down's syndrome, type II diabetes, primary and
secondary systemic amyloidosis, familial amyloid polyneuropathy 1,
glaucoma and age-related macular degeneration. In one particular
embodiment of said aspect, the A.beta. peptide associated condition
is Alzheimer's disease.
[0197] It may be beneficial to administer a therapeutically
effective amount of an A.beta. peptide polypeptide, fusion protein,
conjugate or composition as described herein and at least one
second drug substance, such as an agent selected from the group
consisting of inhibitors of neurotransmitter degradation,
neurotransmitters, acetylcholinesterase inhibitors, NMDA receptor
antagonists, TNF inhibitors, antihistamines, anti-viral agents,
alpha-secretase activators, inhibitors of beta- or gamma-secretase,
inhibitors of .alpha.-synuclein aggregation, inhibitors of tau
aggregation, calcium channel blockers, compounds effective against
oxidative stress, anti-apoptotic compounds, metal chelators,
inhibitors of DNA repair such as pirenzepin and metabolites,
attractants for A.beta. clearing/depleting cellular components,
inhibitors of N-terminal truncated A.beta. including pyroglutamated
A.beta..sub.3-42, anti-inflammatory molecules, atypical
antipsychotics such as clozapine, ziprasidone, risperidone,
aripiprazole and olanzapine, cholinesterase inhibitors (ChEIs) such
as tacrine, rivastigmine, donepezil and galantamine, M1 agonists
and other drugs including any amyloid or tau modifying drug and
nutritive supplements such as vitamin B12, cysteine, acetylcholine
precursor, lecithin, choline, Ginkgo biloba, acetyl-L-carnitine,
idebenone, propentofylline, and xanthine derivatives. In one
embodiment, the at least one second drug substance is selected from
the group consisting of acetylcholinesterase inhibitors, NMDA
receptor antagonists, TNF inhibitors, antihistamines and anti-viral
agents.
[0198] As used herein, the term "co-adminstration" encompasses
concomitant adminstration and adminstration in sequence. Thus, in
one embodiment there is provided a method as defined above further
comprising co-administration of at least one of the agents
described above.
[0199] According to another aspect of the present invention, a
method of separation, removal and/or purification of the A.beta.
peptide is provided. Suitably, such method involves a separation
device, such as chosen among chromatographic media, membranes,
cellulose, silica, agarose, polyacrylamide, magnetic beads,
two-phase systems and other such materials commonly used in
separation. In an embodiment, A.beta. peptide binding polypeptide,
fusion protein or conjugate as described herein is coupled to the
separation device. The thus obtained separation device, having said
A.beta. peptide binding polypeptide, fusion protein or conjugate
coupled thereto, is referred to as an affinity matrix. Thus, in one
aspect of the present disclosure, there is provided an affinity
matrix comprising A.beta. peptide binding polypeptide, fusion
protein or conjugate as described herein.
[0200] The method comprises a step of affinity separation, in which
step an affinity matrix comprising an A.beta. peptide binding
polypeptide, fusion protein or conjugate as described herein is
used.
[0201] For the purposes of purification of A.beta. peptide from a
sample, the sample containing A.beta. to be purified is suitably
applied to such an affinity matrix under conditions permissive for
binding of A.beta. peptide to the matrix. Thereafter, the affinity
matrix is washed under conditions such that the binding of the
A.beta. peptide to the matrix is maintained, but most, ideally all,
other proteins and contaminants bound to the matrix are washed
away. In an elution step, the matrix is treated such that A.beta.
peptide is released from the matrix in an A.beta. peptide enriched
fraction denoted "A.beta. peptide fraction", which may be
recovered.
[0202] If, conversely, the purpose of the separation is the removal
of A.beta. peptide, essentially the same steps as above are
suitably followed, with some exceptions.
[0203] The sample containing A.beta. peptide to be removed is
suitably applied to an affinity matrix under conditions that are
permissive for binding of A.beta. peptide to the matrix.
Thereafter, the affinity matrix is washed under conditions such
that the binding of A.beta. peptide to the matrix is maintained,
but most, ideally all, other proteins are recovered in the
flow-through, thus obtaining a "depleted fraction" with a
substantial reduction in A.beta. peptide content, which is
recovered. The non-A.beta. constituents of the sample may be
retained and used and/or processed further.
[0204] Another related aspect of the disclosure is a method for
reducing the content of A.beta. peptide in a portion of a body
fluid of a human, comprising the steps:
[0205] a) providing a portion of a body fluid from a human;
[0206] b) applying the portion to an affinity matrix as described
herein, under conditions permissive for binding of the A.beta.
peptide to said affinity matrix, thereby causing a reduction of the
content of A.beta. peptide in the portion of body fluid; and
[0207] c) returning at least a part of said portion of body fluid
to said human.
[0208] The method according to the disclosure may be directed to
reducing the content of A.beta. peptide in a body fluid of a
subject afflicted by an A.beta. peptide associated condition
selected from the group consisting of dementia, cognitive
impairment, Alzheimer's disease, cerebral amyloid angiopathy,
Down's syndrome, type II diabetes, primary and secondary systemic
amyloidosis, familial amyloid polyneuropathy 1, glaucoma and
age-related macular degeneration, such as Alzheimer's disease. The
body fluid may for example be whole blood, plasma or serum. Thus,
by using the method according to the invention, subjects afflicted
by said A.beta. peptide associated condition could be treated by
extracorporeal removal of A.beta..
[0209] While the invention has been described with reference to
various exemplary aspects and embodiments, it will be understood by
those skilled in the art that various changes may be made and
equivalents may be substituted for elements thereof without
departing from the scope of the invention. In addition, many
modifications may be made to adapt a particular situation or
molecule to the teachings of the invention without departing from
the essential scope thereof. Therefore, it is intended that the
invention not be limited to any particular embodiment contemplated,
but that the invention will include all embodiments falling within
the scope of the appended claims.
BRIEF DESCRIPTION OF THE FIGURES
[0210] FIGS. 1A-1X are a listing of the amino acid sequences of
examples of A.beta. peptide binding polypeptides of the present
disclosure (SEQ ID NO:1-106, 115 and 118-553), the albumin binding
domain PP013 (SEQ ID NO:107); a control polypeptide with affinity
for an irrelevant target (Z.sub.Taq; SEQ ID NO:108); human
A.beta..sub.1-42 peptide (SEQ ID NO:109); human A.beta..sub.1-40
peptide (SEQ ID NO:110); and amino acid sequences for control
peptides Z.sub.A.beta.3 (SEQ ID NO:111), (Z.sub.A.beta.3).sub.2
(SEQ ID NO:112), (Z.sub.A.beta.3A12).sub.2 (SEQ ID NO:113), albumin
binding polypeptide ABP (SEQ ID NO:114), (Z.sub.Taq).sub.2-PP013
(SEQ ID NO:116) and the albumin binding domain GA3 from
streptococcal Protein G, "ABD" (SEQ ID NO:117) used in selection,
screening and/or characterization of the binding polypeptides of
the disclosure. The deduced A.beta. peptide binding motifs BM1 and
BM2 of the A.beta. peptide binding polypeptides disclosed herein
extend from residue 4 to residue 25 and from residue 64 to residue
85, respectively, in the sequences with SEQ ID NO:1-17, 19-106 and
118-553. In SEQ ID NO:18, BM1 and BM2 extend from residue 4 to
residue 25 and from residue 69 to residue 90, respectively.
[0211] FIG. 2 is a structural overview of the design of the
truncated head-to-tail linked dimeric asymmetric library (A),
denoted ZASlib, and symmetric library Z (B), denoted ZSYMlib, shown
in complex with the A.beta..sub.1-40 peptide (white mesh ribbon)
(Protein Data Bank entry 2OTK). Randomized positions in respective
library are indicated, with numbering referring to the respective
position in a full-length Z variant.
[0212] FIGS. 3A and 3B are a schematic comparison between a
head-to-tail dimer of the first-generation A.beta. peptide
Z.sub.A.beta.3, herein denoted (Z.sub.A.beta.3).sub.2 (SEQ ID
NO:112) and the two affinity maturation libraries (A) ZASlib and
(B) ZSYMlib. Amino acid sequence positions that are randomized in
the libraries are indicated by X and the allowed amino acids in
these positions for each library are indicated.
[0213] FIG. 4 are scatter plots showing the results of
flow-cytometric sorting of (A) ZASlib and (B) ZSYMlib. Fluorescence
intensity on the X axes corresponds to the surface expression level
monitored via HSA binding to the albumin binding polypeptide ABP
(SEQ ID NO:114) expressed as an integral part of the
surface-exposed proteins. Fluorescence intensity on the Y axes
corresponds to A.beta. binding. The scatter plots show the
staphylococcal library before flow-cytometric sorting in cycles 1,
2, 3 and 4, respectively, with regions used for gating outlined in
each plot. Note that the concentration of biotinylated A.beta.
peptide is decreased in each sorting cycle and that off-rate
selection was performed in the last cycle, resulting in decreasing
signal intensities on average in subsequent rounds.
[0214] FIGS. 5A and 5B show the amino acid sequences of
(Z.sub.A.beta.3A12).sub.2 (SEQ ID NO:113) together with the
affinity matured A.beta. peptide binding polypeptides isolated by
FACS from the asymmetric library (ABPP001, 005, 009, 013, 014, 016,
018, 020, 021, 025, 026, 028, 033, 035, 037, 040, 042, 048, 049 and
050, corresponding to SEQ ID NO:1, 5, 9, 13, 14, 16, 18, 20, 21,
25, 26, 28, 33, 35, 37, 40, 42, 48, 49 and 50, respectively) and
from the symmetric library (ABPP053, 054, 059, 060, 061, 062, 070,
075, 078, 084, 089, 090, 095, 096, 097, 100 and 104, corresponding
to SEQ ID NO:53, 54, 59, 60, 61, 62, 70, 75, 78, 84, 89, 90, 95,
96, 97, 100 and 104, respectively). The (S.sub.4G).sub.2 (SEQ ID
NO:633) linker is outlined between the first and second moieties.
Randomized positions are indicated by dots and positions that have
been mutated in the original scaffold are shown. The number of
times each clone was isolated is shown to the right of each
sequence.
[0215] FIG. 6 shows bar graphs of the results from flow-cytometric
analysis of on-cell off-rate ranking of (Z.sub.A.beta.3A12).sub.2
(SEQ ID NO:113) and 37 of the unique affinity matured A.beta.
peptide binding polypeptides isolated by FACS (ABPP001, 005, 009,
013, 014, 016, 018, 020, 021, 025, 026, 028, 033, 035, 037, 040,
042, 048, 049, 050, 053, 054, 059, 060, 061, 062, 070, 075, 078,
084, 089, 090, 095, 096, 097, 100 and 104). The A.beta. peptide
binding polypeptide variants are represented on the X axis, and the
ratio between the mean fluorescence intensity (MFI) corresponding
to A.beta. peptide binding and MFI corresponding to surface
expression level is represented on the Y axis. The arrowheads
indicate the eight clones selected for further
characterization.
[0216] FIG. 7 shows an SDS-PAGE gel of cell lysates after SEC
purification of the eight selected A.beta. peptide binding
polypeptides (ABPP070, 084 and 095 corresponding to SEQ ID NO:70,
84 and 95, respectively, as well as ABPP018, 028, 035, 049 and 050
corresponding to SEQ ID NO:18, 28, 35, 49 and 50, respectively) as
well as of (Z.sub.A.beta.3A12).sub.2 (SEQ ID NO:113). All purified
A.beta. peptide binding polypeptides showed a correct size of
approximately 14 kDa as estimated from the gel. M indicates lanes
with the size marker.
[0217] FIG. 8 shows a sensorgram obtained from affinity
measurements of ABPP095 (SEQ ID NO:95) to biotinylated
A.beta..sub.1-40 on a neutravidin sensor chip using surface plasmon
resonance (SPR) analysis.
[0218] FIG. 9 shows circular dichroism (CD) spectra of ABPP095 (SEQ
ID NO:95). Panel A shows a graph of a variable temperature
measurement (VTM) spectrum obtained at 221 nm while heating ABPP095
from 20 to 90.degree. C. Panel B shows CD spectra of ABPP095 at
wavelengths ranging from 195 to 250 nm at 20.degree. C., before and
after variable temperature measurement (VTM).
[0219] FIG. 10 shows the results from SDS-PAGE analysis of protein
fractions from ABPP095-PP013 (SEQ ID NO:115) mediated capture of
A.beta. peptide from PBS spiked with 50 pg/ml A.beta..sub.1-42. The
gel was silver stained for visualization of protein bands. Lane M,
molecular weight marker; lane 1, eluate from [HSA
Sepharose/ABPP095-PP013] incubated with A.beta..sub.1-42 peptide;
lane 2, reference ABPP095-PP013; lane 3, reference A.beta..sub.1-42
peptide.
[0220] FIG. 11 shows the results from the SDS-PAGE analysis of
purified ABPP095-PP013 (SEQ ID NO:115) and (Z.sub.Taq).sub.2-PP013
(SEQ ID NO:116), respectively. Panel A: lane 1, molecular marker;
lane 2, 20 .mu.g reduced ABPP095-PP013 protein; lane 3, 20 .mu.g
non-reduced ABPP095-PP013 protein. Panel B: lane 1, molecular
marker; lane 2, 10 .mu.g reduced (Z.sub.Taq).sub.2-PP013
protein.
[0221] FIGS. 12A and 12B show results from the behavioral tests
described in Example 8. FIG. 12A shows bar graphs of maximum
velocity (Vmax), average velocity (Vmean), distance travelled and
resting time obtained from 10 (2.times.Tg) sweAPP/PS1 mice treated
with ABPP095-PP013 (SEQ ID NO:115) and 10 (2.times.Tg) sweAPP/PS1
mice treated with (Z.sub.Taq).sub.2-PP013 (SEQ ID NO:116; control)
as measured by the locomotor activity test. FIG. 12B shows
bargraphs of running speed obtained from 10 (2.times.Tg) sweAPP/PS1
mice treated with ABPP095-PP013 and 10 (2.times.Tg) sweAPP/PS1 mice
treated with (Z.sub.Taq).sub.2-PP013 measured by the rotarod
test.
[0222] FIG. 13 shows the results from the radial arm maze test
obtained from 10 (2.times.Tg) sweAPP/PS1 mice treated with
ABPP095-PP013, 10 (2.times.Tg) sweAPP/PS1 mice treated with
(Z.sub.Taq).sub.2-PP013 (control) and 10 wild type control mice.
The number of errors is shown on the y-axis and the day of testing
on the x-axis.
[0223] FIG. 14 shows accumulation of amyloid plaques in motor
cortex of (2.times.Tg) sweAPP/PS1 mice. Panels A and B show
representative A.beta.-immunostained (6E10/4G8) brain sections from
mice treated with (Z.sub.Taq).sub.2-PP013 (control) and
ABPP095-PP013, respectively. Panel C shows bar graphs of the
histological quantification of cortical amyloid plaque burden in
ABPP095-PP013 treated mice and control-treated mice.
[0224] FIG. 15 shows accumulation of amyloid plaques in dentate
gyrus of the hippocampus of (2.times.Tg) sweAPP/PS1 mice. Panels A
and B show representative A.beta. immunostained (6E10/4G8) brain
sections from mice treated with (Z.sub.Taq).sub.2-PP013 (control)
and ABPP095-PP013, respectively. Panel C shows bar graphs of the
histological quantification of hippocampal amyloid plaque burden in
ABPP095-PP013 treated mice and control-treated mice.
[0225] FIG. 16 shows astrogliosis in the hippocampus of
(2.times.Tg) sweAPP/PS1 mice. Panels A and B show representative
GFAP-immunostained brain sections from mice treated with
(Z.sub.Taq).sub.2-PP013 (control) and ABPP095-PP013, respectively.
Panel C shows bar graphs of the semi-quantitative analysis of
astrocytes in ABPP095-PP013 treated mice as compared to
control-treated mice.
[0226] FIG. 17 shows a diffusion time analysis by fluorescence
correlation spectroscopy. The diffusion time (.tau..sub.D),
reflecting the size of the analyzed component, is presented over
time for Alexa467-labeled A.beta..sub.1-40 peptide in the absence
(black dots) and presence (grey rings) of ABPP095.
EXAMPLES
Summary
[0227] The following Examples disclose the development of novel
A.beta. peptide binding polypeptides based on staphylococcal
display technology. The genes encoding the A.beta. peptide binding
polypeptides described herein were sequenced, and the corresponding
amino acid sequences are listed in FIG. 1A-1X and denoted by the
identifiers SEQ ID NO:1-106 and 118-553. The Examples also describe
the generation, characterization and functionality of the A.beta.
peptide binding polypeptides.
[0228] To clarify, the term "Z variant", as used herein, denotes
target binding polypeptides derived from protein Z. Thus, the
A.beta. peptide binding polypeptides as described herein comprise
dimers of a first Z variant unit and a second Z variant unit,
wherein said units may be Z variants which are truncated at the
N-terminal end.
Example 1
Design and Cloning of Affinity Maturation Libraries
Summary
[0229] This Example describes the design and cloning of two
affinity maturation libraries, ZASlib and ZSYMlib, based on dimers
of the previously identified first generation A.beta. peptide
binding polypeptide variant Z.sub.A.beta.3 (SEQ ID NO:111).
Materials and Methods
[0230] Two SlonoMax.RTM. head-to-tail dimer libraries of
double-stranded DNA were purchased from Sloning BioTechnology GmbH
(Pucheim, Germany). Library oligonucleotides encoded the truncated
helix 1 plus helix 2 and 3 of the first Z variant unit and helix 1
and 2 of the second Z variant unit, making up the first and second
moieties of the A.beta. peptide binding polypeptides (asymmetric
library: 5'-GCG GGT GGG GAG NNN NNN TAT NNN NNN AAC TTA AAC GCG NNN
CAA CTG TGT GCC TTC ATC NNN AGT TTA GAA GAT GAC CCA AGC CAA NNN GCT
AAC TTG TTG GCA GAA GCT AAA AAG CTA AAT GAT GCT CAG GCG CCG GCG AGC
AGC AGC AGC GGG AGC AGC AGC AGC GGG CGC GCG AGT GCG GGT CGC GAG NNN
GTT TAT TTA NNN AAC TTA AAC GCG NNN CAA CTG TGT GCC NNN ATC NNN AGT
NNN GAA GAT GAC NNN AGC CAA NNN GCT AAC TT-3' (SEQ ID NO:556;
randomized codons denoted NNN); symmetric library: 5'-GCG GGT NNN
GAG NNN GTT TAT NNN NNN AAC TTA AAC GCG NNN CAA CTG TGT GCC TTC ATC
NNN AGT TTA GAA GAT GAC NNN AGC CAA NNN GCT AAC TTG TTG GCA GAA GCT
AAA AAG CTA AAT GAT GCT CAG GCG CCG GCG AGC AGC AGC AGC GGG AGC AGC
AGC AGC GGG CGC GCG AGT GCG GGT NNN GAG NNN GTT TAT NNN NNN AAC TTA
AAC GCG NNN CAA CTG TGT GCC TTC ATC NNN AGT TTA GAA GAT GAC NNN AGC
CAA NNN GCT AAC TT-3' (SEQ ID NO:557; randomized codons denoted
NNN). The genes were flanked with XhoI and NheI restriction sites
for subcloning into the staphylococcal vector. The libraries were
PCR amplified in 8 cycles using Phusion DNA polymerase (Finnzymes,
Espoo, Finland), and the final PCR products were purified using a
QIAquick PCR purification kit (Qiagen GmbH). Purified PCR products
were digested by XhoI and NheI-HF (New England Biolabs) restriction
enzymes and purified by preparative gel electrophoresis (2% agarose
gel) using QIAquick gel extraction kit (Qiagen GmbH). The S.
carnosus expression vector pSCZ1 (Lofblom et al (2007) J Appl
Microbiol 102(3):736-747) was digested by the same enzymes and
purified by preparative gel electrophoresis as described above.
Purified library fragments were ligated into the vector using T4
DNA ligase (New England Biolabs) at a 1:5 molar ratio of vector to
insert, followed by phenol-chloroform extraction and ethanol
precipitation for purification and concentration of DNA fragments.
Next, the library-encoding plasmids were transformed into
electrocompetent E. coli SS320 cells (Lucigen Corporation,
Middleton, USA) by electroporation. Individual clones were PCR
amplified for subsequent library sequence validation using BigDye
Thermo Cycle Sequencing reactions and an ABI Prism 3700 instrument
(Applied Biosystems, Foster City, Calif.) Library plasmids were
subsequently prepared using a Jetstar Maxi Kit (Genomed), purified
by phenol-chloroform extraction and concentrated by isopropanol
precipitation. Finally, the libraries (hereafter denoted Sc:ZASlib
and Sc:ZSYMlib) were transformed into electrocompetent S. carnosus
as previously described (Lofblom et al (2007), supra).
[0231] To verify that the libraries were functionally displayed on
the staphylococcal surface, cells were incubated with fluorescently
labeled human serum albumin (HSA; Kabi Pharmacia) and analyzed
using flow cytometry. Labeling of HSA with Alexa Fluor 647
succinimidyl ester (Invitrogen) was performed according to the
supplier's recommendations.
Results
[0232] In order to improve affinity for the A.beta. peptide, an
affinity maturation of A.beta. peptide binding polypeptides was
performed. Two affinity maturation libraries were designed based on
a head-to-tail dimer of the first-generation A.beta. peptide
binding polypeptide Z.sub.A.beta.3 (SEQ ID NO:111). The libraries
were designed as single-chain dimers to enable independent
engineering of respective domain as well as to make them less
susceptible to reducing conditions. Previous data show that
truncating the first eleven, unstructured, amino acid residues from
the N-terminus of a dimer of the first-generation A.beta. peptide
binding polypeptide Z.sub.A.beta.3 has positive effects on the
affinity for A.beta. peptide (Lindgren et al (2010) Protein Sci
19(12):2319-2329; Lindberg et al (2013) Biotechnol J 8(1):139-145).
This portion of the first Z variant unit was therefore excluded in
the libraries. In the second Z variant unit, the corresponding
portion was instead substituted by a flexible (S.sub.4G).sub.2 (SEQ
ID NO:633) linker, in order to allow correct folding of the dimeric
construct. The design of the randomizations in both libraries was
based on structural analysis of the interaction of the first
generation A.beta. peptide binding polypeptide with the A.beta.
peptide, and on the sequence output from the selection of first
generation A.beta. peptide binding polypeptides (Gronwall et al
(2007), supra; Hoyer et al (2008), supra).
[0233] Different approaches were used for randomization of the
libraries. In one library, different positions on the two Z variant
units were targeted for randomization (asymmetric randomization),
whereas in the other library, the same positions in both Z variant
units were randomized (symmetric randomization). In the first
approach, 15 asymmetrically distributed positions in both Z variant
units were partially randomized (library denoted ZASlib; FIG. 2A).
In the second approach, 16 symmetrically distributed positions
(eight in each Z variant unit) were partially randomized (library
denoted ZSYMlib; FIG. 2B). Each randomized position was spiked to
50-75% with the codon for the original amino acid, combined with a
mix of codons for 1-4 other amino acids, resulting in a relatively
low mutation frequency. To enable the construction of complex
randomized DNA oligos, sets of trinucleotide building blocks were
combined during production (Slonomics.RTM. technology). This method
provided complete freedom when selecting codons for the library and
allowed randomizations with minimal biases compared to degenerate
codons and error-prone PCR approaches. The designs resulted in
theoretical library complexities of 8.1.times.10.sup.7 (ZASlib) and
2.33.times.10.sup.7 (ZSYMlib) individual oligonucleotides. The
library designs are outlined in FIG. 3.
[0234] The oligonucleotide mixtures encoding the two libraries were
subcloned into the staphylococcal display vector pSCZ1 in fusion to
an albumin binding polypeptide (ABP; SEQ ID NO:114) (Lofblom
(2007), supra) and transformed into S. carnosus to generate
libraries displayed on bacteria, with diversities of approximately
1.times.10.sup.8 (ZASlib) and 1.3.times.10' (ZSYMlib). Sequence
analysis of individual library members revealed a distribution of
codons in accordance with the theoretical design, and no occurrence
of undesired codons was observed. However, about 10% of ZSYMlib did
not have a complete (S.sub.4G).sub.2 linker. To verify that the
libraries were functionally displayed on the staphylococcal
surface, cells were incubated with fluorescently labeled HSA and
analyzed using flow cytometry. Around 60-67% of the population
displayed surface-exposed recombinant proteins, as assessed by
functional albumin binding reporter protein. When incubated with
labeled A.beta..sub.1-40, both libraries demonstrated a fraction of
clones having retained target binding (FIG. 4, cycle 1).
Example 2
Flow Cytometric Sorting of Affinity Maturation Libraries and
Sequencing of Isolated Polypeptides
Summary
[0235] This Example describes the cloning of the affinity
maturation libraries of Example 1 into a vector for staphylococcal
display, and subsequent sorting and selection of A.beta. peptide
binding polypeptides by flow cytometry utilizing increased
stringency conditions in each sorting cycle. Sequencing of isolated
variants lead to the identification of 51 unique variants from the
asymmetric library (SEQ ID NO:1-51) and 55 unique variants from the
symmetric library (SEQ ID NO:52-106).
Materials and Methods
[0236] At least ten times the library size of either library was
inoculated to tryptic soy broth supplemented with yeast extract
(TSB+Y; Merck, Darmstadt, Germany) and 20 .mu.g/ml chloramphenicol,
and grown overnight at 37.degree. C. and 150 rpm. The following
day, cells were harvested by centrifugation (6000 rpm, 6 min,
4.degree. C.) and washed in phosphate-buffered saline supplemented
with 0.1% Pluronic.RTM. F108 NF Surfactant (PBSP; pH 7.4; BASF
Corporation) before addition of C-terminally biotinylated
A.beta..sub.1-40 peptide (SEQ ID NO:110; AnaSpec). Cells were
incubated with gentle mixing at room temperature for 45 minutes
until equilibrium was reached. Subsequently, cells were washed with
ice-cold PBSP and labeled with streptavidin conjugated with
phycoerythrin (SAFE; Invitrogen) as well as HSA conjugated with
Alexa Fluor 647 for 30 minutes on ice at concentrations of 5
.mu.g/ml and 150 nM, respectively. After a final washing step in
ice-cold PBSP, cells were resuspended in ice-cold PBSP prior to
sorting. Cells were sorted in four cycles with an increased
stringency in each cycle, using a MoFlo.RTM. Astrios flow cytometer
(Beckman Coulter). In the first cycle of selection, library cells
were incubated with 50 nM C-terminally biotinylated
A.beta..sub.1-40 peptide, and with 20 nM and 10 nM, respectively,
in the second and third cycles. In the last cycle, the cells were
subjected to an off-rate selection procedure.
[0237] In the final, off-rate selection cycle, enriched library
cells were incubated with 25 nM C-terminally biotinylated
A.beta..sub.1-40 peptide for 45 minutes to reach equilibrium
binding, washed in ice-cold PBSP, incubated with 100 nM unlabeled
A.beta..sub.1-40 peptide for 6 h and then labeled with HSA-Alexa
Fluor 647 and SAFE prior to FACS sorting. In each sorting cycle,
approximately ten times the library size was analyzed in the flow
cytometer and the top fraction of cells (approximately 0.35%) with
the highest ratio of A.beta..sub.1-40-binding to cell surface
expression fluorescence was gated and sorted directly into TSB+Y.
Sorted cells were inoculated to TSB+Y supplemented with
chloramphenicol (10 .mu.g/ml) for overnight amplification at
37.degree. C. with 150 rpm shaking, prior to the next cycle of
FACS.
[0238] After the final cycle of sorting, cells were spread onto
agar plates containing 10 .mu.g/ml chloramphenicol. 96 randomly
picked colonies from each sorted library were sequenced using
BigDye Thermo Cycle Sequencing reactions and an ABI Prism 3700
(Applied Biosystems).
Results
[0239] Staphylococcal display technology in combination with
fluorescence-activated cell sorting (FACS) is well suited for
affinity maturation purposes, since it offers fine affinity
discrimination between individual library members (Lofblom (2007)
supra; Kronqvist (2008) Protein Eng Des Sel 21(4):247-255; Maim et
al (2013) PLoS One 8(5):e62791). In order to isolate high-affinity
A.beta. peptide binding polypeptides, the two displayed libraries
were subjected to four cycles of FACS. Staphylococcal cells were
incubated with biotinylated A.beta..sub.1-40 peptide for 45 min at
room temperature to reach binding equilibrium. Cells were
thereafter washed and incubated on ice with fluorescently labeled
HSA and streptavidin-conjugated phycoerythrin, for detection and
normalization of the target binding with cell surface expression
level as described previously (Kronqvist et al (2008) supra). After
additional washing, library cells demonstrating the highest
target-binding to surface-expression ratio in the flow cytometer
were gated (as outlined in FIG. 4) and isolated for amplification
and subsequent rounds of sorting. In each cycle, the selection
stringency was increased by changing sorting parameters and gates,
as well as by decreasing the target concentration to 50 nM, 20, nM
and 10 nM in cycles 1, 2 and 3, respectively. In the fourth and
final cycle, an off-rate selection approach was used to favor
binders with the slowest dissociation. As shown by flow-cytometric
analysis after each cycle of selection, the sorting resulted in
enrichment of A.beta. binding clones (FIGS. 4A and 4B). After four
cycles of sorting, individual colonies were sequenced for
identification. Out of a total of 192 sequences (96 per library),
51 unique polypeptide variants (SEQ ID NO:1-51) were identified in
the output from the asymmetric library and 55 unique polypeptide
variants (SEQ ID NO:52-106) were identified in the output from the
symmetric library (FIG. 1). Interestingly, one isolated clone
(ABPP018, SEQ ID NO:18) from the asymmetric library contained a
linker consisting of three 54G repeats instead of the two that were
intended by design.
Example 3
On-Cell Screening for A.beta. Binding
Summary
[0240] In this Example, the affinity of 37 isolated polypeptide
variants for A.beta. peptide was analyzed by flow cytometry.
Materials and Methods
[0241] 37 clones (ABPP001, 005, 009, 013, 014, 016, 018, 020, 021,
025, 026, 028, 033, 035, 037, 040, 042, 048, 049 and 050,
corresponding to SEQ ID NO:1, 5, 9, 13, 14, 16, 18, 20, 21, 25, 26,
28, 33, 35, 37, 40, 42, 48, 49 and 50, respectively; and ABPP053,
054, 059, 060, 061, 062, 070, 075, 078, 084, 089, 090, 095, 096,
097, 100 and 104, corresponding to SEQ ID NO:53, 54, 59, 60, 61,
62, 70, 75, 78, 84, 89, 90, 95, 96, 97, 100 and 104, respectively),
each occurring more than once in the selection results of Example
2, were individually inoculated to TSB+Y with chloramphenicol (10
.mu.g/ml) and grown overnight at 37.degree. C. and 150 rpm.
10.sup.6 overnight-cultured cells were pelleted by centrifugation
and washed in PBSP before resuspension in 1 nM biotinylated
A.beta..sub.1-40 (AnaSpec). After 45 min incubation at room
temperature with gentle mixing, cells were washed with ice-cold
PBSP and labeled with SAPE and HSA-Alexa Fluor 647 for 30 minutes
on ice. Next, cells were washed and resuspended in ice-cold PBSP,
and subjected to flow-cytometric analysis. The mean fluorescence
intensities (MFI) from A.beta..sub.1-40 binding and cell surface
expression were measured in a Gallios flow cytometer (Beckman
Coulter). The head-to-tail homodimer of the first-generation binder
(Z.sub.A.beta.3).sub.2 (SEQ ID NO:112) as well as the head-to-tail
homodimer (Z.sub.A.beta.3A12).sub.2 (SEQ ID NO:113), in which
eleven N-terminal amino acid residues were truncated, were included
in the analysis for comparison. The experiment was carried out in
duplicates on different days using freshly prepared solutions.
Results
[0242] 37 candidates, isolated from the libraries as described in
Example 2, were individually subjected to flow-cytometric analysis
to screen for the highest A.beta. binding signals. In total, 19
variants from ZASlib (ABPP001, 005, 009, 013, 014, 016, 020, 021,
025, 026, 028, 033, 035, 037, 040, 042, 048, 049 and 050,
respectively) and 17 from ZSYMlib (ABPP053, 054, 059, 060, 061,
062, 070, 075, 078, 084, 089, 090, 095, 096, 097, 100 and 104,
respectively), appearing multiple times in the sequence analysis,
as well as the variant ABPP018 (SEQ ID NO:18) from the asymmetric
library containing a longer linker, were included in the assay.
Recombinant bacteria were incubated with 1 nM biotinylated
A.beta..sub.1-40 and secondary reagents as described above. The
samples were subsequently analyzed for A.beta. peptide binding in a
flow cytometer, and the ratio of the signal from A.beta. peptide
binding to the signal from surface expression level (FL-2/FL-6) was
determined. The head-to-tail homodimer of Z.sub.A.beta.3 with an
N-terminal truncation (Z.sub.A.beta.3A12).sub.2 (SEQ ID NO:113) was
included in the analysis for comparison. All variants demonstrated
higher binding-signals than the control, indicating an improved
affinity for the A.beta. peptide.
Example 4
On-Cell Off-Rate Ranking and Determination of Binding
Summary
[0243] In this Example, the 37 polypeptide variants expressed on
the staphylococcal cell surface and studied in Example 3, were
ranked based on their affinity for A.beta. peptide as measured by
their off-rate.
Materials and Methods
[0244] Individual staphylococcal clones (ABPP001, 005, 009, 013,
014, 016, 018, 020, 021, 025, 026, 028, 033, 035, 037, 040, 042,
048, 049, 050, 053, 054, 059, 060, 061, 062, 070, 075, 078, 084,
089, 090, 095, 096, 097, 100 and 104) were inoculated to TSB+Y with
chloramphenicol (10 .mu.g/ml) and grown overnight at 37.degree. C.
and 150 rpm. 10.sup.6 cells were cultured overnight, pelleted by
centrifugation and washed in PBSP before resuspension in 100 nM
unlabeled A.beta..sub.1-40 in PBSP. After 6 h incubation at room
temperature with gentle mixing, cells were washed with ice-cold
PBSP and incubated with 25 nM C-terminally biotinylated
A.beta..sub.1-40 peptide (AnaSpec) in PBSP at room temperature for
45 min. Subsequently, cells were washed with ice-cold PBSP and
labeled with SAPE and HSA-Alexa Fluor 647 for 30 minutes on ice.
The cells were washed and resuspended in ice-cold PBSP, prior to
flow-cytometric analysis. The mean fluorescence intensities (MFI)
from A.beta..sub.1-40 binding and cell surface expression were
measured in a Gallios flow cytometer (Beckman Coulter). The
head-to-tail homodimer (Z.sub.A.beta.3A12).sub.2 (SEQ ID NO:113)
was included in the analysis for comparison. The experiment was
carried out in duplicates on different days using freshly prepared
solutions.
Results
[0245] In order to limit the number of A.beta. peptide binding
variants for further analysis, the 37 unique clones were ranked by
off-rate measurements while expressed on the staphylococcal cell
surface. After incubation of binding variants with biotinylated
A.beta..sub.1-40, unlabeled A.beta. peptide was added in 4-fold
excess and dissociation was allowed. Then, the ratio between
A.beta. peptide binding and surface expression level was determined
(FL-2/FL-6). All clones demonstrated considerably slower
dissociation from the A.beta. peptide compared to
(Z.sub.A.beta.3A12).sub.2 (SEQ ID NO:113) (FIG. 6).
[0246] Eight variants were chosen for further study (ABPP070, 084,
095, 018, 028, 035, 049 and 050 corresponding to SEQ ID NO:70, 84,
95, 18, 28, 35, 49 and 50, respectively). Four of these
demonstrated the highest A.beta. binding from affinity screening,
whereas four others exhibited the best off-rate. The selected
clones are indicated by arrows in FIG. 6.
Example 5
Expression and Purification of Soluble A.beta. Peptide Binding
Polypeptides
Summary
[0247] In this Example, the eight polypeptides selected in Example
4, i.e. ABPP070, 084, 095, 018, 028, 035, 049 and 050, were
expressed and purified by immobilized metal ion affinity
chromatography (IMAC).
Materials and Methods
[0248] Eight affinity maturated A.beta. binding polypeptides and
(Z.sub.A.beta.3A12).sub.2 (SEQ ID NO:113) were produced and
purified for further characterization. The DNA sequences encoding
the polypeptides were amplified from colonies by PCR, using primers
that introduced an upstream NdeI and downstream XhoI restriction
site. The DNA sequences were subsequently subcloned into the NdeI
and XhoI digested expression vector pET26b+ (Novagen), generating
constructs with a C-terminal Hiss tag. The plasmids were
transformed into BL21 E. coli cells by heat shock. Cells were
cultured in TSB at 37.degree. C. and when OD.sub.600 reached
approximately 1, expression was induced by addition of IPTG to a
final concentration of 1 mM. After overnight incubation at
25.degree. C., the cells were harvested by centrifugation (4000
rpm, 8 minutes, 4.degree. C.). Cells were then lysed by sonication
and cell debris was removed by centrifugation (16,000 rpm, 20
minutes, 4.degree. C.). A.beta. peptide binding polypeptides were
purified by IMAC using a HisPur.TM. Cobalt resin (Thermo
Scientific) under native conditions (wash buffer: 20 mM
NaHPO.sub.4, 300 mM NaCl, 15 mM imidazole, pH 7.4, 0.45 .mu.m
filtered; elution buffer: 20 mM NaHPO.sub.4, 300 mM NaCl, 150 mM
imidazole, pH 7.4, 0.45 .mu.m filtered). Next, potential multimers
were removed by size exclusion chromatography (SEC) on an
AKTAexplorer 10 using a Superdex 75 gel filtration column (GE
Healthcare) and PBS as running buffer. The molecular weight and
purity of the A.beta. peptide binding polypeptides were
subsequently verified by LC/MS (Agilent Technologies 6520
ESI-Q-TOF) and SDS-PAGE under both reducing and non-reducing
conditions. The protein concentration was determined by absorbance
measurement at 280 nm.
Results
[0249] Eight A.beta. peptide binding polypeptides (ABPP070, 084,
095, 018, 028, 035, 049 and 050, corresponding to SEQ ID NO:70, 84,
95, 18, 28, 35, 49 and 50, respectively) from the on-cell ranking
were subcloned to an expression vector and produced in E. coli as
C-terminally His.sub.6 tagged polypeptides. The polypeptides were
purified by IMAC followed by SEC to remove potential multimeric
complexes. SDS-PAGE analysis of the eluted fractions under both
reducing and oxidizing conditions demonstrated pure proteins having
the correct size of approximately 14 kDa (FIG. 7).
Example 6
Characterization of A.beta. Peptide Binding Polypeptides
Summary
[0250] In this Example, the affinity for A.beta. peptide of the
polypeptides expressed in Example 5, i.e. ABPP070, 084, 095, 018,
028, 035, 049 and 050, was analyzed using surface plasmon resonance
(SPR) and ABPP095 was further characterized by circular dichroism
(CD) spectroscopy.
Materials and Methods
[0251] Biosensor analysis of off-rate and affinity: The affinities
of the purified A.beta. peptide binding polypeptides were
determined using an SPR-based biosensor assay on a ProteOn XPR36
instrument (Bio Rad Laboratories, CA, USA). N-terminally
biotinylated A.beta..sub.1-40 peptide (AnaSpec) was injected over a
Neutravidin sensor chip (Bio Rad Laboratories) for immobilization
(immobilization levels were approximately 50 RU). Duplicate samples
of each polypeptide were injected at concentrations ranging from
6.25 nM to 50 nM over immobilized A.beta. peptides. The flow rate
was 50 .mu.l/min and the association and dissociation was followed
for 300 seconds and 2 hours, respectively. HBS-EP was used as
running buffer and 0.5% SDS was used for regeneration. In all
experiments, subtraction of responses from each sample over a blank
surface was performed to minimize buffer contributions. The on and
off rates were determined by non-linear regression to a Langmuir
1:1 model using the Proteon Manager Software (BioRad
Laboratories).
[0252] Circular dichroism spectroscopy: The secondary structure
content of the affinity-maturated A.beta. peptide binding
polypeptide ABPP095 (SEQ ID NO:95) was analyzed at a concentration
of 0.2 mg/ml by CD spectroscopy using a Jasco J-810
spectropolarimeter (Jasco Scandinavia AB, Molndal, Sweden) in a
cell with an optical path-length of 1 mm. The thermal stability was
measured at 221 nm while heating ABPP095 from 20 to 90.degree. C.
(5.degree. C./min). A CD spectrum at 250-195 nm was obtained at
20.degree. C. before and after heating to 90.degree. C.
Results
[0253] Biosensor analysis of off-rate and affinity: The eight
purified A.beta. peptide binding polypeptides were subjected to
affinity and off-rate determination using an SPR-based biosensor
assay (Table 4). A dilution series of four different concentrations
of each binder was injected over a Neutravidin chip surface with
immobilized biotinylated A.beta. peptides. The rate constants of
the interactions were determined for all candidates by non-linear
regression using a one-site binding model and then used for
calculation of K.sub.D values. ABPP095 demonstrated the highest
affinity to A.beta..sub.1-40, exhibiting an approximate
dissociation constant K.sub.D of 340 pM, corresponding to a 50-fold
improved affinity as compared to the reported 17 nM affinity for
the first-generation binder (Hoyer, 2008, supra). Dissociation was
followed during 2 h, and the off-rate for ABPP095 was determined to
3.2.times.10.sup.-5 s.sup.-1 (FIG. 8).
TABLE-US-00018 TABLE 4 Mean kinetic data for binding to A.beta.
peptide SEQ K.sub.D k.sub.a k.sub.d ID (M, (M.sup.-1s.sup.-1,
(s.sup.-1, NO: mean .+-. SD).sup.1 mean .+-. SD).sup.1 mean .+-.
SD).sup.1 ABPP070.sup.2 70 5.9 .times. 10.sup.-10 .+-. 7.2 .times.
10.sup.4 .+-. 4.2 .times. 10.sup.-5 .+-. 0.4 0.6 0.05 ABPP084 84
1.4 .times. 10.sup.-9 .+-. 1.1 .times. 10.sup.5 .+-. 1.5 .times.
10.sup.-4 .+-. 1.4 0.9 0.04 ABPP095.sup.2 95 3.4 .times. 10.sup.-10
.+-. 9.4 .times. 10.sup.4 .+-. 3.2 .times. 10.sup.-5 .+-. 0.0 0.2
0.04 ABPP018 18 9.7 .times. 10.sup.-10 .+-. 9.1 .times. 10.sup.4
.+-. 8.8 .times. 10.sup.-5 .+-. 0.0 0.3 0.02 ABPP028 28 1.02
.times. 10.sup.-9 .+-. 8.8 .times. 10.sup.4 .+-. 9.0 .times.
10.sup.-5 .+-. 0.1 0.6 0.04 ABPP035 35 2.6 .times. 10.sup.-9 .+-.
2.8 .times. 10.sup.4 .+-. 6.7 .times. 10.sup.-5 .+-. 0.9 0.9 0.09
ABPP049 49 1.15 .times. 10.sup.-9 .+-. 1.3 .times. 10.sup.5 .+-.
1.5 .times. 10.sup.-4 .+-. 0.0 0.2 0.01 ABPP050 50 6.9 .times.
10.sup.-9 .+-. 1.6 .times. 10.sup.3 .+-. 7.1 .times. 10.sup.-5 .+-.
6.1 1.3 0.40 .sup.1 Analyzed in duplicates .sup.2 7600 seconds`
dissociation in biosensor experiments
[0254] Circular Dichroism Spectroscopy: To further characterize the
A.beta. peptide binding polypeptide with the highest affinity
(ABPP095), its thermal stability and secondary structure content
was analyzed with circular dichroism spectroscopy. The analysis
indicated that the A.beta. peptide binding polypeptide had retained
a similar secondary structure as the first-generation binder (Hoyer
et al (2008), supra). To determine the thermal stability of the
polypeptide, the sample was heated from 20 to 90.degree. C., while
monitoring the helical content, resulting in a Tm value of
approximately 43.degree. C. (FIG. 9A), which is similar to the
original variant Z.sub.A.beta.3 (Hoyer et al (2008), supra).
Additionally, a CD spectrum was obtained after the variable
temperature measurement in order to assess the reversibility of
unfolding after heat treatment. The spectrum revealed a perfect
overlap with the spectrum from the unheated sample, suggesting that
ABPP095 refolded completely after heating to 90.degree. C. (FIG.
9B).
Example 7
Capture of A.beta. Peptide from Spiked E. coli Lysate
Summary
[0255] In this Example, the ability of ABPP095 (SEQ ID NO:95) to
capture A.beta. peptide in a complex solution was studied using
affinity analysis.
Materials and Methods
[0256] ABPP095 was genetically fused on the C-terminal via a 10
amino acid flexible linker to a 46 amino acid albumin binding
domain denoted PP013 (SEQ ID NO:107). Genes were ligated into a PET
based expression vector under the control of the T7 promoter and
containing a gene for kanamycin resistance. DNA sequence
verification, plasmid preparation and cultivation were done
essentially as described above. Protein purification was performed
essentially as previously described (Jonsson et al (2008) Prot Eng
Des Sel 21(8):515-527), but the affinity chromatography matrix was
equipped with an anti-[albumin binding domain] ligand (produced in
house) instead of human serum albumin.
[0257] 700 .mu.g of ABPP095-PP013 (SEQ ID NO:115) was
non-covalently coupled to 1 ml HSA-Sepharose 4 fast flow (GE
Healthcare, Uppsala, Sweden) by batch binding at room temperature
for 20 min with gentle mixing. HSA-Sepharose samples with bound
polypeptides were then incubated with PBS spiked with 50 pg/ml
A.beta..sub.1-42 peptide for 1.5 h with gentle mixing. Sepharose
was pelleted by centrifugation and washed with 50 ml PBS prior to
elution (0.3 M HAc, pH 2.8) of captured A.beta..sub.1-42 peptide
from the HSA-Sepharose. Eluates were lyophilized using SpeedVac
(Savant Instruments, Milford, Mass., USA) and dissolved in reducing
sample buffer (20 mM Tris-HCl pH 8.0, 1 mM EDTA, 2.5% SDS, 5%
.beta.-mercaptoethanol, 0.01% BFB). The samples were heated at
96.degree. C. for 10 min and loaded onto a 12% bis-Tris NuPAGE gel
(Invitrogen, Carlsbad, Calif., USA) using MES running buffer (50 mM
MES, 50 mM Tris base, 0.1 SDS, 1 mM EDTA, pH 7.3). Molecular
weights were estimated using SeaBlue2 Pre-stained marker
(Invitrogen). The gel was stained by silver stain
(SilverXpress.RTM. Silver Staining Kit, Invitrogen), according to
the manufacturer's instructions.
Results
[0258] The ability of ABPP095-PP013 (SEQ ID NO:115) to capture
A.beta. peptide was studied using affinity analysis. It is likely
that the potency of the A.beta. peptide binding polypeptide would
benefit from a long in vivo half-life, and it has previously been
demonstrated that fusion of molecules to an albumin binding domain
dramatically improves the time in circulation by binding to serum
albumin (Andersen et al (2011) J Biol Chem 286(7):5234-5241; Orlova
et al (2013) J Nucl Med 54(6):961-968). ABPP095 was therefore fused
to PP013, and the assay was designed to assess whether the
polypeptide could capture A.beta. while simultaneously interacting
with albumin. ABPP095-PP013 was first bound to HSA-Sepharose and
then incubated with PBS containing A.beta..sub.1-42 (50 pg/ml).
This concentration of A.beta..sub.1-42 peptides was chosen to
reflect physiological levels of A.beta..sub.1-42 peptides in the
blood, although levels have been reported to vary significantly
between patients (Kou et al (2000), supra; Du et al (2005), supra;
Pesini et al (2012), supra). SDS-PAGE analysis of the elutate from
the capture demonstrated that ABPP095 was capable of efficient and
selective capture of A.beta..sub.1-42 peptides (FIG. 10). Higher
molecular range bands in the eluate were probably due to unspecific
binding of proteins to the HSA-Sepharose. Nevertheless, the
unspecific binding did not appear to significantly affect the
capacity of the A.beta. peptide binding polypeptide to bind
A.beta..
Example 8
Locomotor and Cognitive Testing of Alzheimer's Disease Model Mice
Treated with A.beta. Peptide Binding Polypeptides
Summary
[0259] In this Example, the sensorimotor and cognitive abilities of
Alzheimer's disease model mice treated with ABPP095 (SEQ ID NO:95)
fused to an albumin binding domain is compared to Alzheimer's
disease model mice treated with a dimer of the control polypeptide
Z.sub.Taq (SEQ ID NO:108) fused to said albumin binding domain.
Materials and Methods
[0260] Protein production: ABPP095-PP013 (SEQ ID NO:115) and
(Z.sub.Taq).sub.2-PP013 (SEQ ID NO:116) were prepared as described
in Example 7. To remove multimers, additional purification was
performed by size exclusion chromatography using a HiLoad 16/60
column in an AKTA system (GE Healthcare) and PBS as running buffer.
Potential residual endotoxins were removed by passing proteins
through 1 ml EndoTrap columns (Hyglos) according to the supplier's
recommendations. The proteins were eluted in DPBS (Gibco, Life
Technologies) at a concentration of 1 mg/ml. Molecular weight and
purity of the proteins were subsequently verified by LC/MS and
SDS-PAGE under both reducing and non-reducing conditions. A large
amount of protein (20 .mu.g) in reduced as well as non-reduced form
was loaded on the SDS-PAGE gel in order to detect potential
multimers. The protein concentration was determined by absorbance
measurement at 280 nm.
[0261] Treatment of transgenic mice: Animal studies were approved
by the New York University School of Medicine Institutional Animal
Care and Use Committee and were consistent with the recommendations
of the American Veterinary Association. To model vascular amyloid
deposition and amyloid related pathology, the well-characterized
double-transgenic (2.times.Tg) sweAPP/PS1 mouse model was used. The
model allows expression of the APPK670N/M671 L mutations ("the
Swedish mutation") as well as the PS1M146V mutation, and has an
early onset of AD pathology. Twenty animals were divided into two
groups and received intraperitoneal (i.p.) injections of 100 .mu.g
(in a volume of 100 .mu.l) of ABPP095-PP013 protein or the
irrelevant (Z.sub.Taq).sub.2-PP013 protein three times per week for
13 weeks, beginning at 3-4 months of age. 10 mice were treated with
ABPP095-PP013 protein and 10 mice were treated with the negative
control (Z.sub.Taq).sub.2-PP013 protein. During the treatment,
veterinary staff monitored animals for any signs of toxicity, such
as changes in body weight, physical appearance, and altered
behavior. At 6-7 months, the mice went through extensive behavioral
testing and were subsequently killed for tissue analyses at 7-8
months of age.
[0262] Behavioral studies: Before cognitive testing, exploratory
locomotor activity and accelerating rotarod performance tests were
performed, in order to measure motor coordination and balance. This
was done in order to exclude that any observed effects in the
performance of the cognitive tasks were due to differences in
sensorimotor abilities.
[0263] Locomotor activity: A Hamilton-Kinder Smart-frame Photobeam
System was used to record activity of the mice over a designated
period of time. After habituation in a circular open field chamber
(70 cm in diameter) for 15 min, each mouse was allowed to explore
the environment for 15 min. Horizontal movements of the mice were
automatically recorded by a video camera mounted above the chamber.
After each session, the field was cleaned with water and 30%
ethanol. Results were reported as distance traveled (cm), average
and maximum travel velocity (cm/s) and mean resting time (s) for
each animal.
[0264] Rotarod performance: Each animal was placed on a rod
apparatus, with a rod diameter of 3.6 cm, to assess differences in
motor coordination and balance (Rotarod 7650 accelerating model;
Ugo Basile) between mice treated with ABPP095-PP013 protein and
mice treated with (Z.sub.Taq).sub.2-PP013 protein. The animals were
habituated to the apparatus for two training trials in order to
reach a baseline level of performance. The animals were
subsequently tested in three additional trials with 15 minute
intervening periods. The rotarod was set to an initial speed of 1.0
rpm, which was gradually raised every 30 s by 0.5 rpm. A soft foam
cushion was placed under the rod to prevent injury from falling.
The rod was cleaned with water and 30% ethanol after each session.
To assess the performance, the total time on the rod and the speed
of the rod were recorded when the mouse fell or inverted (by
clinging) from the top of the rotating barrel.
[0265] Radial arm maze: Spatial memory was assessed using an
eight-arm radial maze with eight 30 cm long arms originating from a
central space. 0.25 ml of 0.1% aqueous saccharine solution was
placed at the end of each arm. In addition to (2.times.Tg)
sweAPP/PS1 mice treated with ABPP095-PP013 and the (2.times.Tg)
sweAPP/PS1 mice treated with (Z.sub.Taq).sub.2-PP013, an
age-matched control group of wild type mice (n=10) was included in
this experiment.
[0266] Before testing, the mice were deprived of water for 24 h and
their access to water was restricted to 1-2 h per day for the
duration of testing. The animal entered and exited the maze through
the central area. Clear Plexiglas guillotine doors, operated by a
remote pulley system, controlled access to the arms from the
central area. After two days of adaptation to the maze, water
restricted mice were given one training session per day for 10-11
consecutive days. Prior to each day's testing, the mice were
adapted to the room with the lights on for 15 min. The animals were
also adapted to the instrument and permitted to enter all arms
until the eight rewards (baits containing saccharine) had been
consumed. The number of errors (entry into previously visited arms)
was recorded.
[0267] Statistical analysis: Data from the exploratory locomotor
activity test, the accelerating rotarod performance test and the
radial arm maze test was analyzed using two-way ANOVA, repeated
measures and a Neuman Keuls (Statistica) or a Bonferroni (GraphPad,
San Diego, Calif.) post hoc test.
Results
[0268] Protein production: It was expected that the potency of an
A.beta. peptide binding polypeptide as a therapeutic agent would
benefit from a long in vivo half-life. It has previously been
demonstrated that fusing Z variant molecules, with affinities for
other, non-related targets, to an affinity-matured albumin binding
domain significantly improved the in vivo half life by binding to
serum albumin. Therefore, in preparation for in vivo analysis, the
high-affinity A.beta.-specific ABPP095 polypeptide and a dimer of
the control polypeptide Z.sub.Taq were subcloned and expressed in
fusion with a C-terminal deimmunized high-affinity albumin binding
domain PP013. A 10 amino acid linker separating the polypeptide and
albumin binding domain was used. Z.sub.Taq was cloned as a
head-to-tail dimer to mimic the format of ABPP095. Approximately
300 mg of each protein was produced and purified for in vivo
studies. Multimers were removed by size exclusion chromatography
and potential endotoxins were removed using EndoTrap columns. The
molecular weight and purity of the proteins were verified by LC/MS
and SDS-PAGE (FIG. 11) and the protein concentration was
determined.
[0269] Behavioral studies: Groups of (2.times.Tg) sweAPP/PS1 mice
treated with ABPP095-PP013 or with (Z.sub.Taq).sub.2-PP013
(control), were assessed in both cognitive and sensorimotor tests.
No significant differences were observed between the groups in
locomotor activity in terms of distance traveled, rest time,
maximum speed, and mean velocity (FIG. 12A). In correlation with
this, no difference was observed in the rotarod performance test
between transgenic mice treated with ABPP095-PP013 and transgenic
mice with (Z.sub.Taq).sub.2-PP013 (control) (FIG. 12B). Thus, the
performance in the cognitive test was not confounded by the
differences or abnormalities in motor function.
[0270] In the radial arm maze, (2.times.Tg) sweAPP/PS1 mice treated
with ABPP095-PP013, as well as wild type mice used as control,
showed a significantly better performance compared to the
transgenic mice injected with control polypeptide. No differences
in performance were observed between the wild-type controls and the
transgenic mice treated with ABPP095-PP013 (FIG. 13). Two-way
repeated measures ANOVA revealed a significant cognition
improvement benefit of ABPP095-PP013 treatment as compared to
injection of (Z.sub.Taq).sub.2-PP013. Thus, treatment with
ABPP095-PP013 significantly improved the cognitive abilities of the
transgenic Alzheimer's disease model mice as evaluated in the
radial arm maze.
Example 9
Histologic Analysis of Alzheimer's Disease Model Mice Treated with
A.beta. Peptide Binding Polypeptide
Summary
[0271] This Example describes histological analyses of (2.times.Tg)
sweAPP/PS1 mice, treated with the A.beta. peptide binding
polypeptide ABPP095-PP013 or control polypeptide and sacrificed
after the behavioral study described in Example 8. The results
demonstrated that ABPP095-PP013 substantially reduced the brain
amyloid burden, and that this effect correlated with behavioral
amelioration in the treated (2.times.Tg) sweAPP/PS1 mice.
Materials and Methods
[0272] Mice were anesthetized with sodium pentobarbital (150 mg/kg
i.p.), perfused transaortically and the brains were immediately
removed and processed. The right hemisphere was immersion-fixed
overnight in periodate-lysine-paraformaldehyde. Following fixation,
the brain was moved to a phosphate buffer solution containing 20%
glycerol and 2% dimethylsulfoxide (DMSO) and stored at 4.degree. C.
until sectioned. Serial corona) brain sections (40 .mu.m) were cut,
placed in ethylene glycol cryoprotectant and stored at -20.degree.
C. until used. Sections were stained for immunohistochemical
analysis with i) a mixture of the anti-A.beta. monoclonal
antibodies 6E10 and 4G8 (Covagen Research Products Inc), ii)
polyclonal anti-glial fibrillary acidic protein (GFAP) antibody
(DAKO) to detect astrocytes. The staining was performed as
previously described (Boutajangout et al (2009) J Alzheimers Dis
18:961-72; Scholzova et al (2009) J Neuroscience 29:1846-54; Liu et
al (2014) J Neurochem 128:577-91).
[0273] Amyloid burden was quantified by a Bioquant stereology image
analysis system (BIOQUANT Image Analysis Corporation) using a
random unbiased sampling scheme. Total A.beta. burden (defined as
the percentage of test area occupied by A.beta. immunoreactivity)
was quantified for the cortex and hippocampus on corona) plane
sections stained with a mixture of the anti-A.beta. monoclonal
antibodies 6E10 and 4G8.
[0274] The assessment of sections stained with the GFAP antibody
was based on a semi-quantitative analysis, using methods previously
published (Yang et al (2011) J Alzheimers Dis 24:269-285;
Scholtzova et al (2009) supra; Sadowski et al (2006) Proc Natl Acad
Sci 103:18787-92). Prior to analysis, brains were observed through
the microscope and given a rating from 0-4, in increments of 0.5,
depending on the degree of pathology and/or the activation stage of
the glial cells. Approximately five cortical sections and six
hippocampal sections were analyzed per animal. The rating was based
on the number of reactive neuronal bodies and processes.
Astrogliosis was analyzed at 10.times. magnifications in the
cortex, hippocampus and thalamus region. The rating for
astrogliosis was based on the extent of GFAP immuoreactivity
(number of GFAP immunoreactive cells and complexity of astrocytic
branching). The rating was done by an observer blinded to the
treatment status of the mice.
[0275] All statistical analyses of total amyloid burden and
astrogliosis were performed using Prism 6.0 (Graphpad, San Diego,
Calif., USA).
Results
[0276] Histological quantification of amyloid burden: Total amyloid
burden in the cortex and dentate gyrus of the hippocampus of
ABPP095-PP013 treated mice versus (Z.sub.Taq).sub.2-PP013 (control)
treated mice were quantified by stereological techniques using
random unbiased sampling on the serial sections immunostained with
a mixture of 6E10 and 4G8 antibodies. A.beta. immunostaining showed
a significant reduction in A.beta. accumulation in both cortical
(FIG. 14A-C) and hippocampal (FIG. 15A-C) sections of the
ABPP095-PP013 treated mice compared to the control group (p=0.03
and p=0.05 in cortex and hippocampus, respectively).
[0277] Neuroinflammatory response after treatment: A concern when
administrating therapeutics that target A.beta. depositions is the
risk of increased brain inflammation, ultimately resulting in
neuronal dysfunction or death. To evaluate the effect of
ABPP095-PP013 treatment on brain inflammation, the extent of
gliosis in the hippocampus and cortex was quantitatively examined
by immunohistochemical staining. Histological observation of
astrocytes, which were stained with an anti-GFAP antibody, revealed
that there was a slight reduction in astrogliosis in the
ABPP095-PP013 treated mice compared to the control treated group,
although the observed differences were not significant (FIG.
16A-C). Most importantly, no increased risk of brain inflammation
was observed in association with ABPP095-PP013 treatment.
Example 10
Extraction of Soluble and Insoluble A.beta. from Alzheimer's
Disease Model Mice Treated with A.beta. Peptide Binding
Polypeptides
Summary
[0278] This Example describes the assessment of total soluble
A.beta. and A.beta. aggregates/oligomers in the brain of
(2.times.Tg) sweAPP/PS1 mice sacrificed after the behavioral study
described in Example 8 and studied histologically as described in
Example 9.
Materials and Methods
[0279] The left hemisphere of each brain was weighed and
homogenized (10% w/v) in tissue homogenization buffer (20 mM Tris
base, pH 7.4, 250 mM sucrose, 1 mM EDTA, 1 mM EGTA) with 100 mM
phenylmethylsulphonyl fluoride, complete protease inhibitor
cocktail and PhosSTOP phosphatase inhibitor cocktail (Roche) added
immediately before homogenization. The extraction of soluble
A.beta. and total A.beta. was done according to the method
published previously (Boutajangout et al (2009) supra; Scholtzova
et al (2009) supra; Goni et al (2010) PLoS One 5:e13391; Yang et al
2011 supra; Liu et al 2014, supra).
[0280] In brief, a first brain homogenate (200 .mu.l) was mixed
with an equal volume of cold 0.4% diethylamine (DEA)/100 mM NaCl,
and subsequently centrifuged at 100000 g for 1 h at 4.degree. C.
The obtained supernatant was neutralized with 1/10 volume of 0.5 M
Tris, pH 6.8, flash-frozen on dry ice and stored at -80.degree. C.
until used as soluble A.beta. fraction.
[0281] A second homogenate (200 .mu.l) was added to 440 .mu.l of
cold formic acid (FA) and sonicated for 1 min on ice. Subsequently,
400 .mu.l of this solution was centrifuged at 100000 g for 1 h at
4.degree. C. and 210 .mu.l of the resulting supernatant was diluted
into 4 ml of FA neutralization solution (1 M Tris base, 0.5 M
Na.sub.2HPO.sub.4, 0.05% NaN.sub.3), flash-frozen on dry ice and
stored at -80.degree. C. until used for total A.beta.
measurement.
[0282] Levels of soluble and total A.beta. using DEA and FA
extracts, respectively, are determined by a sandwich Enzyme-Linked
Immuno Sorbent Assay (ELISA) for instance as described in
Scholtzova et al (2009, supra).
[0283] Preparations of samples and measurements of levels of
A.beta. oligomers and A.beta. aggregates in the brain are
determined for example as described in Scholtzova et al (2014, Acta
Neuropathologica Communications 2:101) and Liu et al 2014, supra)
including western blot analysis using antibodies specific for
A.beta. oligomers/aggregates (for example A11 polyclonal antibody
(Invitrogen)) and analysis by a Human Aggregated A.beta. ELISA kit
(Invitrogen) according to the manufacturer's instructions.
Results
[0284] The results are expected to show reduced levels of total and
soluble A.beta. as well as reduced levels of A.beta.
aggregates/oligomers in brains from ABPP095-PP013 treated mice
compared to brains from mice treated with the control polypeptide
(Z.sub.Taq).sub.2-PP013.
Example 11
Measurement of Plasma Levels of A.beta. Peptide Binding
Polypeptides
Summary
[0285] This Example describes the assessment of plasma levels of
ABPP095-PP013 and (Z.sub.Taq).sub.2-PP013_in the (2.times.Tg)
sweAPP/PS1 mice studied in Example 8.
Materials and Methods
[0286] The mice were bled before the commencement of the behavioral
study described in Example 8 as well as periodically throughout the
study. Plasma levels of ABPP095-PP013 and (Z.sub.Taq).sub.2-PP013
are detected by ELISA using plates (Immulon 2HB; Thermo Electron
Corp) coated with 0.5 .mu.g/well of A.beta..sub.1-40 peptide
(synthesized at the Keck Foundation at Yale University). Detection
is performed using a primary polyclonal goat anti-Z IgG (in-house
produced) at a dilution of 1/1000 and subsequently by a labeled
secondary antibody, for example an anti-goat IgG-HRP (Jackson).
Plates are developed with TMB substrate and the reactions are
stopped with 2M H.sub.2SO.sub.4 followed by absorbance measurements
at 450 nm.
Results
[0287] The results are expected to show the levels of ABPP095-PP013
and (Z.sub.Taq).sub.2-PP013 over time in plasma samples obtained
from treated (2.times.Tg) sweAPP/PS1 mice.
Example 12
Inhibition of A.beta. Aggregation by A.beta. Peptide Binding
Polypeptides Studied by Thioflavin Fluorescence Assay
Summary
[0288] In Alzheimer's disease (AD), the A.beta. peptide is prone to
aggregate. The course of events can be studied in vitro with
methods such as the thioflavin T (ThT) fluorescence assay
(Hellstrand et al (2009), ACS Chem. Neurosci. 1:13-18). This assay
measures changes of in fluorescence intensity of ThT upon binding
to amyloid fibrils. The enhanced fluorescence can be observed by
fluorescence spectroscopy. The capacity of A.beta. peptide binding
polypeptides as disclosed herein to inhibit A.beta. peptide
aggregation is tested in the ThT assay.
Materials and Methods
[0289] Monomeric A.beta..sub.1-40 or A.beta..sub.1-42 at various
concentrations is mixed with 20 .mu.M ThT (Sigma) in a 20 mM sodium
phosphate buffer supplemented with 200 .mu.M EDTA. The preparation
is done in black polysterene ELISA plates with clear bottom and on
ice. The aggregation is triggered by placing 96 well plates at
37.degree. C. and shaking at 100 rpm. The ThT fluorescence is
measured using 440 and 480 nm excitation and emission filters,
respectively, in an Enspire plate reader (Perkin Elmer). The
fluorescence is initially measured over a time period to find out
the optimal A.beta. peptide concentration and the time needed to
reach maximal aggregation. The ability of an A.beta. peptide
binding polypeptide as disclosed herein to block aggregation is
tested by mixing the A.beta. peptide binding polypeptide at various
concentrations together with the optimized A.beta.:ThT ratio and
then reading the fluorescence as described above.
Results
[0290] The results are expected to show that A.beta. peptide
binding polypeptides as disclosed herein block the aggregation of
A.beta. peptide in vitro as measured by the ThT aggregation
assay.
Example 13
Inhibition of A.beta. Aggregation by A.beta. Peptide Binding
Polypeptides Studied by Fluorescence Correlation Spectroscopy
(FCS)
Materials and Methods
[0291] Sample preparation: A.beta..sub.40 (AlexoTech AB, Umea,
Sweden) was freshly prepared by dissolving the peptide powder on
ice in 10 mM NaOH to a concentration of 1.0 mg/ml, followed by
dilution in 20 mM HEPES, pH 7.0 as required. Fluorescently labeled
opioid peptides, custom synthesized and purified >98%
(Biomatik), were added to the freshly prepared solution of
unlabeled A.beta..sub.50 and the concentration was confirmed by FCS
immediately after mixing. The time course of A.beta..sub.40
aggregation was monitored in a solution containing 10 .mu.M
unlabeled A.beta..sub.40, 100 nM A.beta..sub.40-Alexa488 and 100 nM
A.beta..sub.40-Alexa647 in 20 mM HEPES, pH 7.0, T=20.degree. C. in
the presence or absence of 20 .mu.M of the A.beta. binding
polypeptide ABPP095 (SEQ ID NO:95).
[0292] FCS measurements: FCS measurements were performed on a
uniquely modified ConfoCor3 instrument (Carl Zeiss, Jena, Germany)
consisting of an inverted microscope for transmitted light and
epifluorescence (Axiovert 200 M); a VIS-laser module comprising the
Ar/ArKr (458, 477, 488 and 514 nm), HeNe 543 nm and HeNe 633 nm
lasers; and the scanning module LSM 510 META. The instrument was
modified to enable detection using silicon Avalanche Photo
Detectors (SPCM-AQR-1X; Perkin Elmer, USA) for imaging and FCS
(Vukojevic et al (2008), Proc Natl Acad Sci 105:18176-18181). The
C-Apochromat 40.times., NA=1.2, water immersion UV-VIS-IR objective
was used throughout. Alexa488 was excited using the 488 nm line of
the Ar/ArKr laser, whereas the HeNe633 laser was used to excite
Alexa647. The main dichroic beam splitter HFT 488/543/633 was used
to separate the incident and fluorescence light. Fluorescence
signals were separated using a secondary dichroic beam splitter
(NFT 635) and further spectrally narrowed before detection using a
band pass filter (BP 505-610) for Alexa488 and a long pass filter
(LP 650) for Alexa647.
[0293] Fluorescence intensity fluctuations were recorded at
approximately 10 min intervals in arrays of 30 consecutive
measurements, each measurement lasting 10 s, and analyzed by
temporal autocorrelation analysis. Autocorrelation curves were
analyzed using the online software for data analysis and the
generated autocorrelation curves were fitted with a model for free
3D diffusion of one component. Fitting was performed using the
dedicated routine that is part of the running software and the
quality of fitting was evaluated by residuals analysis.
Results
[0294] Fluorescence correlation spectroscopy was applied to study
A.beta..sub.40 aggregation in solution over time, in the presence
or absence of the A.beta. binding polypeptide ABPP095. The results
showed that the propensity of A.beta..sub.40 to aggregate decreased
in the presence of ABPP095. A diffusion time analysis, monitoring
changes in the distribution of diffusion times, which reflects
changes in aggregate size, is presented in FIG. 17. In the absence
of ABPP095, a spread in diffusion times (I.sub.D) appeared after
about 1 h, whereas in the presence of ABPP095 the diffusion times
remained low.
Example 14
Intranasal Administration of A.beta. Peptide Binding
Polypeptides
Summary
[0295] Intranasal drug administration is an attractive route for
delivery of compounds to the central nervous system (CNS). Efficacy
of intranasally administered proteins has been demonstrated in a
variety of rodent disease models (Thorne et al, (2004),
Neuroscience, 127:481-496; De Rosa et al (2005), Proc. Natl. Acad.
Sci. 102:3811-3816; Topkuru et al (2013), Stroke, 44:3189-3194;
Lochhead et al, (2015), J Cereb. Blood Flow Met. 35:371-381) as
well as in humans (reviewed in Lochhead and Thorne (2012), Adv.
Drug Deliv. Rev. 64:614-628). Intranasal administration is
therefore evaluated for delivery of A.beta. peptide binding
polypeptides to the brain and cerebrospinal fluid (CSF).
Furthermore, in contrast to systemic injection, intranasal
administration also avoids high exposure in the blood circulation,
thus lowering the probability of systemic side effects.
Materials and Methods
[0296] Prebleeds of all animals, such as Sprague Dawley rats (from
an accredited vendor), are collected ten days before dosing.
Intranasal administration is carried out under isoflurane
anesthesia. The animals are placed in a supine position and a total
of 60 .mu.l (1000 .mu.g) A.beta. peptide binding polypeptide, alone
or in fusion with the albumin binding domain PP013 (SEQ ID NO:107),
is administered by pipette in 10 .mu.l drops, treating each nare
every 2.5 min over a total of 10 min. 3 mM Pz-peptide
(4-phenylazobenzoxy-carbonyl-Pro-Leu-Gly-Pro-DArg; Bachem) may be
included in the A.beta. peptide binding polypeptide formulation as
a penetration enhancer. Animals are anaesthetized and sacrificed at
0.5, 2, 6, and 24 h after the first intranasal instillation. Blood
samples are collected and the rats are then anesthetized using
ketamine+xylazine/medetomidine for collection of CSF. After
euthanization the rats are perfused with ice cold PBS and the
olfactory bulbs are carefully harvested. The tissues are weighed,
frozen on dry ice and stored at -80.degree. C. until analysis.
Brain tissues are prepared by addition of a lysis buffer followed
by sonication. Cell debris is removed by centrifugation and the
supernatant and CSF are subjected to an ELISA based determination
of A.beta. peptide binding polypeptide concentrations, for example
as described in Example 11. The same ELISA method is applied to
analyze serum from collected blood samples to evaluate the systemic
exposure.
Results
[0297] The results are expected to show significant concentrations
of A.beta. peptide binding polypeptide in the olfactory bulb. The
concentration in CSF and blood are expected to be low in
concordance with Furrer et al (2009, J Neuroimmunol 215:65-72) and
Thorne et al (supra). The olfactory bulb is connected with the
nasal cavity through the olfactory system, and the brainstem is
connected with the nasal passages through the peripheral trigeminal
system. The delivery of A.beta. peptide binding polypeptide to the
CNS is expected to be much more efficient following intranasal
administration than following a dose adjusted (for instance
adjusted to obtain the same systems exposure) intravenous injection
where penetration across the blood brain barrier is necessary.
Example 15
Characterization of A.beta. Peptide Binding Polypeptides with an
Alternative Scaffold
Summary
[0298] This Example describes the adaptation of the binding parts
of A.beta. peptide binding polypeptide ABPP095 in alternative
scaffold sequences, as well as the analysis of such alternative
sequences by circular dichroism (CD) spectroscopy and surface
plasmon resonance (SPR).
Materials and Methods
[0299] Cloning of scaffold mutated variants of ABPP095: ABPP095
with a C-terminal Hiss tag (as used in Example 5 and 6) was further
modified at scaffold positions, symmetrically within both moieties.
Introduced mutations are listed in Table 5. Synthetic genes were
ordered from DNA2.0. From the delivered plasmids, the DNA sequences
encoding the polypeptides were cleaved and subsequently ligated
into the expression vector pET26b+(Novagen) using the NdeI and XhoI
restriction sites. The generated constructs with a C-terminal Hiss
tag were in the general format ABPP095#-LEHHHHHH.
TABLE-US-00019 TABLE 5 Scaffold mutations within ABPP095 SEQ ID
Mutations Mutations Designation NO: in moiety 1 in moiety 2
ABPP095a 551 N41S, D42E N101S, D102E ABPP095b 552 A31S, N32E, A91S,
N92E, A35S, N41S, A95S, N101S, D42E, A43S D102E, A103S ABPP095c 553
N12T, S22K, N72T, S82K, A31S, N32E, A91S, N92E, A35S, N41S, A95S,
N101S, D42E, A43S D102E, A103S
[0300] Production of scaffold mutated variants of ABPP095: E. coli
T7E2 cells (GeneBridges) were transformed with plasmids containing
the gene fragments of each respective ABPP095 variant and
cultivated in a fermenter (GRETA4, Belach Bioteknik AB) at
37.degree. C. in approximately 940 ml of TSB-YE medium supplemented
with 50 .mu.g/ml kanamycin. In order to induce protein expression,
IPTG was added to a final concentration of 0.17 mM at OD.sub.600=2
and the cultivation was incubated at 37.degree. C. for another 5 h.
The cells were harvested by centrifugation.
[0301] Approximately 4 g of each cell pellet was re-suspended in 13
ml [20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4]
and Benzonase.RTM. (Merck, cat. no. 1.01654.0001) was added to a
concentration of approximately 90 U/ml. The cell suspension was
transferred to 10 g Lysing Matrix B (MP Biomedicals, cat. no.
116540429) and the cells were disrupted using a FastPrep.RTM.-24
Instrument (MP Biomedicals). Cell debris was removed by
centrifugation and each supernatant was applied on a 1 ml His
GraviTrap IMAC column (GE Healthcare, cat. no. 11-0033-99).
Contaminants were removed by washing with wash buffer (20 mM sodium
phosphate, 0.5 M NaCl, 60 mM imidazole, pH 7.4) and the ABPP095
variants were subsequently eluted with 3 ml elution buffer (20 mM
sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4). For
reduction of disulfides, dithiothreitol (DTT) was added to a final
concentration of 20 mM followed by incubation at RT for 1 h. Buffer
exchange to [20 mM Tris-HCl, 150 mM NaCl, pH 8.0] was carried out
using PD-10 Desalting columns (GE Healthcare, cat. no. 17-0851-01)
and the samples were diluted to 0.2 mg/ml in the same buffer.
Reduced glutathione (GSH; Sigma, cat. no. G-4251) and oxidized
glutathione (GSSG; Sigma, cat. no. G-4376) was added to a
concentration of 2 mM and 0.4 mM, respectively, followed by
overnight incubation in the dark at 4.degree. C. Each sample was
further purified by reverse phase chromatography (RPC) using a 1 ml
Resource RPC column (GE Healthcare, cat. no 17-1181-01) connected
to an AKTAexplorer 10 system (GE Healthcare). Acetonitrile (ACN)
was supplemented to a final concentration of 10% and the sample was
loaded on the Resource RPC column equilibrated with Solvent A (10%
ACN, 0.1% trifluoroacetic acid (TFA) in Milli-Q water). The column
was washed with 5 CV of Solvent A prior to elution by a 0-60%
linear gradient of Solvent B (80% ACN, 0.1% TFA in Milli-Q water)
over 18 column volumes. Finally, buffer exchange to DPBS (Corning,
cat. no. 21-031-CVR) was carried out using PD-10 Desalting
columns.
[0302] For each protein, the concentration was determined by
measuring the absorbance at 280 nm, using a NanoDrop.RTM. ND-1000
spectrophotometer and the extinction coefficient of the protein.
The purity was analyzed by SDS-PAGE stained with Coomassie Blue and
the identity of each purified ABPP095 variant was confirmed using
HPLC-MS analysis (HPLC-MS 1100; Agilent Technologies).
[0303] Circular dichroism spectroscopy: CD analysis was performed
essentially as described in Example 6, but using a protein
concentration of 0.5 mg/ml.
[0304] Biacore kinetic analysis: Rough kinetic constants (k.sub.a
and k.sub.d) and affinities (K.sub.D) for human A.beta..sub.1-40,
(hA.beta..sub.1-40), human A.beta..sub.1-42 (hA.beta..sub.1-42) and
mouse A.beta..sub.1-40 (mA.beta..sub.1-40) were determined for
ABPP095 (SEQ ID NO:95), its scaffold mutated derivatives ABPP095a
(SEQ ID NO:551), ABPP095b (SEQ ID NO:552) and ABPP095c (SEQ ID
NO:553) as well as for ABPP095-PP013 (SEQ ID NO:115) using a
Biacore 2000 instrument (GE Healthcare). N-terminally biotinylated
hA.beta..sub.1-40 (AnaSpec, cat. no. AS-24648), hA.beta..sub.1-42
(AnaSpec, cat. no. AS-24641-01) and mA.beta..sub.1-40 (AnaSpec,
cat. no. AS-61717-01) were immobilized in separate flow cells on a
streptavidin coated chip (Biacore Sensor Chip SA, cat. no.
BR100032) and using HBS-EP as running buffer (0.01 M HEPES pH 7.4,
0.15 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20, GE Healthcare,
cat. no. BR100188). The ligand immobilization levels on the
surfaces were 17 RU for hA.beta..sub.1-40, 16 RU for
hA.beta..sub.1-42 and 22 RU for mA.beta..sub.1-40. One flow cell
surface on the chip was used as blank during analyte injections.
HBS-EP was used as running buffer and the flow rate was 30
.mu.l/min. The analytes, i.e. A.beta. peptide binding polypeptides,
were each diluted in HBS-EP buffer to concentrations of 630, 210,
70, 23 and 8 nM and injected for 6 min, followed by dissociation in
running buffer for 40 min. After dissociation, the surfaces were
regenerated with 10 mM glycin pH 2.0. Kinetic constants were
calculated from the sensorgrams using the Langmuir 1:1 model of
BiaEvaluation software 4.1 (GE Healthcare).
Results
[0305] Production of scaffold mutated variants of ABPP095: All
variants were expressed as soluble gene products in E. coli.
SDS-PAGE analysis of each final protein preparation showed >98%
purity. The correct oxidized monomer identity was confirmed by
HPLC-MS analysis.
[0306] CD analysis: The melting temperatures (Tm) were determined
using variable temperature measurements and showed that the Tm of
the scaffold mutated variants were similar to the Tm of ABPP095
(Table 6). Reversible folding was observed for all mutated variants
when overlaying spectra measured at 20.degree. C. before and after
heating to 90.degree. C.
TABLE-US-00020 TABLE 6 Melting temperatures of scaffold mutated
ABPP095 variants SEQ Tm Designation ID NO: (.degree. C.) ABPP095 95
43 ABPP095a 551 45 ABPP095b 552 45 ABPP096c 553 44
[0307] SPR kinetic analysis: The interactions of ABPP095, its
scaffold mutated derivatives ABPP095a, ABPP095b and ABPP095c as
well as ABPP095-PP013, with hA.beta..sub.1-40, hA.beta..sub.1-42
and mA.beta..sub.1-40 were analyzed in a Biacore instrument by
injecting various concentrations of the A.beta. peptide binding
polypeptides over a surface containing the respective A.beta.
peptide. All tested A.beta. peptide binding polypeptides showed
binding to the respective A.beta. peptide variant. A summary of the
kinetic parameters (K.sub.D, k.sub.a and k.sub.d) obtained using a
1:1 interaction model is given in Table 7.
TABLE-US-00021 TABLE 7 Kinetic data for binding to different
A.beta. peptides SEQ ID K.sub.D k.sub.a k.sub.d Designation NO:
Target (M) (M.sup.-1s.sup.-1) (s.sup.-1) ABPP095 95
hA.beta..sub.1-40 5.0 .times. 10.sup.-10 7.5 .times. 10.sup.4 3.8
.times. 10.sup.-5 ABPP095a 551 hA.beta..sub.1-40 4.0 .times.
10.sup.-9 7.4 .times. 10.sup.4 3.0 .times. 10.sup.-4 ABPP095b 552
hA.beta..sub.1-40 1.3 .times. 10.sup.-8 5.0 .times. 10.sup.4 6.8
.times. 10.sup.-4 ABPP095c 553 hA.beta..sub.1-40 2.3 .times.
10.sup.-9 8.1 .times. 10.sup.4 1.9 .times. 10.sup.-4 ABPP095- 115
hA.beta..sub.1-40 2.7 .times. 10.sup.-9 1.7 .times. 10.sup.4 4.7
.times. 10.sup.-5 PP013 ABPP095 95 hA.beta..sub.1-42 3.3 .times.
10.sup.-10 1.2 .times. 10.sup.5 4.1 .times. 10.sup.-5 ABPP095a 551
hA.beta..sub.1-42 5.0 .times. 10.sup.-9 1.2 .times. 10.sup.5 6.2
.times. 10.sup.-4 ABPP095b 552 hA.beta..sub.1-42 1.5 .times.
10.sup.-8 1.0 .times. 10.sup.5 1.5 .times. 10.sup.-3 ABPP095c 553
hA.beta..sub.1-42 2.6 .times. 10.sup.-9 1.4 .times. 10.sup.5 3.6
.times. 10.sup.-4 ABPP095- 115 hA.beta..sub.1-42 2.3 .times.
10.sup.-9 2.2 .times. 10.sup.4 4.9 .times. 10.sup.-5 PP013 ABPP095
95 mA.beta..sub.1-40 1.8 .times. 10.sup.-10 1.0 .times. 10.sup.5
1.9 .times. 10.sup.-5 ABPP095a 551 mA.beta..sub.1-40 2.5 .times.
10.sup.-9 1.0 .times. 10.sup.5 2.6 .times. 10.sup.-4 ABPP095b 552
mA.beta..sub.1-40 8.7 .times. 10.sup.-9 7.6 .times. 10.sup.4 6.6
.times. 10.sup.-4 ABPP095c 553 mA.beta..sub.1-40 1.5 .times.
10.sup.-9 1.0 .times. 10.sup.5 1.5 .times. 10.sup.-4 ABPP095- 115
mA.beta..sub.1-40 1.3 .times. 10.sup.-9 2.4 .times. 10.sup.4 3.0
.times. 10.sup.-5 PP013
Example 16
Analysis of Additional A.beta. Peptide Binding Polypeptides
Materials and Methods
[0308] Subcloninq of A.beta. peptide binding polypeptides for
screening: Separate mixes of clones from each library (i.e the
symmetric and the asymmetric library, respectively) obtained from
the third rounds of cell sorting described in Example 2, were PCR
amplified, restricted using endonucleases AccI and XhoI and ligated
into the phagemid vector pAY02592 (essentially as described in
Gronwall et al., supra) restricted using the same endonucleases.
Transformations of the ligations were made into electrocompetent E.
coli ER2738 (Lucigen).
[0309] Sequencing: All individual clones were sequenced. PCR
fragments were amplified from single colonies, sequenced and
analyzed essentially as described in WO2009/077175.
[0310] Production of A.beta. peptide binding polypeptides for
ELISA: Sequenced A.beta. peptide binding polypeptides were produced
by inoculating single colonies from the bulk cloning in 1.2 ml
TSB-YE medium supplemented with 100 .mu.g/ml ampicillin and 0.1 mM
IPTG in deep-well plates (Nunc, cat. no. 278752). The plates were
incubated for 24 h at 37.degree. C. Cells were pelleted by
centrifugation, re-suspended in 150 .mu.l PBST 0.05% (PBS
supplemented with 0.05% Tween-20) and incubated at 82.degree. C. in
a water bath for 20 min. Cell suspensions were transferred to
96-well filtration plates (MerckMillipore, cat. no. MSNANLY50) and
spun in a centrifuge at 1500.times.g for 4 min and the permeates
were collected in new 96-well plates.
[0311] The supernatant of the periplasmic extract contained the
A.beta. peptide binding polypeptides as fusions to the albumin
binding domain corresponding to GA3 of protein G from Streptococcus
strain G148, here denoted ABD, expressed as
AQHDEALE-[ABPP###]-VDYV-[ABD]-YVPG (Gronwall et al., supra).
ABPP### refers to individual, 107 amino acid residue A.beta.
peptide binding polypeptides.
[0312] ELISA analysis of A.beta. peptide binding polypeptides: The
binding of A.beta. peptide binding polypeptides to A.beta..sub.1-40
was analyzed in a sandwich ELISA. Half-area 96-well ELISA plates
(Costar, cat. no. 3690) were coated at 4.degree. C. overnight with
2 .mu.g/ml of an anti-ABD goat antibody (produced in-house) diluted
in coating buffer (50 mM sodium carbonate, pH 9.6; Sigma, cat. no.
C3041). The wells were blocked with 100 .mu.l of PBSC (PBS
supplemented with 0.5% casein; Sigma, cat. no. C8654) and 50 .mu.l
periplasmic solution was added to the wells and incubated for 30
min at RT. The wells were washed 4 times with PBST 0.05% whereafter
50 .mu.l of N-terminally biotinylated hA.beta..sub.1-40 (AnaSpec,
cat. no. AS-24648) at a concentration of 2 nM in PBSC was added to
each well. The plates were incubated for 75 min at RT followed by 4
washes and addition of streptavidin conjugated HRP (Thermo
Scientific, cat. no. N100) diluted 1:30 000 in PBSC. The plates
were incubated for 30 min whereafter they were washed as described
above. The plates were developed by addition of 50 .mu.l 1-Step
Ultra TMB (Thermo Scientific, cat. no. 34028) according to the
manufacturer's recommendations.
[0313] The periplasmic fraction of the A.beta. peptide binding
polypeptide ABPP095 (SEQ ID NO:95) was used in duplicate as
positive control on each ELISA plate. A negative control was
created using ABD periplasm assayed against biotinylated
hA.beta..sub.1-40. The absorbance at 450 nm was measured using a
multi-well plate reader (EnSpire; Perkin Elmer).
[0314] ELISA EC50 analysis of A.beta. peptide binding polypeptides:
A selection of A.beta. peptide binding polypeptides was subjected
to an analysis of the response against a dilution series of
biotinylated hA.beta..sub.1-40 using ELISA essentially as described
above. The assay was performed in 384-well high binding assay
plates (Greiner, cat. no. 781061). Biotinylated protein was added
at a concentration of 150 nM and diluted stepwise 1:5 down to 240
pM. All A.beta. peptide binding polypeptides were also assayed
without added target protein as a background control. Periplasm
samples containing the A.beta. peptide binding polypeptide ABPP095
were included and analyzed as a positive control. As a negative
control, periplasm containing ABD only was assayed against the same
target. Obtained values were analyzed using GraphPad Prism 5 and
non-linear regression and EC50 values (the half maximal effective
concentration) were calculated.
[0315] SPR analysis of periplasmic samples: A selection of A.beta.
peptide binding polypeptides was subjected to an analysis of the
response against biotinylated hA.beta..sub.1-40 using a Biacore
2000 instrument (GE Healthcare). Biotinylated anti-ABD goat
antibody (produced in-house) and N-terminally biotinylated
hA.beta..sub.1-40 (AnaSpec cat. no. AS-24648) were immobilized in
separate flow cells on a streptavidin coated chip (Biacore Sensor
Chip SA, cat. no. BR100032) using HBS-EP as running buffer (0.01 M
HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20, GE
Healthcare, cat. no. BR100188). The ligand immobilization levels on
the surfaces were approximately 2000 RU for the anti-ABD antibody
and 225 RU for hA.beta..sub.1-40. One flow cell surface on the chip
was used as blank during analyte injections. HBS-EP was used as
running buffer and the flow rate was 25 .mu.l/min. The analytes,
i.e. periplasmic preparations of A.beta. peptide binding
polypeptides as described above, were each diluted 15.times. in
HBS-EP buffer and injected for 10 min, followed by dissociation in
running buffer for 5 min. After dissociation, the surfaces were
regenerated with 2 pulses of 10 mM glycin pH 2.0. ABPP095 was
included as first sample, in the middle and as last sample, as a
reference in the analysis to ensure the stability of the surfaces
and was in total run 6 times. Samples of ABD were included as blank
control for the hA.beta..sub.1-40 surface.
Results
[0316] Subcloninq of A.beta. peptide binding polypeptides for
screening: From the third round of cell sorting described in
Example 2, clones were subcloned in bulk to the vector pAY02592.
The outcomes of the two different libraries were kept separate and
clones were transformed to E. coli ER2738.
[0317] Sequencing: Sequencing was performed after subcloning of
individual colonies for identification. Out of a total of 1116
sequences (558 per library), 185 new unique polypeptide variants
were identified in the output from the asymmetric library and 254
unique polypeptide variants were identified in the output from the
symmetric library. Of these, 5 variants were identified in the
output from both libraries. In addition, 51 polypeptide variants
(21 from the asymmetric library and 30 from the symmetric library)
already identified in the first screening described in Example 2
were picked up again.
[0318] ELISA analysis of A.beta. peptide binding polypeptides: The
clones obtained after subcloning in bulk of the outcome after three
rounds of cell sorting were produced in 96-well plates and screened
for A.beta. binding activity in ELISA. The average response of the
negative controls was 0.063 AU. The majority of the A.beta. peptide
binding polypeptides were found to give a response of 0.2 AU or
higher (corresponding to at least 3.times. the negative control)
against hA.beta..sub.1-40 at a concentration of 2 nM. The amino
acid sequences of those among these polypeptides which were newly
identified (not included after the first screen of Example 2) are
listed in FIG. 1A-1X as SEQ ID NO:118-550. Polypeptide variants
identified in the output from the asymmetric library are listed in
FIG. 1A-X as SEQ ID NO:139-142, 155-159 and 376-550, and
polypeptide variants identified in the output from the symmetric
library are listed in FIG. 1A-X as SEQ ID NO:118-138, 143-154 and
160-374. The polypeptides with SEQ ID NO:229, 233, 248, 321 and 334
were identified in the output from both libraries.
[0319] ELISA EC50 analysis of A.beta. peptide binding polypeptides:
A subset of A.beta. peptide binding polypeptides was selected based
on the result in the ELISA experiment described above and subjected
to a target titration in ELISA format. Periplasm samples were
incubated with a serial dilution of biotinylated hA.beta..sub.1-40.
A periplasm sample with the A.beta. peptide binding polypeptide
ABPP095 (SEQ ID NO:95) was also assayed as a positive control.
Obtained values were analyzed and their respective EC50 values were
calculated (Table 8).
TABLE-US-00022 TABLE 8 EC50 values of polypeptides binding to
hA.beta..sub.1-40 SEQ ID EC50 Designation NO: (M) ABPP107 118 4.8
.times. 10.sup.-9 ABPP108 119 5.8 .times. 10.sup.-9 ABPP109 120 5.0
.times. 10.sup.-9 ABPP111 122 1.4 .times. 10.sup.-8 ABPP112 123 4.6
.times. 10.sup.-9 ABPP113 124 3.8 .times. 10.sup.-9 ABPP115 126 3.9
.times. 10.sup.-9 ABPP116 127 3.7 .times. 10.sup.-9 ABPP117 128 6.4
.times. 10.sup.-9 ABPP118 129 5.2 .times. 10.sup.-9 ABPP119 130 3.9
.times. 10.sup.-9 ABPP120 131 3.5 .times. 10.sup.-9 ABPP121 132 5.9
.times. 10.sup.-8 ABPP122 133 5.2 .times. 10.sup.-9 ABPP123 134 5.4
.times. 10.sup.-9 ABPP124 135 1.3 .times. 10.sup.-8 ABPP125 136 4.3
.times. 10.sup.-9 ABPP126 137 5.9 .times. 10.sup.-9 ABPP127 138 5.7
.times. 10.sup.-9 ABPP128 139 5.2 .times. 10.sup.-9 ABPP129 140 9.0
.times. 10.sup.-9 ABPP130 141 6.0 .times. 10.sup.-9 ABPP131 142 6.8
.times. 10.sup.-9 ABPP132 143 8.1 .times. 10.sup.-9 ABPP133 144 4.2
.times. 10.sup.-7 ABPP134 145 2.7 .times. 10.sup.-8 ABPP135 146 6.3
.times. 10.sup.-9 ABPP136 147 7.9 .times. 10.sup.-9 ABPP137 148 8.4
.times. 10.sup.-9 ABPP139 150 8.4 .times. 10.sup.-9 ABPP140 151 4.1
.times. 10.sup.-8 ABPP143 154 9.3 .times. 10.sup.-9 ABPP144 155 1.6
.times. 10.sup.-8 ABPP145 156 9.4 .times. 10.sup.-9 ABPP146 157 8.6
.times. 10.sup.-9 ABPP147 158 7.9 .times. 10.sup.-9 ABPP148 159 6.0
.times. 10.sup.-9 ABPP049 49 7.1 .times. 10.sup.-9 ABPP051 51 4.6
.times. 10.sup.-9 ABPP055 55 2.8 .times. 10.sup.-9 ABPP059 59 1.3
.times. 10.sup.-8 ABPP064 64 4.0 .times. 10.sup.-9 ABPP065 65 1.3
.times. 10.sup.-8 ABPP072 72 8.5 .times. 10.sup.-9 ABPP078 78 3.0
.times. 10.sup.-8 .sup.1 Analyzed in duplicate
[0320] SPR analysis of periplasmic samples: The interactions of a
subset of Z variants with anti-ABD goat antibody and
hA.beta..sub.1-40 were analyzed in a Biacore instrument by
injecting dilutions of periplasmic samples. The responses against
the two surfaces were monitored and are shown in Table 9. The
response against the anti-ABD goat antibody shows the relative
amount of Z variant in the periplasmic sample and the response
against the hA.beta..sub.1-40 shows the binding capability of each
variant in that relative dilution.
TABLE-US-00023 TABLE 9 Relative responses in SPR analysis SEQ
Anti-ABD hA.beta..sub.1-40 ID surface (Response Designation NO:
(Response RU) RU) ABPP107 118 42.2 15.6 ABPP108 119 41.5 15.2
ABPP109 120 42.0 20.2 ABPP110 121 37.5 13.8 ABPP111 122 35.6 14.2
ABPP112 123 48.1 12.1 ABPP113 124 42.0 20.2 ABPP114 125 50.9 34.1
ABPP115 126 39.7 13.3 ABPP116 127 40.0 15.1 ABPP117 128 31.6 9.7
ABPP118 129 41.2 19.2 ABPP119.sup.1 130 31.8 .+-. 1.5 15.6 .+-. 1.5
ABPP120 131 37.7 13.1 ABPP121 132 47.4 47.5 ABPP122 133 47.6 30.1
ABPP123 134 38.8 15.5 ABPP124 135 41.0 23.3 ABPP125 136 38.6 18.4
ABPP126 137 37.8 17.8 ABPP127 138 31.9 9.1 ABPP128 139 48.7 33.0
ABPP129 140 43.6 35.8 ABPP130 141 44.6 21.0 ABPP131 142 34.7 18.9
ABPP132 143 45.3 23.0 ABPP133 144 38.9 14.5 ABPP134 145 40.8 10.3
ABPP135 146 35.0 14.9 ABPP136 147 39.5 13.7 ABPP137 148 35.2 15.3
ABPP138 149 37.8 10.1 ABPP139 150 32.7 10.4 ABPP140 151 36.5 12.3
ABPP141 152 31.5 10.5 ABPP142 153 32.2 9.5 ABPP143 154 31.7 15.8
ABPP144 155 32.1 10.0 ABPP145 156 34.3 15.2 ABPP146 157 36.7 15.2
ABPP147 158 39.2 17.0 ABPP148 159 28.7 10.2 ABPP035 35 31.8 8.6
ABPP049 49 30.6 13.7 ABPP051 51 51.5 21.4 ABPP055.sup.1 55 37.0
.+-. 1.1 18.2 .+-. 4.9 ABPP059 59 39.5 15.4 ABPP064 64 38.8 12.0
ABPP065 65 39.1 8.4 ABPP070 70 40.7 16.6 ABPP072 72 39.7 10.1
ABPP078 78 38.6 12.5 ABPP095.sup.2 95 40.6 .+-. 2.2 24.6 .+-. 0.5
ABD.sup.1,3 117 19.8 .+-. 1.3 2.0 .+-. 0.3 .sup.1 Analyzed 2 times,
average presented .sup.2 Analyzed 6 times, average presented .sup.3
Lower molecular weight, hence lower RU expected
Itemized List of Embodiments
[0321] 1. A.beta. peptide binding polypeptide, which comprises
[0322] a first moiety comprising a first A.beta. peptide binding
motif BM1, [0323] a second moiety comprising a second A.beta.
peptide binding motif BM2, which motifs may be the same or
different, and [0324] a linker,
[0325] wherein each one of said binding motifs BM1 and BM2 consists
of an amino acid sequence selected from
TABLE-US-00024 i) (SEQ ID NO: 632)
EX.sub.2X.sub.3YX.sub.5X.sub.6NLX.sub.9A
X.sub.11QLCAX.sub.161X.sub.18X.sub.19X.sub.20 ED
wherein, independently from each other,
[0326] X.sub.2 is selected from I, M, Q, R, T and Y;
[0327] X.sub.3 is selected from H and V;
[0328] X.sub.5 is selected from F, I and L;
[0329] X.sub.6 is selected from P and T;
[0330] X.sub.9 is selected from N and T;
[0331] X.sub.11 is selected from D and H;
[0332] X.sub.16 is selected from F and I;
[0333] X.sub.18 is selected from N, Q and R;
[0334] X.sub.19 is selected from K and 5; and
[0335] X.sub.20 is selected from F, I, L and R;
[0336] and [0337] ii) an amino acid sequence which has at least 95%
identity to the sequence defined in i).
[0338] 2. A.beta. peptide binding polypeptide according to item 1,
wherein sequence i) in BM1 consists of the amino acid sequence
TABLE-US-00025 (SEQ ID NO: 558)
EX.sub.2X.sub.3YX.sub.5X.sub.6NLX.sub.9A
X.sub.11QLCAFIX.sub.18X.sub.19L ED
wherein, independently from each other,
[0339] X.sub.2 is selected from I, M, Q, R and Y;
[0340] X.sub.3 is selected from H and V;
[0341] X.sub.5 is selected from F, I and L;
[0342] X.sub.6 is selected from P and T;
[0343] X.sub.9 is selected from N and T;
[0344] X.sub.11 is selected from D and H;
[0345] X.sub.18 is selected from N, Q and R; and
[0346] X.sub.19 is selected from K and S.
[0347] 3. A.beta. peptide binding polypeptide according to item 2,
wherein, independently from each other, X.sub.2 is further selected
from I, Q, R and Y; and X.sub.5 is further selected from F and
L.
[0348] 4. A.beta. peptide binding polypeptide according to item 2,
wherein X.sub.2 is further selected from I, M and R.
[0349] 5. A.beta. peptide binding polypeptide according to any
preceding item, wherein sequence i) in BM2 consists of the amino
acid sequence
TABLE-US-00026 (SEQ ID NO: 560) EX.sub.2VYX.sub.5PNLX.sub.9A
X.sub.11QLCAX.sub.16IX.sub.18X.sub.19X.sub.20 ED
wherein, independently from each other,
[0350] X.sub.2 is selected from I, M, Q, R and T;
[0351] X.sub.5 is selected from F and L;
[0352] X.sub.9 is selected from N and T;
[0353] X.sub.11 is selected from D and H;
[0354] X.sub.16 is selected from F and I;
[0355] X.sub.18 is selected from N, Q and R;
[0356] X.sub.19 is selected from K and 5; and
[0357] X.sub.20 is selected from F, I, L and R.
[0358] 6. A.beta. peptide binding polypeptide according to item 5,
wherein, independently from each other, X.sub.2 is further selected
from R and T; and X.sub.5 is L.
[0359] 7. A.beta. peptide binding polypeptide according to item 5,
wherein, independently from each other, X.sub.2 is further selected
from I, M, Q and R; X.sub.16 is F; and X.sub.20 is L.
[0360] 8. A.beta. peptide binding polypeptide according to any
preceding item, wherein at least one of said first and second
moieties comprises the amino acid sequence
X.sub.AGX.sub.B-[BM]
wherein BM is BM1 or BM2 as defined in any one of items 1-7, and,
independently of each other,
[0361] X.sub.A is selected from A and 5; and
[0362] X.sub.B is selected from G and R.
[0363] 9. A.beta. peptide binding polypeptide according to any one
of items 1-7, wherein at least one of first and second moieties
comprises a binding module amino acid sequence, Bmod, selected
from
TABLE-US-00027 iii) (SEQ ID NO: 562)
[BM]DX.sub.aSQX.sub.bX.sub.cX.sub.dLLX.sub.e
EAKKLX.sub.fX.sub.gX.sub.hQA PX.sub.i
wherein BM is BM1 or BM2 as defined in any one of items 1-7, and,
independently of each other,
[0364] X.sub.a is selected from P, Q, R and 5;
[0365] X.sub.b is selected from N, Q, R and 5;
[0366] X.sub.c is selected from A and 5;
[0367] X.sub.d is selected from K, N and E;
[0368] X.sub.e is selected from A, S and C;
[0369] X.sub.f is selected from E, N and 5;
[0370] X.sub.g is selected from D, E and 5;
[0371] X.sub.h is selected from A and 5; and
[0372] X.sub.i is selected from no amino acid, A and K,
and [0373] iv) an amino acid sequence which has at least 97%
identity to a sequence defined in iii).
[0374] 10. A.beta. peptide binding polypeptide according to item 9,
wherein sequence iii) consists of the amino acid sequence
TABLE-US-00028 (SEQ ID NO: 563) [BM]DX.sub.aSQX.sub.bAX.sub.dLLA
EAKKLNDAQA PX.sub.i
wherein BM is BM1 or BM2 as defined in any one of items 1-7, and,
independently of each other,
[0375] X.sub.a is selected from P, Q, R and 5;
[0376] X.sub.b is selected from N, Q, R and 5;
[0377] X.sub.d is selected from K and N; and
[0378] X.sub.i is selected from no amino acid, A and K.
[0379] 11. A.beta. peptide binding polypeptide according to item
10, wherein sequence iii) consists of an amino acid sequence
selected from
TABLE-US-00029 (SEQ ID NO: 564) [BM]-DPSQSANLLAEAKKLNDAQAP; (SEQ ID
NO: 565) [BM]-DPSQQANLLAEAKKLNDAQAP; (SEQ ID NO: 566)
[BM]-DSSQSANLLAEAKKLNDAQAP; (SEQ ID NO: 567)
[BM]-DPSQQAKLLAEAKKLNDAQAP; (SEQ ID NO: 568)
[BM]-DPSQNANLLAEAKKLNDAQAP; (SEQ ID NO: 569)
[BM]-DRSQQANLLAEAKKLNDAQAP; (SEQ ID NO: 570)
[BM]-DQSQRANLLAEAKKLNDAQAP; (SEQ ID NO: 571)
[BM]-DQSQQANLLAEAKKLNDAQAP; (SEQ ID NO: 572)
[BM]-DRSQSANLLAEAKKLNDAQAP; (SEQ ID NO: 573)
[BM]-DRSQRANLLAEAKKLNDAQAP; and (SEQ ID NO: 574)
[BM]-DRSQNANLLAEAKKLNDAQAP.
[0380] 12. A.beta. peptide binding polypeptide according to item
10, wherein said first moiety comprises a Bmod in which sequence
iii) is an amino acid sequence selected from
TABLE-US-00030 (SEQ ID NO: 575) [BM1]-DPSQSANLLAEAKKLNDAQAPA; (SEQ
ID NO: 576) [BM1]-DPSQQANLLAEAKKLNDAQAPA; (SEQ ID NO: 577)
[BM1]-DSSQSANLLAEAKKLNDAQAPA; (SEQ ID NO: 578)
[BM1]-DPSQRANLLAEAKKLNDAQAPA; (SEQ ID NO: 579)
[BM1]-DPSQQAKLLAEAKKLNDAQAPA; (SEQ ID NO: 580)
[BM1]-DPSQNANLLAEAKKLNDAQAPA; and (SEQ ID NO: 581)
[BM1]-DRSQNANLLAEAKKLNDAQAPA.
[0381] 13. A.beta. peptide binding polypeptide according to item
10, wherein said second moiety comprises a Bmod in which sequence
iii) is an amino acid sequence selected from
TABLE-US-00031 (SEQ ID NO: 582) [BM2]-DPSQQANLLAEAKKLNDAQAPK; (SEQ
ID NO: 583) [BM2]-DPSQSANLLAEAKKLNDAQAPK; (SEQ ID NO: 584)
[BM2]-DRSQQANLLAEAKKLNDAQAPK; (SEQ ID NO: 585)
[BM2]-DQSQRANLLAEAKKLNDAQAPK; (SEQ ID NO: 586)
[BM2]-DQSQQANLLAEAKKLNDAQAPK; (SEQ ID NO: 587)
[BM2]-DPSQRANLLAEAKKLNDAQAPK; (SEQ ID NO: 588)
[BM2]-DRSQSANLLAEAKKLNDAQAPK; (SEQ ID NO: 589)
[BM2]-DRSQRANLLAEAKKLNDAQAPK; (SEQ ID NO: 590)
[BM2]-DSSQSANLLAEAKKLNDAQAPK; and (SEQ ID NO: 591)
[BM2]-DPSQNANLLAEAKKLNDAQAPK.
[0382] 14. A.beta. peptide binding polypeptide according to any one
of items 8-13, wherein at least one of first and second moieties
comprises the amino acid sequence
X.sub.AGX.sub.B-[BMod]
wherein BMod is as defined in any one of items 9-13, and,
independently of each other,
[0383] X.sub.A is selected from A and 5; and
[0384] X.sub.B is selected from G and R.
[0385] 15. A.beta. peptide binding polypeptide according to item
14, wherein at least one of said first and second moieties
comprises an amino acid sequence selected from
TABLE-US-00032 (SEQ ID NO: 592) AGG-[BM]-DPSQSANLLAEAKKLNDAQAP;
(SEQ ID NO: 593) AGG-[BM]-DPSQQANLLAEAKKLNDAQAP; (SEQ ID NO: 594)
AGG-[BM]-DSSQSANLLAEAKKLNDAQAP; (SEQ ID NO: 595)
AGG-[BM]-DPSQRANLLAEAKKLNDAQAP; (SEQ ID NO: 596)
AGR-[BM]-DPSQSANLLAEAKKLNDAQAP; (SEQ ID NO: 597)
AGG-[BM]-DPSQQAKLLAEAKKLNDAQAP; (SEQ ID NO: 598)
AGG-[BM]-DPSQNANLLAEAKKLNDAQAP; (SEQ ID NO: 599)
AGR-[BM]-DPSQQANLLAEAKKLNDAQAP; (SEQ ID NO: 600)
AGR-[BM]-DRSQQANLLAEAKKLNDAQAP; (SEQ ID NO: 601)
AGR-[BM]-DQSQRANLLAEAKKLNDAQAP; (SEQ ID NO: 602)
AGR-[BM]-DQSQQANLLAEAKKLNDAQAP; (SEQ ID NO: 603)
AGR-[BM]-DPSQRANLLAEAKKLNDAQAP; (SEQ ID NO: 604)
AGR-[BM]-DRSQSANLLAEAKKLNDAQAP; (SEQ ID NO: 605)
SGG-[BM]-DPSQSANLLAEAKKLNDAQAP; (SEQ ID NO: 606)
AGR-[BM]-DRSQRANLLAEAKKLNDAQAP; (SEQ ID NO: 607)
AGR-[BM]-DPSQNANLLAEAKKLNDAQAP; (SEQ ID NO: 608)
AGR-[BM]-DSSQSANLLAEAKKLNDAQAP; and (SEQ ID NO: 609)
AGR-[BM]-DRSQNANLLAEAKKLNDAQAP.
[0386] 16. A.beta. peptide binding polypeptide according to item
14, wherein said first moiety comprises an amino acid sequence
selected from
TABLE-US-00033 (SEQ ID NO: 610) AGG-[BM1]-DPSQSANLLAEAKKLNDAQAPA;
(SEQ ID NO: 611) AGG-[BM1]-DPSQQANLLAEAKKLNDAQAPA; (SEQ ID NO: 612)
AGG-[BM1]-DSSQSANLLAEAKKLNDAQAPA; (SEQ ID NO: 613)
AGG-[BM1]-DPSQRANLLAEAKKLNDAQAPA; (SEQ ID NO: 614)
AGR-[BM1]-DPSQSANLLAEAKKLNDAQAPA; (SEQ ID NO: 615)
AGG-[BM1]-DPSQQAKLLAEAKKLNDAQAPA; and (SEQ ID NO: 616)
AGG-[BM1]-DPSQNANLLAEAKKLNDAQAPA; (SEQ ID NO: 617)
AGR-[BM1]-DPSQNANLLAEAKKLNDAQAP; (SEQ ID NO: 618)
AGR-[BM1]-DSSQSANLLAEAKKLNDAQAP; and (SEQ ID NO: 619)
AGR-[BM1]-DRSQNANLLAEAKKLNDAQAP.
[0387] 17. A.beta. peptide binding polypeptide according to item
14, wherein said second moiety comprises an amino acid sequence
selected from
TABLE-US-00034 (SEQ ID NO: 620) AGR-[BM2]-DPSQQANLLAEAKKLNDAQAPK;
(SEQ ID NO: 621) AGR-[BM2]-DPSQSANLLAEAKKLNDAQAPK; (SEQ ID NO: 622)
AGR-[BM2]-DRSQQANLLAEAKKLNDAQAPK; (SEQ ID NO: 623)
AGR-[BM2]-DQSQRANLLAEAKKLNDAQAPK; (SEQ ID NO: 624)
AGR-[BM2]-DQSQQANLLAEAKKLNDAQAPK; (SEQ ID NO: 625)
AGR-[BM2]-DPSQRANLLAEAKKLNDAQAPK; (SEQ ID NO: 626)
AGR-[BM2]-DRSQSANLLAEAKKLNDAQAPK; (SEQ ID NO: 627)
AGG-[BM2]-DPSQSANLLAEAKKLNDAQAPK; (SEQ ID NO: 628)
SGG-[BM2]-DPSQSANLLAEAKKLNDAQAPK; (SEQ ID NO: 629)
AGR-[BM2]-DRSQRANLLAEAKKLNDAQAPK; (SEQ ID NO: 630)
AGG-[BM2]-DSSQSANLLAEAKKLNDAQAPK; and (SEQ ID NO: 631)
AGG-[BM2]-DPSQNANLLAEAKKLNDAQAPK.
[0388] 18. A.beta. peptide binding polypeptide according to any one
of items 1-17, wherein said linker is arranged between said first
moiety and said second moiety.
[0389] 19. A.beta. peptide binding polypeptide according to item
18, wherein said linker is a flexible linker comprising at least
one amino acid residue selected from the group consisting of
glycine, serine and threonine.
[0390] 20. A.beta. peptide binding polypeptide according to item
19, wherein said linker has a general formula selected from
(G.sub.nS.sub.m).sub.p and (S.sub.nG.sub.m).sub.p,
[0391] wherein, independently,
[0392] n=1-7,
[0393] m=0-7,
[0394] n+m.ltoreq.8 and
[0395] p=1-10.
[0396] 21. A.beta. peptide binding polypeptide according to item
20, wherein n=1-5.
[0397] 22. A.beta. peptide binding polypeptide according to any one
of items 20-21, wherein m=0-5.
[0398] 23. A.beta. peptide binding polypeptide according to any one
of items 20-22, wherein p=1-5.
[0399] 24. A.beta. peptide binding polypeptide according to any one
of items 20-23, wherein n=4, m=1 and p=2-3.
[0400] 25. A.beta. peptide binding polypeptide according to item
24, wherein said linker is selected from (S.sub.4G).sub.2 (SEQ ID
NO:633) and (S.sub.4G).sub.3 (SEQ ID NO:634).
[0401] 26. A.beta. peptide binding polypeptide according to item
25, wherein said linker is (S.sub.4G).sub.2 (SEQ ID NO:633).
[0402] 27. A.beta. peptide binding polypeptide according to item
25, wherein said linker is (S.sub.4G).sub.3 (SEQ ID NO:634).
[0403] 28. A.beta. peptide binding polypeptide according to item
18, wherein said linker comprises an amino acid sequence selected
from the group consisting of VEVDNKFNKEMAS (SEQ ID NO:635),
VDNKFNKEMAS (SEQ ID NO:636), VEVDNKFNKE (SEQ ID NO:637), VDNKFNKE
(SEQ ID NO:638), AEAKYAKE (SEQ ID NO:639), ADNNFNK (SEQ ID NO:640),
ADNKFNK (SEQ ID NO:641), ADAQQNNFNK (SEQ ID NO:642), AQHDE (SEQ ID
NO:643), VDNKFNK (SEQ ID NO:644), AEAKYAK (SEQ ID NO:645), VDAKYAK
(SEQ ID NO:646), ADAKYAK (SEQ ID NO:647).
[0404] 29. A.beta. peptide binding polypeptide according to any one
of items 20-28, wherein said linker further comprises 1-5
additional amino acid residues at the N- or C-terminal end of said
linker, such as 1-4 additional amino acid residues, such as 1-3
additional amino acid residues, such as 3 additional amino acid
residues.
[0405] 30. A.beta. peptide binding polypeptide according to item
29, wherein said additional amino acid residues are selected from
the group consisting of AS, MAS and RAS.
[0406] 31. A.beta. peptide binding polypeptide according to any
preceding item, comprising a first moiety as defined in item 12 and
a second moiety as defined in item 13.
[0407] 32. A.beta. peptide binding polypeptide according any
preceding item, comprising a first moiety as defined in item 16 and
a second moiety as defined in item 17.
[0408] 33. A.beta. peptide binding polypeptide according to any
preceding item, wherein [0409] sequence i) of BM1 corresponds to
amino acid residues 4-25 in any one of SEQ ID NO:1-106 and 118-553,
and [0410] sequence i) of BM2 corresponds to amino acid residues
64-85 in any one of SEQ ID NO:1-17, 19-106 and 118-553 or to amino
acid residues 69-90 in SEQ ID NO:18.
[0411] 34. A.beta. peptide binding polypeptide according to item
33, wherein [0412] sequence i) of BM1 corresponds to amino acid
residues 4-25 in any one of SEQ ID NO:1, 5, 9, 13, 14, 16, 18, 20,
21, 25, 26, 28, 33, 35, 37, 40, 42, 48, 49, 50, 51, 53, 54, 55, 59,
60, 61, 62, 64, 65, 70, 72, 75, 78, 84, 89, 90, 95, 96, 97, 100,
104 and 118-159, and [0413] sequence i) of BM2 corresponds to amino
acid residues 64-85 in any one of SEQ ID NO:1, 5, 9, 13, 14, 16,
20, 21, 25, 26, 28, 33, 35, 37, 40, 42, 48, 49, 50, 51, 53, 54, 55,
59, 60, 61, 62, 64, 65, 70, 72, 75, 78, 84, 89, 90, 95, 96, 97,
100, 104 and 118-159 or to amino acid residues 69-90 in SEQ ID
NO:18.
[0414] 35. A.beta. peptide binding polypeptide according to item
34, wherein [0415] sequence i) of BM1 corresponds to amino acid
residues 4-25 in any one of SEQ ID NO:18, 28, 35, 49, 50, 51, 55,
59, 64, 65, 70, 72, 78, 84, 95 and 118-159, and [0416] sequence i)
of BM2 corresponds to amino acid residues 64-85 in any one of SEQ
ID NO:28, 35, 49, 50, 51, 55, 59, 64, 65, 70, 72, 78, 84, 95 and
118-159 or to amino acid residues 69-90 in SEQ ID NO:18.
[0417] 36. A.beta. peptide binding polypeptide according to item
35, wherein [0418] sequence i) of BM1 corresponds to amino acid
residues 4-25 in any one of SEQ ID NO:18, 70 and 95, and [0419]
sequence i) of BM2 corresponds to any one of amino acid residues
64-85 in SEQ ID NO:70 and 95 or to amino acid residues 69-90 in SEQ
ID NO:18.
[0420] 37. A.beta. peptide binding polypeptide according to item
36, wherein [0421] sequence i) of BM1 corresponds to amino acid
residues 4-25 in SEQ ID NO:95 and [0422] sequence i) of BM2
corresponds to amino acid residues 64-85 in SEQ ID NO:95.
[0423] 38. A.beta. peptide binding polypeptide according to any
preceding item, wherein [0424] the first moiety comprises amino
acid residues 1-25 in any one of SEQ ID NO:1-106 and 118-553, and
[0425] the second moiety comprises amino acid residues 61-85 in any
one of SEQ ID NO:1-17, 19-106 and 118-553 or amino acid residues
66-90 in SEQ ID NO:18.
[0426] 39. A.beta. peptide binding polypeptide according to item
38, wherein [0427] the first moiety comprises amino acid residues
1-25 in any one of SEQ ID NO:1, 5, 9, 13, 14, 16, 18, 20, 21, 25,
26, 28, 33, 35, 37, 40, 42, 48, 49, 50, 51, 53, 54, 55, 59, 60, 61,
62, 64, 65, 70, 72, 75, 78, 84, 89, 90, 95, 96, 97, 100, 104 and
118-159, and [0428] the second moiety comprises amino acid residues
61-85 in any one of SEQ ID NO:1, 5, 9, 13, 14, 16, 20, 21, 25, 26,
28, 33, 35, 37, 40, 42, 48, 49, 50, 51, 53, 54, 55, 59, 60, 61, 62,
64, 65, 70, 72, 75, 78, 84, 89, 90, 95, 96, 97, 100, 104 and
118-159 or amino acid residues 66-90 in SEQ ID NO:18.
[0429] 40. A.beta. peptide binding polypeptide according to item
39, wherein [0430] the first moiety comprises amino acid residues
1-25 in any one of SEQ ID NO:18, 28, 35, 49, 50, 51, 55, 59, 64,
65, 70, 72, 78, 84, 95 and 118-159, and [0431] the second moiety
comprises amino acid residues 61-85 in any one of SEQ ID NO:28, 35,
49, 50, 51, 55, 59, 64, 65, 70, 72, 78, 84, 95 and 118-159 or amino
acid residues 66-90 in SEQ ID NO:18.
[0432] 41. A.beta. peptide binding polypeptide according to item
40, wherein [0433] the first moiety comprises amino acid residues
1-25 in any one of SEQ ID NO:18, 70 and 95, and [0434] the second
moiety comprises amino acid residues 61-85 in any one of SEQ ID
NO:70 and 95 or amino acid residues 66-90 in SEQ ID NO:18.
[0435] 42. A.beta. peptide binding polypeptide according to item
41, wherein [0436] the first moiety comprises amino acid residues
1-25 in SEQ ID NO:95, and [0437] the second moiety comprises amino
acid residues 61-85 in SEQ ID NO:95.
[0438] 43. A.beta. peptide binding polypeptide according to any one
of items 9-14, wherein [0439] sequence iii) in Bmod of the first
moiety corresponds to amino acid residues 4-47 in any one of SEQ ID
NO:1-106 and 118-553, and [0440] sequence iii) in Bmod of the
second moiety corresponds to amino acid residues 64-107 in any one
of SEQ ID NO:1-17, 19-106 and 118-553 or amino acid residues 69-112
in SEQ ID NO:18.
[0441] 44. A.beta. peptide binding polypeptide according to item
43, wherein [0442] sequence iii) in Bmod of the first moiety
corresponds to amino acid residues 4-47 in any one of SEQ ID NO:1,
5, 9, 13, 14, 16, 18, 20, 21, 25, 26, 28, 33, 35, 37, 40, 42, 48,
49, 50, 51, 53, 54, 55, 59, 60, 61, 62, 64, 65, 70, 72, 75, 78, 84,
89, 90, 95, 96, 97, 100, 104 and 118-159, and [0443] sequence iii)
in Bmod of the second moiety corresponds to amino acid residues
64-107 in any one of SEQ ID NO:1, 5, 9, 13, 14, 16, 20, 21, 25, 26,
28, 33, 35, 37, 40, 42, 48, 49, 50, 51, 53, 54, 55, 59, 60, 61, 62,
64, 65, 70, 72, 75, 78, 84, 89, 90, 95, 96, 97, 100, 104 and
118-159 or amino acid residues 69-112 in SEQ ID NO:18.
[0444] 45. A.beta. peptide binding polypeptide according to item
44, wherein [0445] sequence iii) in Bmod of the first moiety
corresponds to amino acid residues 4-47 in any one of SEQ ID NO:18,
28, 35, 49, 50, 51, 55, 59, 64, 65, 70, 72, 78, 84, 95 and 118-159,
and [0446] sequence iii) in Bmod of the second moiety corresponds
to amino acid residues 64-107 in any one of SEQ ID NO:28, 35, 49,
50, 51, 55, 59, 64, 65, 70, 72, 78, 84, 95 and 118-159 or amino
acid residues 69-112 in SEQ ID NO:18.
[0447] 46. A.beta. peptide binding polypeptide according to item
45, wherein [0448] sequence iii) in Bmod of the first moiety
corresponds to amino acid residues 4-47 in any one of SEQ ID NO:18,
70 and 95, and [0449] sequence iii) in Bmod of the second moiety
corresponds to amino acid residues 64-107 in any one of SEQ ID
NO:70 and 95 or amino acid residues 69-112 in SEQ ID NO:18.
[0450] 47. A.beta. peptide binding polypeptide according to item
46, wherein [0451] sequence iii) in Bmod of the first moiety
corresponds to amino acid residues 4-47 in SEQ ID NO:95, and [0452]
sequence iii) in Bmod of the second moiety corresponds to amino
acid residues 64-107 in SEQ ID NO:95.
[0453] 48. A.beta. peptide binding polypeptide according to any one
of items 33-47, wherein [0454] the first moiety comprises an amino
acid sequence corresponding to amino acid residues 4-25, 1-25, 4-47
or 1-47 from any one of SEQ ID NO:1-106 and 118-553; and [0455] the
second moiety comprises an amino acid sequence corresponding to
amino acid residues 64-85, 61-85, 64-107 or 61-107 from any one of
SEQ ID NO:1-17, 19-106 and 118-553 or to amino acid residues 69-90,
66-90, 69-112 or 66-112 from SEQ ID NO:18; [0456] wherein both said
amino acid sequences comprised in each of the first and second
moieties are from the same SEQ ID NO.
[0457] 49. A.beta. peptide binding polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:1-106
and 118-553; such as from the group consisting of SEQ ID NO:1, 5,
9, 13, 14, 16, 18, 20, 21, 25, 26, 28, 33, 35, 37, 40, 42, 48, 49,
50, 51, 53, 54, 55, 59, 60, 61, 62, 64, 65, 70, 72, 75, 78, 84, 89,
90, 95, 96, 97, 100, 104 and 118-159; such as from the group
consisting of SEQ ID NO:18, 28, 35, 49, 50, 51, 55, 59, 64, 65, 70,
72, 78, 84, 95 and 118-159, such as from the group consisting of
SEQ ID NO 18, 70 and 95.
[0458] 50. A.beta. peptide binding polypeptide according to item
49, comprising the amino acid sequence SEQ ID NO:95.
[0459] 51. A.beta. peptide binding polypeptide according to any
preceding item, which is capable of binding to A.beta. peptide such
that the K.sub.D value of the interaction is at most
1.times.10.sup.-7 M, such as at most 1.times.10.sup.-8 M, such as
at most 1.times.10.sup.-9 M, such as at most 1.times.10.sup.-10
M.
[0460] 52. A.beta. peptide binding polypeptide, which constitutes a
fragment of a polypeptide according to any preceding item, which
fragment retains binding affinity for an A.beta. peptide.
[0461] 53. A.beta. peptide binding polypeptide according to any
preceding item, which comprises at least one additional amino acid
residue at at least one of the N terminus and the C terminus.
[0462] 54. A.beta. peptide binding polypeptide according to item
53, which comprises additional amino acid residues at both the N
terminus and the C terminus.
[0463] 55. A.beta. peptide binding polypeptide according any one of
items 53-54, wherein said at least one additional amino acid
residue improves or simplifies production, purification,
stabilization in vivo or in vitro, coupling or detection of the
polypeptide.
[0464] 56. Fusion protein or conjugate, comprising [0465] a first
part consisting of an A.beta. peptide binding polypeptide according
to any preceding item; and [0466] a second part consisting of a
polypeptide having a desired biological activity.
[0467] 57. Fusion protein or conjugate according to item 56,
wherein said desired biological activity is a therapeutic
activity.
[0468] 58. Fusion protein or conjugate according to item 56 or 57,
wherein said desired biological activity is a binding activity.
[0469] 59. Fusion protein or conjugate according to item 58,
wherein said binding activity of said second part is binding to
A.beta. peptide.
[0470] 60. Fusion protein or conjugate according to item 58,
wherein said binding activity is an albumin binding activity, which
increases the in vivo half-life of the fusion protein or
conjugate.
[0471] 61. Fusion protein or conjugate according to item 60,
wherein said second part comprises the albumin binding domain of
streptococcal protein G or a derivative thereof, such as comprising
the amino acid sequence SEQ ID NO:107, SEQ ID NO:117 or a
derivative thereof.
[0472] 62. Fusion protein or conjugate according to item 58 or 59,
wherein said binding activity acts to block a biological
activity.
[0473] 63. Fusion protein or conjugate according to item 58 or 59,
wherein said binding activity acts to stimulate a biological
activity.
[0474] 64. Fusion protein or conjugate according to item 58,
wherein said binding activity is binding to a target selected from
the group consisting of Tau, apolipoprotein E (ApoE),
beta-secretase 1 (BACE1), gamma-secretase, transferrin receptor
(TfR), complement factor C1s, presenilin 1, presenilin 2,
nicastrin, alpha-synuclein and Bri.
[0475] 65. Fusion protein or conjugate according to item 56,
wherein said desired biological activity is an enzymatic
activity.
[0476] 66. Fusion protein or conjugate according to item 57,
wherein the second part is a therapeutically active
polypeptide.
[0477] 67. Fusion protein or conjugate according to any one of
items 56-57 and 65-66, wherein the second part is selected from the
group consisting of human endogenous enzymes, hormones, growth
factors, chemokines, cytokines and lymphokines.
[0478] 68. Fusion protein or conjugate according to any one of
items 56-57 and 65-66, wherein the second part is selected from the
group consisting of antibodies and antigen binding fragments
thereof.
[0479] 69. Fusion protein or conjugate according to item 68,
wherein said antibody or antigen binding fragment thereof is
selected from the group consisting of full-length antibodies, Fab
fragments, Fab' fragments, F(ab').sub.2 fragments, Fc fragments, Fv
fragments, single chain Fv fragments, (scFv).sub.2 and domain
antibodies.
[0480] 70. Fusion protein or conjugate according to item 68 or 69,
wherein said antibody or antigen binding fragment thereof has an
affinity for a target selected from the group consisting of Tau,
apolipoprotein E (ApoE), beta-secretase 1 (BACE1), gamma-secretase,
transferrin receptor (TfR), complement factor C1s, presenilin 1,
presenilin 2, nicastrin, alpha-synuclein and Bri.
[0481] 71. Fusion protein or conjugate according to any one of
items 56-70, wherein the second part mediates transport of the
fusion protein or conjugate to the cerebrospinal fluid or brain,
for example being selected from the group consisting of
transferrin, ghrelin, insulin and leptin.
[0482] 72. A.beta. peptide binding polypeptide, fusion protein or
conjugate according to any preceding item, further comprising a
label.
[0483] 73. A.beta. peptide binding polypeptide, fusion protein or
conjugate according to item 72, wherein said label is selected from
fluorescent dyes and metals, chromophoric dyes, chemiluminescent
compounds and bioluminescent proteins, enzymes, radionuclides and
radioactive particles.
[0484] 74. A.beta. peptide binding polypeptide, fusion protein or
conjugate according to any preceding item, comprising a chelating
environment provided by a polyaminopolycarboxylate chelator
conjugated to the A.beta. peptide binding polypeptide via a thiol
group of a cysteine residue or an amine group of a lysine
residue.
[0485] 75. A polynucleotide encoding a polypeptide according to any
one of items 1-71.
[0486] 76. Expression vector comprising a polynucleotide according
to item 75.
[0487] 77. Host cell comprising an expression vector according to
item 76.
[0488] 78. Method of producing a polypeptide according to any one
of items 1-71, comprising [0489] culturing a host cell according to
item 77 under conditions permissive of expression of said
polypeptide from said expression vector, and [0490] isolating said
polypeptide.
[0491] 79. Composition comprising an A.beta. peptide binding
polypeptide, fusion protein or conjugate according to any one of
items 1-74 and at least one pharmaceutically acceptable excipient
or carrier.
[0492] 80. Composition according to item 79, further comprising at
least one additional active agent.
[0493] 81. Composition according to item 80, wherein said
additional active agent is selected from the group consisting of
inhibitors of neurotransmitter degradation, neurotransmitters,
acetylcholinesterase inhibitors, NM DA receptor antagonists, TNF
inhibitors, antihistamines, anti-viral agents, alpha-secretase
activators, inhibitors of beta- or gamma-secretase, inhibitors of
.alpha.-synuclein aggregation, inhibitors of tau aggregation,
calcium channel blockers, compounds effective against oxidative
stress, anti-apoptotic compounds, metal chelators, inhibitors of
DNA repair such as pirenzepin and metabolites, attractants for
A.beta. clearing/depleting cellular components, inhibitors of
N-terminal truncated A.beta. including pyroglutamated
A.beta..sub.3-42, anti-inflammatory molecules, atypical
antipsychotics such as clozapine, ziprasidone, risperidone,
aripiprazole and olanzapine, cholinesterase inhibitors (ChEIs) such
as tacrine, rivastigmine, donepezil and galantamine, M1 agonists
and other drugs including any amyloid or tau modifying drug and
nutritive supplements such as vitamin B12, cysteine, acetylcholine
precursor, lecithin, choline, Ginkgo biloba, acetyl-L-carnitine,
idebenone, propentofylline, and xanthine derivatives.
[0494] 82. Composition according to item 81, wherein said
additional active agent is selected from the group consisting of
acetylcholinesterase inhibitors, NMDA receptor antagonists, TNF
inhibitors, antihistamines and anti-viral agents.
[0495] 83. A.beta. peptide binding polypeptide, fusion protein or
conjugate according to any one of items 1-74 or composition
according to any one of items 80-82 for administration via a route
selected from the group consisting of oral, intravenous,
intraperitoneal, subcutaneous, intrathecal (e.g. via the Ommaya
reservoir), pulmonary, transdermal, intramuscular, intranasal,
buccal, sublingual or suppository administration, such as selected
from the group consisting of oral, intrathechal, intravenous and
intranasal administration.
[0496] 84. A.beta. peptide binding polypeptide, fusion protein or
conjugate according to any one of items 1-74 or a composition
according to any one of items 80-82 for use as a medicament, as a
diagnostic agent or as a prognostic agent.
[0497] 85. A.beta. peptide binding polypeptide, fusion protein,
conjugate or composition for use as a medicament according to item
84, whereby said polypeptide, fusion protein, conjugate or
composition reduces the amount of A.beta. peptide in blood.
[0498] 86. A.beta. peptide binding polypeptide, fusion protein,
conjugate or composition for use as a medicament according to item
84, whereby said polypeptide, fusion protein, conjugate or
composition slows down, stops or reverses an equilibrium between
A.beta. monomers and A.beta. aggregates.
[0499] 87. A.beta. peptide binding polypeptide, fusion protein,
conjugate or composition for use as a medicament according to item
84, whereby said polypeptide, fusion protein, conjugate or
composition prevents or reverses A.beta. aggregate formation.
[0500] 88. A.beta. peptide binding polypeptide, fusion protein,
conjugate or composition for use according to any one of items
84-87 in the treatment, diagnosis or prognosis of an A.beta.
peptide associated condition, for example selected from the group
consisting of amyloidosis, which refers to a group of diseases and
disorders associated with amyloid plaque formation including
secondary amyloidosis and age-related amyloidosis including, but
not limited to, neurological disorders or conditions characterized
by a loss of cognitive memory capacity such as, for example,
Alzheimer's disease (AD), mild cognitive impairment (MCI), Lewybody
dementia, Down's syndrome, hereditary cerebral hemorrhage with
amyloidosis (Dutch type), the Guam Parkinson-dementia complex; as
well as other diseases which are based on or associated with
amyloid-like proteins such as cerebral amyloid angiopathy, primary
and secondary systemic amyloidosis, familial amyloid polyneuropathy
1, progressive supranuclear palsy, multiple sclerosis, Creutzfeld
Jacob disease, Parkinson's disease, HIV-related dementia, ALS
(amyotropic lateral sclerosis), inclusion-body myositis (IBM), type
II diabetes, and senile cardiac amyloidosis; and various eye
diseases including glaucoma, macular degeneration, drusen-related
optic neuropathy, and cataract due to beta-amyloid deposition.
[0501] 89. A.beta. peptide binding polypeptide, fusion protein,
conjugate or composition for use according to item 88, wherein the
A.beta. peptide associated condition is Alzheimer's disease.
[0502] 90. Method for detecting an A.beta. peptide in a sample,
comprising [0503] providing a sample suspected to contain A.beta.
peptide, [0504] contacting said sample with an A.beta. peptide
binding polypeptide, fusion protein or conjugate according to any
one of items 1-74 or a composition according to any one of items
80-82, and [0505] detecting binding of the A.beta. peptide binding
polypeptide, fusion protein, conjugate or composition,
[0506] wherein detection of binding indicates the presence of
A.beta. peptide in the sample.
[0507] 91. Method for determining the presence of A.beta. peptide
in a subject, the method comprising the steps: [0508] contacting
the subject, or a sample isolated from the subject, with an A.beta.
peptide binding polypeptide, fusion protein or conjugate according
to any one of items 1-74 or a composition according to any one of
items 80-82, and [0509] obtaining a value corresponding to the
amount of A.beta. peptide binding polypeptide, fusion protein,
conjugate or composition that has bound in said subject or to said
sample.
[0510] 92. Method according to item 91, further comprising a step
of comparing said value to a reference.
[0511] 93. Method according to item 91 or 92, wherein said subject
is a mammalian subject, such as a human subject.
[0512] 94. Method according to any one of items 91-93, wherein the
sample is a biological fluid sample, such as a sample selected form
the group consisting of a whole blood sample, a plasma sample and a
serum sample.
[0513] 95. Method according to any one of items 91-94, wherein the
sample is a tissue sample.
[0514] 96. Method according to any one of items 91-95, performed in
vivo.
[0515] 97. Method according to any one of items 91-95, performed in
vitro.
[0516] 98. Method of treatment of an A.beta. peptide associated
condition, comprising administering, to a subject in need thereof,
an effective amount of an A.beta. peptide binding polypeptide,
fusion protein or conjugate according to any one of items 1-74 or a
composition according to any one of items 80-82.
[0517] 99. Method according to item 98, wherein said A.beta.
peptide associated condition is selected from the group consisting
of amyloidosis, which refers to a group of diseases and disorders
associated with amyloid plaque formation including secondary
amyloidosis and age-related amyloidosis including, but not limited
to, neurological disorders or conditions characterized by a loss of
cognitive memory capacity such as, for example, Alzheimer's disease
(AD), mild cognitive impairment (MCI), Lewybody dementia, Down's
syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch
type), the Guam Parkinson-dementia complex; as well as other
diseases which are based on or associated with amyloid-like
proteins such as cerebral amyloid angiopathy, primary and secondary
systemic amyloidosis, familial amyloid polyneuropathy 1,
progressive supranuclear palsy, multiple sclerosis, Creutzfeld
Jacob disease, Parkinson's disease, HIV-related dementia, ALS
(amyotropic lateral sclerosis), inclusion-body myositis (IBM), type
II diabetes, and senile cardiac amyloidosis; and various eye
diseases including glaucoma, macular degeneration, drusen-related
optic neuropathy, and cataract due to beta-amyloid deposition.
[0518] 100. Method according to item 99, wherein said A.beta.
peptide associated condition is Alzheimer's disease.
[0519] 101. Affinity matrix comprising an A.beta. peptide binding
polypeptide, fusion protein or conjugate according to any one of
items 1-74.
[0520] 102. Method of separation of A.beta. peptide present in a
sample from other constituents in the sample, comprising a step of
affinity separation, in which step an A.beta. peptide binding
polypeptide according to any one of items 1-74 is used.
[0521] 103. Method according to item 102, comprising the steps:
[0522] applying the sample to an affinity matrix according to item
101 under conditions permissive for binding of A.beta. peptide to
the affinity matrix; [0523] washing the affinity matrix for removal
of substances not bound thereto; and [0524] eluting the bound
A.beta. peptide from the affinity matrix, thus obtaining an A.beta.
peptide fraction with an enriched A.beta. peptide content; and
[0525] recovering said A.beta. peptide fraction.
[0526] 104. Method according to item 102, comprising the steps:
[0527] applying the sample to an affinity matrix according to item
101 under conditions permissive for binding of A.beta. peptide to
the affinity matrix; [0528] washing the affinity matrix for
recovery of substances not bound thereto, thus obtaining a depleted
fraction with a substantially reduced A.beta. peptide content; and
[0529] recovering said depleted fraction.
[0530] 105. Method for reducing the content of A.beta. peptide in a
portion of a body fluid of a human, comprising the steps: [0531]
providing a portion of a body fluid from a human; [0532] applying
the portion of a body fluid to an affinity matrix according to item
101 under conditions permissive for binding of A.beta. peptide to
the affinity matrix, thereby causing a reduction of the content of
A.beta. peptide in the portion of body fluid; and [0533] returning
at least a part of said portion of body fluid to said human.
[0534] 106. Method according to item 105, wherein said body fluid
is whole blood.
[0535] 107. Method according to item 105, wherein said body fluid
is plasma.
[0536] 108. Method according item 105, wherein said body fluid is
serum.
[0537] 109. Method according to any one of items 105-108, wherein
said sample of a body fluid is obtained from a subject suffering
from an A.beta. peptide associated condition is selected from the
group consisting of amyloidosis, which refers to a group of
diseases and disorders associated with amyloid plaque formation
including secondary amyloidosis and age-related amyloidosis
including, but not limited to, neurological disorders or conditions
characterized by a loss of cognitive memory capacity such as, for
example, Alzheimer's disease (AD), mild cognitive impairment (MCI),
Lewybody dementia, Down's syndrome, hereditary cerebral hemorrhage
with amyloidosis (Dutch type), the Guam Parkinson-dementia complex;
as well as other diseases which are based on or associated with
amyloid-like proteins such as cerebral amyloid angiopathy, primary
and secondary systemic amyloidosis, familial amyloid polyneuropathy
1, progressive supranuclear palsy, multiple sclerosis, Creutzfeld
Jacob disease, Parkinson's disease, HIV-related dementia, ALS
(amyotropic lateral sclerosis), inclusion-body myositis (IBM), type
II diabetes, and senile cardiac amyloidosis; and various eye
diseases including glaucoma, macular degeneration, drusen-related
optic neuropathy, and cataract due to beta-amyloid deposition.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20210355180A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20210355180A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References