U.S. patent application number 14/023586 was filed with the patent office on 2016-08-18 for single domain antibodies capable of modulating bace activity.
This patent application is currently assigned to VIB VZW. The applicant listed for this patent is KATHOLIEKE UNIVERSITEIT LEUVEN, K.U. LEUVEN R&D, VIB VZW, VRIJE UNIVERSITEIT BRUSSEL. Invention is credited to Bart De Strooper, Els Marjaux, Serge Muyldermans, Lujia Zhou.
Application Number | 20160237166 14/023586 |
Document ID | / |
Family ID | 52625849 |
Filed Date | 2016-08-18 |
United States Patent
Application |
20160237166 |
Kind Code |
A9 |
De Strooper; Bart ; et
al. |
August 18, 2016 |
SINGLE DOMAIN ANTIBODIES CAPABLE OF MODULATING BACE ACTIVITY
Abstract
Described are single domain antibodies with a specificity for
BACE1. More specifically, described are single variable-domain
antibodies derived from camelids that bind to BACE1 and are capable
of inhibiting the activity of BACE1. The antibodies can be used for
research and medical applications. Specific applications include
the use of BACE1-specific antibodies for the treatment of
Alzheimer's disease.
Inventors: |
De Strooper; Bart; (Leuven,
BE) ; Marjaux; Els; (Attenrode-Wever, BE) ;
Zhou; Lujia; (Leuven, BE) ; Muyldermans; Serge;
(Hoeilaart, BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
VIB VZW
KATHOLIEKE UNIVERSITEIT LEUVEN, K.U. LEUVEN R&D
VRIJE UNIVERSITEIT BRUSSEL |
Gent
Leuven
Brussel |
|
BE
BE
BE |
|
|
Assignee: |
VIB VZW
Gent
BE
VRIJE UNIVERSITEIT BRUSSEL
Brussel
BE
KATHOLIEKE UNIVERSITEIT LEUVEN, K.U. LEUVEN R&D
Leuven
BE
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20150071940 A1 |
March 12, 2015 |
|
|
Family ID: |
52625849 |
Appl. No.: |
14/023586 |
Filed: |
September 11, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12736389 |
Dec 27, 2010 |
8568717 |
|
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PCT/EP2009/053985 |
Apr 3, 2009 |
|
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14023586 |
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61041965 |
Apr 3, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/40 20130101;
C07K 2317/569 20130101; C07K 2317/565 20130101; C07K 2317/22
20130101; C07K 2317/76 20130101; C07K 2317/92 20130101 |
International
Class: |
C07K 16/40 20060101
C07K016/40 |
Claims
1. An isolated single domain antibody, devoid of a light chain,
wherein the single domain antibody specifically binds beta
secretase (BACE1) and is able to inhibit cleavage of beta amyloid
precursor protein (APP) as determined in a cellular assay.
2. The isolated single domain antibody of claim 1, wherein the
single domain antibody is derived from camelids.
3. A medicament comprising: the isolated single domain antibody of
claim 1; and at least one pharmaceutically acceptable carrier.
4. A medicament comprising: the isolated single domain antibody of
claim 2; and at least one pharmaceutically acceptable carrier.
5. A method of treating a subject suffering from Alzheimer's
disease, the method comprising: administering the single domain
antibody of claim 1 to the subject, so as to treat the subject.
6. A method of treating a subject suffering from Alzheimer's
disease, the method comprising: administering the single domain
antibody of claim 2 to the subject, so as to treat the subject.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/736,389, filed Dec. 27, 2010, pending,
which application is a national stage filing under 35 U.S.C.
.sctn.371 of International Application No. PCT/EP2009/053985, filed
on Apr. 3, 2009, and published in English on Oct. 8, 2009 as WO
2009/121948 A2, which application claims the benefit of U.S. Patent
Application Ser. No. 61/041,965, filed Apr. 3, 2008.
TECHNICAL FIELD
[0002] Described are single domain antibodies with a specificity
for BACE1. More specifically, described are single variable-domain
antibodies derived from camelids that bind to BACE1 and are capable
of inhibiting the activity of BACE1. The antibodies can be used for
research and medical applications. Specific applications include
the use of BACE1-specific antibodies for the treatment of
Alzheimer's disease.
BACKGROUND
[0003] Alzheimer's disease ("AD") is a devastating
neurodegenerative disease that affects millions of elderly patients
worldwide and is the most common cause of nursing home admittance.
AD is clinically characterized by progressive loss of memory,
orientation, cognitive function, judgment and emotional stability.
With increasing age, the risk of developing AD increases
exponentially, so that by age 85, some 20% to 40% of the population
is affected. Memory and cognitive function deteriorate rapidly
within the first five years after diagnosis of mild to moderate
impairment, and death due to disease complications is an inevitable
outcome. Definitive diagnosis of AD can only be made post-mortem,
based on histopathological examination of brain tissue from the
patient. Two histological hallmarks of AD are the occurrence of
neurofibrillar tangles of hyperphosphorylated tau protein and of
proteinaceous amyloid plaques, both within the cerebral cortex of
AD patients. The amyloid plaques are composed mainly of a peptide
of 37 to 43 amino acids designated "beta-amyloid," also referred to
as "beta amyloid," "amyloid beta," "A.beta." or "Abeta." It is now
clear that the Abeta peptide is derived from a type 1 integral
membrane protein, termed "beta amyloid precursor protein" (also
referred to as "APP") through two sequential proteolytic events.
First, the APP is hydrolyzed at a site N-terminal of the
transmembrane alpha helix by a specific proteolytic enzyme referred
to as "beta-secretase" (the membrane-bound protease BACE1). The
soluble N-terminal product of this cleavage event diffuses away
from the membrane, leaving behind the membrane-associated
C-terminal cleavage product, referred to as "C99." The protein C99
is then further hydrolyzed within the transmembrane alpha helix by
a specific proteolytic enzyme referred to as "gamma-secretase."
This second cleavage event liberates the Abeta peptide and leaves a
membrane-associated "stub." The Abeta peptide thus generated is
secreted from the cell into the extracellular matrix where it
eventually forms the amyloid plaques associated with AD.
[0004] Despite intensive research during the last 100 years,
prognosis of AD patients now is still quite the same as that of
patients a century ago, since there is still no real cure
available. There are two types of drugs approved by the U.S. Food
and Drug Administration and used in clinics today to treat AD:
Acetylcholinesterase (AchE) inhibitors and Memantine. There is
ample evidence in the art that the amyloid beta peptide, the main
component of the amyloid plaques that are specific to the AD
etiology, has a key role in the development of AD disease.
Therefore, one of the most favorite strategies to lower A.beta. is
to diminish its production by .gamma.- and .beta.-secretase
inhibitors. One strategy was the development of gamma-secretase
inhibitors; however, such inhibitors often result in serious side
effects since gamma-secretase is involved in the proteolytic
processing of at least 30 proteins.
[0005] Yet another attractive strategy is the development of BACE1
inhibitors. BACE1 is produced as a prepropeptide. The 21-amino acid
signal peptide translocates the protease into the ER where the
signal peptide is cleaved off and from where BACE1 is then directed
to the cell surface. After its passage through the trans-Golgi
network (TGN), part of BACE1 is targeted to the cell surface from
where it is internalized into early endosomal compartments. BACE1
then either enters a direct recycling route to the cell surface or
is targeted to late endosomal vesicles destined for the lysosomes
or for the TGN. At the TGN, it might be retransported to the cell
membrane. Given its long half-life and fast recycling rate, mature
BACE1 may cycle multiple times between cell surface, endosomal
system and TGN during the course of its lifespan. BACE1 inhibitory
antibodies are described in US20060034848.
SUMMARY OF THE DISCLOSURE
[0006] Herein, we sought to develop alternative inhibitors of the
activity of BACE1 through the generation of single chain antibodies
with a specificity for BACE1. In the resulting collection of
binders of BACE1, we identified inhibitors of BACE1. In particular,
these BACE1-specific camelid antibodies capable of inhibiting BACE1
activity can be used for the treatment of Alzheimer's disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1: Amino acid sequence alignment of the variable domain
of the BACE1-specific dromedary HCAbs (listed, in order, Nb_B1,
Nb_B2, Nb_B3, Nb_B5, Nb_B8, Nb_B9, Nb_B10, Nb_B11, Nb_B12, Nb_B15,
Nb_B16, Nb_B21, Nb_B25, and Nb_B26, corresponding with SEQ ID
NOS:1-14, respectively; and Nb_B4, Nb_B6, Nb_B7, Nb_B13, Nb_B14 and
Nb_B24, corresponding with SEQ ID NOS:38-43, respectively).
V.sub.HH hallmark residues (F/Y.sub.37, E/Q.sub.44, R.sub.45 and
G.sub.47) are indicated in bold, whereas residues characteristic
for a VH-motif (L.sub.11, V.sub.37, G.sub.44, L.sub.45 and
W.sub.47) are labeled in italics. Cysteine residues other than the
canonical C.sub.22 and C.sub.92 are underlined. Numbering and
grouping of residues into either framework or CDR regions are as
defined by Kabat (Kabat et al., 1991).
[0008] FIG. 2: Capacity of the different BACE1 binders to recognize
their antigen. Panel A: A RaPID plot representing the kinetic rate
values k.sub.on (M.sup.-1s.sup.-1) and k.sub.off (s.sup.-1) for the
NANOBODY.RTM.-immunogen interactions as determined by surface
plasmon resonance (BIAcore). The ratio of k.sub.off to k.sub.on
gives the dissociation constant or K.sub.D. Kinetic constants were
measured at pH 7.0 (black spots) and pH 5.0 (gray spots). The
majority of the BACE1 binders has K.sub.D values between 10 nM and
100 nM at both pH conditions. Panel B: Capacity of the different
BACE1 binders to pull down BACE1 from cell lysates. Lysates of
BACE1-transfected COS cells were incubated with equal amounts (2
.mu.g) of the various recombinant his-tagged anti-BACE1
NANOBODIES.RTM.. Following pull down of the NANOBODIES.RTM.,
samples were subjected to SDS-PAGE and analyzed by Western blotting
using anti-BACE1 (ProSci). Nb_BCIILP and Nb_A.beta.3, raised
against beta-lactamase BCII 569/H (Conrath et al., 2001a) and
A.beta. peptide, respectively, were used as negative controls. Only
part of the NANOBODIES.RTM., raised against non-glycosylated BACE1
ectodomain, are able to efficiently capture glycosylated BACE1 from
COS cell lysates.
[0009] FIG. 3: Effect of Nb_B26 and Nb_B9 on APP processing in
cells. Panel A: Schematic representation of the cDNA construct used
to express V.sub.HHs into mammalian cells. The construct consists
of the V.sub.HH cDNA, fused at its C-terminus to the signal peptide
of BACE1, to ensure ER translocation, and at its C-terminus it is
fused to a myc-epitope tag. Panel B: Nb_B9, but not Nb_B26,
adversely affects .beta.-site APP processing upon transient
overexpression. COS-B1 cells, stably expressing low levels of
BACE1, were co-transfected with APP.sub.Sw and either Nb_B26 or
Nb_B9. Control cells were either transfected with empty vector or
with APP.sub.Sw alone. Two days after transfection, cells were
lysed and total protein extracts were analyzed by Western blotting
using anti-myc, anti-BACE1 (ProSci) and B63.1, to detect V.sub.HHs,
BACE1 and APP full length and CTFs (C83 and C99), respectively. One
representative experiment is shown. Panel C: Western blots as the
one shown in Panel C were probed with GARIR800, an infrared-coupled
secondary antibody, and then scanned on an ODYSSEY.RTM. scanner.
The signal intensity of the APP CTFs was quantified using the
ODYSSEY.RTM. Application Software v1.2.15 (LI-COR). The ratio of
.beta.-CTF to total CTFs (mean.+-.SEM, n=8 to 10), normalized to
the ratio of non-transfected cells (set as 1), shows that Nb_B9
could consistently decrease activity by about 30% (t-test,
p<0.001), whereas Nb_B26 had no impact on APP processing.
[0010] FIG. 4: V.sub.HH Nb_B9 inhibits .beta.-secretase cleavage of
APP by adding to the medium of cultured cells. Neuroblastoma cells
SH-SY5Y/APPwt were treated with 3 .mu.M NANOBODIES.RTM. for 24
hours, sAPP.alpha. and sAPPI.beta. from conditioned medium were
analyzed by Western blot. Cells treated with NANOBODY.RTM. B9 (SEQ
ID NO:6) showed a significant decrease in sAPPI.beta.
producing.
[0011] FIG. 5: Amino acid sequence alignment of BACE1-specific
V.sub.HHs isolated from dromedary and llama V.sub.HH libraries (SEQ
ID NOS:6, and 15-28). Numbering and grouping of residues into
either framework or CDR regions are as defined by Kabat (Kabat et
al., 1991).
[0012] FIG. 6: Western blot analysis of sAPPI.beta. and sAPP.alpha.
from conditioned medium of neuroblastoma cells SH-SY5Y/APPwt
treated with NANOBODIES.RTM. by adding to the medium at final
concentration of 20 .mu.M. Cells treated with NANOBODY.RTM. B9 (SEQ
ID NO:6), 10C4 (SEQ ID NO:22), and 4A2 (SEQ ID NO:26) showed a
significant decrease in sAPPI.beta. producing.
[0013] FIG. 7: The inhibition effects of different NANOBODIES.RTM.
(10 .mu.M) on BACE1 activity in FRET assay at a concentration of 10
.mu.M. In this cell-free enzymatic assay, Nb_B9 (SEQ ID NO:6),
Nb_10C4 (SEQ ID NO:22), Nb_4A2 (SEQ ID NO:26), and Nb_1B3 (SEQ ID
NO:15) significantly modulate BACE1 activity.
[0014] FIG. 8: Dose-response curve of NANOBODIES.RTM. 10C4 (Panel
A), 4A2 (Panel B), and B9 (Panel C) on BACE1 cleavage activity in
FRET assay using a small peptide substrate. Nb_10C4 and Nb_4A2
significantly inhibit BACE1 activity. Nb_B9 significantly increases
BACE1 activity.
[0015] FIG. 9: Dose-response curve of Nb_B9 on BACE1 cleavage
activity in MBP-ELISA using a big peptide substrate. In this
cell-free enzymatic assay, Nb_B9 significantly inhibits BACE1
activity.
[0016] FIG. 10: V.sub.HHs Nb_B9 (SEQ ID NO:6) and Nb_4A2 (SEQ ID
NO:26) inhibit BACE1 cleavage of APPwt in primary cultured mouse
neurons, as reflected in a decrease of A.beta., sAPP.beta. and
CTF.beta.. Primary cultured neurons from wild-type mice were
transduced with APPwt by Semliki Forest Virus (SFV), and then
treated with purified Nb_B9 and Nb_4A2 by adding to the medium at a
final concentration of 20 .mu.M (V.sub.HHs were first dissolved in
PBS), neurons treated with PBS were used as a negative control.
After a 16-hour treatment, conditioned medium and cell extract were
analyzed by Western blot for APP-FL, CTF.beta., CTF.alpha.,
A.beta., sAPP.beta. and sAPP.alpha./.beta..
[0017] FIG. 11: Dose response curve of V.sub.HHs Nb_B9 (SEQ ID
NO:6) inhibiting BACE1 in primary cultured mouse neurons
established by metabolic labeling assays after a 6-hour treatment.
Primary cultured neurons from wild-type mice were transduced with
APPwt by Semliki Forest Virus (SFV), and treated with purified
V.sub.HH B9 by adding to the medium serial dilutions (V.sub.HH B9
was first dissolved and diluted in PBS). Neuron cultures were
metabolic labeled for 6 hours, APP-FL and CTF.beta. from cell
extracts were analyzed by phosphorimaging, while sAPP.beta.,
A.beta. and sAPP.alpha. from conditioned medium were analyzed by
Western blot.
DETAILED DESCRIPTION
[0018] Described are BACE1 single variable-domain antibodies that
can be used in research and medical applications. More
specifically, described is the detection of BACE1 overexpression
and to the treatment of Alzheimer's disease using BACE1 single
domain antibodies. As used herein, the antibodies are devoid of any
light chain but comprise at least one heavy chain antibody. In a
particular embodiment, the variable domain of a heavy chain
antibody is derived from camelids. Such a variable domain heavy
chain antibody is herein designated as a NANOBODY.RTM. or a
V.sub.HH antibody. NANOBODY.RTM., NANOBODIES.RTM., and
NANOCLONE.RTM. are trademarks of Ablynx NV (Belgium).
[0019] Thus, in a first embodiment, provided is a single
variable-domain antibody, devoid of a light chain, specifically
binding to BACE1. In a particular embodiment, the single domain
antibody is derived from camelids. In the family of "camelids,"
immunoglobulins devoid of light polypeptide chains are found.
"Camelids" comprise old-world camelids (Camelus bactrianus and
Camelus dromaderius) and new world camelids (for example, Lama
paccos, Lama glama and Lama vicugna).
[0020] In another embodiment, provided is a single domain antibody
derived from camelids, which amino acid sequence comprises SEQ ID
NOS:1-28. The amino acid sequences of the dromedary/llama
NANOBODIES.RTM. (also designated as V.sub.HH antibodies) are
depicted in FIGS. 1 and 5. NANOBODY.RTM. B1 (Nb_B1) corresponds
with SEQ ID NO:1, NANOBODY.RTM. B2 (Nb_B2) corresponds with SEQ ID
NO:2, NANOBODY.RTM. B3 (Nb_B3) corresponds with SEQ ID NO:3,
NANOBODY.RTM. B5 (Nb_B5) corresponds with SEQ ID NO:4,
NANOBODY.RTM. B8 (Nb_B8) corresponds with SEQ ID NO:5,
NANOBODY.RTM. B9 (Nb_B9) corresponds with SEQ ID NO:6,
NANOBODY.RTM. B10 (Nb_B10) corresponds with SEQ ID NO:7,
NANOBODY.RTM. 11 (Nb_B11) corresponds with SEQ ID NO:8,
NANOBODY.RTM. 12 (Nb_B12) corresponds with SEQ ID NO:9,
NANOBODY.RTM. 15 (Nb_B15) corresponds with SEQ ID NO:10,
NANOBODY.RTM. 16 (Nb_B16) corresponds with SEQ ID NO:11,
NANOBODY.RTM. 21 (Nb_B21) corresponds with SEQ ID NO:12,
NANOBODY.RTM. 25 (Nb_B25) corresponds with SEQ ID NO:13,
NANOBODY.RTM. 26 (Nb_B26) corresponds with SEQ ID NO:14,
NANOBODY.RTM. 1B3 (Nb_1B3) corresponds with SEQ ID NO:15,
NANOBODY.RTM. 10C2 (Nb_10C2) corresponds with SEQ ID NO:16,
NANOBODY.RTM. 12B6 (Nb_12B6) corresponds with SEQ ID NO:17,
NANOBODY.RTM. 10B5 (Nb_10B5) corresponds with SEQ ID NO:18,
NANOBODY.RTM. 13A5 (Nb_13A5) corresponds with SEQ ID NO:19,
NANOBODY.RTM. 2C6 (Nb2C6) corresponds with SEQ ID NO:20,
NANOBODY.RTM. 6A4 (Nb_6A4) corresponds with SEQ ID NO:21,
NANOBODY.RTM. 10C4 (Nb_10C4) corresponds with SEQ ID NO:22,
NANOBODY.RTM. 13B6 (Nb_13B6) corresponds with SEQ ID NO:23,
NANOBODY.RTM. 1A4 (Nb_1A4) corresponds with SEQ ID NO:24,
NANOBODY.RTM. 2B6 (Nb_2B6) corresponds with SEQ ID NO:25,
NANOBODY.RTM. 4A2 (Nb_4A2) corresponds with SEQ ID NO:26,
NANOBODY.RTM. 1D4 (Nb_1D4) corresponds with SEQ ID NO:27 and
NANOBODY.RTM. 9D3 (Nb_9D3) corresponds with SEQ ID NO:28.
[0021] In yet another embodiment, the single domain antibody is
capable of inhibiting the activity of BACE1. It is understood that
"inhibition of the activity" is equivalent with the wording
"down-regulating the activity." Generally, "inhibition" means that
the activity of BACE1 is inhibited by at least 30%, at least 40%,
at least 50%, at least 60%, at least 70%, at least 80%, at least
90%, at least 95% or even 96%, 97%, 98%, 99% or even 100%.
Inhibition of BACE1 can be determined as mentioned herein further
in the examples.
[0022] In yet another embodiment, the single domain antibody is
capable of inhibiting the activity of BACE1 and it comprises at
least one of the complementarity-determining regions (CDRs) with an
amino acid sequence selected from the group comprising SEQ ID
NOS:29-37.
[0023] In yet another embodiment, the single domain antibody is
capable of preventing the uptake of pro-BACE1 and its amino acid
sequence comprises SEQ ID NOS:6, 22 or 26.
[0024] It should be noted that the term "NANOBODY.RTM." as used
herein in its broadest sense is not limited to a specific
biological source or to a specific method of preparation. For
example, the NANOBODIES.RTM. hereof can generally be obtained: (1)
by isolating the V.sub.HH domain of a naturally occurring heavy
chain antibody; (2) by expression of a nucleotide sequence encoding
a naturally occurring V.sub.HH domain; (3) by "humanization" of a
naturally occurring V.sub.HH domain or by expression of a nucleic
acid encoding a such humanized V.sub.HH domain; (4) by
"camelization" of a naturally occurring VH domain from any animal
species and, in particular, from a mammalian species, such as from
a human being, or by expression of a nucleic acid encoding such a
camelized VH domain; (5) by "camelization" of a "domain antibody"
or "Dab" as described in the art, or by expression of a nucleic
acid encoding such a camelized VH domain; (6) by using synthetic or
semi-synthetic techniques for preparing proteins, polypeptides or
other amino acid sequences known per se; (7) by preparing a nucleic
acid encoding a NANOBODY.RTM. using techniques for nucleic acid
synthesis known per se, followed by expression of the nucleic acid
thus obtained; and/or (8) by any combination of one or more of the
foregoing. One preferred class of NANOBODIES.RTM. corresponds to
the V.sub.HH domains of naturally occurring heavy chain antibodies
directed against BACE1. As further described herein, such V.sub.HH
sequences can generally be generated or obtained by suitably
immunizing a species of camelid with BACE1 (i.e., so as to raise an
immune response and/or heavy chain antibodies directed against
BACE1), by obtaining a suitable biological sample from the camelid
(such as a blood sample, serum sample or sample of B-cells), and by
generating V.sub.HH sequences directed against BACE1, starting from
the sample, using any suitable technique known per se. Such
techniques will be clear to the skilled person.
[0025] Alternatively, such naturally occurring V.sub.HH domains
against BACE1 can be obtained from naive libraries of Camelid
V.sub.HH sequences, for example, by screening such a library using
BACE1 or at least one part, fragment, antigenic determinant or
epitope thereof using one or more known screening techniques per
se. Such libraries and techniques are, for example, described in
WO9937681, WO0190190, WO03025020 and WO03035694. Alternatively,
improved synthetic or semi-synthetic libraries derived from naive
V.sub.HH libraries may be used, such as V.sub.HH libraries obtained
from naive V.sub.HH libraries by techniques such as random
mutagenesis and/or CDR shuffling, such as, for example, described
in WO0043507. Yet another technique for obtaining V.sub.HH
sequences directed against BACE1 involves suitably immunizing a
transgenic mammal that is capable of expressing heavy chain
antibodies (i.e., so as to raise an immune response and/or heavy
chain antibodies directed against BACE1), obtaining a suitable
biological sample from the transgenic mammal (such as a blood
sample, serum sample or sample of B-cells), and then generating
V.sub.HH sequences directed against BACE1 starting from the sample,
using any suitable technique known per se. For example, for this
purpose, the heavy chain antibody-expressing mice and the further
methods and techniques described in WO02085945 and in WO04049794
can be used.
[0026] A particularly preferred class of NANOBODIES.RTM. hereof
comprises NANOBODIES.RTM. with an amino acid sequence that
corresponds to the amino acid sequence of a naturally occurring
V.sub.HH domain, but that has been "humanized," i.e., by replacing
one or more amino acid residues in the amino acid sequence of the
naturally occurring V.sub.HH sequence (and, in particular, in the
framework sequences) by one or more of the amino acid residues that
occur at the corresponding position(s) in a VH domain from a
conventional four-chain antibody from a human being. This can be
performed in a manner known per se, which will be clear to the
skilled person, for example, on the basis of the further
description herein and the prior art on humanization referred to
herein. Again, it should be noted that such humanized
NANOBODIES.RTM. of the invention can be obtained in any suitable
manner known per se (i.e., as indicated under points (1)-(8) above)
and, thus, are not strictly limited to polypeptides that have been
obtained using a polypeptide that comprises a naturally occurring
V.sub.HH domain as a starting material.
[0027] Another particularly preferred class of NANOBODIES.RTM. of
the invention comprises NANOBODIES.RTM. with an amino acid sequence
that corresponds to the amino acid sequence of a naturally
occurring VH domain, but that has been "camelized," i.e., by
replacing one or more amino acid residues in the amino acid
sequence of a naturally occurring VH domain from a conventional
four-chain antibody by one or more of the amino acid residues that
occur at the corresponding position(s) in a V.sub.HH domain of a
heavy chain antibody. Such "camelizing" substitutions are
preferably inserted at amino acid positions that form and/or are
present at the VH-VL interface, and/or at the so-called Camelidae
hallmark residues, as defined herein (see, for example, WO9404678).
Preferably, the VH sequence that is used as a starting material or
starting point for generating or designing the camelized
NANOBODY.RTM. is preferably a VH sequence from a mammal, more
preferably, the VH sequence of a human being, such as a VH3
sequence. However, it should be noted that such camelized
NANOBODIES.RTM. of the invention can be obtained in any suitable
manner known per se (i.e., as indicated under points (1)-(8) above)
and, thus, are not strictly limited to polypeptides that have been
obtained using a polypeptide that comprises a naturally occurring
VH domain as a starting material. For example, both "humanization"
and "camelization" can be performed by providing a nucleotide
sequence that encodes a naturally occurring V.sub.HH domain or VH
domain, respectively, and then changing, in a manner known per se,
one or more codons in the nucleotide sequence in such a way that
the new nucleotide sequence encodes a "humanized" or "camelized"
NANOBODY.RTM. of the invention, respectively. This nucleic acid can
then be expressed in a manner known per se, so as to provide the
desired NANOBODY.RTM. of the invention.
[0028] Alternatively, based on the amino acid sequence of a
naturally occurring V.sub.HH domain or VH domain, respectively, the
amino acid sequence of the desired humanized or camelized
NANOBODY.RTM. of the invention, respectively, can be designed and
then synthesized de novo using techniques for peptide synthesis
known per se. Also, based on the amino acid sequence or nucleotide
sequence of a naturally occurring V.sub.HH domain or VH domain,
respectively, a nucleotide sequence encoding the desired humanized
or camelized NANOBODY.RTM. hereof, respectively, can be designed
and then synthesized de novo using techniques for nucleic acid
synthesis known per se, after which the nucleic acid thus obtained
can be expressed in a manner known per se, so as to provide the
desired NANOBODY.RTM. of the invention. Other suitable methods and
techniques for obtaining the NANOBODIES.RTM. hereof and/or nucleic
acids encoding the same, starting from naturally occurring VH
sequences or preferably V.sub.HH sequences, will be clear from the
skilled person, and may, for example, comprise combining one or
more parts of one or more naturally occurring VH sequences (such as
one or more FR sequences and/or CDR sequences), one or more parts
of one or more naturally occurring V.sub.HH sequences (such as one
or more FR sequences or CDR sequences), and/or one or more
synthetic or semi-synthetic sequences, in a suitable manner, so as
to provide a NANOBODY.RTM. hereof or a nucleotide sequence or
nucleic acid encoding the same.
[0029] According to one non-limiting aspect hereof, a NANOBODY.RTM.
may be as defined herein, but with the proviso that it has at least
"one amino acid difference" (as defined herein) in at least one of
the framework regions compared to the corresponding framework
region of a naturally occurring human VH domain, and, in
particular, compared to the corresponding framework region of
DP-47. More specifically, according to one non-limiting aspect of
the invention, a NANOBODY.RTM. may be as defined herein, but with
the proviso that it has at least "one amino acid difference" (as
defined herein) at at least one of the Hallmark residues (including
those at positions 108, 103 and/or 45) compared to the
corresponding framework region of a naturally occurring human VH
domain, and, in particular, compared to the corresponding framework
region of DP-47. Usually, a NANOBODY.RTM. will have at least one
such amino acid difference with a naturally occurring VH domain in
at least one of FR2 and/or FR4, and, in particular, at at least one
of the Hallmark residues in FR2 and/or FR4 (again, including those
at positions 108, 103 and/or 45). Also, a humanized NANOBODY.RTM.
hereof may be as defined herein, but with the proviso that it has
at least "one amino acid difference" (as defined herein) in at
least one of the framework regions compared to the corresponding
framework region of a naturally occurring V.sub.HH domain. More
specifically, according to one non-limiting aspect hereof, a
NANOBODY.RTM. may be as defined herein, but with the proviso that
it has at least "one amino acid difference" (as defined herein) at
at least one of the Hallmark residues (including those at positions
108, 103 and/or 45) compared to the corresponding framework region
of a naturally occurring V.sub.HH domain. Usually, a NANOBODY.RTM.
will have at least one such amino acid difference with a naturally
occurring V.sub.HH domain in at least one of FR2 and/or FR4, and,
in particular, at at least one of the Hallmark residues in FR2
and/or FR4 (again, including those at positions 108, 103 and/or
45). As will be clear from the disclosure herein, it is also within
the scope hereof to use natural or synthetic analogs, mutants,
variants, alleles, homologs and orthologs (herein collectively
referred to as "analogs") of the NANOBODIES.RTM. hereof as defined
herein, and, in particular, analogs of the NANOBODIES.RTM. of SEQ
ID NOS:6, 22 or 26. Thus, according to one embodiment, the term
"NANOBODY.RTM. hereof" in its broadest sense also covers such
analogs. Generally, in such analogs, one or more amino acid
residues may have been replaced, deleted and/or added, compared to
the NANOBODIES.RTM. hereof as defined herein. Such substitutions,
insertions or deletions may be made in one or more of the framework
regions and/or in one or more of the CDRs, and, in particular,
analogs of the CDRs of the NANOBODIES.RTM. of SEQ ID NOS:6, 22 or
26, the CDRs corresponding with SEQ ID NOS:29-37 (see Table 1,
FIGS. 1 and 5).
TABLE-US-00001 TABLE 1 CDRs of BACE1-specific NANOBODIES .RTM. Nb
CDR1 CDR2 CDR3 Nb_B9 EYTYGYCSMG TITSDGSTSYV KTCANKLGAKFIS (SEQ ID
(SEQ ID NO: DSVKG (SEQ (SEQ ID NO: NO: 6) 29) ID NO: 30) 31)
Nb_10C4 GYTYSTCSMA SIRNDGSTAYA RIGVGPGGTCSIYA (SEQ ID (SEQ ID NO:
DSVKG (SEQ PY (SEQ ID NO: NO: 22) 32) ID NO: 33) 34) Nb_4A2
GFTFETQYMT SINSGGTIKYY GQWAGVGAASS (SEQ ID (SEQ ID NO: ANSSVKG (SEQ
(SEQ ID NO: NO: 26) 35) ID NO: 36) 37)
[0030] When such substitutions, insertions or deletions are made in
one or more of the framework regions, they may be made at one or
more of the Hallmark residues and/or at one or more of the other
positions in the framework residues. Substitutions, insertions or
deletions at the Hallmark residues are generally less preferred
(unless these are suitable humanizing substitutions as described
herein). By means of non-limiting examples, a substitution may, for
example, be a conservative substitution (as described herein)
and/or an amino acid residue may be replaced by another amino acid
residue that naturally occurs at the same position in another
V.sub.HH domain. Thus, any one or more substitutions, deletions or
insertions, or any combination thereof, that either improve the
properties of the NANOBODY.RTM. hereof or that at least do not
detract too much from the desired properties or from the balance or
combination of desired properties of the NANOBODY.RTM. of the
invention (i.e., to the extent that the NANOBODY.RTM. is no longer
suited for its intended use) are included within the scope of the
disclosure.
[0031] A skilled person will generally be able to determine and
select suitable substitutions, deletions or insertions, or suitable
combinations thereof, based on the disclosure herein and optionally
after a limited degree of routine experimentation, which may, for
example, involve introducing a limited number of possible
substitutions and determining their influence on the properties of
the NANOBODIES.RTM. thus obtained. For example, and depending on
the host organism used to express the NANOBODY.RTM. or polypeptide
of the disclosure, such deletions and/or substitutions may be
designed in such a way that one or more sites for
post-translational modification (such as one or more glycosylation
sites) are removed, as will be within the ability of the person
skilled in the art.
[0032] Alternatively, substitutions or insertions may be designed
so as to introduce one or more sites for attachment of functional
groups (as described herein), for example, to allow site-specific
pegylation. One preferred class of analogs of the NANOBODIES.RTM.
hereof comprise NANOBODIES.RTM. that have been humanized (i.e.,
compared to the sequence of a naturally occurring NANOBODY.RTM. of
the invention). As mentioned in the background art cited herein,
such humanization generally involves replacing one or more amino
acid residues in the sequence of a naturally occurring V.sub.HH
with the amino acid residues that occur at the same position in a
human VH domain, such as a human VH3 domain.
[0033] Examples of possible humanizing substitutions or
combinations of humanizing substitutions will be clear to the
skilled person, from the possible humanizing substitutions
mentioned in the background art cited herein, and/or from a
comparison between the sequence of a NANOBODY.RTM. and the sequence
of a naturally occurring human VH domain. The humanizing
substitutions should be chosen such that the resulting humanized
NANOBODIES.RTM. still retain the favorable properties of
NANOBODIES.RTM. as defined herein and, more preferably, such that
they are as described for analogs in the preceding paragraphs. A
skilled person will generally be able to determine and select
suitable humanizing substitutions or suitable combinations of
humanizing substitutions, based on the disclosure herein and
optionally after a limited degree of routine experimentation, which
may, for example, involve introducing a limited number of possible
humanizing substitutions and determining their influence on the
properties of the NANOBODIES.RTM. thus obtained. Generally, as a
result of humanization, the NANOBODIES.RTM. of the disclosure may
become more "human-like," while still retaining the favorable
properties of the NANOBODIES.RTM. of the invention as described
herein. As a result, such humanized NANOBODIES.RTM. may have
several advantages, such as a reduced immunogenicity, compared to
the corresponding naturally occurring V.sub.HH domains.
[0034] Again, based on the disclosure herein and optionally after a
limited degree of routine experimentation, the skilled person will
be able to select humanizing substitutions or suitable combinations
of humanizing substitutions that optimize or achieve a desired or
suitable balance between the favorable properties provided by the
humanizing substitutions on the one hand and the favorable
properties of naturally occurring V.sub.HH domains on the other
hand. Examples of such modifications, as well as examples of amino
acid residues within the NANOBODY.RTM. sequence that can be
modified in such a manner (i.e., either on the protein backbone but
preferably on a side chain), methods and techniques that can be
used to introduce such modifications and the potential uses and
advantages of such modifications will be clear to the skilled
person. For example, such a modification may involve the
introduction (e.g., by covalent linking or in another suitable
manner) of one or more functional groups, residues or moieties into
or onto the NANOBODY.RTM. of the invention, and, in particular, of
one or more functional groups, residues or moieties that confer one
or more desired properties or functionalities to the NANOBODY.RTM.
of the invention.
[0035] Examples of such functional groups and of techniques for
introducing them will be clear to the skilled person, and can
generally comprise all functional groups and techniques mentioned
in the general background art cited hereinabove, as well as the
functional groups and techniques known per se for the modification
of pharmaceutical proteins and, in particular, for the modification
of antibodies or antibody fragments (including ScFvs and single
domain antibodies), for which reference is, for example, made to
Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co.,
Easton, Pa. (1980). Such functional groups may, for example, be
linked directly (for example, covalently) to a NANOBODY.RTM. of the
invention, or optionally, via a suitable linker or spacer, as will
again be clear to the skilled person.
[0036] One of the most widely used techniques for increasing the
half-life and/or reducing immunogenicity of pharmaceutical proteins
comprises attachment of a suitable pharmacologically acceptable
polymer, such as poly(ethyleneglycol) (PEG) or derivatives thereof
(such as methoxypoly(ethyleneglycol) or mPEG). Generally, any
suitable form of pegylation can be used, such as the pegylation
used in the art for antibodies and antibody fragments (including,
but not limited to, (single) domain antibodies and ScFvs);
reference is made to, for example, Chapman, Nat. Biotechnol.
54:531-545 (2002); by Veronese and Harris, Adv. Drug Deliv. Rev.
54:453-456 (2003); by Harris and Chess, Nat. Rev. Drug Discov. 2
(2003); and in WO04060965. Various reagents for pegylation of
proteins are also commercially available, for example, from Nektar
Therapeutics, USA. Preferably, site-directed pegylation is used, in
particular, via a cysteine-residue (see, for example, Yang et al.,
Protein Engineering 16, 10:761-770 (2003). For example, for this
purpose, PEG may be attached to a cysteine residue that naturally
occurs in a NANOBODY.RTM. hereof. A NANOBODY.RTM. hereof may be
modified so as to suitably introduce one or more cysteine residues
for attachment of PEG, or an amino acid sequence comprising one or
more cysteine residues for attachment of PEG may be fused to the N-
and/or C-terminus of a NANOBODY.RTM. hereof, all using techniques
of protein engineering known per se to the skilled person.
Preferably, for the NANOBODIES.RTM. and proteins of the disclosure,
a PEG is used with a molecular weight of more than 5000, such as
more than 10,000 and less than 200,000, such as less than 100,000;
for example, in the range of 20,000-80,000.
[0037] Another, usually less preferred modification comprises
N-linked or O-linked glycosylation, usually as part of
co-translational and/or post-translational modification, depending
on the host cell used for expressing the NANOBODY.RTM. or
polypeptide of the disclosure. Yet another modification may
comprise the introduction of one or more detectable labels or other
signal-generating groups or moieties, depending on the intended use
of the labeled NANOBODY.RTM.. Suitable labels and techniques for
attaching, using and detecting them will be clear to the skilled
person and, for example, include, but are not limited to,
fluorescent labels (such as fluorescein, isothiocyanate, rhodamine,
phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and
fluorescamine and fluorescent metals such as Eu or others metals
from the lanthanide series), phosphorescent labels,
chemiluminescent labels or bioluminescent labels (such as luminal,
isoluminol, theromatic acridinium ester, imidazole, acridinium
salts, oxalate ester, dioxetane or GFP and its analogs),
radio-isotopes, metals, metal chelates or metallic cations or other
metals or metallic cations that are particularly suited for use in
in vivo, in vitro or in situ diagnosis and imaging, as well as
chromophores and enzymes (such as malate dehydrogenase,
staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol
dehydrogenase, alpha-glycerophosphate dehydrogenase, triose
phosphate isomerase, biotinavidin peroxidase, horseradish
peroxidase, alkaline phosphatase, asparaginase, glucose oxidase,
beta-galactosidase, ribonuclease, urease, catalase,
glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholine
esterase). Other suitable labels will be clear to the skilled
person and, for example, include moieties that can be detected
using NMR or ESR spectroscopy. Such labeled NANOBODIES.RTM. and
polypeptides of the disclosure may, for example, be used for in
vitro, in vivo or in situ assays (including immunoassays known per
se, such as ELISA, RIA, EIA and other "sandwich assays," etc.) as
well as in vivo diagnostic and imaging purposes, depending on the
choice of the specific label.
[0038] As will be clear to the skilled person, another modification
may involve the introduction of a chelating group, for example, to
chelate one of the metals or metallic cations referred to above.
Suitable chelating groups include, for example, without limitation,
diethyl-enetriaminepentaacetic acid (DTPA) or
ethylenediaminetetraacetic acid (EDTA).
[0039] Yet another modification may comprise the introduction of a
functional group that is one part of a specific binding pair, such
as the biotin-(strept)avidin binding pair. Such a functional group
may be used to link the NANOBODY.RTM. of the invention to another
protein, polypeptide or chemical compound that is bound to the
other half of the binding pair, i.e., through formation of the
binding pair. For example, a NANOBODY.RTM. hereof may be conjugated
to biotin, and linked to another protein, polypeptide, compound or
carrier conjugated to avidin or streptavidin. For example, such a
conjugated NANOBODY.RTM. may be used as a reporter, for example, in
a diagnostic system where a detectable signal-producing agent is
conjugated to avidin or streptavidin. Such binding pairs may, for
example, also be used to bind the NANOBODY.RTM. of the invention to
a carrier, including carriers suitable for pharmaceutical purposes.
One non-limiting example is the liposomal formulations described by
Cao and Suresh, Journal of Drug Targeting 8, 4:257 (2000). Such
binding pairs may also be used to link a therapeutically active
agent to the NANOBODY.RTM. hereof.
[0040] It is expected that the NANOBODIES.RTM. and polypeptides of
the disclosure will generally bind to all naturally occurring or
synthetic analogs, variants, mutants, alleles, parts and fragments
of BACE1, or at least to those analogs, variants, mutants, alleles,
parts and fragments of BACE1, that contain one or more antigenic
determinants or epitopes that are essentially the same as the
antigenic determinant(s) or epitope(s) to which the NANOBODIES.RTM.
and polypeptides of the disclosure bind in BACE1 (e.g., in
wild-type BACE1). Again, in such a case, the NANOBODIES.RTM. and
polypeptides of the disclosure may bind to such analogs, variants,
mutants, alleles, parts and fragments with an affinity and/or
specificity that are the same as, or different from (i.e., higher
than or lower than), the affinity and specificity with which the
NANOBODIES.RTM. hereof bind to (wild-type) BACE1.
[0041] It is also included within the scope hereof that the
NANOBODIES.RTM. and polypeptides of the disclosure bind to some
analogs, variants, mutants, alleles, parts and fragments of BACE1,
but not to others. Also, in determining the degree of sequence
identity between two amino acid sequences, the skilled person may
take into account so-called "conservative" amino acid
substitutions, which can generally be described as amino acid
substitutions in which an amino acid residue is replaced with
another amino acid residue of similar chemical structure and which
has little or essentially no influence on the function, activity or
other biological properties of the polypeptide. Such conservative
amino acid substitutions are well known in the art, for example,
from WO04037999, WO9849185, WO0046383 and WO0109300; and
(preferred) types and/or combinations of such substitutions may be
selected on the basis of the pertinent teachings from WO04037999,
as well as WO9849185. Such conservative substitutions preferably
are substitutions in which one amino acid within the following
groups (a)-(e) is substituted by another amino acid residue within
the same group: (a) small aliphatic, nonpolar or slightly polar
residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged
residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c)
polar, positively charged residues: His, Arg and Lys; (d) large
aliphatic, nonpolar residues: Met, Leu, His, Val and Cys; and (e)
aromatic residues: Phe, Tyr and Trp. Particularly preferred
conservative substitutions are as follows: Ala into Gly or into
Ser; Arg into Lys; Asn into Gln or into His; Asp into Glu; Cys into
Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into
Asn or into GIn; His into Leu or into Val; Leu into His or into
Val; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or
into His; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr
into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Val, into His
or into Leu.
[0042] Polypeptide therapeutics and, in particular, antibody-based
therapeutics, have significant potential as drugs because they have
exquisite specificity to their target and a low inherent toxicity.
However, it is known by the skilled person that an antibody that
has been obtained for a therapeutically useful target requires
additional modification in order to prepare it for human therapy,
so as to avoid an unwanted immunological reaction in a human
individual upon administration. The modification process is
commonly termed "humanization." It is known by the skilled artisan
that antibodies raised in species, other than in humans, require
humanization to render the antibody therapeutically useful in
humans ((1) CDR grafting: Protein Design Labs: U.S. Pat. No.
6,180,370, U.S. Pat. No. 5,693,761; Genentech U.S. Pat. No.
6,054,297; Celltech: EP626390, U.S. Pat. No. 5,859,205; (2)
Veneering: Xoma: U.S. Pat. No. 5,869,619, U.S. Pat. No. 5,766,886,
U.S. Pat. No. 5,821,123). There is a need for a method for
producing antibodies that avoids the requirement for substantial
humanization or that completely obviates the need for
humanization.
[0043] There is a need for a new class of antibodies that have
defined framework regions or amino acid residues and that can be
administered to a human subject without the requirement for
substantial humanization, or the need for humanization at all.
According to one aspect of the invention, NANOBODIES.RTM. are
polypeptides that are derived from heavy chain antibodies and whose
framework regions and complementary determining regions are part of
a single domain polypeptide. Examples of such heavy chain
antibodies include, but are not limited to, naturally occurring
immunoglobulins devoid of light chains. Such immunoglobulins are
disclosed in WO9404678, for example. The antigen-binding site of
this unusual class of heavy chain antibodies has a unique structure
that comprises a single variable domain. For clarity reasons, the
variable domain derived from a heavy chain antibody naturally
devoid of light chain is known herein as a V.sub.HH or V.sub.HH
domain or NANOBODY.RTM.. Such a V.sub.HH domain peptide can be
derived from antibodies raised in Camelidae species, for example,
in camel, dromedary, llama, alpaca and guanaco. Other species
besides Camelidae (e.g., shark, pufferfish) may produce functional
antigen-binding heavy chain antibodies naturally devoid of light
chain. V.sub.HH domains derived from such heavy chain antibodies
are within the scope of the invention.
[0044] Camelidae antibodies express a unique, extensive repertoire
of functional heavy chain antibodies that lack light chains. The
V.sub.HH molecules derived from Camelidae antibodies are the
smallest intact antigen-binding domains known (approximately 15
kDa, or ten times smaller than conventional IgG) and, hence, are
well suited toward delivery to dense tissues and for accessing the
limited space between macromolecules. Other examples of
NANOBODIES.RTM. include NANOBODIES.RTM. derived from VH domains of
conventional four-chain antibodies that have been modified by
substituting one or more amino acid residues with
Camelidae-specific residues (the so-called camelization of heavy
chain antibodies, WO9404678). Such positions may preferentially
occur at the VH-VL interface and at the so-called Camelidae
hallmark residues (WO9404678), comprising positions 37, 44, 45, 47,
103 and 108. NANOBODIES.RTM. correspond to small, robust and
efficient recognition units formed by a single immunoglobulin (Ig)
domain.
[0045] A "fragment of a NANOBODY.RTM." as used herein refers to
less than 100% of the sequence (e.g., 99%, 90%, 80%, 70%, 60%, 50%,
40%, 30%, 20%, 10% etc.), but comprising 5, 6, 7, 8, 9, 10, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids.
A fragment is preferably of sufficient length such that the
interaction of interest is maintained with affinity of
1.times.10.sup.6 M or better. A "fragment" as used herein also
refers to optional insertions, deletions and substitutions of one
or more amino acids that do not substantially alter the ability of
the target to bind to a NANOBODY.RTM. raised against the wild-type
target. The number of amino acid insertions, deletions or
substitutions is preferably up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,
62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids. One embodiment of
the present invention relates to a polypeptide comprising at least
one NANOBODY.RTM. wherein one or more amino acid residues have been
substituted without substantially altering the antigen binding
capacity.
[0046] In a particular embodiment, the antibody of the invention is
bivalent and formed by bonding together, chemically or by
recombinant DNA techniques, two monovalent single domains of heavy
chains. In another particular embodiment, the antibody of the
invention is bi-specific and formed by bonding together two
variable domains of heavy chains, each with a different specificity
(i.e., one with a specificity for BACE1 and the other one with a
specificity for a neuron, such as, for example, ICAM5 or
telencephalin). Similarly, polypeptides comprising multivalent or
multi-specific single domain antibodies are included here as
non-limiting examples.
[0047] In yet another embodiment, a single domain antibody that is
capable of preventing the uptake of BACE1 can be used as a
medicament. In yet another embodiment, a single domain antibody
that comprises at least one of the complementarity-determining
regions (CDRs) with an amino acid sequence selected from the group
comprising SEQ ID NOS:29-37 can be used as a medicament. In yet
another embodiment, a single domain antibody, which amino acid
comprises SEQ ID NOS:6, 22 or 26, can be used as a medicament.
[0048] In yet another embodiment, a single domain antibody that is
capable of preventing the uptake of pro-BACE1 can be used for the
manufacture of a medicament to treat diseases associated with an
overexpression of BACE1. An example of a disease where an
overexpression of BACE1 occurs is Alzheimer's disease. In general,
"therapeutically effective amount," "therapeutically effective
dose" and "effective amount" means the amount needed to achieve the
desired result or results (inhibiting BACE1 binding; treating or
preventing Alzheimer's disease). One of ordinary skill in the art
will recognize that the potency and, therefore, an "effective
amount" can vary for the NANOBODY.RTM. that inhibits BACE1 binding
used in the invention. One skilled in the art can readily assess
the potency of the NANOBODY.RTM.. By "pharmaceutically acceptable"
is meant a material that is not biologically or otherwise
undesirable, i.e., the material may be administered to an
individual along with the compound without causing any undesirable
biological effects or interacting in a deleterious manner with any
of the other components of the pharmaceutical composition in which
it is contained.
[0049] The term "medicament to treat" relates to a composition
comprising antibodies as described above and a pharmaceutically
acceptable carrier or excipient (both terms can be used
interchangeably) to treat or to prevent diseases as described
herein. The administration of a NANOBODY.RTM. as described above or
a pharmaceutically acceptable salt thereof may be by way of oral,
inhaled or parenteral administration. In particular embodiments,
the NANOBODY.RTM. is delivered through intrathecal or
intracerebroventricular administration. The active compound may be
administered alone or preferably formulated as a pharmaceutical
composition.
[0050] An amount effective to treat Alzheimer's disease that
expresses the antigen recognized by the NANOBODY.RTM. depends on
the usual factors, such as the nature and severity of the disorder
being treated and the weight of the mammal. However, a unit dose
will normally be in the range of 0.01 to 50 mg, for example, 0.01
to 10 mg, or 0.05 to 2 mg of NANOBODY.RTM. or a pharmaceutically
acceptable salt thereof. Unit doses will normally be administered
once or more than once a day, for example, two, three, or four
times a day, more usually one to three times a day, such that the
total daily dose is normally in the range of 0.0001 to 1 mg/kg;
thus a suitable total daily dose for a 70 kg adult is 0.01 to 50
mg, for example, 0.01 to 10 mg or more, usually 0.05 to 10 mg.
[0051] In certain embodiments, the compound or a pharmaceutically
acceptable salt thereof is administered in the form of a unit-dose
composition, such as a unit dose oral, parenteral, or inhaled
composition. Such compositions are prepared by admixture and are
suitably adapted for oral, inhaled or parenteral administration
and, as such, may be in the form of tablets, capsules, oral liquid
preparations, powders, granules, lozenges, reconstitutable powders,
injectable and infusable solutions or suspensions or suppositories
or aerosols. Tablets and capsules for oral administration are
usually presented in a unit dose, and contain conventional
excipients such as binding agents, fillers, diluents, tableting
agents, lubricants, disintegrants, colorants, flavorings, and
wetting agents. The tablets may be coated according to well-known
methods in the art. Suitable fillers for use include cellulose,
mannitol, lactose and other similar agents. Suitable disintegrants
include starch, polyvinylpyrrolidone and starch derivatives such as
sodium starch glycolate. Suitable lubricants include, for example,
magnesium stearate. Suitable pharmaceutically acceptable wetting
agents include sodium lauryl sulphate. These solid oral
compositions may be prepared by conventional methods of blending,
filling, tableting, or the like. Repeated blending operations may
be used to distribute the active agent throughout those
compositions employing large quantities of fillers. Such operations
are, of course, conventional in the art.
[0052] Oral liquid preparations may be in the form of, for example,
aqueous or oily suspensions, solutions, emulsions, syrups, or
elixirs, or may be presented as a dry product for reconstitution
with water or other suitable vehicle before use. Such liquid
preparations may contain conventional additives such as suspending
agents, for example, sorbitol, syrup, methyl cellulose, gelatin,
hydroxyethylcellulose, carboxymethyl cellulose, aluminium stearate
gel or hydrogenated edible fats, emulsifying agents, for example,
lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles
(which may include edible oils), for example, almond oil,
fractionated coconut oil, oily esters such as esters of glycerine,
propylene glycol, or ethyl alcohol; preservatives, for example,
methyl or propyl p-hydroxybenzoate or sorbic acid, and, if desired,
conventional flavoring or coloring agents. Oral formulations also
include conventional sustained release formulations, such as
tablets or granules having an enteric coating.
[0053] Preferably, compositions for inhalation are presented for
administration to the respiratory tract as a snuff or an aerosol or
solution for a nebulizer, or as a microfine powder for
insufflation, alone or in combination with an inert carrier such as
lactose. In such a case, the particles of active compound suitably
have diameters of less than 50 microns, preferably less than 10
microns, for example, between 1 and 5 microns, such as between 2
and 5 microns. A favored inhaled dose will be in the range of 0.05
to 2 mg, for example, 0.05 to 0.5 mg, 0.1 to 1 mg or 0.5 to 2
mg.
[0054] For parenteral administration, fluid unit dose forms are
prepared containing a compound of the present invention and a
sterile vehicle. The active compound, depending on the vehicle and
the concentration, can be either suspended or dissolved. Parenteral
solutions are normally prepared by dissolving the compound in a
vehicle and filter sterilizing before filling into a suitable vial
or ampoule and sealing. Advantageously, adjuvants such as a local
anesthetic, preservatives and buffering agents are also dissolved
in the vehicle. To enhance the stability, the composition can be
frozen after filling into the vial and the water removed under
vacuum. Parenteral suspensions are prepared in substantially the
same manner except that the compound is suspended in the vehicle
instead of being dissolved and sterilized by exposure to ethylene
oxide before suspending in the sterile vehicle.
[0055] Advantageously, a surfactant or wetting agent is included in
the composition to facilitate uniform distribution of the active
compound. Where appropriate, small amounts of bronchodilators, for
example, sympathomimetic amines such as isoprenaline, isoetharine,
salbutamol, phenylephrine and ephedrine; xanthine derivatives such
as theophylline and aminophylline; corticosteroids such as
prednisolone; and adrenal stimulants such as ACTH, may be included.
As is common practice, the compositions will usually be accompanied
by written or printed directions for use in the medical treatment
concerned.
[0056] In yet another embodiment, one or more single domain
antibodies of the invention can be linked (optionally, via one or
more suitable linker sequences) to one or more (such as two and
preferably one) amino acid sequences that allow the resulting
polypeptide of the invention to cross the blood brain barrier. In
particular, the one or more amino acid sequences that allow the
resulting polypeptides of the invention to cross the blood brain
barrier may be one or more (such as two and preferably one)
NANOBODIES.RTM., such as the NANOBODIES.RTM. described in WO
02/057445, of which FC44 (SEQ ID NO:189 of WO 06/040153) and FC5
(SEQ ID NO:190 of WO 06/040154) are preferred examples.
[0057] The present invention further provides a pharmaceutical
composition for use in the treatment and/or prophylaxis of
herein-described disorders, which comprises a pharmaceutically
acceptable salt thereof, or a pharmaceutically acceptable solvate
thereof, and, if required, a pharmaceutically acceptable carrier
thereof.
[0058] It should be clear that the therapeutic method hereof for
addressing Alzheimer's disease can also be used in combination with
any other AD disease therapy known in the art, such as
gamma-secretase inhibitors or other beta-secretase inhibitors.
[0059] In a particular embodiment, the single domain antibodies
hereof can be used for the preparation of a diagnostic assay. BACE1
can be detected in a variety of cells and tissues, especially in
brain cells and tissues, wherein the degree of expression
corroborates with the severity of Alzheimer's disease. Therefore,
there is provided a method of in situ detecting localization and
distribution of BACE1 expression in a biological sample. The method
comprises the step of reacting the biological sample with a
detectable BACE1 NANOBODY.RTM. and detecting the localization and
distribution of the detectable NANOBODY.RTM.. The term "biological
sample" refers to cells and tissues, including, but not limited to,
brain cells and tissues. The term further relates to body
fluids.
[0060] Therefore, there is provided a method of detecting BACE1
protein in a body fluid of a patient. The method comprises the
steps of reacting the body fluid with an anti-BACE1 NANOBODY.RTM.
hereof and monitoring the reaction. The body fluid is, for example,
plasma, urine, cerebrospinal fluid, pleural effusions or saliva.
Monitoring the reaction may be effected by having the NANOBODY.RTM.
labeled with a detectable moiety, or to use its constant region as
an inherent detectable moiety, to which a second antibody, which
includes a detectable moiety, can specifically bind. CSF BACE1 can,
for example, be detected in patients suffering from Alzheimer's
disease. According to a preferred embodiment of the present
invention, reacting the body fluid with the anti-BACE1
NANOBODY.RTM. is effected in solution.
[0061] Alternatively, reacting the body fluid with the anti-BACE1
NANOBODY.RTM. is effected on a substrate capable of adsorbing
proteins present in the body fluid, all as well known in the art of
antibody-based diagnosis. Further, according to the disclosure,
there is provided a method of detecting the presence, absence or
level of BACE1 protein in a biological sample. The method comprises
the following steps. First, proteins are extracted from the
biological sample, thereby a plurality of proteins are obtained.
The protein extract may be a crude extract and can also include
non-proteinaceous material. Second, the proteins are size
separated, e.g., by electrophoresis, gel filtration, etc. Fourth,
the size-separated proteins are interacted with an anti-BACE1
NANOBODY.RTM.. Finally, the presence, absence or level of the
interacted anti-BACE1 NANOBODY.RTM. is detected. In case of gel
electrophoresis, the interaction with the NANOBODY.RTM. is
typically performed following blotting of the size-separated
proteins onto a solid support (membrane).
[0062] The following examples more fully illustrate the disclosure.
Starting materials and reagents disclosed below are known to those
skilled in the art, and are available commercially or can be
prepared using well-known techniques.
EXAMPLES
1. Generation and Isolation of BACE1-Specific NANOBODIES.RTM.
[0063] To generate BACE1-specific antibodies, a dromedary was
immunized six times with recombinant human BACE1 over a period of
about six weeks. After this period, a BACE1-specific humoral
response, as assessed by ELISA, was observed for each of the three
different IgG subclasses that exist in Camelidae, namely the
conventional IgG1 molecules and the heavy chain-only subclasses
IgG2 and IgG3 (IgG classes reviewed in Conrath et al., 2003). The
variable chain of the HCAbs (V.sub.HH), which contains the
antigen-binding fragment, was amplified from isolated dromedary
lymphocytes and cloned into a pHEN4 phagemid vector to generate a
library of 4.times.10.sup.7 individual transformants. After
rescuing this bank with M13K07 helper phages, the V.sub.HH
repertoire was expressed on the surface of bacteriophages. With
these phages, BACE1-specific V.sub.HHs could be isolated from the
whole V.sub.HH pool using panning, an in vitro selection technique
(reviewed in Smith and Petrenko, 1997). For this, the phages were
incubated onto a solid phase passively coated with the immunogen.
After washing, bound phages were eluted and used to infect
exponentially growing E. coli TG1 cells to produce new virions.
These virions were used in a next selection round in order to
enrich for BACE1-specific binders. After two to three consecutive
rounds of panning, individual colonies were randomly picked and
incubated with IPTG to induce expression of the NANOBODIES.RTM..
The V.sub.HH protein fragments were extracted from the bacterial
periplasm and tested individually by ELISA for their ability to
interact with BACE1. The positive scoring clones were sequenced
and, as such, twenty different specific NANOBODIES.RTM. were
identified.
2. Sequence Analysis of the BACE1-Binders
[0064] Fourteen out of the twenty selected BACE1-binders are
clearly derived from V.sub.HH germ-line genes: Nb_B1, Nb_B2, Nb_B3,
Nb_B5, Nb_B8, Nb_B9, Nb_B10, Nb_B11, Nb_B12, Nb_B15, Nb_B16,
Nb_B21, Nb_B25, and Nb_B26, corresponding with SEQ ID NOS:1-14,
respectively (FIG. 1). The amino acid sequence of their framework-2
(FR2) region resembles that of a typical V.sub.HH FR2 (Muyldermans
et al., 1994), with residues F/Y, E/Q, R/C and G at positions 37,
44, 45 and 47, respectively (numbering according to Kabat et al.,
1991). However, the remaining six antibody fragments, Nb_B4, Nb_B6,
Nb_B7, Nb_B13, Nb_B14 and Nb_B24 (corresponding with SEQ ID
NOS:38-43, respectively), seem to originate from conventional
antibody germ-line genes, since they contain the
V.sub.37G.sub.44L.sub.45W.sub.47 tetrad, a typical hallmark that
distinguishes the variable domain of the heavy chain of
conventional antibodies (VH) from V.sub.HH fragments at the
germline level. These hallmark residues are critically required in
H.sub.2-L.sub.2 antibodies for the association of the heavy chain
with a light chain.
[0065] Due to the high sequence similarity of the six V.sub.H-like
NANOBODIES.RTM., it is most likely they are all derived from one
and the same B cell lineage. The differences in amino acid sequence
could be the result of the ongoing somatic hypermutation of the
antibody gene fragments in maturing B cells and the subsequent
antigen-driven selection, a continuous process leading to ever
better fitting antibodies. The six V.sub.H-like NANOBODIES.RTM.
also differ from the other binders in that they contain a leucine
residue at position 11 in their framework-1 (FR1), another
characteristic of V.sub.H genes that is important for the
interaction with a light chain (Lesk and Chothia, 1988; Padlan,
1994).
[0066] In a typical V.sub.HH FR1, this Leu residue is often
replaced by a smaller and hydrophilic residue, usually a serine, as
seen in the 14 BACE1 binders with a true V.sub.HH motif. The
V.sub.H-like molecules all have a short CDR3 of only six amino
acids, whereas, the other binders have significantly larger H3
loops, ranging from 13 to 21 residues, with an average length of
17. This is consistent with the average V.sub.HH-CDR3 length of 15
to 16 residues reported before in literature (reviewed in
Muyldermans and Lauwereys, 1999).
[0067] In general, the CDR2 and CDR1 of V.sub.HHs consist of 16 to
17 and 10 residues, respectively, but about 30% of dromedary
V.sub.HH cDNAs were reported to be off-sized. This does not
adversely affect their function, but instead, even increases the
antigen-binding repertoire (Nguyen et al., 2000). Unusual CDR1 and
CDR2 lengths are also observed for our BACE1 binders. The CDR2 of
Nb_B15 contains 19 residues due to a tandem repetition of two amino
acids, whereas, that of Nb_B21 consists of 18 residues. Aberrant
CDR1 sizes are found in Nb_B1, Nb_B15 and Nb_B16 due to an
insertion, a deletion of one amino acid and a deletion of two
residues, respectively. Finally, Nb_B25 has an unusually long
framework-3 region with a tandem repetition of two amino acid
residues. Deviating lengths in the BACE1-binders are due to changes
located at three typical V.sub.HH insertion/deletion hot spots,
surrounding residues 30.+-.3, 54.+-.3 and 74.+-.1. These hot spots
can be found within or at the border of peculiar DNA sequences,
such as palindromic sequences (corresponding to residues 30-33 and
54-57) or heptamer-like sequences of an Ig recombination signal
(often found at residues 76-78) (Nguyen et al., 2000).
[0068] Besides the conserved disulphide bridge between Cys.sub.22
and Cys.sub.92, extra non-canonical cysteine residues do not
frequently occur in conventional antibodies, although they are not
totally excluded. However, an additional pair of cysteines is
encountered in 75% of reported dromedary V.sub.HHs (Arbabi
Ghahroudi et al., 1997; Lauwereys et al., 1998; Conrath et al.,
2001a; Saerens et al., 2004). One of these extra cysteine residues
is typically located within the CDR3 loop, whereas, the other one
can be found either on position 30, 32 or 33 within the CDR1 or at
position 45 in FR2. Since the V.sub.HH CDR3 loop folds back onto
the CDR1-FR2 region, the two cysteine residues come into contact
distance and are likely engaged into an interloop disulphide bond
that cross-links the antigen-binding loops (Desmyter et al., 1996).
Such a bond reduces flexibility of the long CDR3 loop and thus
provides increased stability. Besides, the interloop bond might
lead to a constrained, but new conformation of the CDR loops,
thereby increasing the antigen-binding repertoire.
[0069] Compared to the percentages known from literature, there is
a low incidence of additional cysteines in the BACE1 binders. A
putative additional disulphide bond is only present in four out of
the 14 NANOBODIES.RTM. with V.sub.HH motif. Nb_B25 has a cysteine
residue at position 33 within the CDR1; Nb_B9 has one at position
32; and in Nb_B5, an additional bridge will probably be formed
between Cys.sub.45 and the CDR3. A cysteine at position 53, as seen
in Nb_B12, has been described so far for neither dromedary, nor
llama V.sub.HHs.
3. Defining Affinities of the BACE1-Binders for their Immunogen at
pH 7.0 and pH 5.0
[0070] The cDNAs of the 20 isolated BACE1-binders were subcloned
into the pHEN6 prokaryotic expression vector and expressed in E.
coli WK6 cells to produce his-tagged soluble proteins. The
recombinant V.sub.HHs were subsequently purified by Ni-NTA affinity
chromatography, followed by size-exclusion chromatography. The
expression levels of the distinct clones varied between 1 and 15 mg
per liter of culture medium. The affinity of all V.sub.HHs for
BACE1 was determined quantitatively using the surface plasmon
resonance technology on Biacore 3000. Each of the different
V.sub.HHs was injected at concentrations ranging from 0 to 0.5
.mu.M on a chip onto which BACE1 was coupled. Binding was evaluated
both at pH 7.0 and pH 5.0. Measurements at pH 5.0 were included
because a firm interaction between BACE1 and a V.sub.HH should be
preserved at this pH, since the endosomal compartment with its
slightly acidic content was reported to be the major subcellular
site of .beta.-site cleavage of APP (Koo, 1994). Besides, BACE1 was
shown to have optimal .beta.-secretase activity at about pH 5.0 in
vitro (Sinha et al., 1999; Vassar et al., 1999; Yan et al., 1999;
Lin et al., 2000). The dissociation constants obtained for all
V.sub.HHs vary between 4 and 669 nM at pH 7.0 and between 4.2 nM
and 6.8 .mu.M at pH 5.0 (FIG. 2, Panel A). The majority of the
binders have dissociation constants between 10 and 100 nM at both
pH conditions.
4. Capacity of the Different V.sub.HHs to Pull Down Native
BACE1
[0071] For the immunization of the dromedary, the isolation of the
BACE1-specific binders during panning and the in vitro affinity
measurements by Biacore, we used recombinant human soluble BACE1,
completely devoid of carbohydrate chains. This recombinant protein,
supplied by Dr. S. Masure (Johnson & Johnson, Beerse, Belgium)
was obtained from an insect cell expression system using slightly
truncated BACE1 cDNA in which the four N-glycosylation sites and
the whole membrane anchor were removed (Bruinzeel et al., 2002). It
is not unthinkable that epitopes that are easily accessible in the
"naked" BACE1, used for the immunization, are shielded by glycan
chains or other post-translational modifications of BACE1 proteins
generated in mammalian cells. Therefore, we wondered whether the
selected binders would all be able to recognize glycosylated BACE1
expressed in mammalian cells.
[0072] To test this, 2 .mu.g of his-tagged V.sub.HH molecules were
incubated with 4 .mu.g of total protein extract from COS cells
transiently transfected with human BACE1 cDNA. Nickel-beads were
subsequently used to pull down the V.sub.HH molecules together with
the bound proteins. After extensive washing, bound proteins were
eluted, separated by SDS-PAGE and BACE1 protein was detected by
Western blotting using a rabbit polyclonal BACE1-specific antibody
(ProSci, 2253) (FIG. 2, Panel B). V.sub.HH proteins raised against
either A.beta. (Nb_A.beta.3) or beta-lactamase BCII 569/H
(Nb_BCIILP) (Conrath et al., 2001a), were used as negative controls
and were unable to capture BACE1 from the cell lysate, as expected.
Five binders, Nb_B7, Nb_B9, Nb_B10, Nb_B13 and Nb_B24 have the
highest efficacy to pull down BACE1 compared to the other
NANOBODIES.RTM.. For Nb_B3, Nb_B8, Nb_B12 and Nb_B21 at the best a
trifling trace of coprecipitated BACE1 can be detected after
overnight exposure.
[0073] Note that in the group of the 6 V.sub.H-like NANOBODIES.RTM.
(Nb_B4, Nb_B6, Nb_B7, Nb_B13, Nb_B14 and Nb_B24), huge differences
are observed in the ability of each NANOBODY.RTM. to bind to
glycosylated BACE1, even though they probably originated from the
same B-cell lineage. Despite a high overall sequence similarity of
about 90%, Nb_B13 and Nb_B14 share only 70% of amino acids in their
antigen-binding CDR regions and this difference apparently is
sufficient to adversely affect the affinity of Nb_B14 for its
antigen when compared to Nb_B13.
5. Effect of Ectopic Expression of the NANOBODIES.RTM. on BACE
Inhibition
[0074] In a next step, we decided to express some of the V.sub.HHs
into mammalian cells. Thereto, COST-B1 cells, stably expressing low
levels of BACE1, were co-transfected with APPSw and either Nb_B26
or Nb_B9. Control cells were either transfected with empty vector
or with APPSw alone. The cDNAs (of Nb_B26 or Nb_B9) were cloned
into a eukaryotic expression vector, downstream of the signal
peptide of BACE1 and with a myc-epitope tag at its C-terminus (FIG.
3, Panel A). The signal sequence ensures translocation of the newly
formed protein into the secretory pathway, where the V.sub.HH
should encounter its antigen, the ectodomain of BACE1. APP.sub.Sw
and the two V.sub.HHs were co-transfected into COS cells stably
expressing low levels of human BACE1 (COS-B1 cells). These cells
have detectable, but not saturated, levels of .beta.-secretase
activity and are easily transfected using liposome-based
transfection reagents. Two days after transfection, cell extracts
were prepared, proteins were separated by SDS-PAGE and transferred
to a nitrocellulose membrane. Using rabbit polyclonal B63.1 as a
primary antibody and GARIR, an infrared-coupled secondary antibody,
the APP C-terminal fragments were visualized and quantified by the
ODYSSEY.RTM. Infrared Imaging System (FIG. 3, Panels B and C).
Again, Nb_B26 had no effect on APP processing. The ratio of
.beta.-CTF on total APP CTFs is equal to that of non-treated cells
(FIG. 3, Panel C). Nb_B9 consistently decreased .beta.-secretase
activity by about 30% (p<0.001), even though it was expressed at
much lower levels than Nb_B26 (FIG. 3, Panel B). This decrease
occurred in the absence of any effect on BACE1 protein levels,
ruling out the possibility that the NANOBODY.RTM. affects BACE1
protein stability.
6. Effect of Addition of Extracellular NANOBODIES.RTM. on APP
Processing in Cells
[0075] In a next step, NANOBODY.RTM. Nb_B9 was tested as to whether
it could also affect APP processing when added to culture medium of
cells. At least part of BACE1 is directed to the plasma membrane
before being targeted to endosomes, so BACE1-specific antibodies
could potentially bind to the ectodomain at the cell surface and be
smuggled inside cells by co-internalization with their antigen.
Since .beta.-secretase cleavage of wild-type APP predominantly
occurs within the endosomal compartments, neutralizing the enzyme's
activity from the plasma membrane might be sufficient to decrease
.beta.-site APP proteolysis. SH-SY5Y cells, neuroblastoma cells
with relatively high endogenous BACE1 activity, were infected with
recombinant adenoviruses containing the cDNA encoding either human
APP wild-type or the FAD APP.sub.Sw mutant. The FAD mutant was
included since it is a much better BACE1 substrate, which enables
easier detection of A.beta. and .beta.-CTF. However, the majority
of APP.sub.Sw has been shown to be cleaved at the .beta.-site in
the secretory pathway before reaching the plasma membrane (Martin
et al., 1995; Thinakaran et al., 1996), so BACE1-neutralizing
V.sub.HHs binding at the cell surface might not be capable of
preventing .beta.-site APP.sub.Sw cleavage.
[0076] Two days after adenoviral infection, the SH-SY5Y cells were
radioactively labeled and incubated with 2 .mu.M Nb_B9 for six
hours. The conditioned medium was used to immunoprecipitate
secreted A.beta., whereas, APP full-length and C-terminal fragments
were pulled down from cell lysates. APP fragments were separated by
SDS-PAGE. Gels were fixed, dried and analyzed by phosphorimaging.
The presence of Nb_B9 caused clear detectable change in amounts of
.beta.-CTF or A.beta. compared to non-treated cells (FIG. 4).
7. Isolation of Other BACE1-Specific NANOBODIES.RTM.
[0077] Further, a new screening of the V.sub.HH phage libraries was
performed using a different panning strategy, while Nb_B9 was
included for further analysis. Phage pannings of the two V.sub.HH
libraries were performed using biotinylated antigen (the ectodomain
of human BACE1). After three rounds of consecutive panning, 500
single colonies were randomly picked for phage ELISA screening. One
hundred fifty-eight out of 500 colonies scored positive in phage
ELISA screening. The positive colonies were further screened by
periplasmic extract ELISA; 44 colonies out the 158 colonies were
scored positive. The positive colonies isolated from periplasmic
extract ELISA screening were analyzed by PCR and restriction enzyme
digestion to group them according to restriction pattern and for
further sequencing analysis. Fourteen new V.sub.HHs were identified
from the screening.
[0078] The alignment of the V.sub.HHs sequence was listed in FIG.
5. Among these V.sub.HHs, ten clones (1B3, 10C2, 12B6, 10B5, 13A5,
2C6, 6A4, 10C4, 13B6 and 1A4 (SEQ ID NOS:15-24, respectively)) were
isolated from the dromedary libraries, and four clones (2B6, 4A2,
1D4 and 9D3 (SEQ ID NOS:25-28, respectively)) were isolated from
the llama library. The cDNA of these clones were subcloned into
expression vector pHEN6 and V.sub.HH antibodies were then purified
for functional assay tests.
8. BACE1-Specific NANOBODIES.RTM. Inhibit BACE1 Activity in a
Cellular Assay and Modulate BACE1 Activity in a Cell-Free Enzymatic
Assay
[0079] All 15 V.sub.HHs (14 new V.sub.HHs+Nb_B9) were first tested
in a cellular assay by adding them to the medium of SH-SY5Y cells
stably expressing APPwt at a final concentration of 20 .mu.M. As
shown in FIG. 6, cells treated with V.sub.HHs B9, 10C4, 4A2 for 24
hours were shown to decrease sAPPI.beta. generation while
sAPP.alpha. levels in the conditioned medium remained the same as
that of control, suggesting BACE1 activity was inhibited by these
V.sub.HHs in the cellular assay.
[0080] In parallel, the capacity of the V.sub.HHs to modulate
.beta.-secretase activity was tested in an in vitro
.beta.-secretase assay that is based on the Fluorescence Resonance
Energy Transfer (FRET) technology. This assay makes use of a
synthetic peptide substrate that mimics the BACE1 cleavage site of
APP and is coupled to a fluorophore on its N-terminus and a
fluorescence acceptor on its C-terminus. The light emitted by the
fluorophore is absorbed by the fluorescence acceptor as long as
these two moieties are in close proximity. Only upon proteolysis,
when recombinant BACE1 is added to the synthetic substrate, energy
transfer no longer occurs and the amount of light emitted, which is
linearly related to the amount of cleaved product and, hence, to
the .beta.-secretase activity, can be measured. All V.sub.HHs were
tested by this BACE1 FRET assay at a final concentration of 10
.mu.M.
[0081] As shown in FIG. 7, 10C4 and 4A2, the two candidate BACE1
inhibitors identified in the cellular assays, inhibited BACE1
activity in the FRET assay. Interestingly, B9, the candidate
inhibitor isolated from the cellular assay, was shown to increase
260% of BACE1 activity in the FRET assay. Another clone, 1B3 was
also shown to increase 125% of BACE1 activity in the FRET assay,
although it had no apparent effect on BACE1 in the cellular assay.
The remaining V.sub.HHs had no or negligible effects on BACE1
activity in the FRET assay.
[0082] The dose-response curves of 10C4, 4A2 and B9 on BACE1
activity were established by FRET assay. As shown in FIG. 8, 10C4
could inhibit maximal .about.70% of BACE1 activity and the IC50 was
150 nM. 4A2 could inhibit maximal .about.40% of BACE1 activity and
the IC50 was 1.2 .mu.M. B9 could increase BACE1 activity up to 3.5
times with .about.100 nM concentration, and the EC50 in the
response curve was 4.1 nM.
[0083] The contradictory results from B9 modulating BACE1 activity
in opposite ways in the cellular assay and the FRET assay
implicates that B9 might have different effects on BACE1 cleavage
of a large substrate or a small substrate (APP as the cellular
substrate for BACE1 contains 695 amino acids while the peptide
substrate in FRET assay contains only ten amino acids). Therefore,
it was tested whether B9 could inhibit BACE1 cleavage of a big
substrate in another cell-free enzymatic assay MBP-ELISA, which
uses the maltose binding protein connected to the C-terminal 125
amino acids of APPswe (MBP-APPswe-C125) as BACE1 substrate. As
shown in FIG. 9, B9 inhibited BACE1 cleavage of MBP-APPswe-C125 in
a dose-dependent manner, and could inhibit up to 95% of BACE1
activity. The results of this assay indicate that B9 is an
inhibitor of BACE1 when using a big peptide substrate. So, V.sub.HH
Nb_B9, instead of being an active site binder, was more likely a
steric inhibitor for BACE1. V.sub.HH Nb_B9 could bind to an
allosteric site on BACE1, thus stimulating BACE1 cleavage of small
substrates that can still reach the cleavage site, but blocking
access of big substrates, like APP to BACE1 by steric
hindrance.
9. Affinity Analysis of the BACE1-Specific NANOBODIES.RTM.
[0084] The binding affinities of V.sub.HHs B9, 10C4 and 4A2 to
human BACE1 ectodomain were analyzed by Biacore. As shown in Table
2 (left), B9 had the best affinity among the three inhibitory
V.sub.HHs, with a Kd of 3.67 nM at pH 7.0. 10C4 and 4A2 had
affinities of 74.7 nM and 48.2 nM, respectively, which are all
within the normal range of affinities for V.sub.HH antibodies.
[0085] Further, it was tested if the affinities of the V.sub.HHs
were stable at pH 4.5, at which BACE1 has its optimal activity. As
shown in Table 2 (right), there was no significant change in the
affinities of the three V.sub.HHs at pH 4.5 compared to that in
neutral pH, indicating that all three V.sub.HHs have binding
affinities to BACE1 that were acidic stable.
TABLE-US-00002 TABLE 2 V.sub.HH affinities to human BACE1 at pH 7.0
and pH 4.5 pH 7.0 pH 4.5 k.sub.on k.sub.off K.sub.D k.sub.on
k.sub.off K.sub.D (M.sup.-1s.sup.-1) (s.sup.-1) (nM)
(M.sup.-1s.sup.-1) (s.sup.-1) (nM) Nb_B9 2.67E+05 9.80E-04 3.67
6.62E+05 1.30E-03 1.96 Nb_4A2 4.79E+05 2.31E-02 48.2 3.97E+05
8.41E-03 21.2 Nb_10C4 1.06E+05 7.92E-03 74.7 4.51E+05 1.25E-02
27.7
[0086] The cross-reactivity of three V.sub.HHs to mouse BACE1 was
investigated in anticipation of tests in primary cultures of mouse
neurons. As shown in Table 3, both at neutral pH and acidic pH
condition, all three V.sub.HHs cross-reacted with mouse BACE1, and
their affinities to mouse BACE1 were within the same range of
affinities as those measured with human BACE1.
TABLE-US-00003 TABLE 3 V.sub.HH affinities to mouse BACE1 at pH 7.0
and pH 4.5 pH 7.0 pH 4.5 k.sub.on k.sub.off K.sub.D k.sub.on
k.sub.off K.sub.D (M.sup.-1s.sup.-1) (s.sup.-1) (nM)
(M.sup.-1s.sup.-1) (s.sup.-1) (nM) Nb_B9 5.02E+05 8.90E-04 1.77
1.06E+06 1.18E-03 1.11 Nb_4A2 1.65E+05 5.81E-03 35.2 2.51E+05
2.16E-03 8.61 Nb_10C4 1.90E+05 7.97E-03 41.9 9.84E+05 9.88E-03
10
10. BACE1-Specific NANOBODIES.RTM. Inhibit BACE1 Cleavage of APPwt
in Primary Cultured Mouse Neurons
[0087] V.sub.HHs Nb_B9 and Nb_4A2 were tested in the neuronal cell
culture assay (FIGS. 10 and 11). Primary cultured neurons from
wild-type mice were transduced with APPwt by Semliki Forest Virus
(SFV), and then treated with purified V.sub.HH Nb_B9 or Nb_4A2 by
adding to the medium serial dilutions (V.sub.HHs were first
dissolved and diluted in PBS). Neuron cultures were metabolic
labeled for six hours. CTF.beta., sAPP.beta. and A.beta. were later
analyzed as readout of BACE1 activity.
[0088] As shown in FIG. 10, Nb_B9 and Nb_4A2 inhibited BACE1
cleavage of APP reflected in the decrease of A13, sAPP.beta., and
CTF.beta. signals, while full-length APP and sAPP.alpha. levels
remained at the same level as that of the control. The
dose-response curve of Nb_B9 in neuron assay (FIG. 11) was
established by quantification of the CTF.beta. level, which showed
Nb_B9 inhibited BACE1 activity in a dose-dependent manner, with
maximal inhibition effect around 57% BACE1 activity and the IC50
was around 500 nM.
11. Validation of BACE1-Inhibitory NANOBODIES.RTM. in Mouse
Model
[0089] Camel single domain antibodies, the minimal-sized
antibodies, which have superior properties for intracellular
expression and function, including solubility, stability and
functionality without the requirement for association between heavy
and light chains of conventional antibodies, are candidate
therapeutic molecules for in vivo gene delivery. The BACE1
inhibitory V.sub.HH Nb_B9 is tested in a transgenic mouse model of
Alzheimer's disease through viral vector-mediated gene delivery.
Adeno-associated virus (AAV), one of the most effective vehicles
for gene delivery to the central nervous system, is used in this
experimentation. The cDNA of V.sub.HH Nb_B9, fused with a signal
peptide from BACE1 at its N-terminal and a Myc-tag at its
C-terminal, was constructed into an AAV vector. The AAV vector used
here contains a hybrid cytomegalovirus/chicken .beta.-actin
promoter and a wood-chuck post-transcriptional regulatory element,
which is an optimized cassette for driving protein expression in
neurons (Bjorklund et al., 2000).
[0090] For in vivo testing, Dutch-mutant APP transgenic mice are
used, which overexpress E693Q-mutated human APP under the control
of a neuron-specific Thyl promoter element (Herzig et al., 2004).
The E693Q Dutch mutation site on human APP is 21 amino acid
residues behind BACE1 cleavage site, which does not interfere with
APP processing by BACE1. Transgenic mice overexpressing the
Dutch-mutant APP generate predominantly A.beta.40 peptide, which is
used as readout for BACE1 activity for in vivo test of V.sub.HH
Nb_B9. Two administrative routes, including stereotactic injection
to the hippocampus region of adult mouse brain (Fukuchi et al.,
2006) and intracranial injection to neonatal mouse brain (Levites
et al., 2006) are used for the delivery of AAV vector packaged
V.sub.HH Nb_B9. AAV vector packaged GFP and V.sub.HH Nb_B24 are
used as negative controls.
Materials and Methods
Cell Culture
[0091] COS, BHK, MEF, CHO, HEK-APP.sub.Sw, N2A and HeLa cells were
cultured at 37.degree. C. in a 5% CO.sub.2 environment in
Dulbecco's modified Eagle's medium/nutrient mixture F-12 (1:1)
(Gibco) supplemented with 10% (v/v) Fetal Bovine Serum (FBS)
(Hyclone). The HEK-APP.sub.Sw cells were kindly provided by Prof C.
Haass (Adolf Butenandt Institute, Ludwig-Maximilians University,
Munich, Germany). For transient liposome-based transfections, a mix
of FuGENE 6 (Roche Applied Science) and plasmid DNA with a ratio of
3:1 (in .mu.l and .mu.g, respectively) was added to a culture dish
containing a 50% to 80% confluent monolayer of cells, according to
the manufacturer's instructions. COS-hBACE1 stable cells were
obtained after transient transfection of COS cells with
pcDNA3.1zeo-hBACE1 and selection in 400 .mu.g/ml zeocin
(Invitrogen). SH-SY5Y cells were grown in DMEM GLuTAMAX.RTM. 4500
mg/l D-glucose, 1 mM Sodium pyruvate (Gibco), supplemented with 15%
(v/v) FBS.
[0092] Primary cortical neuronal cultures were isolated from E14
mouse embryos (according to Goslin and Banker, 1991). Briefly,
dissected brain cortices were trypsinized with 0.25% trypsin in
HBSS medium (Gibco), pelleted and transferred to DMEM (Invitrogen,
San Diego, Calif.), supplemented with 10% (v/v) FBS and dissociated
by passing them through Pasteur pipettes of decreasing diameters.
Dispersed cells were collected by centrifugation and plated on
poly-L-lysine (Sigma)-coated dishes and maintained in neurobasal
medium (Gibco) supplemented with 0.5 .mu.M L-glutamine (Invitrogen)
and 2% (v/v) B27 Serum-free Supplement (Gibco). Cytosine
arabinoside (5 .mu.M) was added 24 hours after plating to prevent
proliferation of glial cells.
Metabolic Labeling and Immunoprecipitation of APP Fragments
[0093] Cells were washed in Met-free or Met/Cys-free medium (GIBCO)
and radioactively labeled in the appropriate medium containing,
respectively, 100 .mu.Ci .sup.35S Met or .sup.35S Met/Cys (Trans
.sup.35S Label, MP Biomedicals, Irvine, Calif.). In case of
incubations with FK-506, rapamycin and Nb_B26 (2.1 .mu.M),
compounds were added to the labeling medium. After six hours
incubation, the culture supernatant was collected as a source of
secreted A.beta. or sAPPI.beta. and centrifuged to remove detached
cells. Cells were lysed in DIP buffer (20 mM Tris-HCl pH 7.4, 150
mM NaCl, 1% TRITON.RTM. X-100, 1% sodium desoxycholate, 0.1% SDS),
except for the HEK-APP.sub.Sw cells, which were lysed in Tris
buffered saline (TBS: 150 mM NaCl, 20 mM Tris-HCl, pH 7.5),
containing 1% TRITON.RTM. X-100 and a cocktail of protease
inhibitors (Complete, Roche). This lysis buffer still allows
determination of protein concentration (Bio-Rad Protein Assay) to
analyze efficiency of RNA interference on equal amounts of protein
extract.
[0094] APP full-length and APP C-terminal stubs were precipitated
from cell extracts using the APP C-terminal antibodies B63.1, B11/4
or B12/6 (1:200) and immunocomplexes were captured by protein
G-sepharose. For A.beta. species, samples of the cell-conditioned
medium were incubated with either B7/8 or 4G8 (1:200). For the
neurons overexpressing different BACE1 mutants, BACE1 proteins were
precipitated from cell extracts with B45.1.
[0095] Immunoprecipitates were washed extensively in DIP buffer,
followed by one washing step in TBS 1/3, eluted in LDS sample
buffer (Invitrogen) supplemented with 1% .beta.-mercapto ethanol
and separated on 10% NuPAGE.RTM. gel (Novex) run in MES buffer for
the APP fragments and MOPS for the BACE1 mutants. Gels were fixed,
dried and exposed to a phosphor-imaging screen. Intensity of
radioactive bands was quantified using PhosphorImaging (Typhoon,
PerkinElmer) and the IMAGEQUANT.RTM. software package.
[0096] To detect sAPP.beta., samples of conditioned medium were
subjected to SDS-PAGE and Western blotting using B53/4
antibody.
Deglycosylation Experiments
[0097] Cells were harvested in Dulbecco's PBS (GIBCO), pelleted and
lysed in 100 mM phosphate buffer at pH 5.8 for EndoH treatment (46%
of 0.2 M NaH.sub.2PO.sub.4, 4% of 0.2 M Na.sub.2HPO.sub.4 and 50%
water) and pH 7.4 for EndoF (9.5% of 0.2 M NaH.sub.2PO.sub.4, 40.5%
of 0.2 M Na.sub.2HPO.sub.4 and 50% water), supplemented with 0.1%
SDS, 0.5% TRITON.RTM. X-100, 0.5% .beta.-mercapto-ethanol and
protease inhibitors (Complete, Roche). Lysates were first denatured
by heating them for 10 minutes at 70.degree. C. and then treated
with EndoH (1 unit/30 .mu.l, Roche Applied Science) or EndoF (1
unit/30 .mu.l, Roche Applied Science) for 19 hours at 37.degree. C.
and analyzed by SDS-PAGE and Western blotting.
Generation of Recombinant GST-Fusion Proteins
[0098] pGEX-4T-1 plasmids (Pharmacia) encoding GST fusion proteins
were introduced in BL21-competent cells (Merck Eurolab) and
expression of the GST proteins was induced by 0.1 mM isopropyl
.beta.-D-thiogalactopyranoside (IPTG, Promega). Recombinant
proteins were released from the bacteria by sonication in a
Tris-saline buffer (150 mM NaCl, 10 mM Tris) containing a protease
inhibitor cocktail (1 mM EDTA, 14 .mu.g/ml aprotinin, 2 .mu.g/ml
pepstatin), 100 .mu.g/ml lysozyme, 5 mM DTT and 0.5%
N-laurylsarcosine (sarcosyl) (Frangioni and Neel, 1993). After
centrifugation at 12500 rpm (Beckman J2-21M/E) to remove insoluble
bacterial debris, TRITON.RTM. X-100 was added to a final
concentration of 1% to neutralize the effects of the ionic
detergent sarcosyl.
Immunization of a Dromedary and Llama and Analysis of the Immune
Response
[0099] The immunization of dromedary and llama, the isolation of
BACE1-binders and affinity measurements were done in collaboration
with Prof. S. Muyldermans, VUB, Belgium.
[0100] With weekly intervals, a dromedary and llama were immunized
six times subcutaneously with 150 .mu.g of pure recombinant human
BACE1 mixed with GERBU adjuvant (GERBU Biochemicals). The immunogen
used for the immunization of the dromedary was provided by Dr. S.
Masure (Johnson & Johnson Pharmaceutical Research &
Development, Beerse, Belgium). In order to obtain large amounts of
active recombinant BACE1, insect cells were infected with
baculoviruses encoding BACE1 ectodomain (sBACE1) in which the four
putative N-glycosylation sites were removed by substituting the
respective Asn codons for Gln codons (Bruinzeel et al., 2002). The
lack of glycosylation made it possible to produce a large,
homogeneous pool of BACE1.
[0101] A llama was immunized with a different source of BACE1. In
this case, the antigen was purified from sBACE1-overexpressing
HEK293 cells (obtained from Prof. N. Mertens, Protein Service
Facility, VIB, UGent) and, hence, resembled much better native,
mature and thus fully glycosylated BACE1.
[0102] Forty-five days after the first injection, anticoagulated
blood was collected. BACE1-specific antibody titers for each IgG
subclass were analyzed using ELISA. The three individual IgG
subclasses were first purified from serum based on their
differential absorption on Protein A and Protein G and distinct
elution conditions (Conrath et al., 2001a). Solid-phase coated
BACE1 protein was incubated with serial dilutions of the different
IgG subclasses and bound IgGs were subsequently detected with a
rabbit anti-dromedary IgG antiserum and anti-rabbit IgG-alkaline
phosphatase conjugates (Saerens et al., 2004).
Construction of a V.sub.HH Gene Fragment Library
[0103] Peripheral lymphocytes were isolated from the
dromedary/llama sera (LYMPHOPREP.RTM., NYCOMED.RTM.) and total RNA
was extracted (according to Chomczynski and Sacchi, 1987). After
RT-PCR with a dN.sub.6 primer, the cDNA obtained was used as
template for the amplification of a DNA fragment spanning the IgG
variable domain until the CH2 domain, using primers CALL001 and
CALL002 (see Table 4). These primers anneal to the IgG leader
sequence and the CH2 exon of the heavy chain of all three IgG
subclasses existing in dromedary, respectively. Using agarose gel
extraction, the 600 bp fragment coming from heavy chain-only
antibodies (V.sub.HH-CH2, without CH1 domain) was separated from
the 900 bp fragment derived from conventional antibodies
(V.sub.H-CH1-CH2 exons). V.sub.HH gene fragments were then
amplified by PCR on the 600 bp DNA with a pair of nested primers,
AE6 and FR4FOR (see Table 4). AE6 anneals to the V.sub.HH
framework-1 and contains a Pst I site, whereas FR4FOR with a Not I
site is complementary to the framework-4. The different V.sub.HH
fragments were ligated into a pHEN4 phagemid vector and transformed
into E. coli TG1 cells to create a library of 4.times.10.sup.7
transformants. Colony PCR screening showed that approximately 90%
of the colonies were transformed with a phagemid vector containing
an insert with the size expected for a V.sub.HH fragment.
TABLE-US-00004 TABLE 4 Sequences of the different primers used for
the V.sub.HH gene fragment library construction Primer Sequence (5'
to 3') SEQ ID NO: CALL001 GTCCTGGCTGCTCTTCTACAAGG 44 CALL002
GGTACGTGCTGTTGAACTGTTCC 45 AE6 GATGTGCAGCTGCAGGAGTCTGGAGGAGG 46
FR4FOR GGACTAGTGCGGCCGCTGCAGACGGTGAC 47 CTGGGT
Selection of BACE1-Specific V.sub.HH Fragments
[0104] The V.sub.HH repertoire was expressed onto the surface of
phages after rescuing the library with M13K07 helper phages.
Specific V.sub.HHs against BACE1 were enriched by three consecutive
rounds of in vitro selection, a technique also known as panning
(Smith and Petrenko, 1997). For this, the V.sub.HHs were incubated
on a solid phase coated with antigen. Unbound phages were washed
away in PBS plus 0.05% TWEEN.RTM. 20 and bound phages were eluted
with 100 mM triethylamine (pH 10.0). Eluted phage particles were
immediately neutralized with 1 M Tris-HCl (pH 7.5) and used to
re-infect exponentially growing E. coli TG1 cells. After the second
and third round of selection, individual colonies were randomly
picked.
Enzyme-Linked Immunosorbent Assay (ELISA)
[0105] Expression of the selected V.sub.HH was induced with 1 mM
IPTG. The recombinant soluble C-terminally Hemagglutinin
(HA)-tagged V.sub.HHs (the gene encoding the HA-epitope is included
in the pHEN4 phagemid vector) were extracted from the periplasm by
an osmotic shock (200 mM Tris-HCl pH 8.0, 250 mM sucrose, 0.5 mM
EDTA) (Skerra and Pluckthun, 1988) and tested for their capacity to
recognize their antigen in ELISA tests. Maxisorb 96-well plates
(Nunc) were coated overnight with BACE1 protein (100 .mu.l of 1
.mu.g/ml in PBS) at 4.degree. C. Residual binding sites were
blocked for two hours at room temperature with 1% (w/v) casein
dissolved in PBS. This antigen-coated solid phase was then
incubated with the different periplasmic extracts for one hour at
room temperature. After washing, the solid phase was successively
incubated with mouse anti-HA, alkaline phosphatase-conjugated
anti-mouse (Sigma) and 2 mg/ml p-nitrophenyl phosphate (Sigma).
Signals were analyzed at 410 nm.
Expression and Purification of V.sub.HHs
[0106] The V.sub.HH genes of the clones scoring positive in ELISA
were subcloned into the expression vector pHEN6, using Pst I and
BstE II. Thereby, the HA-epitope tag at the C-terminus of the
V.sub.HH molecules was replaced by a his6-tag. E. coli WK6 cells
were transformed with the pHEN6 plasmids and expression of the
recombinant soluble V.sub.HH proteins was induced by IPTG (Saerens
et al., 2004). Soluble V.sub.HH molecules were extracted from the
bacteria using an osmotic shock (Skerra and Pluckthun, 1988). The
his-tagged recombinant proteins were then captured on a
nickel-nitrilotriacetic acid superflow Sepharose column
(QIAGEN.RTM.), eluted with an acetate buffer (pH 4.7), and
additionally purified by size-exclusion chromatography.
BIAcore Measurements
[0107] The kinetic constants and affinity of the V.sub.HH-antigen
interactions were determined by surface plasmon resonance
technology on a Biacore 3000 (Biacore AB). Purified V.sub.HH
molecules, in a concentration range of 0-500 nM in Hepes Buffered
Saline pH 7.0 or citrate buffer pH 5.0, were injected at 30
.mu.l/minute onto BACE1 (500 resonance units), immobilized on a CM5
chip (according to De Genst et al., 2005). The kinetic and
equilibrium constants (k.sub.on, k.sub.off and K.sub.D) were
determined with the BIAevaluation v3.1 software (Biacore AB).
In Vitro FRET-Based Analysis of .beta.-Secretase Activity
[0108] To determine whether the V.sub.HHs affect BACE1 activity, an
in vitro BACE1 FRET assay kit was used (Panvera P2985). This assay
uses a synthetic BACE1 substrate that emits light upon cleavage.
The amount of total fluorescence is linearly related to the
cleavage rate of the substrate and hence to .beta.-secretase
activity. Reaction mixtures containing 20 nM of recombinant BACE1
enzyme and 250 nM of synthetic substrate were incubated with an
excess of each V.sub.HH (2.2 .mu.M) or the BACE1 inhibitor STA-200
(Enzyme System Products, 2.2 .mu.M) in 50 mM sodium acetate, pH 4.5
at room temperature, protected from light. After two hours,
fluorescence was measured at 595 nm using VICTOR 1420 multilabel
counter (Perkin Elmer Life Sciences). For each V.sub.HH, the
background signal, emitted by a mix containing V.sub.HH and
substrate but no enzyme, was subtracted from the signal measured
for the mix containing V.sub.HH, substrate and BACE1. As an
alternative source of .beta.-secretase activity, microsomal
membranes were generated from HeLa cells ectopically expressing
BACE1 as described hereinbefore. The resulting microsomal pellet
was resuspended in 50 mM sodium acetate, pH 4.5. Fifty .mu.g of
microsomal proteins were mixed with 250 nM of the synthetic BACE1
substrate and 2.2 .mu.M of V.sub.HH or STA-200. The enzymatic
reaction and analysis were performed as before except that
reactions were gently mixed every ten minutes during the two-hour
incubation.
[0109] In another approach, FRET peptide substrate
MCA-S-E-V-N-L-D-A-E-F--R-K(Dnp)-R--R-R-R-NH2 (SEQ ID NO:48) was
synthesized by Ana Spec Inc. (San Jose, Calif., USA). Enzyme human
BACE1 (1-460): IgGFc was purified from HEK293 cells according to
the protocol described previously (Yang et al., 2004). For the
reaction, enzyme was diluted in reaction buffer (50 mM Ammonium
Acetate, pH 4.6, 3% BSA, 0.7% TRITON.RTM. X-100) at a concentration
of 1 .mu.g/ml, and substrate was diluted in reaction buffer at a
concentration of 125 .mu.M. Twenty .mu.l V.sub.HH (diluted in
reaction buffer) were mixed with 30 .mu.l enzyme dilution and 50
.mu.l substrate dilution in 96-well black polystyrene plates
(Costar). The plates were read immediately for baseline signal with
Envision (355 nm excitation, 430 nm emission, 1 second/well),
followed by incubation overnight in the dark at room temperature.
The plates were read the following morning using the same reader
protocol; the FRET signal (-baseline signal) was used as the
readout of enzyme activity in each reaction.
Co-Precipitation of Human BACE1 with his-Tagged V.sub.HHs Using
Ni-Beads
[0110] BACE1-overexpressing COS cells were lysed in PBS containing
1% TRITON.RTM. X-100 and protease inhibitors (1 .mu.g/ml pepstatin,
14 .mu.g/ml aprotinin, 0.5 mM PEFABLOC.RTM.). One hundred .mu.g of
this protein extract were incubated overnight at 4.degree. C. with
2 .mu.g of his-tagged V.sub.HH proteins and Ni-PDC beads (Affiland)
in binding buffer (342 mM NaCl, 16.2 mM Na.sub.2HPO.sub.4, 6.7 mM
KCl, 3.7 mM KH.sub.2PO.sub.4 with 1% TRITON.RTM. X-100) with the
same protease inhibitors as used for cell lysis. The precipitates
were washed in binding buffer supplemented with 10 mM imidazole, to
reduce unspecific interactions, eluted using 300 .mu.M imidazole
and resolved by SDS-PAGE. BACE1 was visualized by Western blotting
using a polyclonal rabbit anti-BACE1 antibody (ProSci Inc).
Phage Libraries Panning with Biotin-Labeled Antigen
[0111] Pannings of V.sub.HH phage library were performed with
biotin-streptavidin system. Purified BACE1 ectodomain protein was
labeled with Sulfo-NHS-SS-Biotin (Pierce) according to the
manufacturer's protocol. V.sub.HH libraries were rescued with
M13K07 helper phage to generate phages. For panning, 10.sup.11
phages were blocked with 1% BSA in 400 .mu.l panning buffer (50 mM
Tris-HCl pH 7.5, 150 mM NaCl, and 0.05% TWEEN.RTM. 20 for 30
minutes at RT. One hundred .mu.l biotinylated BACE1 was added to
phage to a final concentration at 200 nM. Phage and biotinylated
BACE1 were incubated for one hour at RT with rotation. Meanwhile,
40 .mu.l immobilized streptavidin (Pierce) were blocked in 1% BSA.
After one-hour incubation, pre-blocked immobilized streptavidin
were added to phage-biotinylated BACE1 solution and incubated for
40 minutes at RT with rotation. After incubation, immobilized
streptavidin were spinned down by centrifugation at 3000 rpm for
one minute, the supernatant containing unbound phage were
discarded. The immobilized streptavidin were washed five times with
1 ml panning buffer; each wash lasted five minutes with rotation.
After wash, 50 mM DTT were added to immobilized streptavidin and
incubated for 40 minutes at RT with rotation. The immobilized
streptavidin were spinned down and the supernatants containing
eluted phages were used to re-infect E. coli TG1 cells for the next
round of phage panning After three rounds of consecutive panning,
recovered phages were used to infect E. coli TG1 cells and plated
out at 10.sup.-4 to 10.sup.-6 dilution, and single colonies were
picked for further analysis.
Phage ELISA Screening
[0112] To generate phage particles with V.sub.HHs displayed on the
surface for ELISA screening, single colonies from phage panning
were inoculated in 2 ml 2.times.TY medium supplemented with 50
.mu.g/ml ampicillin and 1% glucose in 24-well plates at 37.degree.
C. for eight hours with shaking at 220 rpm. After an eight-hour
incubation, 5.times.10.sup.8 pfu M13K07 helper phages were added to
infect each well of bacterials. Infected bacterials were grown at
37.degree. C. overnight with shaking at 220 rpm. The next morning,
bacterials were spinned down by centrifugation at 3000 rpm for 20
minutes; supernatants containing phage particles were transferred
into 24-well plates. Twenty percent PEG 6000/2.5 M NaCl were added
to the supernatants using 1/6 volume to precipitate phage particles
at 4.degree. C. for 30 minutes. Phage particles were later
retrieved by centrifugation at 3000 rpm for 30 minutes, and pellets
were resuspended in 100 .mu.l PBS.
[0113] For ELISA assay of phage particles, BACE1 ectodomain protein
was coated on 48 wells of each 96-well microtiter plate at 100
ng/well at 4.degree. C. overnight, the non-coated wells were used
for control. The next morning, microtiter plates were blocked with
3% mild for one hour at RT. After blocking, 100 .mu.l phage
particles were added to each coated and non-coated well, and were
incubated for two hours at RT. Plates were then washed five times
with washing buffer (PBS, 0.05% TWEEN.RTM. 20). After wash,
HRP-conjugated anti-M13 antibody (Amersham) was added to each well
using 1:3000 dilution in 3% milk and incubated for one hour. Plates
were then washed five times with washing buffer. After wash,
developing substrate 0.02 mg/ml ABTS (Sigma) supplemented with 0.3%
H.sub.2O.sub.2 (Sigma) were added to microtiter plates and
incubated for 30 minutes at RT. Plates were read at OD405 nm with
an ELISA reader.
Periplasmic Extract ELISA Screening
[0114] Expression vector pHEN4 containing a PelB (Pectate lysase)
signal sequence before the V.sub.HH cDNAs, thus V.sub.HHs are
exported to the periplasmic space after expressed in bacterial
system. To generate periplasmic extract containing V.sub.HH
proteins for ELISA test, single colonies from phage panning were
inoculated in 1 ml Terrific Broth (TB) medium supplemented with 100
.mu.g/ml ampicillin in 24-well plates at 37.degree. C. with shaking
at 220 rpm. When OD.sub.600 reached 0.6, 1 mM IPTG was added to the
culture to induce the expression of V.sub.HH proteins. Bacterials
were further incubated for 15 hours at 28.degree. C. for protein
expression. After the incubation, bacterials were harvested by
centrifugation at 3000 rpm for 20 minutes, cell pellets were
dissolved in TES solution (20 mM Tris-HCl pH 7.4, 1 mM EDTA, 250 mM
sucrose) and incubated on ice for 30 minutes. The osmotic shock was
given by adding 1.5.times. volume TES/4 to the bacterials and
incubated on ice for 45 minutes. Supernatants containing V.sub.HH
proteins were collected by centrifugation at 300 rpm for 20 minutes
and further used for ELISA. The ELISA assays followed the same
protocol described above for phage ELISA. To detect V.sub.HH
proteins generated with a C-terminal HA tag, anti-HA monoclonal
antibody (Amersham) was used as primary antibody and alkaline
phosphatase conjugated goat anti-mouse antibody (Amersham) were
used as secondary antibody. ELISA plates were developed with
p-Nitrophenyl-phosphate (PNPP) substrate (Sigma) and read at OD 405
nm with an ELISA reader.
Adeno-Associated Virus (AAV) Construction and Preparation
[0115] For AAV generation, a standard method was followed (Levites
et al., 2006). Briefly, the cDNA of V.sub.HH Nb_B9, fused with a
signal peptide from BACE1 at its N-terminal and a Myc-tag at its
C-terminal, was constructed into an AAV vector containing a hybrid
cytomegalovirus/chicken .beta.-actin promoter and a woodchuck
post-transcriptional regulatory element. AAVs were generated by
plasmid transfection with helper plasmids in HEK293T cells.
Forty-eight hours after transfection, the cells were harvested and
lysed in the presence of 0.5% sodium deoxycholate and 50 U/ml
Benzonase (Sigma) by freeze thawing, and the virus was isolated
using a discontinuous iodixanol gradient purified on a HITRAP.RTM.
HQ column (Amersham Biosciences). The genomic titer of virus was
determined by quantitative PCR.
Mice
[0116] All animal experiments were in compliance with protocols
approved by the local Animal Care and Use Committees. Dutch-mutant
APP transgenic mice (C57BL/6J-TgN(Thy-APP.sub.E693D)) were kindly
provided by the laboratory of Mathias Jucker, University of
Tubingen, Germany.
Stereotaxic Injections
[0117] In the first series of experiments, AAV vectors expressing
V.sub.HH Nb_B9 and GFP (negative control), and V.sub.HH Nb_B24
(negative control) were administrated directly into the hippocampus
of three-month-old Dutch-mutant APP transgenic mice. Mice were
anesthetized with avertin and placed in a stereotaxic apparatus.
AAV preparations were injected bilaterally (2 .mu.l per site) into
the CA3 region of the hippocampus (-2.0 mm antero-posterior from
bregma, +/-2.3 mm medio-lateral from bregma, and 1.7 mm below
dura). Mice were then individually housed and allowed to recover
from surgery. Their brains were processed for analyses five weeks
after treatment.
Neonatal Injections
[0118] The procedure was described previously (Levites et al.,
2006). Briefly, postnatal day 0 (P0) pups were cryoanesthetized on
ice for five minutes. AAV preparations (2 .mu.l) were injected
intracerebroventricularly into both hemispheres using a 10 ml
Hamilton syringe with a 30 gauge needle. The pups were then placed
on a heating pad with their original nesting materials for three to
five minutes and returned to their mother for further recovery.
Their brains were processed for analyses three months after
injection.
Tissue Preparation and Biochemical Analysis of A.beta.
[0119] To analyze A.beta., the hippocampus (from stereotaxic
injections) and the whole brains (from neonatal injections) were
homogenized in Tissue Protein Extraction reagent (Pierce)
supplemented with COMPLETE.TM. protease inhibitor and phosphatase
inhibitor tablets (Roche Applied Science). The homogenized samples
were centrifuged at 4.degree. C. for one hour at 100,000.times.g,
and the supernatant was used for immunoblot analysis and for
A.beta. ELISA measurements using ELISA kits (The Genetics
Company).
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Sequence CWU 1
1
481126PRTCamelus dromedarius 1Asp Val Gln Leu Gln Glu Ser Gly Gly
Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val
Ala Ser Gly Trp Thr Tyr Ser Ser Asn 20 25 30 Ser Leu Ser Met Ala
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Gly Val Ala
Thr Ile Thr Ser Tyr Val Gly Arg Thr Tyr Tyr Ala Asp 50 55 60 Ser
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp His Ala Lys Ser Thr 65 70
75 80 Val Tyr Leu Gln Ile Asp Ser Leu Lys Pro Glu Asp Thr Ala Thr
Tyr 85 90 95 Tyr Cys Ala Ala Glu Tyr Leu Gly Gly Ser Phe Leu Ser
Thr Gly Ala 100 105 110 Tyr Lys Tyr Trp Gly Gln Gly Thr Gln Val Thr
Val Ser Ser 115 120 125 2127PRTCamelus dromedarius 2Asp Val Gln Leu
Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Tyr Ser Pro Tyr 20 25 30
Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35
40 45 Ala Ala Ile Arg Lys Gly Ile Gly Thr Thr Tyr Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Phe Ser Gln Asp Asp Ala Lys Asn
Thr Met Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Ile Tyr Phe Cys 85 90 95 Ala Val Gly His Tyr Arg Ala Tyr Ala
Thr Thr Ser Phe Asp Pro Arg 100 105 110 Arg Tyr Asp Tyr Trp Gly Gln
Gly Thr Gln Val Thr Val Ser Ser 115 120 125 3125PRTCamelus
dromedarius 3Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln
Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr
Thr Tyr Asn Ile Tyr 20 25 30 Thr Met Ala Trp Phe Arg Gln Ala Pro
Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Gly Ile Tyr Ser Pro Gly
Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr
Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met
Asn Ser Leu Ala Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala
Ala Arg Gly Gly Leu Leu Ser Arg Val Leu Lys Glu Ala Gly Tyr 100 105
110 Asn Ala Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125
4125PRTCamelus dromedarius 4Asp Val Gln Leu Gln Glu Ser Gly Gly Gly
Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Tyr Thr Phe Thr Lys Tyr 20 25 30 Pro Met Gly Trp Phe Arg
Gln Ala Pro Gly Lys Glu Cys Glu Leu Val 35 40 45 Ser Ser Ile Ile
Ser Gly Gly Val Thr Thr Tyr Ala Ser Ser Val Lys 50 55 60 Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85
90 95 Ala Gln Tyr Pro Tyr Ser Ser Ser Trp Pro Arg Cys Pro Phe Arg
Ile 100 105 110 Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125 5123PRTCamelus dromedarius 5Asp Val Gln Leu Gln Glu Ser
Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Arg Ser Gly Gly Thr Val Ser Ile Pro 20 25 30 Tyr Met Ala
Trp Phe Arg Gln Gly Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala
Ala Ile Tyr Asp Gly Arg Ala Lys Thr Tyr Ala Gly Ser Leu Gln 50 55
60 Gly Arg Phe Thr Ile Ser Gln Asp Asn Asp Lys Asn Thr Leu Tyr Leu
65 70 75 80 Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr
Cys Ala 85 90 95 Ala Gly Asn Gly Gly Gly Asn Trp Leu Arg Pro Ser
Glu Tyr Asn Tyr 100 105 110 Trp Gly Lys Gly Thr Gln Val Thr Val Ser
Ser 115 120 6121PRTCamelus dromedarius 6Asp Val Gln Leu Gln Glu Ser
Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Glu Tyr Thr Tyr Gly Tyr Cys 20 25 30 Ser Met Gly
Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Ser
Thr Ile Thr Ser Asp Gly Ser Thr Ser Tyr Val Asp Ser Val Lys 50 55
60 Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Lys Tyr Tyr
Cys Tyr 85 90 95 Thr Lys Thr Cys Ala Asn Lys Leu Gly Ala Lys Phe
Ile Ser Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser 115
120 7127PRTCamelus dromedarius 7Asp Val Gln Leu Gln Glu Ser Gly Gly
Gly Ser Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Tyr Phe Tyr Ser Arg Trp 20 25 30 Tyr Met Gly Trp Phe
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile
Asn Ser Gly Gly Ser Ile Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr
Cys 85 90 95 Ala Ala Ala Leu Ser Arg Val Pro Gly Phe Phe Pro Leu
Phe Pro Ser 100 105 110 Gln Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val
Thr Val Ser Ser 115 120 125 8129PRTCamelus dromedarius 8Asp Val Gln
Leu Gln Glu Ser Gly Gly Gly Ser Ala Gln Ala Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Glu Val Ser Gly Tyr Thr Tyr Ser Gly Tyr 20 25
30 Phe Met Gly Trp Phe Arg Gln Ala Pro Gly Glu Glu Arg Glu Gly Val
35 40 45 Ala Ala Ile Asp Thr Asn Gly Gly Arg Thr Trp Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser His Asp Asn Ala Glu
Ser Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Gln Pro Glu Asp
Thr Ala Ile Tyr Phe Cys 85 90 95 Ala Ala Arg Arg Pro Pro Gly Gly
Ser Trp Tyr Pro Pro Pro Leu Arg 100 105 110 Lys Tyr Ser Tyr Asn Phe
Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser
9128PRTCamelus dromedarius 9Asp Val Gln Leu Gln Glu Ser Gly Gly Gly
Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala
Ser Gly Phe Thr Tyr Arg Arg Tyr 20 25 30 Phe Met Gly Trp Phe Arg
Gln Ala Pro Gly Lys Glu Arg Glu Ala Val 35 40 45 Ala Thr Met Phe
Ser Cys Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ala Thr Gln Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80
Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 85
90 95 Ala Ala Ala Ser Gly Cys Trp Tyr Asp Gly Ser Pro Ala Ala Arg
Ser 100 105 110 Val Asp Val Ser Phe Trp Gly His Gly Thr Gln Val Thr
Val Ser Ser 115 120 125 10129PRTCamelus dromedarius 10Asp Val Gln
Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Tyr Ser Tyr Tyr 20 25
30 Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala
35 40 45 Ala Ile Ala Ile Val Asn Ser Gly Gly Gly Arg Thr Tyr Tyr
Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Gly Asn
Asp Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Lys Pro
Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Ala Ala Arg Ser Leu Ser
Trp Tyr Ser His Pro Leu Leu Gln 100 105 110 Pro Ser Gln Phe Asn Asn
Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser
11128PRTCamelus dromedarius 11Asp Val Gln Leu Gln Glu Ser Gly Gly
Gly Ser Val Gln Ala Glu Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr
Ala Ser Gly Tyr Thr Tyr Ser Leu Met 20 25 30 Gly Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Val 35 40 45 Ile Asn Ser
Gly Val Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys Gly 50 55 60 Arg
Phe Thr Ile Ser Gln Asp Asn Ala Lys Ser Thr Val Tyr Leu Gln 65 70
75 80 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala
Ala 85 90 95 Arg Arg Ser Trp Phe Thr Gly Met Thr Thr Thr Gln Ala
Leu Asp Pro 100 105 110 Asp Trp Phe Ser Tyr Trp Gly Gln Gly Thr Gln
Val Thr Val Ser Ser 115 120 125 12125PRTCamelus dromedarius 12Asp
Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ser Glu Met Asn
20 25 30 Arg Phe Ala Trp Leu Arg Gln Ala Pro Gly Lys Asp Arg Glu
Val Val 35 40 45 Ala Val Ile Phe Pro Thr Ala Arg Gly Ala Lys Phe
Tyr Ser Asp Ser 50 55 60 Val Asn Gly Arg Phe Thr Ile Ser Gln Asp
Thr Ala Lys Asn Thr Val 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Glu
Pro Glu Asp Thr Ala Met Tyr Phe 85 90 95 Cys Ala Ala Ser Ala Asn
Ala Met Thr Gly Phe Gln Pro Ser Gly Tyr 100 105 110 Thr Tyr Trp Gly
Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 13129PRTCamelus
dromedarius 13Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Thr Val Gln
Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr
Thr Tyr Arg Ser Tyr 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro
Gly Lys Glu Arg Glu Glu Val 35 40 45 Ala Ser Ile Asn Ser Asp Gln
Gly Ser Thr Arg Tyr Ala Ala Ser Val 50 55 60 Lys Gly Arg Phe Thr
Ser Ser Gln Asp Asn Ala Asn Asn Thr Val Thr 65 70 75 80 Val Tyr Leu
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr
Cys Ala Ala Asn Asp Gly Cys Ala Tyr Arg Val Tyr Arg Gly Gly 100 105
110 Ala Tyr Gly Tyr Asn Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125 Ser 14129PRTCamelus dromedarius 14Asp Val Gln Leu Gln
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Tyr
Met Gly Trp Phe Arg Gln Ala Pro Gly Arg Glu Arg Glu Glu Val 35 40
45 Thr Gly Ile Thr Gln Ile Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
Val Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala
Ile Tyr Tyr Cys 85 90 95 Ala Lys Leu Arg Arg Pro Phe Tyr Tyr Pro
Leu Leu Glu Arg Pro Ser 100 105 110 Glu Gly Asp Phe Asp Tyr Trp Gly
Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser 15126PRTCamelus
dromedarius 15Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln
Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Tyr
Thr Asp Ser Thr Tyr 20 25 30 Tyr Met Ala Trp Phe Arg Gln Ala Pro
Gly Lys Glu Arg Gly Gly Val 35 40 45 Ala Thr Leu Ala Ser Arg Tyr
Asp Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr
Ile Ser Gln Asp Arg Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met
Asn Ser Leu Lys Pro Glu Asp Thr Gly Ile Tyr Tyr Cys 85 90 95 Ala
Ala Ser Pro Arg Arg Pro Gly Phe Phe Pro Leu Asp Pro Ser Gln 100 105
110 Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
125 16127PRTCamelus dromedarius 16Gln Val Gln Leu Gln Glu Ser Gly
Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Arg Asp Ile Leu Thr Leu Tyr 20 25 30 Tyr Met Gly Trp
Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala
Ile Ser Ser Asp Ile Ile Phe Thr Ser Tyr Ala Asn Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Asp Lys Asn Thr Val Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr
Tyr Cys 85 90 95 Ala Ala Ala Ser Thr Trp Val Pro Gly Phe Phe Pro
Leu Phe Ala Ser 100 105 110 Gln Tyr Asn Ser Trp Gly Gln Gly Thr Gln
Val Thr Val Ser Ser 115 120 125 17127PRTCamelus dromedarius 17Gln
Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Asp Ile Leu Thr Leu Tyr
20 25 30 Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
Gly Val 35 40 45 Ala Ala Ile Ser Ser Asp Ile Ile Phe Thr Ser Tyr
Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu
Asp Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro
Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Ala Ala Ser Thr Trp
Val Pro Gly Phe Phe Pro Leu Phe Ala Ser 100 105 110 Gln Tyr Asn Ser
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125
18125PRTCamelus dromedarius 18Gln Val Gln Leu Gln Glu Ser Gly Gly
Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
His Ser Asn Thr Tyr Pro Thr Tyr Met 20 25 30 Gly Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala 35 40 45 Ile Tyr Thr
Gly Asp Gly Thr Thr Tyr Tyr Gly Asp Ser Val Lys Gly 50 55 60 Arg
Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 65 70
75 80 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala
Ala
85 90 95 Ala Leu Ser Arg Val Pro Gly Phe Phe Pro Leu Phe Pro Ser
Gln Tyr 100 105 110 Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser
Ser 115 120 125 19125PRTCamelus dromedarius 19Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala His Ser Asn Thr Tyr Pro Thr Tyr Met 20 25 30 Gly
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala 35 40
45 Ile Tyr Thr Gly Asp Gly Thr Thr Tyr Tyr Gly Asp Ser Val Lys Gly
50 55 60 Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
Leu Gln 65 70 75 80 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr
Tyr Cys Ala Ala 85 90 95 Ala Leu Ser Arg Val Pro Gly Phe Phe Pro
Leu Phe Pro Ser Gln Tyr 100 105 110 Asn Tyr Trp Gly Gln Gly Thr Gln
Val Thr Val Ser Ser 115 120 125 20127PRTCamelus dromedarius 20Gln
Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ser Ser Val Tyr
20 25 30 Tyr Ile Ser Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
Gly Val 35 40 45 Ala Ala Ile Asn Ser Gly Gly Gly Ile Thr Phe Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn
Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro
Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Ala Ala Leu Ser Arg
Val Pro Gly Phe Phe Pro Leu Phe Pro Ser 100 105 110 Gln Tyr Asn Tyr
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125
21115PRTCamelus dromedarius 21Gln Val Gln Leu Gln Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Tyr Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile
Asn Ser Gly Gly Gly Thr Thr Tyr Ser Thr Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95 Ala Thr Asp Ser Thr Gly Ser His Arg Gly Gln Gly Thr
Gln Val Thr 100 105 110 Val Ser Ser 115 22124PRTCamelus dromedarius
22Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1
5 10 15 Phe Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Thr
Cys 20 25 30 Ser Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg
Glu Leu Val 35 40 45 Ser Ser Ile Arg Asn Asp Gly Ser Thr Ala Tyr
Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Gln Asp Asn
Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro
Glu Asp Thr Ala Met Tyr Tyr Cys Asn 85 90 95 Ile Arg Ile Gly Val
Gly Pro Gly Gly Thr Cys Ser Ile Tyr Ala Pro 100 105 110 Tyr Trp Gly
Glu Gly Thr Gln Val Thr Val Ser Ser 115 120 23127PRTCamelus
dromedarius 23Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln
Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile
Ser Arg Ser Thr Tyr 20 25 30 Phe Met Gly Trp Phe Arg Gln Ala Pro
Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Val Ile Asn Tyr Gly Thr
Thr Thr Pro Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr
Val Ser Arg Asp Ser Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Arg Met
Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala
Ala Ala Ser Thr Trp Val Pro Gly Phe Phe Pro Leu Phe Ala Ser 100 105
110 Gln Tyr Asn Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115
120 125 24128PRTCamelus dromedarius 24Gln Val Gln Leu Gln Glu Ser
Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Ala Thr Ala Ser Asp Tyr 20 25 30 Cys Met Gly
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala
Ala Ile Ser Arg Gly Gly Met Thr Tyr His Val Asp Ser Val Arg 50 55
60 Gly Arg Phe Thr Ile Ser Arg Asn Asn Ala Gln Asn Thr Val Tyr Leu
65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Thr Tyr Ser
Cys Ala 85 90 95 Ala Val Ser Cys Ala Gly Ala Trp Phe Ala Asn Arg
Ala Leu Arg Glu 100 105 110 Ser Ala Phe Thr Tyr Trp Gly Pro Gly Thr
Gln Val Thr Val Ser Ser 115 120 125 25120PRTLama glama 25Gln Val
Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ile Phe Asp Leu Arg 20
25 30 Asp Met Gly Trp Tyr Arg Gln Val Pro Gly Lys Gln Arg Glu Leu
Val 35 40 45 Ala Ala Ile Thr Ser Gly Gly Thr Ser Asn Tyr Ala Asp
Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp
Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Ala Lys Asn Phe Phe Ser Ala
Ser Gly Tyr Phe Leu Tyr Trp Gly Lys 100 105 110 Gly Thr Gln Val Thr
Val Ser Ser 115 120 26121PRTLama glama 26Gln Val Gln Leu Gln Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Thr Gln 20 25 30 Tyr Met
Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Tyr Val 35 40 45
Ser Ser Ile Asn Ser Gly Gly Thr Ile Lys Tyr Tyr Ala Asn Ser Ser 50
55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
Leu 65 70 75 80 Tyr Leu Gln Met Asn Asn Leu Arg Pro Glu Asp Thr Ala
Ile Tyr Tyr 85 90 95 Cys Gln Leu Gly Gln Trp Ala Gly Val Gly Ala
Ala Ser Ser Arg Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 27122PRTLama glama 27Gln Val Gln Leu Gln Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Trp Met Tyr Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile
Ser Thr Glu Gly Gly Ser Thr Arg Tyr Ala Gly Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr 65 70
75 80 Leu Gln Met Asp Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ser Lys Gly Thr Gly Pro Phe Thr Asp Ile Arg Ser Thr
Gly Ser Arg 100 105 110 Gly Lys Gly Thr Gln Val Thr Val Ser Ser 115
120 28122PRTLama glama 28Gln Val Gln Leu Gln Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met Tyr Trp Phe Arg
Gln Ala Pro Gly Lys Gly Leu Glu Arg Val 35 40 45 Ser Ala Ile Asn
Phe Gly Gly Asp Val Thr Tyr Tyr Thr Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Thr Lys Gly Leu Ser Pro Tyr Arg Asp Leu Glu Ser Ser Gly Ser
Arg 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
2910PRTCamelus dromedarius 29Glu Tyr Thr Tyr Gly Tyr Cys Ser Met
Gly 1 5 10 3016PRTCamelus dromedarius 30Thr Ile Thr Ser Asp Gly Ser
Thr Ser Tyr Val Asp Ser Val Lys Gly 1 5 10 15 3113PRTCamelus
dromedarius 31Lys Thr Cys Ala Asn Lys Leu Gly Ala Lys Phe Ile Ser 1
5 10 3210PRTCamelus dromedarius 32Gly Tyr Thr Tyr Ser Thr Cys Ser
Met Ala 1 5 10 3316PRTCamelus dromedarius 33Ser Ile Arg Asn Asp Gly
Ser Thr Ala Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 3416PRTCamelus
dromedarius 34Arg Ile Gly Val Gly Pro Gly Gly Thr Cys Ser Ile Tyr
Ala Pro Tyr 1 5 10 15 3510PRTLama glama 35Gly Phe Thr Phe Glu Thr
Gln Tyr Met Thr 1 5 10 3618PRTLama glama 36Ser Ile Asn Ser Gly Gly
Thr Ile Lys Tyr Tyr Ala Asn Ser Ser Val 1 5 10 15 Lys Gly
3711PRTLama glama 37Gly Gln Trp Ala Gly Val Gly Ala Ala Ser Ser 1 5
10 38115PRTCamelus dromedarius 38Asp Val Gln Leu Gln Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Glu Phe Thr Phe Gly Ser Tyr 20 25 30 Trp Met Tyr Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gln
Ile Asn Ala Arg Gly Ser Thr Ile Tyr Tyr Val Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Thr Asp Ser Arg Gly Thr His Lys Gly Gln Gly
Thr Gln Val Thr 100 105 110 Val Ser Ser 115 39115PRTCamelus
dromedarius 39Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Arg
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ala Asn Tyr 20 25 30 Trp Leu Tyr Trp Val Arg Asp Ala Pro
Gly Lys Gly Ile Glu Trp Val 35 40 45 Ser Gln Ile Gly Pro Ser Gly
Arg Ser Thr Tyr Tyr Ala Asp Ala Val 50 55 60 Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Lys Thr Leu Tyr 65 70 75 80 Leu Gln Met
Asn Ser Leu Lys Pro Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala
Thr Ser Ser Gly Gly Asn Glu Arg Gly Gln Gly Thr Gln Val Thr 100 105
110 Val Ser Ser 115 40115PRTCamelus dromedarius 40Asp Val Gln Leu
Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr 20 25 30
Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Gln Val Asn Ser Asp Gly Gly Ser Thr Tyr Tyr Val Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Thr Leu Tyr 65 70 75 80 Leu His Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asp Ser Ser Gly Arg Tyr Arg
Gly Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115
41115PRTCamelus dromedarius 41Asp Val Gln Leu Gln Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met Tyr Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln Ile
Asn Ser Ser Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Thr Gly Ser Ala Gly Gln Gly Lys Gly Gln Gly Thr
Gln Val Thr 100 105 110 Val Ser Ser 115 42115PRTCamelus dromedarius
42Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn
Tyr 20 25 30 Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Gln Ile Asp Gly Gly Gly Arg Lys Thr Tyr
Tyr Ala Asp Ser Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys
Pro Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Thr Asp Ser Ala
Gly Ser His Arg Gly Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser
115 43115PRTCamelus dromedarius 43Asp Val Gln Leu Gln Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Pro Phe Ser Val Tyr 20 25 30 Trp Met Tyr Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gln
Ile Asp Ser Gly Gly Tyr Thr Thr Tyr Tyr Thr Asp Ser Val 50 55 60
Lys Gly Arg Phe Ser Ala Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Thr Asp Ser Ile Gly Ser Asn Lys Gly Gln Gly
Thr Gln Val Thr 100 105 110 Val Ser Ser 115 4423DNAArtificial
SequencePrimer CALL001 44gtcctggctg ctcttctaca agg
234523DNAArtificial SequencePrimer CALL002 45ggtacgtgct gttgaactgt
tcc 234629DNAArtificial SequenceAE6 46gatgtgcagc tgcaggagtc
tggaggagg 294735DNAArtificial SequencePrimer FR4FOR 47ggactagtgc
ggccgctgca gacggtgacc tgggt 354815PRTArtificial SequenceFRET
peptide substrate 48Ser Glu Val Asn Leu Asp Ala Glu Phe Arg Lys Arg
Arg Arg Arg 1 5 10 15
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