U.S. patent application number 13/079886 was filed with the patent office on 2012-02-16 for method of detecting progression of a neurodementing disease.
This patent application is currently assigned to Philipps-Universitaet Marburg. Invention is credited to Michael BACHER, Richard DODEL, Marilena MANEA, Michael PRZYBYLSKI, Raluca STEFANESCU.
Application Number | 20120040382 13/079886 |
Document ID | / |
Family ID | 39609117 |
Filed Date | 2012-02-16 |
United States Patent
Application |
20120040382 |
Kind Code |
A1 |
DODEL; Richard ; et
al. |
February 16, 2012 |
METHOD OF DETECTING PROGRESSION OF A NEURODEMENTING DISEASE
Abstract
Isolated, monoclonal, human, anti-.beta.-amyloid antibodies are
provided which bind to dimeric forms of Ab with higher affinity
than to monomeric forms of Ab and when bound to an A.beta.
polypeptide comprising A.beta.(21-37) shield A.beta.(21-37) from
proteolytic digestion. The antibodies were shown to inhibit fibril
formation and reduce plaque size in vivo and to not bind brain
vessel walls. Accordingly, the antibodies are useful in human and
veterinary medicine for the treatment and prophylaxis of
Alzheimer's disease and other neurodementing diseases. Methods of
detecting or measuring the progression of a neurodementing disease
also are provided.
Inventors: |
DODEL; Richard; (Weimar a.d.
Lahn, DE) ; BACHER; Michael; (Coelbe, DE) ;
PRZYBYLSKI; Michael; (Trebur, DE) ; STEFANESCU;
Raluca; (Konstanz, DE) ; MANEA; Marilena;
(Konstanz, DE) |
Assignee: |
Philipps-Universitaet
Marburg
Marburg
DE
|
Family ID: |
39609117 |
Appl. No.: |
13/079886 |
Filed: |
April 5, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12013185 |
Jan 11, 2008 |
7939075 |
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13079886 |
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60884526 |
Jan 11, 2007 |
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60884513 |
Jan 11, 2007 |
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60981675 |
Oct 22, 2007 |
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60981667 |
Oct 22, 2007 |
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Current U.S.
Class: |
435/7.95 ;
435/7.92; 436/501 |
Current CPC
Class: |
A61P 25/28 20180101;
A61P 9/00 20180101; C07K 2317/21 20130101; C07K 2317/56 20130101;
A61P 25/00 20180101; C07K 16/18 20130101; A61K 2039/505 20130101;
C07K 2317/565 20130101 |
Class at
Publication: |
435/7.95 ;
436/501; 435/7.92 |
International
Class: |
G01N 33/566 20060101
G01N033/566 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 11, 2007 |
EP |
07000507.9 |
Jan 11, 2007 |
EP |
07000521.0 |
Oct 22, 2007 |
EP |
07119002.9 |
Oct 22, 2007 |
EP |
07119026.8 |
Claims
1-17. (canceled)
18. A method of detecting or measuring the progression of a
neurodementing disease in a patient, comprising (A) measuring in a
sample from said patient an antibody titer against a first A.beta.
peptide, wherein the first A.beta. peptide comprises at least the
sequence according to A.beta.(30-37) and at most the sequence
according to A.beta.(12-40); (B) measuring in a sample from said
patient an antibody titer against a second A.beta. peptide wherein
the second A.beta. peptide comprises at least the sequence
according to A.beta.(4-10) and at most the sequence according to
A.beta.(1-20); and (C) comparing the titers from steps (A) and
(B).
19. The method of claim 18, wherein said neurodementing disease is
selected from the group consisting of Alzheimer's disease, Down's
syndrome, dementia with Lewy bodies, fronto-temporal dementia,
cerebral amyloid angiopathy and amyloidoses.
20. The method of claim 18, wherein said neurodementing disease is
Alzheimer's disease.
21. The method of claim 18, wherein the first A.beta. peptide
comprises at least the sequence according to A.beta.(21-37).
22. The method of claim 18, further comprising comparing said
patient titers with titers determined for normal and AD patients
whereby a higher titer against the first A.beta. peptide correlates
with a lower risk of development and/or progression of Alzheimer's
disease.
23. The method of claim 18, further comprising comparing said
patient titers with titers determined for normal and AD patients
whereby a higher titer against the first A.beta. peptide, relative
to the titer against the second A.beta. peptide correlates with a
lower risk of development and/or progression of Alzheimer's
disease.
24. The method of claim 18, further comprising comparing said
patient titers with titers determined for normal and AD patients
whereby a higher titer against the second A.beta. peptide
correlates with a higher risk of development and/or progression of
Alzheimer's disease.
25. The method of claim 18, further comprising comparing said
patient titers with titers determined for normal, and AD patients
whereby a higher titer against the second A.beta. peptide, relative
to the titer against the first A.beta. peptide, correlates with a
higher risk of development and/or progression of Alzheimer's
disease.
26. A method of detecting or measuring the progression of a
neurodementing disease in a patient, comprising A) obtaining a
first sample from said patient at a given time point; B) obtaining
a second sample from said patient at later time point; C) measuring
in said first and second samples the antibody titer against an
epitope comprising at least A.beta.(30-37) and at most
A.beta.(12-40); and D) comparing the titers of said first and
second samples.
27. A method of detecting or measuring the progression of a
neurodementing disease in a patient, comprising A) obtaining a
first sample from said patient at a given time point; B) obtaining
a second sample from said patient at later time point; C) measuring
in said first and second samples the antibody titer against an
epitope comprising at least A.beta.(4-10) and at most
A.beta.(1-20); and D) comparing the titers of said first and second
samples.
28. A method of detecting or measuring the progression of a
neurodementing disease in a patient, comprising A) obtaining a
first sample from said patient at a given time point; B) obtaining
a second sample from said patient at later time point; C) measuring
in said first and second samples the antibody titer against an
epitope comprising A.beta.(30-37); and D) comparing the titers of
said first and second samples.
29. (canceled)
Description
INFORMATION ON RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 60/884,513, filed Jan. 11, 2007, and to U.S.
Provisional Application No. 60/884,526 filed Jan. 11, 2007, and to
U.S. Provisional Application No. 60/981,667, filed Oct. 22, 2007,
and to U.S. Provisional Application No. 60/981,675, filed Oct. 22,
2007, and to European Patent Application No. 07000507.9, filed Jan.
11, 2007, and to European Patent Application No. 07000521.0, filed
Jan. 11, 2007, and to European Patent Application No. 07119002.9,
filed Oct. 22, 2007, and to European Patent Application No.
07119026.8, filed Oct. 22, 2007, all of which are hereby
incorporated by reference in their entireties.
BACKGROUND
[0002] Alzheimer's disease (AD), the most common form of dementia
among elderly population (prevalence: 1000/100,000; >65 years),
represents the fourth leading cause of death in the developed
world. Cortical atrophy, neuronal loss, region-specific amyloid
deposition, neuritic plaques, and neurofibrillary tangles are key
neuropathological features in the AD brain. These alterations are
thought to be linked to cognitive decline which clinically defines
AD. Within these markers, neuritic plaques are amyloid
immunoreactive, thioflavin positive, and accompanied by
astrogliosis, microgliosis, cytoskeletal changes, and synaptic
loss. The degree of neuritic degeneration within plaques correlates
with clinical parameters of dementia. Neuritic plaques are
spherical, multicellular lesions that are usually found in moderate
to large numbers in limbic structures and associated neocortices of
the AD brain. These plaques are comprised of extracellular deposits
of amyloid-.beta.peptide(s) (A.beta.) that include abundant amyloid
fibrils intermixed with non-fibrillary forms of the peptide. Such
plaques also contain variable numbers of activated microglia that
are often situated very near the fibrillar amyloid core, as well as
reactive astrocytes surrounding the core.
[0003] The major constituent of the neuritic plaque, .beta.-amyloid
polypeptide (A.beta.), arises from a larger precursor protein, the
amyloid precursor protein (APP) (Kang, et al., 1987; Tanzi, et al.,
1987). A.beta. is produced by normal cells and can be detected as a
circulating peptide in the plasma and cerebrospinal fluid (CSF) of
healthy humans. Although the physiological role of the amyloid
precursor protein (APP) in the brain is not well understood,
missense mutations in APP confer autosomal dominant inheritance of
AD (FAD), and shed light on potentially important pathogenic
mechanism(s). The accumulation of A.beta., a 39-42 amino acid
proteolytic product of APP, in neuritic plaques, structures which
at autopsy fulfill the neuropathological criteria for a definitive
diagnosis of AD, is thought to be causative for disease
progression. A major A.beta. cleavage product of APP is the
A.beta.(1-42) polypeptide, but A.beta. peptides shorter at the
C-terminus (39 to 41) are also produced by the proteolytic
(.gamma.-secretase) cleavage in the membrane. The N-terminal part
of A.beta.(1-42) is localized in the extracellular region of APP,
and the major C-terminal part of the A.beta. peptide is contained
within the transmembrane domain.
[0004] Missense mutations, in APP associated with FAD, occur in
proximity to the A.beta. domain and result in an increase in the
production of the 4 kDa A.beta. peptide. In A.beta., it has been
postulated that increased synthesis and/or a decreased clearance of
A.beta. may lead to amyloid plaque deposition and subsequently to
the neuropathological changes associated with the disease. In vitro
studies, using synthetic A.beta. peptide(s), have shown that
neurotoxicity is dependent on A.beta. being fibrillar and
predominantly in a .beta.-pleated sheet conformation.
[0005] The accumulation of extracellular plaques containing the
neurotoxic amyloid peptide fragment (A.beta.) of .beta.-amyloid
precursor protein (APP), as the major product, is one of the
characteristics of Alzheimer's disease (AD). Although APP has been
recognized as a key molecule for AD, the molecular (patho-)
physiological degradation and proteolytic pathways of APP, and
cellular interactions and biochemical fate of A.beta. peptide(s)
are still unclear. Despite the lack of details on degradation
pathways and cellular transport for the formation and deposition of
A.beta.-derived plaques, recent studies towards the development of
immunisation methods of AD based on therapeutically active
antibodies produced from A.beta.(1-42) have yielded initial success
in transgenic mouse models of Alzheimer's disease. Several reports
have demonstrated that antibodies generated by immunization with
A.beta.(1-42) are capable of inhibiting the formation of
A.beta.-plaques by disaggregating A.beta.-fibrils, and improve the
impairments in the spatial memory of mice. The transgenic APPV717F
mouse (TG mouse) is a well characterized model of AD-like plaque
pathology with age- and region-dependent deposits of A.beta.(1-40)
and A.beta.(1-42) (Games, et al., 1995). Recently, Schenk et al.
and others investigated alterations in the deposition of AD in
APPV717F TG mouse following immunization with pre-aggregated
A.beta.(1-42) or administration of antibodies against A.beta.(Bard,
et al., 2000; Schenk, et al., 1999). Both immunization and
administration of AD antibodies significantly attenuated amyloid
plaque deposition, neuritic dystrophy, and astrogliosis. In these
studies, increased titers of mouse anti-human A.beta.-antibodies
were necessary for the observed reduction in plaque burden. These
findings raise the possibility that formation and clearance of an
A.beta.-antigen: antibody complex may decrease brain A.beta.
deposition either following antibody generation within the central
nervous system or by peripheral antibody transport across the
blood-brain-barrier (BBB). Furthermore, passive immunization
appears to reduce brain A.beta. burden by altering A.beta.
equilibrium between the CNS and plasma (DeMattos, et al., 2001).
Remarkably, active or passive immunization significantly reverses
behavioral and memory impairment in APPV717F mouse or other APP
transgenic mice (Dodart, et al., 2002; Janus, et al., 2000; Morgan,
et al., 2000). These results suggest that immunization may prevent
memory deficits possibly by altering a soluble pool of A.beta..
Thus, treatment of AD patients with active or passive immunization
is one of several emerging therapeutic approaches targeting the
production, clearance, and aggregation of the AD peptide.
[0006] Based on these results, a clinical trial using an active
immunization procedure [A.beta.(1-42) peptide and/or preaggregates
thereof; adjuvant: QS21] was initiated for treatment of patients
with established AD. Unfortunately, severe side-effects developed
("meningoencephalitis") and the clinical trial was stopped. A
subgroup of AD patients (n=30) treated with active immunization in
this clinical trial, was analyzed (Hock, et al., 2002; Hock, et
al., 2003). The authors demonstrated that (i) immunization induces
the production of antibodies against A.beta.(1-42) and (ii) in
patients where a production of antibodies was observable, the
cognitive decline was significantly reduced in comparison to the
untreated control group. The authors concluded that immunization
may be a therapeutic option for AD.
[0007] Recent studies elucidated in more detail the recognition
properties of antibodies produced upon immunization with
A.beta.(1-42). This work resulted in the identification of a
specific A.beta.-epitope recognized by the antibodies generated in
transgenic AD mice (McLaurin et al., 2002; Przybylski et al.,
2003). These results have been obtained by using selective
proteolytic excision technologies (Epitope-Excision) in combination
with high resolution mass spectrometry (FTICR-MS) as bioanalytical
tools of high sensitivity and specificity for the identification of
antigen epitopes (Macht et al 1996; Suckau et al 1992; Macht et al.
2004; see FIGS. 1, 2)). Using mass spectrometric epitope excision
of the immobilized A.beta.-antigen-immune complex, the epitope was
identified to consist of the residues (4-10) (FRHDSGY) of
A.beta.(1-42). The selectivity of this recognition structure was
ascertained by elucidation of the identical epitope from AD
plaques, A.beta.(1-42) extracts from A.beta.-protofibrils,
chemically synthesised A.beta.(1-42), and other
(A.beta.-independent) polypeptides comprising the N-terminal
A.beta. sequence (Przybylski et al. 2003).
[0008] Naturally occurring anti-A.beta. autoantibodies
(A.beta.-autoantibodies) were identified by Du et al. in both the
blood and the CSF from non-immunized humans (Du, et al., 2001).
These antibodies specifically recognize human A.beta. as has been
shown by immunoprecipitation (Du, et al., 2001) and ELISA.
Furthermore, the antibodies readily recognize synthetic
A.beta.(1-40) as well as human A.beta. deposited in the brain of
PDAPP transgenic mice. In addition, fibrillation/oligomerization
and neurotoxicity of A.beta.-peptides were reduced in the presence
of A.beta.-autoantibodies (Du, et al., 2003).
[0009] Furthermore, it has been investigated whether there is a
difference of the A.beta.-autoantibody concentration in patients
with Alzheimer's disease compared to controls. Interestingly, a
significant difference among the two groups was found, resulting in
a substantially decreased titer (approximately 15-20-fold) of
antibodies against A.beta. in patients with Alzheimer's disease.
These results have been confirmed recently by other groups (Weksler
et al., 2004). Antibodies against A.beta. can also be detected in
commercially available intravenous IgG preparations (IVIgG). The
treatment of patients with different neurological diseases with
these intravenous immunoglobulin preparations led to the reduction
of A.beta. concentration in the CSF (Dodel, et al., 2002). The
substantial effect of the A.beta.-autoantibodies in preventing, and
protecting against A.beta.-plaque deposition was also established
in young (4 months) APP-transgenic (TgCRND8) mice. Additionally, in
a pilot trial with 5 patients with AD, utilizing IVIgG, total
A.beta. was reduced significantly in the CSF and increased in the
serum upon delivery of IVIgG (Dodel, et al., 2004). In the five
investigated patients no cognitive deterioration was observed
during the six months observation period. These results have been
confirmed by a recent pilot study involving 8 AD patients, who were
treated with IVIgG (Relkin et al., 2006)
[0010] However, administering IVIgG to a patient with AD is not
convenient and associated with high costs, as the fraction of
therapeutic A.beta. autoantibodies is low. The vast majority of IgG
in this preparation is not A.beta. specific and may result in
undesirable effects. Furthermore, the sources for IVIgG are
limited, which is an unacceptable disadvantage in view of the
prevalence of patients with Alzheimer's disease.
[0011] Methods of detecting and monitoring the progression AD and
other neurodementing diseases similarly are inadequate. Current AD
diagnostics fall into three groups: (i) determinations for genetic
risk factors or mutations (mainly for FAD cases, but not for
sporadic AD diagnostics); (ii) neuroimaging methods; and (iii)
diagnostics based on biochemical/biological markers. Present work
on the development of diagnostic procedures based on biomarkers
have been mainly focused on CSF, which has the principal
disadvantage that such methods require elaborate, invasive
material. A major problem associated with brain-derived biomarkers
is that clinically examined controls often also include subjects
with preclinical AD pathology. Further, current available
biomarkers have the major disadvantage of low specificity. Similar
disadvantages have been noted for a series of proteins expressed in
the frontal cortex, identified by brain proteomics approaches, as
potential brain biomarkers arising from presumed alterations of
blood brain barrier in AD.
[0012] Studies on biomarkers in plasma and serum have been
performed mainly with determinations of SP (senile plaques) and NFT
(neurofibrillary tangles) components, e.g. the A.beta. peptides
A.beta.(1-40) (SEQ ID NO: 1) and A.beta.(1-42) (found with elevated
levels) and hyperphosphorylated Tau-protein. However the
specificity of A.beta. determinations, and application for early
and differential diagnostics has been considered uncertain, the
same is the case for protein Tau determinations which has been
described as a marker of already progressing neurodegeneration.
[0013] There exists, therefore, a need for improved methods of
treating and detecting neurodementing diseases such as AD.
SUMMARY
[0014] In one aspect, isolated, monoclonal, human,
anti-.beta.-amyloid antibodies are provided that comprise more than
one amino acid sequence selected from at least two consensus amino
acid sequences of the group consisting of SEQ ID NO: 6, SEQ ID NO:
7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11,
wherein each of said more than one amino acid sequence is from a
different SEQ ID NO, wherein said antibody binds to dimeric forms
of A.beta. with higher affinity than to monomeric forms of
A.beta..
[0015] In one embodiment, the antibody comprises more than two
amino acid sequences selected from at least three consensus amino
acid sequences of the group consisting of SEQ ID NO: 6, SEQ ID NO:
7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11,
wherein each of said more than two amino acid sequences is from a
different SEQ ID NO. In another, the antibody comprises more than
three amino acid sequences selected from at least four consensus
amino acid sequences of the group consisting of SEQ ID NO: 6, SEQ
ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO:
11, wherein each of said more than three amino acid sequences is
from a different SEQ ID NO. In still another embodiment, the
antibody comprises more than four amino acid sequences selected
from at least five consensus amino acid sequences of the group
consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:
9, SEQ ID NO: 10 and SEQ ID NO: 11, wherein each of said more than
four amino acid sequences is from a different SEQ ID NO. In another
embodiment, the antibody comprises an amino acid sequence from each
of the consensus amino acid sequences of SEQ ID NO: 6, SEQ ID NO:
7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11.
[0016] In one embodiment, the antibody comprises the amino acid
sequences of SEQ ID NO: 33, SEQ ID NO: 41; SEQ ID NO: 45 as
specific light chain CDRs CDR1, CDR2 and CDR3 respectively and SEQ
ID NO: 15; SEQ ID NO: 23 and SEQ ID NO: 29 as specific heavy chain
CDRs CDR1, CDR2 and CDR3 respectively, while in another the
antibody comprises the amino acid sequences of SEQ ID NO: 53 and
SEQ ID NO: 60. In other embodiments, the antibody comprises the
amino acid sequences of SEQ ID NO: 145 and SEQ ID NO: 60, SEQ ID
NO: 51 and SEQ ID NO: 60, SEQ ID NO: 52 and SEQ ID NO: 60, SEQ ID
NO: 146 and SEQ ID NO: 60, SEQ ID NO: 53 and SEQ ID NO: 148, SEQ ID
NO: 55 and SEQ ID NO: 61, or SEQ ID NO: 145 and SEQ ID NO: 62. The
antibody also can comprise the CDRs from the amino acid sequences
of SEQ ID NO: 145 and SEQ ID NO: 60, SEQ ID NO: 51 and SEQ ID NO:
60, SEQ ID NO: 52 and SEQ ID NO: 60, SEQ ID NO: 146 and SEQ ID NO:
60, SEQ ID NO: 53 and SEQ ID NO: 148, SEQ ID NO: 55 and SEQ ID NO:
61, or SEQ ID NO: 145 and SEQ ID NO: 62.
[0017] In another aspect the antibody binds to a peptide comprising
A.beta.(21-37), while in another the antibody shields residues of
A.beta.(21-37) from proteolytic digestion when being bound to an
A.beta. polypeptide comprising A.beta.(21-37). In another, the
antibody binds specifically to A.beta.(12-40) or A.beta.(20-37)
when such antibody is coupled to NHS-activated 6-aminohexanoic
acid-coupled sepharose, but does not bind specifically to
A.beta.(17-28), A.beta.(25-35) or A.beta.(31-40).
[0018] The inventive anti-.beta.-amyloid antibodies also can be
formulated as pharmaceutical compositions.
[0019] In another aspect, methods of preventing or treating a
neurodementing disease in a patient comprise administering to the
patient a therapeutically acceptable amount of the inventive
anti-.beta.-amyloid antibodies. Such methods can be used in
preventing or treating neurodementing diseases selected from the
group consisting of Alzheimer's disease, Down's syndrome, dementia
with Lewy bodies, fronto-temporal dementia, cerebral amyloid
angiopathy and amyloidoses. In a preferred embodiment, the
neurodementing disease is Alzheimer's disease.
[0020] In another embodiment, the inventive anti-.beta.-amyloid
antibodies can be used for the manufacture of a medicament in order
to treat a neurodementing disease or to slow or prevent the
progression of a neurodementing disease.
[0021] In another aspect, methods of detecting or measuring the
progression of a neurodementing disease in a patient are provided
that comprise (A) measuring in a sample from said patient an
antibody titer against a first A.beta. peptide, wherein the first
A.beta. peptide comprises at least the sequence according to
A.beta.(30-37) and at most the sequence according to
A.beta.(12-40); (B) measuring in a sample from said patient an
antibody titer against a second A.beta. peptide wherein the second
A.beta. peptide comprises at least the sequence according to
A.beta.(4-10) and at most the sequence according to A.beta.(1-20);
and (C) comparing the titers from steps (A) and (B). In some
embodiments, the first A.beta. peptide comprises at least the
sequence according to A.beta.(21-37). In another embodiment, the
methods further comprise comparing the patient titers with titers
determined for healthy donors and AD patients whereby a higher
titer against the first A.beta. peptide correlates with a lower
risk of development and/or progression of Alzheimer's disease. The
methods also can comprise comparing the patient titers with titers
determined for healthy donors and AD patients whereby a higher
titer against the first A.beta. peptide, relative to the titer
against the second A.beta. peptide correlates with a lower risk of
development and/or progression of Alzheimer's disease.
Alternatively, the methods can comprise comparing the patient
titers with titers determined for healthy donors and AD patients
whereby a higher titer against the second A.beta. peptide
correlates with a higher risk of development and/or progression of
Alzheimer's disease. In another embodiment, the methods further
comprise comparing the patient titers with titers determined for
healthy donors and AD patients whereby a higher titer against the
second A.beta. peptide, relative to the titer against the first
A.beta. peptide, correlates with a higher risk of development
and/or progression of Alzheimer's disease.
[0022] Methods of detecting or measuring the progression of a
neurodementing disease in a patient also are provided that comprise
A) obtaining a first sample from said patient at a given time
point; B) obtaining a second sample from said patient at later time
point; C) measuring in said first and second samples the antibody
titer against an epitope comprising at least A.beta.(30-37) and at
most A.beta.(12-40); and D) comparing the titers of said first and
second samples. Other such methods comprise A) obtaining a first
sample from said patient at a given time point; B) obtaining a
second sample from said patient at later time point; C) measuring
in said first and second samples the antibody titer against an
epitope comprising at least A.beta.(4-10) and at most
A.beta.(1-20); and D) comparing the titers of said first and second
samples. In other embodiments, such methods comprise A) obtaining a
first sample from said patient at a given time point; B) obtaining
a second sample from said patient at later time point; C) measuring
in said first and second samples the antibody titer against an
epitope comprising A.beta.(30-37); and D) comparing the titers of
said first and second samples.
[0023] In another aspect, there is provided a kit comprising (A) a
first A.beta. peptide comprising at least the sequence according to
A.beta.(30-37) and at most the sequence according to
A.beta.(12-40), and (B) a second A.beta. peptide wherein the second
A.beta. peptide comprising at least the sequence according to
A.beta.(4-10) and at most the sequence according to
A.beta.(1-20).
[0024] The following figures form part of the present specification
and are included to further demonstrate certain aspects of the
present invention. The invention may be better understood by
reference to one or more of these figures in combination with the
detailed description of specific embodiments presented herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1: Principle of epitope-excision and epitope extraction
for mass spectrometric epitope identification. Antibody
immunoglobulin with native, disulfide-bonding is generally highly
resistant to proteolytic digestion by endoproteases (e.g., trypsin,
chymotrypsin, AspN-protease), and the epitope region of antigen
polypeptides comprising the epitope-paratope interaction structure
is generally protected from proteolytic degradation in the immune
complex, while the free nonbinding regions are amenable to
digestion. Thus, the epitope sequence remaining bound to the
antibody after proteolytic removal and washing away nonbinding
structures is then dissociated from the antibody and identified by
mass spectrometry. Both electrospray-ionization (ESI) and
matrix-assisted laser desorption-ionization (MALDI) have been found
useful mass spectrometric methods, and have been applied
successfully for epitope identifications. "TFA" means
trifluoroacetic acid. "MALDI-MS" stands for matrix-assisted laser
desorption-ionisation mass spectrometry.
[0026] FIG. 2: Mass spectrometric identification of proteolytic
peptide fragments of free soluble A.beta. peptide. Without the
complexation by antibody binding, digestion of A.beta.(1-40) by
trypsin leads to formation of all peptide fragments expected
according to the proteolytic cleavage specificity (A.beta.(1-5),
A.beta.(6-16), A.beta.(17-28), A.beta.(17-40), A.beta.(1-16)). Mass
spectrometric analysis is performed by high resolution
MALDI-Fouriertransform-ion cyclotron resonance (MALDI-FTICR-MS),
which provides spectra at approximately 100,000 mass resolution
with complete isotope resolution of ions and mass determination
accuracies of typically 1-5 ppm. All FTICR-MS spectra were obtained
with a Bruker (Bruker Daltonik, Bremen, Germany) Apex II 7T FT-ICR
mass spectrometer equipped with an Apollo II
electrospray/nanoelectrospray multiportion source and an external
Scout 100 fully-automated X-Y target stage MALDI source with pulsed
collision gas. The pulsed nitrogen laser is operated at 337 nm.
Ions generated by laser shots were accumulated in the hexapole for
0.5-1 sec at 15 V and extracted at -7 V into the analyzer cell. A
100 mg/ml solution of 2,5-dihydroxybenzoic acid (DHB, Aldrich,
Germany) in acetonitrile: 0.1% TFA in water (2:1) was used as the
matrix. 0.5 .mu.l of sample solution were mixed on the
stainless-steel MALDI sample target and allowed to dry. Typical ESI
conditions were .about.2 kV needle voltage and 100 nA spray
current. Ions were accumulated in a hexapole for 2 sec and then
transferred into the cylindrical ICR cell. "ppm" stands for parts
per million. "m/z" indicates the mass-to-charge-ratio.
[0027] FIG. 3: Epitope Identification of Amyloid Plaque Specific
Antibody by MALDI-FTICR-MS: Mass spectrometric identification of
N-terminal A.beta.-epitope recognized by plaque-specific antibody
produced upon active immunization of transgenic mice with
A.beta.(1-42) or A.beta.(1-42)-derived aggregates. The immobilized,
purified antibody was incubated with A.beta.(1-40), A.beta.(1-42),
and the immune complex subjected to epitope excision by proteases
trypsin, chymotrypsin, Glu-C protease and Asp-N-protease. The left
spectrum shows the fragment, A.beta.(1-16) remaining bound after
trypsin digestion, the right spectrum shows A.beta.(1-11) after
epitope excision using Glu-C-protease. Black small arrows shown in
the A.beta. sequence shown denote cleavages identified by epitope
excision, fat grey arrows denote substrate cleavage sites on
A.beta. that were found shielded upon antibody binding. Identical
A.beta.(4-10) epitope sequences were identified with soluble
A.beta.-plaques and -protofibrils bound as antigens, and from a
mouse anti-A.beta.(1-16) peptide monoclonal antibody
(Bachem-Peninsula Laboratories, San Francisco).
[0028] FIG. 4: Toxicity of A.beta.-oligomers for human
neuroblastoma cells (SH-Sy5y) in absence or presence of
anti-A.beta.(21-37) autoantibody as described in Example 4. OD:
optical density.
[0029] FIG. 5: 1D-Gel electrophoretic separation of polyclonal
plaque-specific antibodies from an AD patient (AD77), isolated by
A.beta.(4-10) epitope-specific affinity chromatography as described
in Example 1. KDa: Molecular weight in kilodalton. IgG:
Immunoglobulin G. DTT: Dithiothreitol.
[0030] FIG. 6: Identification of A.beta.(21-37) as the epitope
recognized by human anti-A.beta.(21-37)-autoantibodies by epitope
excision-mass spectrometry. The upper graph shows the sequence of
A.beta.(1-40), with cleavages by different proteases indicated by
black arrows. Peptide fragments denoted by solid black arrows above
the A.beta.-sequence were identified after epitope excision using
pronase; peptide fragments denoted by black dotted arrows
underneath the A.beta. sequence were found by epitope excision
using trypsin and Glu-C-protease (R5, E11, K16); note that Arg-5 is
completely shielded in the immune complex with the plaque-specific
antibody, while completely amenable to cleavage in the immune
complex with the anti-A.beta.(21-37)-autoantibody. Cleavage
positions, observed in free A.beta., indicated by broken arrows
were found shielded after binding of
anti-A.beta.(21-37)-autoantibody (Glu-C: E22, D23; trypsin: K28).
The MALDI-MS analysis upon partial digestion (2 hrs) with pronase
is shown here for illustration.
[0031] FIG. 7: Isolation of "plaque-specific" antibodies
recognizing the N-terminal A.beta.(4-10) epitope from the serum
A.beta.-autoantibodies of an Alzheimer patient, isolated by
A.beta.(4-10)-epitope specific chromatography. IgG stands for
immunoglobulin G. AD signifies Alzheimer's Disease. Affinity
column: G5A.beta.(4-10). kDa: Molecular weight in kilodalton.
[0032] FIG. 8: Molecular recognition mechanism of plaque-specific,
plaque-disaggregating A.beta.-antibodies recognizing the
A.beta.(4-10) epitope, and the anti-A.beta.(21-37)-autoantibodies
recognizing the A.beta.(21-37) carboxyterminal epitope.
[0033] FIG. 9: Structure of A.beta.(12-40) epitope-specific
affinity column and experimental procedure for isolation of
anti-A.beta.(21-37) autoantibodies. Cys-A.beta.(12-40): Cysteine
coupled A.beta.(12-40) peptide.
[0034] FIG. 10: Analytical scheme and experimental procedures
employed for sequence determination of affinity-isolated
anti-A.beta.(21-37) autoantibodies: N-terminal protein sequence
analysis; 2D-electrophoretic separation, in-gel proteolytic
digestion and high resolution FTICR-MS identification of constant
region sequences; proteolytic digestion and HPLC separation of
peptide fragments, followed by a) Edman sequence determinations; b)
LC-MS/MS sequence determination; c) MALDI-TOFMS identification of
constant region partial sequences; MALDI-FTICR-MS identification of
constant/variable partial sequences.
[0035] FIG. 11: Analytical scheme of experimental procedure
employed for assignment of heavy- and light chain sequence pairs of
serum-IVIgG anti-A.beta.(21-37) autoantibodies.
[0036] FIG. 12: 2-Dimensional SDS-gel electrophoretic separation of
polyclonal anti-M(21-37) autoantibodies isolated from IVIgG; see
FIG. 22 (a-c) for identification and sequence determination of
A.beta.-antibody isoforms. DTT: Dithiothreitol. CHAPS:
3-[(3-Cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate.
[0037] FIG. 13: 1D-SDS-PAGE isolation of heavy and light chains of
serum IVIgG anti-A.beta.(21-37) autoantibody. a) Reduction
(10000.times.DTT); b) Alkylation (3.times. iodoacetamide/DTT). LMW:
low molecular weight protein standard.
[0038] FIG. 14: 1D-Gel electrophoretic separation of HPLC-isolated
heavy and light chains of anti-A.beta.(21-37) autoantibodies
(serum-IVIgG) used for Edman sequence determinations. LMW: low
molecular weight protein standard.
[0039] FIG. 15: 1D-Gel electrophoretic separation and blotting of
serum IVIgG anti-A.beta.(21-37) autoantibody heavy and light chains
on PVDF membranes for Edman sequence determination.
[0040] FIG. 16: HPLC separation of heavy chain tryptic peptides.
Isolated peptide fractions were subjected to a) Edman sequence
analysis, b) LC-MS/MS sequence determination, c) direct
MALDI-TOF-MS and d) MALDI-FTICR-MS analysis.
[0041] FIG. 17: HPLC separation of light chain tryptic peptides.
Isolated peptide fractions were subjected to a) Edman sequence
analysis, b) LC-MS/MS sequence determination, c) direct
MALDI-TOF-MS and d) and MALDI-FTICR-MS analysis.
[0042] FIG. 18 (a-d): Edman sequence determination of HPLC-isolated
heavy chain tryptic peptide, Serum_IVIG_G1_HC(1).sub.--1 c(348-359;
EPQVYTLPPSR). 18a: Standard. 18b: Residue 1. 18c: Residue 9. 18d:
Residue 11.
[0043] FIG. 19 (a-c): LC-MS/MS sequence determination of heavy
chain tryptic HPLC peptides, fraction 27 (a) and HPLC fraction 39,
heavy chain CDR1 peptide v(20-30). FIG. 19a shows the total ion
chromatogram, the peptide fraction isolated at 1.3-2.1 min elution
time is encircled in red. (b) ESI-mass spectrum of the peptide
fraction isolated at 1.3-2.1 min; (c) MS/MS fragment ion analysis
of the doubly charged precursor ion selected, m/z 482.2.
[0044] FIG. 20 (a, b): MALDI-TOF-MS Identification of tryptic HPLC
peptides. a) identification of tryptic peptide, fraction 50, heavy
chain (138-151), mol. mass 1423; b) identification of peptide
isolated in fraction 75, heavy chain (375-396), mol. mass 2544
Da.
[0045] FIG. 21 (a-c): MALDI-FTICR-Mass spectrometric identification
of heavy chain constant region tryptic peptides from HPLC fractions
47, 66, and 96. (a) identification of 3 peptides isolated in
fraction 47 denoted on the molecular ion peaks, (349-359);
(349-364), (137-151); (b) identification of 2 peptides in fraction
66, (260-278) and 279-292); (c) identification of peptide (306-321)
in fraction 96.
[0046] FIG. 22 (a-c): MALDI-FT-ICR Identification of sequences
comprising the serum IVIgG1 heavy chain constant regions, isolated
from 2D-gel bands subjected to in-gel tryptic digestion; spot 4
heavy chain (22a), spot 12 heavy chain (22b) and spot 13 heavy
chain (22c) as illustrated in FIG. 12. Sequence determinations were
performed using the NCBI data base, at a mass accuracy threshold of
5-10 ppm.
[0047] FIG. 23a to c: Table giving an overview about the identified
and sequenced antibodies specific for a C-terminal part of
A.beta.-peptide, in particular specific for A.beta.(21-37). The
table provides the name of the antibody chain sample, the source of
the sample, the type of immunoglobulin chain sequenced, verified
interactions of light and heavy chains (indicated is the name of
the partner chain as connection) as well as confirmed isoforms of
the respective immunoglobulin chain and the type of CDR sequence
identified for CDR1, CDR2 and CDR3. FIG. 23a: IVIG_(1)_A';
IVIG_(2)_B'; IVIG_(3); IVIG_(4)_A; IVIG_(5)_B; IVIG_(6); IVIG_(7);
IVIG_(8); Serum_(9); Serum_(10); Serum_(11); FIG. 23b: IVIG_(12);
IVIG_(13); IVIG_(14); IVIG_(15); IVIG_(16); IVIG_(17); IVIG_(18);
IVIG_(19); IVIG_(20); IVIG_(21). FIG. 23c: Serum_(22); Serum_(23);
Serum_(24); Serum_(25).
[0048] FIG. 24a to q: Table indicating the identified CDR types and
corresponding consensus sequences for all sequenced antibody
chains. The CDR numbers correspond with the numbering as used in
FIG. 23 for CDRs. FIG. 24a CDRs of heavy chains; FIG. 24b: CDRs of
light chains (lambda chain as well as kappa chain); FIG. 24c: CDR
consensus sequence for the CDR1 of the heavy chain, FIG. 24d: CDR
consensus sequence for the CDR2 of the heavy chain, FIG. 24e: CDR
consensus sequence for the CDR3 of the heavy chain; FIG. 24f: CDR
consensus sequences for CDR1 of the kappa light chain, FIG. 24g:
CDR consensus sequences for CDR2 of the kappa light chain, FIG.
24h: CDR consensus sequence for CDR3 of the kappa light chain, FIG.
24i: preferred consensus sequence for CDR1 of the heavy chain, FIG.
24j: more preferred consensus sequence for CDR1 of the heavy chain,
FIG. 24k: more preferred consensus sequence for CDR1 of the heavy
chain. FIG. 24l: preferred consensus sequence for CDR2 of the heavy
chain, FIG. 24m: more preferred consensus sequence for CDR2 of the
heavy chain, FIG. 24n: more preferred consensus sequence for CDR2
of the heavy chain, FIG. 24o: preferred consensus sequence for CDR3
of the heavy chain, FIG. 24p: more preferred consensus sequence for
CDR3 of the heavy chain, FIG. 24q: more preferred consensus
sequence for CDR3 of the heavy chain.
[0049] FIGS. 25 a to l: Amino acid sequences of light chain
variable region sequences of anti-A.beta.(21-37) autoantibodies
from serum-IVIgG and individual serum anti-A.beta.(21-37)
autoantibodies (see FIG. 23 for sequence overview). Annotation of
codes for sequencing methods employed (Edman; Edman-protein
N-terminal; MALDI-TOF-MS; MALDI-FTICR-MS, LC-MS/MS), and of CDR
sequences is indicated on the bottom of each sequence. CDRs are
indicated by boxes.
[0050] FIG. 25a: amino acid sequence of light chain kappa variable
region of sample IVIG_(1)_A' (SEQ ID NO:47).
[0051] FIG. 25b: amino acid sequence of light chain kappa variable
region of sample IVIG_(2)_B' (SEQ ID NO:48).
[0052] FIG. 25c: amino acid sequence of light chain lambda variable
region of sample IVIG_(3) (SEQ ID NO:49).
[0053] FIG. 25d: amino acid sequence of light chain kappa variable
region of sample IVIG_(6) (SEQ ID NO:50).
[0054] FIG. 25e: amino acid sequence of light chain kappa variable
region of sample IVIG_(7) (SEQ ID NO:51).
[0055] FIG. 25f: amino acid sequence of light chain kappa variable
region of sample IVIG_(8) (SEQ ID NO:52).
[0056] FIG. 25g: amino acid sequence of light chain kappa variable
region of sample Serum_(9) (SEQ ID NO:53).
[0057] FIG. 25h: amino acid sequence of light chain kappa variable
region of sample Serum_(10) (SEQ ID NO:54).
[0058] FIG. 25i: amino acid sequence of light chain kappa variable
region of sample Serum_(11) (SEQ ID NO:55).
[0059] FIG. 25j: amino acid sequence of light chain kappa variable
region of sample Serum_(9) (SEQ ID NO:145).
[0060] FIG. 25k: amino acid sequence of light chain kappa variable
region of sample Serum_(9) (SEQ ID NO:146).
[0061] FIG. 25l: amino acid sequence of light chain kappa variable
region of sample Serum_(9) (SEQ ID NO:147).
[0062] FIG. 26 a to q: Amino acid sequences of heavy chain variable
region sequences of anti-A.beta.(21-37) autoantibodies from
serum-IVIgG and individual serum anti-A.beta.(21-37) autoantibodies
(see FIG. 23, sequence overview). Annotation of codes for
sequencing methods employed (Edman; Edman-protein N-terminal;
MALDI-TOF-MS; MALDI-FTICR-MS, LC-MS/MS), and of CDR sequences is
indicated on the bottom of each sequence. CDRs are indicated by
boxes.
[0063] FIG. 26a: amino acid sequence of heavy chain variable region
of sample IVIG_(4)_A (SEQ ID NO:56).
[0064] FIG. 26b: amino acid sequence of heavy chain variable region
of sample IVIG_(5)_B (SEQ ID NO:57).
[0065] FIG. 26c: amino acid sequence of heavy chain variable region
of sample IVIG_(12) (SEQ ID NO:58).
[0066] FIG. 26d: amino acid sequence of heavy chain variable region
of sample IVIG_(13) (SEQ ID NO:59).
[0067] FIG. 26e: amino acid sequence of heavy chain variable region
of sample IVIG_(14) (SEQ ID NO:60).
[0068] FIG. 26f: amino acid sequence of heavy chain variable region
of sample IVIG_(15) (SEQ ID NO:61).
[0069] FIG. 26g: amino acid sequence of heavy chain variable region
of sample IVIG_(16) (SEQ ID NO:62).
[0070] FIG. 26h: amino acid sequence of heavy chain variable region
of sample IVIG_(17) (SEQ ID NO:63).
[0071] FIG. 26i: amino acid sequence of heavy chain variable region
of sample IVIG_(18) (SEQ ID NO:64).
[0072] FIG. 26j: amino acid sequence of heavy chain variable region
of sample IVIG_(19) (SEQ ID NO:65).
[0073] FIG. 26k: amino acid sequence of heavy chain variable region
of sample IVIG_(20) (SEQ ID NO:66).
[0074] FIG. 261: amino acid sequence of heavy chain variable region
of sample IVIG_(21) (SEQ ID NO:67).
[0075] FIG. 26m: amino acid sequence of heavy chain variable region
of sample Serum_(22) (SEQ ID NO:68).
[0076] FIG. 26n: amino acid sequence of heavy chain variable region
of sample Serum_(23) (SEQ ID NO:69).
[0077] FIG. 26o: amino acid sequence of heavy chain variable region
of sample Serum_(24) (SEQ ID NO:70).
[0078] FIG. 26p: amino acid sequence of heavy chain variable region
of sample Serum_(25) (SEQ ID NO:71).
[0079] FIG. 26q: amino acid sequence of heavy chain variable region
of sample IVIG_(14) (SEQ ID NO:148).
[0080] FIG. 27a to c: Amino acid sequences of the constant region
of kappa and lambda light chains of anti-A.beta.(21-37)
autoantibody chains. The mutation of the LC-kappa-constant region
sequence at V192L is indicated in bold letters.
[0081] FIG. 27a: Complete amino acid sequence of constant region
light chain kappa isoform 1 (SEQ ID NO:72).
[0082] FIG. 27b: Complete amino acid sequence of constant region
light chain kappa of isoform 2 (SEQ ID NO:73).
[0083] FIG. 27c: Complete amino acid sequence of constant region
light chain lambda (SEQ ID NO:74).
[0084] FIG. 28a to c: Amino acid sequences and sequence isoforms of
the constant region identified for IVIgG-anti-A.beta.(21-37)
autoantibody heavy chains. Amino acid mutations identified at
F300Y, N301A (N-glycosylation site), F304Y, G331A, D360E, L362M,
S368T, and V401M are indicated by bold letters. The N-glycosylation
consensus sequence and site at, N-301.sup.ST, are indicated by
shaded box and bold grey letter.
[0085] FIG. 28a: Complete amino acid sequence of constant region
heavy chain of isoform 1 (SEQ ID NO:75).
[0086] FIG. 28b: Complete amino acid sequence of constant region
heavy chain of isoform 2 (SEQ ID NO:76).
[0087] FIG. 28c: Complete amino acid sequence of constant region
heavy chain isoform 3 (SEQ ID NO:77).
[0088] FIG. 29 a to p: Complete amino acid sequences of light
chains of anti-A.beta.(21-37) autoantibodies from serum-IVIgG and
individual serum (see FIG. 23 for sample overview). Annotation of
codes for sequencing methods employed (Edman; Edman-protein
N-terminal; MALDI-TOF-MS; MALDI-FTICR-MS, LC-MS/MS), and of CDR
sequences is indicated on the bottom of each sequence. CDRs are
indicated by boxes. Variable sequence domains, and single amino
acid residues in the constant region sequences found with single
site mutations are indicated in bold letters.
[0089] FIG. 29a: complete amino acid sequence of light chain kappa
of sample IVIG_(1)_A', constant region isoform 1 (SEQ ID
NO:78).
[0090] FIG. 29b: complete amino acid sequence of light chain kappa
of sample IVIG_(1)_A', constant region isoform 2 (SEQ ID
NO:79).
[0091] FIG. 29c: complete amino acid sequence of light chain kappa
of sample IVIG_(2)_B', constant region isoform 1 (SEQ ID
NO:80).
[0092] FIG. 29d: complete amino acid sequence of light chain kappa
of sample IVIG_(2)_B', constant region isoform 2 (SEQ ID
NO:81).
[0093] FIG. 29e: complete amino acid sequence of light chain lambda
of sample IVIG_(3) (SEQ ID NO:82).
[0094] FIG. 29f: complete amino acid sequence of light chain kappa
of sample IVIG_(6), constant region isoform 1 (SEQ ID NO:83).
[0095] FIG. 29g: complete amino acid sequence of light chain kappa
of sample IVIG_(6), constant region isoform 2 (SEQ ID NO:84).
[0096] FIG. 29h: complete amino acid sequence of light chain kappa
of sample IVIG_(7), constant region isoform 1 (SEQ ID NO:85).
[0097] FIG. 29i: complete amino acid sequence of light chain kappa
of sample IVIG_(7), constant region isoform 2 (SEQ ID NO:86).
[0098] FIG. 29j: complete amino acid sequence of light chain kappa
of sample IVIG_(8), constant region isoform 1 (SEQ ID NO:87).
[0099] FIG. 29k: complete amino acid sequence of light chain kappa
of sample IVIG_(8), constant region isoform 2 (SEQ ID NO:88).
[0100] FIG. 291: complete amino acid sequence of light chain kappa
of sample Serum_(9), constant region isoform 1 (SEQ ID NO:89).
[0101] FIG. 29m: complete amino acid sequence of light chain kappa
of sample Serum_(9), constant region isoform 2 (SEQ ID NO:90).
[0102] FIG. 29n: complete amino acid sequence of light chain kappa
of sample Serum_(10), constant region isoform 1 (SEQ ID NO:91).
[0103] FIG. 29o: complete amino acid sequence of light chain kappa
of sample Serum_(11), constant region isoform 1 (SEQ ID NO:92).
[0104] FIG. 29p: complete amino acid sequence of light chain kappa
of sample Serum_(11), constant region isoform 2 (SEQ ID NO:93).
[0105] FIGS. 30-1 to 30-44: Complete amino acid sequences of heavy
chains of anti-A.beta.(21-37) autoantibodies from serum-IVIgG and
individual serum (see FIG. 23 for sample overview). Annotation of
codes for sequencing methods employed (Edman; Edman-protein
N-terminal; MALDI-TOF-MS; MALDI-FTICR-MS, LC-MS/MS), and of CDR
sequences is indicated on the bottom of each sequence. CDRs are
indicated by boxes. Variable sequence domains, and single amino
acid residues in the constant region sequences found with single
site mutations are indicated in bold letters. The N-glycosylation
site, N-301 is indicated in bold, grey letter.
[0106] FIG. 30-1: complete amino acid sequence of heavy chain of
sample IVIG_(4)_A, constant region isoform 1 (SEQ ID NO:94).
[0107] FIG. 30-2: complete amino acid sequence of heavy chain of
sample IVIG_(4)_A, constant region isoform 2 (SEQ ID NO:95).
[0108] FIG. 30-3: complete amino acid sequence of heavy chain of
sample IVIG_(4)_A, constant region isoform 3 (SEQ ID NO:96).
[0109] FIG. 30-4: complete amino acid sequence of heavy chain of
sample IVIG_(5)_B, constant region isoform 1 (SEQ ID NO:97).
[0110] FIG. 30-5: complete amino acid sequence of heavy chain of
sample IVIG_(5)_B, constant region isoform 2 (SEQ ID NO:98).
[0111] FIG. 30-6: complete amino acid sequence of heavy chain of
sample IVIG_(5)_B, constant region isoform 3 (SEQ ID NO:99).
[0112] FIG. 30-7: complete amino acid sequence of heavy chain of
sample IVIG_(12)_B, constant region isoform 1 (SEQ ID NO:100).
[0113] FIG. 30-8: complete amino acid sequence of heavy chain of
sample IVIG_(12)_B, constant region isoform 2 (SEQ ID NO:101).
[0114] FIG. 30-9: complete amino acid sequence of heavy chain of
sample IVIG_(12)_B, constant region isoform 3 (SEQ ID NO:102).
[0115] FIG. 30-10: complete amino acid sequence of heavy chain of
sample IVIG_(13), constant region isoform 1 (SEQ ID NO:103).
[0116] FIG. 30-11: complete amino acid sequence of heavy chain of
sample IVIG_(13), constant region isoform 2 (SEQ ID NO:104).
[0117] FIG. 30-12: complete amino acid sequence of heavy chain of
sample IVIG_(13), constant region isoform 3 (SEQ ID NO:105).
[0118] FIG. 30-13: complete amino acid sequence of heavy chain of
sample IVIG_(14), constant region isoform 1 (SEQ ID NO:106).
[0119] FIG. 30-14: complete amino acid sequence of heavy chain of
sample IVIG_(14), constant region isoform 2 (SEQ ID NO:107).
[0120] FIG. 30-15: complete amino acid sequence of heavy chain of
sample IVIG_(14), constant region isoform 3 (SEQ ID NO:108).
[0121] FIG. 30-16: complete amino acid sequence of heavy chain of
sample IVIG_(15), constant region isoform 1 (SEQ ID NO:109).
[0122] FIG. 30-17: complete amino acid sequence of heavy chain of
sample IVIG_(15), constant region isoform 2 (SEQ ID NO:110).
[0123] FIG. 30-18: complete amino acid sequence of heavy chain of
sample IVIG_(15), constant region isoform 3 (SEQ ID NO:111).
[0124] FIG. 30-19: complete amino acid sequence of heavy chain of
sample IVIG_(16), constant region isoform 1 (SEQ ID NO:112).
[0125] FIG. 30-20: complete amino acid sequence of heavy chain of
sample IVIG_(16), constant region isoform 2 (SEQ ID NO:113).
[0126] FIG. 30-21: complete amino acid sequence of heavy chain of
sample IVIG_(16), constant region isoform 3 (SEQ ID NO:114).
[0127] FIG. 30-22: complete amino acid sequence of heavy chain of
sample IVIG_(17), constant region isoform 1 (SEQ ID NO:115).
[0128] FIG. 30-23: complete amino acid sequence of heavy chain of
sample IVIG_(17), constant region isoform 2 (SEQ ID NO:116).
[0129] FIG. 30-24: complete amino acid sequence of heavy chain of
sample IVIG_(17), constant region isoform 3 (SEQ ID NO:117).
[0130] FIG. 30-25: complete amino acid sequence of heavy chain of
sample IVIG_(18), constant region isoform 1 (SEQ ID NO:118).
[0131] FIG. 30-26: complete amino acid sequence of heavy chain of
sample IVIG_(18), constant region isoform 2 (SEQ ID NO:119).
[0132] FIG. 30-27: complete amino acid sequence of heavy chain of
sample IVIG_(18), constant region isoform 3 (SEQ ID NO:120).
[0133] FIG. 30-28: complete amino acid sequence of heavy chain of
sample IVIG_(19), constant region isoform 1 (SEQ ID NO:121).
[0134] FIG. 30-29: complete amino acid sequence of heavy chain of
sample IVIG_(19), constant region isoform 2 (SEQ ID NO:122).
[0135] FIG. 30-30: complete amino acid sequence of heavy chain of
sample IVIG_(19), constant region isoform 3 (SEQ ID NO:123).
[0136] FIG. 30-31: complete amino acid sequence of heavy chain of
sample IVIG_(20), constant region isoform 1 (SEQ ID NO:124).
[0137] FIG. 30-32: complete amino acid sequence of heavy chain of
sample IVIG_(20), constant region isoform 2 (SEQ ID NO:125).
[0138] FIG. 30-33: complete amino acid sequence of heavy chain of
sample IVIG_(20), constant region isoform 3 (SEQ ID NO:126).
[0139] FIG. 30-34: complete amino acid sequence of heavy chain of
sample IVIG_(21), constant region isoform 1 (SEQ ID NO:127).
[0140] FIG. 30-35: complete amino acid sequence of heavy chain of
sample IVIG_(21), constant region isoform 2 (SEQ ID NO:128).
[0141] FIG. 30-36: complete amino acid sequence of heavy chain of
sample IVIG_(21), constant region isoform 3 (SEQ ID NO:129).
[0142] FIG. 30-37: complete amino acid sequence of heavy chain of
sample Serum_(22), constant region isoform 1 (SEQ ID NO:130).
[0143] FIG. 30-38: complete amino acid sequence of heavy chain of
sample Serum_(22), constant region isoform 2 (SEQ ID NO:131).
[0144] FIG. 30-39: complete amino acid sequence of heavy chain of
sample Serum_(23), constant region isoform 1 (SEQ ID NO:132).
[0145] FIG. 30-40: complete amino acid sequence of heavy chain of
sample Serum_(23), constant region isoform 2 (SEQ ID NO:133).
[0146] FIG. 30-41 complete amino acid sequence of heavy chain of
sample Serum_(24), constant region isoform 1 (SEQ ID NO:134).
[0147] FIG. 30-42: complete amino acid sequence of heavy chain of
sample Serum_(24), constant region isoform 3 (SEQ ID NO:135).
[0148] FIG. 30-43 complete amino acid sequence of heavy chain of
sample Serum_(24), constant region isoform 1 (SEQ ID NO:136).
[0149] FIG. 30-44: complete amino acid sequence of heavy chain of
sample Serum_(24), constant region isoform 3 (SEQ ID NO:137).
[0150] FIG. 31: Table illustrating the conserved nature of the
N-terminus of kappa light chain of the antibodies sequenced for the
present invention. Indicated are the 6 types of N-terminal
sequences, consisting of 18 amino acid residues, which were
identified in the kappa light chain sequences of the antibodies of
the present invention.
[0151] FIGS. 32a and b: Scheme of intra- and inter-disulfide
linkages of anti-A.beta.(21-37) autoantibodies for HC-LC-kappa and
HC-LC-lambda connections. HC intradisulfide linkages are C21-C96,
C148-C204, C265-C325, C371-C429; LC-kappa-intradisulfide linkages
are C23-C89, C135-C195; LC-lambda-intradisulfide linkages are
C22-C92, C142-C201; HC-HC interdisulfide linkages are C230-C230,
C233-C33; HC-LC-kappa- and HC-LC-lambda-interdisulfide linkages are
C224-C215 and C224-C219, respectively. 32a: IVIgG_LC(1)_HC(1); 32b:
IVIG_HC(1)_LC.lamda.(3).
[0152] FIG. 33: Western blot showing that the recombinant
anti-A.beta.(21-37) autoantibody CSL-Clone 7 immunoprecipitates
oligomeric forms of A.beta.1-40 as described in Example 6. The
antibody Bam 90.1 (Sigma Aldrich Cat# A8978 binding to
A.beta.(13-28)) was used to detect the immunoprecipitated
A.beta..
[0153] FIG. 34a: Molecular confirmation of epitope recognition
specificity of A.beta.-autoantibody. Illustrated is the affinity of
3 synthetic A.beta.-polypeptides A.beta.(4-10), A.beta.(20-30) and
A.beta.(20-37) towards anti-A.beta.-autoantibodies isolated from
serum of healthy (non-AD control individuals) donors, A and B, by
MALDI-mass spectrometry. Affinity-purified antibodies were
immobilized on NHS-sepharose as described in Example 2A
(A.beta.12-40). Equimolar mixtures (5 .mu.mol mixtures of synthetic
A.beta.-peptides in aqueous PBS buffer solution, pH 7) were bound
to the antibodies after mass spectrometric analysis (MALDI-MS of
peptide mixture, upper panel). MALDI-MS of the supernatant washing
fraction revealed the N-terminal A.beta.(4-10) epitope signal as
the predominant ion (confirming the lack of binding of N-terminal
A.beta.; middle panel), and washing was continued until no MS
signal was detectable. After elution with 0.1% trifluoroacetic
acid, the A.beta.(20-37) peptide was identified as the only
polypeptide capable of binding to the autoantibodies (lower panel).
All MS determinations were made with a Broker Bilflex MALDI-TOF
spectrometer.
[0154] FIG. 34b: Mass spectrograms showing epitope specificity of
A.beta.-autoantibody. Immobilized A.beta.(21-37) autoantibodies
purified from IVIgG according to Example 2A were incubated with a
synthetic A.beta.(12-40) polypeptide. The elution profiles were
analyzed via MS as above. The data show that the A.beta.(21-37)
autoantibodies specifically bound the A.beta.(12-40)
polypeptide.
[0155] FIG. 34c: Mass spectrograms showing epitope specificity of
A.beta.-autoantibody. Immobilized A.beta.(21-37) autoantibodies
purified from IVIgG were incubated with synthetic
A.beta.-polypeptides A.beta.(25-35), A.beta.(17-28) and
A.beta.(31-40). The data show that the A.beta.(21-37)
autoantibodies bound none of the A.beta. partial polypeptides.
[0156] FIG. 34.sub.--d to 34.sub.--l: Mass spectrograms showing
epitope specificity of A.beta.-autoantibody. Immobilized
A.beta.(21-37) autoantibodies purified from IVIgG and immobilized
antibody ACA (see example 5) were incubated with synthetic
polypeptides A.beta.(4-10), A.beta.(17-28), A.beta.(12-40) and
A.beta.(20-37). The data show that both the immobilized ACA
antibody and the immobilized A.beta.(21-37) autoantibodies bind to
A.beta.(1-40) and to A.beta.(12-40) but that only the immobilized
A.beta.(21-37) autoantibodies specifically bind to A.beta.(20-37).
Both immobilized antibodies did not bind A.beta.(17-28).
Specifically, FIG. 34.sub.--d shows that mab ACA does not bind to
A.beta.(4-10), FIG. 34.sub.--e shows that mab ACA does not bind to
A.beta.(17-28), FIG. 34.sub.--f shows that mab ACA does bind to
A.beta.(12-40), FIG. 34.sub.--g shows that mab ACA does not bind to
A.beta.(20-37), FIG. 34.sub.--h shows that mab ACA does bind to
A.beta.(1-40), FIG. 34.sub.--i shows that A.beta.(21-37)
autoantibodies do bind to A.beta.(1-40), FIG. 34j shows that
A.beta.(21-37) autoantibodies do bind to A.beta.(12-40), FIG.
34.sub.--k shows that A.beta.(21-37) autoantibodies do not bind to
A.beta.(17-28) and FIG. 34.sub.--l shows that A.beta.(21-37)
autoantibodies do bind to A.beta.(20-37).
[0157] FIG. 35: Serum ELISA for determination of
anti-A.beta.(21-37) autoantibodies. BSA is bovine serum albumin.
HRP is horseradish peroxidase. OPD is o-phenylenediamine. IgG
stands for immunoglobulin G.
[0158] FIG. 36: ELISA determination of A.beta.-autoantibody (from
IVIgG). IVIgG stands for intravenous IgG preparation. The ELISA was
carried out with A.beta.(1-40) coated on 96-well plate, and
dilutions of A.beta.-antibody were added, and determined with
anti-human horseradish peroxidase-conjugated secondary antibody.
A.beta.-antibody quantifications were performed with a 1
.mu.g/.mu.l stock solution, using a BSA reference curve for
calibration. The percentage indicated represents the
A.beta.-antibody concentrations in IVIgG from two separate ELISA
determinations.
[0159] FIG. 37: Western blot showing that affinity purified IVIgG
according to Example 4 immunoprecipitates oligomeric forms of
A.beta.1-40 as described in Example 6. The antibody Bam 90.1 (Sigma
Aldrich Cat# A8978 binding to A.beta.(13-28)) was used to detect
the immunoprecipitated A.beta..
[0160] FIG. 38: Bar graph representing the mean total plaque area
per antibody used in an AD animal model as described in Example 13.
Black columns represent the plaque area in the cortex, white bars
represent the plaque area in the hippocampus. Plaque area was
measured using the Nikon NIS Elements Software on pictures of
immunostained brain slices of the treated animals. The measured
plaque area of the CSL 360- or CSL Clone 7-treated animals (N=2)
were averaged for both animals for comparison with the
affinity-purified IVIgG-treated animal (N=1).
[0161] FIG. 39: ELISA data showing that anti-A.beta.(21-37)
autoantibody CSL-Clone 7 binds to A.beta.(1-40) and to
A.beta.(12-40) peptides but not to A.beta.(4-10) as discussed in
Example 9D.
[0162] FIG. 40: The effect of 3 different A.beta.-specific
antibodies: A.beta. affinity column purified human IVIgG (as
described in Example 4), the human monoclonal A.beta. autoantibody
CSL Clone 7 (as described in Example 5) and humanized murine
monoclonal antibody raised against a midterminal A.beta. peptide
sequence (AK ACA, as described in Example 5) to inhibit A.beta.
fibril formation as measured by THT fluorescence staining as
described in Example 10. The fluorescence of the THT assay is
proportional to fibrillar Ab and was used to assess fibril
morphology. The fluorescence of A.beta.(1-40) incubated in the
presence of a nonspecific human monoclonal (CSL360) was set to
100%.
[0163] FIG. 41: Dot Blot Analysis as described in Example 11A.
Samples were tested with control antibodies (6E10, Bam90.1, CSL
Clone 7, affinity purified IVIG, ACA), serum from an AD-patient
(AD1), serum from an age matched healthy human individual (K4) as
described in Example 11A
[0164] FIG. 42: IgG from serum samples (one AD positive sample and
one age-matched control sample) after purification on Protein G
(Pierce) were loaded on an A.beta.(1-16) column, washed and eluted
with 100 mM Glycine pH 2.8. The eluate was analyzed in a
Biotin-G.sub.5-A.beta.(4-10) ELISA as described in Example 11B.
[0165] FIG. 43: Binding to A.beta.(1-40 Cys) dimer as opposed to
A.beta.(1-40) monomer as described in Example 12C for the
recombinant A.beta.(21-37) autoantibodies 55/61, 146/61 and the
control antibody ACA
[0166] FIG. 44: Binding to A.beta.(1-40 Cys) dimer as opposed to
A.beta.(1-40) monomer as described in Example 12C for the
recombinant A.beta.(21-37) autoantibodies 54/61, 47/56, 51/60 and
53/60.
[0167] FIG. 45: Binding to A.beta.(1-40 Cys) dimer as opposed to
A.beta.(1-40) monomer as described in Example 12C for the
recombinant A.beta.(21-37) autoantibodies 146/60, 52/60, 53/148 and
145/60.
[0168] FIG. 46: Tricine Gel protein blot analysis of .beta. amyloid
peptides. Protein visualisation of .beta. amyloid peptide was done
using standard coomassie staining techniques as described in the
Novex gel manual (Invitrogen) and deep purple reagent for high
sensitivity. Deep purple (GE, Sweden) was visualised using a
Typhoon scanner as per manufacturer instruction.
[0169] FIG. 47: Western blot comparing the binding of the
antibodies 6E10, ACA and CSL Clone 7 to A.beta.(1-40) monomer,
A.beta.(1-40) oligomer and A.beta.(1-40 Cys) oligomer as described
in Example 12D.
[0170] FIG. 48a: Immunohistochemistry of a human brain sample of a
patient suffering from Alzheimers disease using the 6F3D anti
.beta.-amyloid antibody (Dako) as primary antibody, and the
Vectastain.RTM. M.O.M.-Kit (HRP anti mouse) as a detection system.
A specific immunostaining is detectable in the vessel wall (arrow)
as well as the Alzheimer-plaque (arrowheads). This immunostaining
serves as positive control.
[0171] FIG. 48b: Immunohistochemistry of a human brain sample of a
patient suffering from Alzheimers disease using the ACA antibody as
primary antibody, and the Vectastain.RTM. Elite ABC Kit (HRP anti
human) as detection system. A specific immunostaining is detectable
in the vessel wall (arrow).
[0172] FIG. 48c: Immunohistochemistry of a human brain sample of a
patient suffering from Alzheimers disease using the ACA antibody as
primary antibody, and the Vectastain.RTM. Elite ABC Kit (HRP anti
human) as detection system. A specific immunostaining is detectable
in the vessel wall (arrow) as well as the Alzheimer-plaque
(arrowheads, insert).
[0173] FIG. 48d: Immunohistochemistry of a human brain sample of a
patient suffering from Alzheimers disease and CAA using the
affinity purified IVIgG as primary antibody, and the
Vectastain.RTM. Elite ABC Kit (HRP anti human) as detection system.
No specific immunostaining is detectable in the vessel wall
(arrow).
[0174] FIG. 48e: Immunohistochemistry of a human brain sample of a
patient suffering from Alzheimers disease using the clone 7
antibody as primary antibody, and the Vectastain.RTM. Elite ABC Kit
(HRP anti human) as detection system. No specific immunostaining is
detectable in the vessel wall (arrow).
[0175] FIG. 48f: Immunohistochemistry of a human brain sample of a
patient suffering from Alzheimers disease using the CSL 360
antibody as primary antibody, and the Vectastain.RTM.Elite ABC Kit
(HRP anti human) as detection system. No specific immunostaining is
detectable in the vessel wall (arrow), but the blood in the vessel
lumen show unspecific background staining.
[0176] FIG. 49: Toxicity of A.beta.-oligomers for human
neuroblastoma cells (SH-Sy5y) was tested as described in Example 4.
The experiment was repeated with affinity purified IVIgG (purified
as described above, mab CSL Clone 7 (see Example 5). As a negative
control the antibodies CSL360 (see Example 10) or no antibody was
used. A positive control antibody used was ACA (see Example 10).
Results clearly show a dose dependent effectiveness of protecting
cells from the neurotoxic effects of A.beta. oligomers of both the
affinity purified mab CSL Clone 7 and affinity purified IVIgG.
DETAILED DESCRIPTION
[0177] A.beta.-autoantibodies were isolated from the serum of AD
patients and healthy controls or pooled commercially obtainable
serum immunoglobulin (IVIgG). The cDNA and amino acid sequences of
the variable regions of the heavy and light chains were determined
and all possible pairings of heavy and light chains were expressed
in mammalian cells. A number of these pairings were found to bind
with higher affinity to A.beta. dimers than to A.beta. monomers and
one of these, CSL clone 7, was shown to possess biological
activities potentially useful for the treatment of AD. The
inventors further discovered that the CDRs of anti-A.beta.(21-37)
autoantibodies are highly homologous. Accordingly, consensus CDRs
were determined and used to prepare human anti-.beta.-amyloid
antibodies useful for preventing or treating neurodementing
diseases like AD. The inventors also surprisingly discovered that
AD patients, as compared to healthy controls, have an increased
antibody titer against A.beta.(4-10) and a decreased antibody titer
against A.beta.(21-37). Thus, the inventors discovered not only
means for detecting and measuring the progression of a
neurodementing disease like AD, but also methods for delaying the
onset or progression of AD. Kits for detecting and measuring the
progression of neurodementing diseases, like AD, also are
provided.
DEFINITIONS
[0178] The term "A.beta. polypeptide" as used herein, defines a
polypeptide having the amino acid sequence SEQ ID NO:01 or
fragments thereof. Such fragments in particular comprise
polypeptides having the sequence SEQ ID NO:02.
[0179] The term "antibody", as used herein, comprises also
derivatives and/or fragments of antibodies. Such derivatives or
fragments of antibodies retain the antigen binding properties of
the intact antibody, but which lack some sequences of the intact
antibody, for example the Fc-domain. Examples for such derivatives
or fragments include, but are not limited to, Fab or F(ab').sub.2
fragments, which are obtainable via enzymatic digest of antibodies
with papain or pepsin protease, respectively, single chain variable
fragments (scFv), Fv fragments, minibodies and diabodies.
[0180] The term "autoantibody" or "autoantibodies", as used herein,
refers in general to antibodies which are directed against epitopes
on proteins of the human body and which can be found in the blood
or cerebrospinal fluid of a human subject without prior
immunization with the respective antigen. Meanwhile, the term
"anti-A.beta.(21-37) autoantibody" refers to autoantibodies that
bind to an A.beta. peptide comprising A.beta.(21-37) (SEQ ID NO:2)
and shield said SEQ ID NO 2 from proteolytic digestion. Such
anti-A.beta.(21-37) autoantibodies also bind with a higher affinity
to dimers of A.beta. than to corresponding monomers of A.beta..
[0181] The term "CDR", as used herein, refers to Complementarity
Determining Regions. Usually 3 of such CDR-regions (CDR1, CDR2,
CDR3) can be found in the variable region on the light chain as
well as on the heavy chain of an antibody. Each of these six
hypervariable regions can contribute to the antigen specificity of
the antibody. However, as used herein, the term CDR does not imply
that the molecule referred to is in fact an antibody. Rather, the
term is considered to designate a sequence contributing to the
specific binding of a polypeptide according to the invention to a
C-terminal part of full length A.beta. polypeptide (A.beta.1-40),
in particular contributing to the binding to A.beta.(21-37)
polypeptide. Consequently, also derivatives of antibodies or other
polypeptides engineered for binding to said epitope can exhibit
CDRs.
[0182] The term "consensus CDR" as used herein refers to a single
sequence derived by aligning two or more sequences for a given CDR
according to the Kabat numbering system. (see Kabat, E. A., Wu, T.
T., Perry, H. M., Gottesman, K. S. & Foeller, C. (1991)
Sequences of Proteins of Immunological Interest (Department of
Health and Human Services, Public Health Service, National
Institutes of Health, Bethesda, Md.) NH Publ. No 91-3442 5th Ed.
and R. Kontermann, S. Dubel (eds.), Antibody Engineering; Springer
Lab Manual Series; Springer, Heidelberg 2001, both of which are
hereby incorporated by reference.) Accordingly, for each amino acid
position of the "consensus CDR", the identity of amino acids which
can occur at that position is determined. CDR designations as well
as amino acid insertions are made according to Kabat numbering.
[0183] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy chain
variable domain (VH) connected to a light chain variable domain
(VL) in the same polypeptide chain (VH VL). By using a linker that
is too short to allow pairing between the two domains on the same
chain, the domains are forced to pair with the complementary
domains of another chain and create two antigen-binding sites.
Diabodies are described more fully in, for example, EP 404,097; WO
93/11161.
[0184] The term "epitope" or "epitope peptide", as used herein,
generally refers to a polypeptide comprising the molecular
recognition peptide sequence or structure derived from an antigen,
that is bound by a specific antibody. It is an immunological
determinant group of an antigen which is specifically recognized by
the antibody. An epitope may comprise at least 5, preferably at
least 8 amino acids in a spatial or discontinuous conformation. An
epitope may also comprise a single segment of a polypeptide chain
comprising a continuous linear amino acid sequence with a minimal
length of approx. 5 amino acids.
[0185] "Fv" is the minimum antibody fragment that contains a
complete antigen recognition and binding site. This region consists
of a dimer of one heavy and one light chain variable domain in
tight, non-covalent association. It is in this configuration that
the three CDRs of each variable domain interact to define an
antigen binding site on the surface of the VH VL dimer.
Collectively, the six CDRs confer antigen binding specificity to
the antibody.
[0186] The term "moiety", as used herein, refers to a portion of a
polypeptide with distinct function(s). Such moieties can provide
for a structural or functional feature which is normally not
present in the rest of the polypeptide or which feature is enhanced
by this moiety. Such functional or structural moieties can for
example provide binding, stabilization or detection of the
polypeptide. The moiety can be a polypeptide on its own or can be
any other compound, which provides the desired function(s) to the
polypeptide. Said moiety is stably associated with the polypeptide,
in particular covalently coupled to the polypeptide. The term
moiety, as used herein, does not confer any information about the
size of this portion in comparison to the polypeptide itself. The
moiety can be smaller, equally sized or larger than the polypeptide
it is coupled to.
[0187] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a specific
antigenic site or epitope, in contrast to polyclonal antibody
preparations that typically include different antibodies directed
against different epitopes.
[0188] The terms "neurodementing diseases", "dementing disorders"
or "neurodementia diseases of AD-type", as used herein, refer to
diseases selected from Alzheimer's disease, Down's syndrome,
Dementia with Lewy bodies, fronto-temporal dementia as well as to
disorders such as cerebral amyloid angiopathy and amyloidoses.
[0189] The term "oligomerized", "oligomers" and "oligomeric" refers
to multimers of A.beta. comprising also A.beta. dimers, trimers,
tetramers and higher oligomers but not to A.beta. fibrils.
[0190] The term "plaque-specific" antibody, as used herein, refers
to an antibody directed against the A.beta.(4-10) epitope of
A.beta. polypeptide.
[0191] The term "polypeptide", as used herein, refers to a
polypeptide chain of at least 5 amino acid residues. The term also
refers to an assembly of more than one polypeptide chain, e.g. an
assembly of four polypeptide chains, such as an IgG antibody.
[0192] The term "paratope" or "paratope peptide", as used herein,
generally defines a molecular recognition peptide sequence derived
from a specific monoclonal or polyclonal antibody. This recognition
peptide sequence exerts specific binding properties to an antigen
epitope, and may comprise variable and/or constant region partial
sequences of an antibody.
[0193] "Specifically binding to epitope X" as used herein, refers
to the property of an antibody to bind to a particular epitope,
e.g. epitope X, with a higher affinity than to other epitopes.
[0194] "Specifically binding to oligomeric structures of A.beta.",
as used herein, means that the respective antibody binds to
oligomeric structures of A.beta. with a higher affinity than to
monomeric structures of A.beta..
[0195] A "therapeutically effective" amount of A.beta. antibody
refers to the dosage that provides the specific pharmacological
response for which the antibody is administered in a significant
number of subjects in need of such treatment. A therapeutically
effective amount can be determined by prevention or amelioration of
adverse conditions or symptoms of the neurodementing disease being
treated. The appropriate dosage will vary depending, for example,
on the type, stage, and severity of the disease, as well as on the
mode of administration. It is emphasized that a "therapeutically
effective amount" administered to a particular subject in a
particular instance may not be effective for 100% of patients
treated for a specific disease, even though such dosage is deemed a
"therapeutically effective amount" by skilled practitioners.
[0196] The term "titer" denotes a measurement of the amount or
concentration of a particular antibody in a sample, typically
blood.
[0197] The terms "treatment," "treating," "treat," and the like
refer to obtaining a desired pharmacological and/or physiologic
effect. The effect can be prophylactic in tennis of completely or
partially preventing a disease or symptom thereof and/or can be
therapeutic in terms of a partial or complete stabilization or cure
for a disease and/or adverse effect attributable to the disease.
"Treatment" covers any treatment of a disease in a mammal,
particularly a human, and includes: (a) preventing the disease or
symptom from occurring in a subject which can be predisposed to the
disease or symptom but has not yet been diagnosed as having it; (b)
inhibiting the disease symptom, i.e., arresting its development; or
(c) relieving the disease symptom, i.e., causing regression of the
disease or symptom.
Antibodies
[0198] In one aspect the present invention relates to a polypeptide
binding specifically to the epitope A.beta.(21-37) (SEQ ID NO
2).
[0199] A polypeptide according to the present invention can be for
example an antibody, an antibody fragment, or any other
polypeptide, binding to a C-terminal part of full length AD
polypeptide (A.beta.1-40), in particular to the epitope denoted in
A.beta.(21-37) (SEQ ID NO:2).
[0200] In one aspect, the inventive polypeptides are capable of
binding specifically to a polypeptide comprising the epitope
A.beta.(21-37) (SEQ ID NO 2) of amyloid beta, in particular under
physiological conditions, e.g. pH about 7.4, salt concentrations
about 50 to about 150 mM in PBS. In one embodiment, the
polypeptides have a relative dissociation constant, under in vitro
conditions, (relative KD; reflecting in vitro results but not
necessarily identical values under in vivo conditions) of at least
about 10-5 M, about 10-6 M, about 10-7 M, about 10-8, about 10-9 M,
about 10-10M, about 10-11 M, about 10-12 M, or higher. For example,
the relative dissociation constant of the binding of a polypeptide
to the epitope denoted in A.beta.(21-37) (SEQ ID NO:2) can be
between about 10-8 M to about 10-12 M, in particular around 1 to
50.times.10-9 M. Such dissociation rate constants can be determined
readily using kinetic analysis techniques such as surface plasmon
resonance (BIAcore or Biosensor), using general procedures outlined
by the manufacturer or other methods known in the art.
[0201] In another aspect, it was surprisingly discovered that
polypeptides specific for A.beta.(21-37) are highly homologous.
Thus, polypeptides are provided that comprise sequences selected
from consensus CDR sequences. Accordingly, in one embodiment, a
polypeptide according to the present invention can comprise a CDR
sequence having a sequence as denoted in any of the consensus
sequences SEQ ID NOs: 6 to 8.
[0202] SEQ ID NOs: 6 to 8 represent consensus sequences for CDR
regions of the heavy chain of an antibody having the ability to
bind to SEQ ID NO:2. The consensus sequences are derived from the
sequence information derived from the antibodies, which have been
identified by the inventors to bind specifically to SEQ ID NO:2. In
particular, the identified consensus sequences for the CDR regions
of both the heavy chain (i.e. SEQ ID NOs: 6 to 8 and SEQ ID Nos 153
to 161) and the light chain (i.e. SEQ ID NOs: 9 to 11) were derived
from naturally-occurring, human antibodies isolated as described in
Example 2A.
[0203] SEQ ID NO:6 represents a CDR1 consensus sequence for the
heavy chain, wherein
the amino acid at position 1 of SEQ ID NO:6 which is at the Kabat
position H31 can be Ser, Gly or Asn, the amino acid at position 2
of SEQ ID NO:6 which is the amino acid at the Kabat position 8 H32
is Tyr, the amino acid at position 3 of SEQ ID NO:6 which is the
amino acid at the Kabat position H33 can be Trp or Asp, the amino
acid at position 4 of SEQ ID NO:6 which is the amino acid at the
Kabat position H34 is Met and the amino acid at position 5 of SEQ
ID NO:6 which is the amino acid at the Kabat position H35 can be
Ser or His.
[0204] SEQ ID NO:153 represents a preferred CDR1 consensus sequence
for the heavy chain, wherein
the amino acid at position 1 of SEQ ID 153 which is at the Kabat
position H31 can be Asn or Ser, the amino acid at position 2 of SEQ
ID 153 which is the amino acid at the Kabat position 8 H32 is Tyr,
the amino acid at position 3 of SEQ ID NO: 153 which is the amino
acid at the Kabat position H33 can be Asp or Trp, the amino acid at
position 4 of SEQ ID NO: 153 which is the amino acid at the Kabat
position H34 is Met and the amino acid at position 5 of SEQ ID NO:
153 which is the amino acid at the Kabat position H35 can be His or
Ser.
[0205] SEQ ID NO:154 represents a more preferred CDR1 consensus
sequence for the heavy chain, wherein
the amino acid at position 1 of SEQ ID NO: 154 which is at the
Kabat position H31 of SEQ ID NO:6 is Asn, the amino acid at
position 2 of SEQ ID NO: 154 which is the amino acid at the Kabat
position 8 H32 is Tyr, the amino acid at position 3 of SEQ ID NO:
154 which is the amino acid at the Kabat position H33 is Asp, the
amino acid at position 4 of SEQ ID NO: 154 which is the amino acid
at the Kabat position H34 is Met and the amino acid at position 5
of SEQ ID NO: 154 which is the amino acid at the Kabat position H35
is His.
[0206] SEQ ID NO:155 represents a more preferred CDR1 consensus
sequence for the heavy chain, wherein
the amino acid at position 1 of SEQ ID NO: 155 which is at the
Kabat position H31 is Ser, the amino acid at position 2 of SEQ ID
NO: 155 which is the amino acid at the Kabat position 8 H32 is Tyr,
the amino acid at position 3 of SEQ ID NO: 155 which is the amino
acid at the Kabat position H33 can be Trp or Asp, the amino acid at
position 4 of SEQ ID NO: 155 which is the amino acid at the Kabat
position H34 is Met and the amino acid at position 5 of SEQ ID NO:
155 which is the amino acid at the Kabat position H35 is Ser.
[0207] SEQ ID NO:7 represents a CDR2 consensus sequence for the
heavy chain, wherein
the amino acid at position 1 of SEQ ID NO:7 which is the amino acid
at the Kabat position H50 can be Ser or Arg or Glu, the amino acid
at position 2 of SEQ ID NO:7 which is the amino acid at the Kabat
position H51 can be Val or Ile, the amino acid at position 3 of SEQ
ID NO:7 which is the amino acid at the Kabat position H52 can be
Lys or Gly or Asn, the amino acid at position 4 of SEQ ID NO:7
which is the amino acid at the Kabat position H52a can be Gln or no
amino acid, the amino acid at position 5 of SEQ ID NO:7 which is
the amino acid at the Kabat position H53 can be Asp or Phe or Thr
or Arg, the amino acid at position 6 of SEQ ID NO:7 which is the
amino acid at the Kabat position H54 can be Gly or Phe or Ala or
Ser, the amino acid at position 7 of SEQ ID NO:7 which is the amino
acid at the Kabat position H55 can be Ser or Gly, the amino acid at
position 8 of SEQ ID NO:7 which is the amino acid at the Kabat
position H56 can be Glu or Gly or Arg or Asp or Ala, the amino acid
at position 9 of SEQ ID NO:7 which is the amino acid at the Kabat
position H57 can be Lys or Pro or Ser or Thr or Arg, the amino acid
at position 10 of SEQ ID NO:7 which is the amino acid at the Kabat
position H58 can be Tyr or Leu or Ala or Asn, the amino acid at
position 11 of SEQ ID NO:7 which is the amino acid at the Kabat
position H59 can be Tyr or Ala, the amino acid at position 12 of
SEQ ID NO:7 which is the amino acid at the Kabat position H60 can
be Val or Thr or Ala or Asn, the amino acid at position 13 of SEQ
ID NO:7 which is the amino acid at the Kabat position H61 can be
Asp or Gly or Pro, the amino acid at position 14 of SEQ ID NO:7
which is the amino acid at the Kabat position H62 is Ser, the amino
acid at position 15 of SEQ ID NO:7 which is the amino acid at the
Kabat position H63 can be Val or Leu, the amino acid at position 16
of SEQ ID NO:7 which is the amino acid at the Kabat position H64 is
Lys and the amino acid at position 17 of SEQ ID NO:7 which is the
amino acid at the Kabat position H65 can be Gly or Ser.
[0208] SEQ ID NO:156 represents a preferred CDR2 consensus sequence
for the heavy chain, wherein
the amino acid at position 1 of SEQ ID NO:156 which is the amino
acid at the Kabat position H50 can be Arg or Ser or Glu, the amino
acid at position 2 of SEQ ID NO:156 which is the amino acid at the
Kabat position H51 can be Ile or Val, the amino acid at position 3
of SEQ ID NO:156 which is the amino acid at the Kabat position H52
can be Gly or Lys or Asn, the amino acid at position 4 of SEQ ID
NO:156 which is the amino acid at the Kabat position H52a can be
Gln or no amino acid, the amino acid at position 5 of SEQ ID NO:156
which is the amino acid at the Kabat position H53 can be Thr or Asp
or Arg, the amino acid at position 6 of SEQ ID NO:156 which is the
amino acid at the Kabat position H54 can be or Ala or Gly or Ser,
the amino acid at position 7 of SEQ ID NO:156 which is the amino
acid at the Kabat position H55 can be Gly or Ser, the amino acid at
position 8 of SEQ ID NO:156 which is the amino acid at the Kabat
position H56 can be Arg or Asp or Glu or Ala, the amino acid at
position 9 of SEQ ID NO:156 which is the amino acid at the Kabat
position H57 can be Thr or Arg or Lys, the amino acid at position
10 of SEQ ID NO:156 which is the amino acid at the Kabat position
H58 can be Asn or Tyr, the amino acid at position 11 of SEQ ID
NO:156 which is the amino acid at the Kabat position H59 is Tyr,
the amino acid at position 12 of SEQ ID NO:156 which is the amino
acid at the Kabat position H60 can be Asn, Ala or Val, the amino
acid at position 13 of SEQ ID NO:156 which is the amino acid at the
Kabat position H61 can be Pro or Gly or Asp, the amino acid at
position 14 of SEQ ID NO:156 which is the amino acid at the Kabat
position H62 is Ser, the amino acid at position 15 of SEQ ID NO:156
which is the amino acid at the Kabat position H63 can be Leu or
Val, the amino acid at position 16 of SEQ ID NO:156 which is the
amino acid at the Kabat position H64 is Lys and the amino acid at
position 17 of SEQ ID NO:156 which is the amino acid at the Kabat
position H65 can be Gly or Ser.
[0209] SEQ ID NO:157 represents a more preferred CDR2 consensus
sequence for the heavy chain, wherein
the amino acid at position 1 of SEQ ID NO:157 which is the amino
acid at the Kabat position H50 can be Arg or Glu, the amino acid at
position 2 of SEQ ID NO:157 which is the amino acid at the Kabat
position H51 is Ile, the amino acid at position 3 of SEQ ID NO:157
which is the amino acid at the Kabat position H52 can be Gly or
Asn, the amino acid at position 4 of SEQ ID NO:157 which is the
amino acid at the Kabat position H53 can be Thr or Arg, the amino
acid at position 5 of SEQ ID NO:157 which is the amino acid at the
Kabat position H54 can be Ala or Ser, the amino acid at position 6
of SEQ ID NO:157 which is the amino acid at the Kabat position H55
is Gly, the amino acid at position 7 of SEQ ID NO:157 which is the
amino acid at the Kabat position H56 can be Arg or Asp or Ala, the
amino acid at position 8 of SEQ ID NO:157 which is the amino acid
at the Kabat position H57 can be Thr or Arg, the amino acid at
position 9 of SEQ ID NO:157 which is the amino acid at the Kabat
position H58 can be Asn or Tyr, the amino acid at position 10 of
SEQ ID NO:157 which is the amino acid at the Kabat position H59 is
Tyr, the amino acid at position 11 of SEQ ID NO:157 which is the
amino acid at the Kabat position H60 can be Asn or Ala, the amino
acid at position 12 of SEQ ID NO:157 which is the amino acid at the
Kabat position H61 can be Pro or Gly, the amino acid at position 13
of SEQ ID NO:157 which is the amino acid at the Kabat position H62
is Ser, the amino acid at position 14 of SEQ ID NO:157 which is the
amino acid at the Kabat position H63 can be Leu or Val, the amino
acid at position 15 of SEQ ID NO:157 which is the amino acid at the
Kabat position H64 is Lys and the amino acid at position 16 of SEQ
ID NO:157 which is the amino acid at the Kabat position H65 can be
Gly or Ser.
[0210] SEQ ID NO:158 represents a more preferred CDR2 consensus
sequence for the heavy chain, wherein
the amino acid at position 1 of SEQ ID NO:158 which is the amino
acid at the Kabat position H50 is Ser, the amino acid at position 2
of SEQ ID NO:158 which is the amino acid at the Kabat position H51
is Val, the amino acid at position 3 of SEQ ID NO:158 which is the
amino acid at the Kabat position H52 is Lys, the amino acid at
position 4 of SEQ ID NO:158 which is the amino acid at the Kabat
position H52a is Gln, the amino acid at position 5 of SEQ ID NO:158
which is the amino acid at the Kabat position H53 is Asp, the amino
acid at position 6 of SEQ ID NO:158 which is the amino acid at the
Kabat position H54 is Gly, the amino acid at position 7 of SEQ ID
NO:158 which is the amino acid at the Kabat position H55 is Ser,
the amino acid at position 8 of SEQ ID NO:158 which is the amino
acid at the Kabat position H56 is Glu, the amino acid at position 9
of SEQ ID NO:158 which is the amino acid at the Kabat position H57
is Lys, the amino acid at position 10 of SEQ ID NO:158 which is the
amino acid at the Kabat position H58 is Tyr, the amino acid at
position 11 of SEQ ID NO:158 which is the amino acid at the Kabat
position H59 is Tyr, the amino acid at position 12 of SEQ ID NO:158
which is the amino acid at the Kabat position H60 is Val, the amino
acid at position 13 of SEQ ID NO:158 which is the amino acid at the
Kabat position H61 is Asp, the amino acid at position 14 of SEQ ID
NO:158 which is the amino acid at the Kabat position H62 is Ser,
the amino acid at position 15 of SEQ ID NO:158 which is the amino
acid at the Kabat position H63 is Val, the amino acid at position
16 of SEQ ID NO:158 which is the amino acid at the Kabat position
H64 is Lys and the amino acid at position 17 of SEQ ID NO:158 which
is the amino acid at the Kabat position H65 is Gly.
[0211] SEQ ID NO:8 represents a CDR3 consensus sequence for the
heavy chain, wherein
the amino acid at position 1 of SEQ ID NO:8 which is the amino acid
at the Kabat position H95 can be Asp or Gly, the amino acid at
position 2 of SEQ ID NO:8 which is the amino acid at the Kabat
position H 96 can be Ala or Gly, the amino acid at position 3 of
SEQ ID NO:8 which is the amino acid at the Kabat position H97 can
be Ser or Gly, the amino acid at position 4 of SEQ ID NO:8 which is
the amino acid at the Kabat position H98 can be Ser or Arg, the
amino acid at position 5 of SEQ ID NO:8 which is the amino acid at
the Kabat position H99 is Trp, the amino acid at position 6 of SEQ
ID NO:8 which is the amino acid at the Kabat position H100 can be
Tyr or Ala, the amino acid at position 7 of SEQ ID NO:8 which is
the amino acid at the Kabat position H100a can be Arg or Pro or
Asp, the amino acid at position 8 of SEQ ID NO:8 which is the amino
acid at the Kabat position H100b can be Asp or Leu, the amino acid
at position 9 of SEQ ID NO:8 which is the amino acid at the Kabat
position H100c can be Trp or Gly or Ala, the amino acid at position
10 of SEQ ID NO:8 which is the amino acid at the Kabat position
H100d can be Phe or Ala, the amino acid at position 11 of SEQ ID
NO:8 which is the amino acid at the Kabat position H100e can be Phe
or no amino acid, the amino acid at position 12 of SEQ ID NO:8
which is the amino acid at the Kabat position H101 is Asp and the
amino acid at position 13 of SEQ ID NO:8 which is the amino acid at
the Kabat position H102 can be Pro or Ile.
[0212] SEQ ID NO:159 represents a preferred CDR3 consensus sequence
for the heavy chain, wherein
the amino acid at position 1 of SEQ ID NO:159 which is the amino
acid at the Kabat position H95 can be Gly or Asp, the amino acid at
position 2 of SEQ ID NO:159 which is the amino acid at the Kabat
position H 96 can be Ala or Gly, the amino acid at position 3 of
SEQ ID NO:159 which is the amino acid at the Kabat position H97 can
be Gly or Ser, the amino acid at position 4 of SEQ ID NO:159 which
is the amino acid at the Kabat position H98 can be Arg or Ser, the
amino acid at position 5 of SEQ ID NO:159 which is the amino acid
at the Kabat position H99 is Trp, the amino acid at position 6 of
SEQ ID NO:159 which is the amino acid at the Kabat position H100
can be Ala or Tyr, the amino acid at position 7 of SEQ ID NO:159
which is the amino acid at the Kabat position H100a can be Pro or
Arg or Asp, the amino acid at position 8 of SEQ ID NO:159 which is
the amino acid at the Kabat position H100b can be Leu or Asp, the
amino acid at position 9 of SEQ ID NO:159 which is the amino acid
at the Kabat position H100c can be Gly or Trp or Ala, the amino
acid at position 10 of SEQ ID NO:159 which is the amino acid at the
Kabat position H100d can be Ala or Phe, the amino acid at position
11 of SEQ ID NO:159 which is the amino acid at the Kabat position
H100e can be Phe or no amino acid, the amino acid at position 12 of
SEQ ID NO:159 which is the amino acid at the Kabat position H101 is
Asp and the amino acid at position 13 of SEQ ID NO:159 which is the
amino acid at the Kabat position H102 can be Ile or Pro.
[0213] SEQ ID NO:160 represents a more preferred CDR3 consensus
sequence for the heavy chain, wherein
the amino acid at position 1 of SEQ ID NO:160 which is the amino
acid at the Kabat position H95 is Gly, the amino acid at position 2
of SEQ ID NO:160 which is the amino acid at the Kabat position H 96
is Ala, the amino acid at position 3 of SEQ ID NO:160 which is the
amino acid at the Kabat position H97 is Gly, the amino acid at
position 4 of SEQ ID NO:160 which is the amino acid at the Kabat
position H98 is Arg, the amino acid at position 5 of SEQ ID NO:160
which is the amino acid at the Kabat position H99 is Trp, the amino
acid at position 6 of SEQ ID NO:160 which is the amino acid at the
Kabat position H100 is Ala, the amino acid at position 7 of SEQ ID
NO:160 which is the amino acid at the Kabat position H100a is Pro,
the amino acid at position 8 of SEQ ID NO:160 which is the amino
acid at the Kabat position H100b is Leu, the amino acid at position
9 of SEQ ID NO:160 which is the amino acid at the Kabat position
H100c is Gly, the amino acid at position 10 of SEQ ID NO:160 which
is the amino acid at the Kabat position H100d is Ala, the amino
acid at position 11 of SEQ ID NO:160 which is the amino acid at the
Kabat position H100e is Phe, the amino acid at position 12 of SEQ
ID NO:160 which is the amino acid at the Kabat position H101 is Asp
and the amino acid at position 13 of SEQ ID NO:160 which is the
amino acid at the Kabat position H102 is Ile.
[0214] SEQ ID NO:161 represents a more preferred CDR3 consensus
sequence for the heavy chain, wherein
the amino acid at position 1 of SEQ ID NO:161 which is the amino
acid at the Kabat position H95 is Asp, the amino acid at position 2
of SEQ ID NO:161 which is the amino acid at the Kabat position H 96
can be Gly or Ala, the amino acid at position 3 of SEQ ID NO:161
which is the amino acid at the Kabat position H97 can be Ser or
Gly, the amino acid at position 4 of SEQ ID NO:161 which is the
amino acid at the Kabat position H98 can be Ser or Arg, the amino
acid at position 5 of SEQ ID NO:161 which is the amino acid at the
Kabat position H99 is Trp, the amino acid at position 6 of SEQ ID
NO:161 which is the amino acid at the Kabat position H100 can be
Tyr or Ala, the amino acid at position 7 of SEQ ID NO:161 which is
the amino acid at the Kabat position H100a can be Arg or Asp, the
amino acid at position 8 of SEQ ID NO:161 which is the amino acid
at the Kabat position H100b can be Asp or Leu, the amino acid at
position 9 of SEQ ID NO:161 which is the amino acid at the Kabat
position H100c can be Trp or Ala, the amino acid at position 10 of
SEQ ID NO:161 which is the amino acid at the Kabat position H100d
is Phe, the amino acid at position 11 of SEQ ID NO:161 which is the
amino acid at the Kabat position H101 is Asp and the amino acid at
position 12 of SEQ ID NO:161 which is the amino acid at the Kabat
position H102 can be Pro or Ile.
[0215] In another embodiment, the polypeptide according to the
invention comprises as CDRs all three respective consensus CDR
sequences as denoted in SEQ ID NO:6 to 8.
[0216] In another embodiment, the polypeptide according to the
invention comprises at least two of the respective consensus CDR
sequences as denoted in SEQ ID NO:6 to 8.
[0217] In another embodiment, the polypeptide according to the
invention comprises a CDR sequence having at least two of the
respective consensus sequences as denoted in any of the consensus
sequences SEQ ID NOs: 6 to 11.
[0218] SEQ ID NOs: 9 to 11 represent consensus sequences for CDR
regions of the light chain of an antibody having the ability to
bind to SEQ ID NO:2. The consensus sequences are derived from the
sequence information derived from the antibodies, which have been
identified by the inventors to bind specifically to SEQ ID NO:2. In
particular, the identified consensus sequences for the CDR regions
of the light chain were derived from naturally occurring, human
antibodies isolated as described in Example 2A.
[0219] SEQ ID NO:9 represents a CDR1 consensus sequence for a kappa
light chain immunoglobulin CDR1 region, wherein
the amino acid at position 1 of SEQ ID NO:9 which is the amino acid
at the Kabat position L24 can be an Arg, the amino acid at position
2 of SEQ ID NO:9 which is the amino acid at the Kabat position L25
can be Ala or Glu, the amino acid at position 3 of SEQ ID NO:9
which is the amino acid at the Kabat position L26 can be Ser, the
amino acid at position 4 of SEQ ID NO:9 which is the amino acid at
the Kabat position L27 can be Gln, the amino acid at position 5 of
SEQ ID NO:9 which is the amino acid at the Kabat position L28 can
be Ser or Gly, the amino acid at position 6 of SEQ ID NO:9 which is
the amino acid at the Kabat position L29 can be Val or Ile, the
amino acid at position 7 of SEQ ID NO:9 which is the amino acid at
the Kabat position L30 can be Asn or Arg or Ser, the amino acid at
position 8 of SEQ ID NO:9 which is the amino acid at the Kabat
position L31 can be Ser or Asn, the amino acid at position 9 of SEQ
ID NO:9 which is the amino acid at the Kabat position L32 can be
Tyr, the amino acid at position 10 of SEQ ID NO:9 which is the
amino acid at the Kabat position L33 can be Leu and the amino acid
at position 11 of SEQ ID NO:9 which is the amino acid at the Kabat
position L34 can be Ala.
[0220] SEQ ID NO:10 represents a CDR2 consensus sequence for a
kappa light chain immunoglobulin CDR2 region, wherein
the amino acid at position 1 of SEQ ID NO:10 which is the amino
acid at the Kabat position L50 can be Ala or Gly or Lys or Trp, the
amino acid at position 2 of SEQ ID NO:10 which is the amino acid at
the Kabat position L51 can be Val or Ala, the amino acid at
position 3 of SEQ ID NO:10 which is the amino acid at the Kabat
position L52 can be Ser or Ala, the amino acid at position 4 of SEQ
ID NO:10 which is the amino acid at the Kabat position L53 can be
Thr or Ser or Asn or Ile, the amino acid at position 5 of SEQ ID
NO:10 which is the amino acid at the Kabat position L54 can be Arg
or Leu, the amino acid at position 6 of SEQ ID NO:10 which is the
amino acid at the Kabat position L55 can be Ala or Gln or Phe or
Glu and the amino acid at position 7 of SEQ ID NO:10 which is the
amino acid at the Kabat position L56 can be Thr or Ser.
[0221] SEQ ID NO:11 represents a CDR3 consensus sequence for a
kappa light chain immunoglobulin CDR3 region, wherein
the amino acid at position 1 of SEQ ID NO:11 which is the amino
acid at the Kabat position L89 can be Gln, the amino acid at
position 2 of SEQ ID NO:11 which is the amino acid at the Kabat
position L90 can be Gln, the amino acid at position 3 of SEQ ID
NO:11 which is the amino acid at the Kabat position L91 can be Ala
or Tyr, the amino acid at position 4 of SEQ ID NO:11 which is the
amino acid at the Kabat position L92 can be Gly or Asn, the amino
acid at position 5 of SEQ ID NO:11 which is the amino acid at the
Kabat position L93 can be Ser, the amino acid at position 6 of SEQ
ID NO:11 which is the amino acid at the Kabat position L94 can be
Ser or Phe, the amino acid at position 7 of SEQ ID NO:11 which is
the amino acid at the Kabat position L95 can be Gln or Pro, the
amino acid at position 8 of SEQ ID NO:11 which is the amino acid at
the Kabat position L96 can be Gly or Leu and the amino acid at
position 9 of SEQ ID NO:11 which is the amino acid at the Kabat
position L97 can be Thr.
[0222] In one embodiment, the polypeptide according to the
invention comprises as CDRs for the light chain all three
respective consensus CDR sequences as denoted in SEQ ID NO:6 to
11.
[0223] In one embodiment, the polypeptide according to the
invention comprises at least two CDRs for the light chain selected
from all three respective consensus CDR sequences as denoted in SEQ
ID NO:9 to 11.
[0224] Even more preferred is a polypeptide comprising at least two
CDR sequences selected from the consensus CDR sequences denoted for
the light chain (SEQ ID NO:9 to 11) or at least two of the
consensus CDR sequences denoted for the heavy chain (SEQ ID NO:6 to
8) or at least one CDR from the light chain (SEQ ID NO:9 to 11) and
at least one CDR from the heavy chain (SEQ ID NO:6 to 8).
[0225] In one embodiment, the polypeptide according to the
invention comprises as CDRs for the light chain all three
respective consensus CDR sequences as denoted in SEQ ID NO:9 to
11.
[0226] Even more preferred is a polypeptide comprising at least two
CDR sequences selected from the consensus CDR sequences denoted for
the light chain (SEQ ID NO:9 to 11) and/or at least two of the
consensus CDR sequences denoted for the heavy chain (SEQ ID NO:6 to
8).
[0227] In one embodiment, isolated, monoclonal, anti-.beta.-amyloid
antibodies are provided that comprise more than one amino acid
sequence selected from at least two consensus amino acid sequences
of the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:
8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, wherein each of
said more than one amino acid sequence is from a different SEQ ID
NO, wherein said antibody binds to dimeric forms of A.beta. with
higher affinity than to monomeric forms of A.beta..
[0228] In a further embodiment, the polypeptide according to the
invention comprises as CDR1 on the heavy chain one of the sequences
as denoted in SEQ ID NOs: 13 to 20, as CDR2 on the heavy chain one
of the sequence as denoted in SEQ ID NOs: 21 to 27 and 149, and/or
as CDR3 on the heavy chain one of the sequences as denoted in SEQ
ID NOs: 28 to 32.
[0229] In another embodiment the polypeptide according to the
invention comprises at least two consensus CDR sequences selected
from consensus sequences for CDR 1, CDR 2 and CDR 3 of the heavy
chain and CDR 1, CDR 2 and CDR 3 of the light chain, wherein the
consensus sequences are derived by aligning the sequences of the
following antibody variable regions according to the Kabat
numbering: [0230] a) for CDR1 of the heavy chain, SEQ ID NOs: 56 to
71 and 148 [0231] b) for CDR2 of the heavy chain, SEQ ID NOs: 56 to
71 and 148 [0232] c) for CDR3 of the heavy chain, SEQ ID NOs: 56 to
71 and 148 [0233] d) for CDR1 of the light chain, SEQ ID NOs: 47 to
55 and 145 to 147 [0234] e) for CDR2 of the light chain SEQ ID NOs:
47 to 55 and 145 to 147 [0235] f) for CDR3 of the light chain, SEQ
ID Nos: 47 to 55 and 145 to 147
[0236] SEQ ID NOs 13 to 20 represent the CDR1 sequences, SEQ ID NOs
21 to 27 and 149 represent the CDR2 sequences and SEQ ID NOs 28 to
32 represent the CDR3 sequences found by the inventors to be
present on the heavy chain of antibodies binding to a C-terminal
part of A.beta.(1-40), in particular to A.beta.(21-37).
[0237] In a further embodiment, the polypeptide according to the
present invention comprises as CDR1 on the light chain one of the
sequences as denoted in SEQ ID NOs: 33 to 37, as CDR2 on the light
chain one of the sequence as denoted in SEQ ID NOs: 38 to 43 and
150 to 152, and/or as CDR3 on the light chain one of the sequences
as denoted in SEQ ID NOs: 44 to 46.
[0238] SEQ ID NOs 33 to 37 represent the CDR1 sequences, SEQ ID NOs
38 to 43 and 150 to 152 represent the CDR2 sequences and SEQ ID NOs
44 to 46 represent the CDR3 sequences found by the inventors to be
present on the light chain of antibodies binding to a C-terminal
part of A.beta.(1-40), in particular to A.beta.(21-37).
[0239] In a preferred embodiment, a polypeptide according to the
present invention comprises on the heavy chain a CDR1 sequence
selected from SEQ ID NOs: 13 to 20, a CDR2 sequence selected from
SEQ ID NOs: 21 to 27 and 148, and/or a CDR3 sequence selected from
SEQ ID NOs: 28 to 32, and on the light chain a CDR1 sequence
selected from SEQ ID NOs: 33 to 37, a CDR2 sequence selected from
SEQ ID NOs: 38 to 43 and 150 to 152 and/or a CDR3 sequence selected
from SEQ ID NOs: 44 to 46.
[0240] In particular, a polypeptide according to the present
invention can comprise on the light chain: [0241] a) as CDR1 SEQ ID
NO:34, as CDR2 SEQ ID NO:38 and as CDR3 SEQ ID NO:44, or [0242] b)
as CDR1 SEQ ID NO:33, as CDR2 SEQ ID NO:42 and as CDR3 SEQ ID
NO:44, or [0243] c) as CDR1 SEQ ID NO:37, as CDR2 SEQ ID NO:43 and
as CDR3 SEQ ID NO:46, or [0244] d) as CDR1 SEQ ID NO:34, as CDR2
SEQ ID NO:40 and as CDR3 SEQ ID NO:44, or [0245] e) as CDR1 SEQ ID
NO:35, as CDR2 SEQ ID NO:38 and as CDR3 SEQ ID NO:44, or [0246] f)
as CDR1 SEQ ID NO:33, as CDR2 SEQ ID NO:41 and as CDR3 SEQ ID
NO:44, or [0247] g) as CDR1 SEQ ID NO:33, as CDR2 SEQ ID NO:41 and
as CDR3 SEQ ID NO:45, or [0248] h) as CDR1 SEQ ID NO:34, as CDR2
SEQ ID NO:38 and as CDR3 SEQ ID NO:44, or [0249] i) as CDR1 SEQ ID
NO:36, as CDR2 SEQ ID NO:39 and as CDR3 SEQ ID NO:44.
[0250] In particular, a polypeptide according to the present
invention can also comprise on the heavy chain: [0251] a) as CDR1
SEQ ID NO:13, as CDR2 SEQ ID NO:21 and as CDR3 SEQ ID NO:28, or
[0252] b) as CDR1 SEQ ID NO:14, as CDR2 SEQ ID NO:27 and as CDR3
SEQ ID NO:30, or [0253] c) as CDR1 SEQ ID NO:13, as CDR2 SEQ ID
NO:26 and as CDR3 SEQ ID NO:28, or [0254] d) as CDR1 SEQ ID NO:14,
as CDR2 SEQ ID NO:21 and as CDR3 SEQ ID NO:30, or [0255] e) as CDR1
SEQ ID NO:15, as CDR2 SEQ ID NO:23 and as CDR3 SEQ ID NO:29, or
[0256] f) as CDR1 SEQ ID NO:15, as CDR2 SEQ ID NO:22 and as CDR3
SEQ ID NO:29, or [0257] g) as CDR1 SEQ ID NO:20, as CDR2 SEQ ID
NO:27 and as CDR3 SEQ ID NO:31, or [0258] h) as CDR1 SEQ ID NO:18,
as CDR2 SEQ ID NO:25 and as CDR3 SEQ ID NO:31, or [0259] i) as CDR1
SEQ ID NO:18, as CDR2 SEQ ID NO:27 and as CDR3 SEQ ID NO:31, or
[0260] j) as CDR1 SEQ ID NO:14, as CDR2 SEQ ID NO:27 and as CDR3
SEQ ID NO:31, or [0261] k) as CDR1 SEQ ID NO:14, as CDR2 SEQ ID
NO:21 and as CDR3 SEQ ID NO:31, or [0262] l) as CDR1 SEQ ID NO:16,
as CDR2 SEQ ID NO:21 and as CDR3 SEQ ID NO:31, or [0263] m) as CDR1
SEQ ID NO:19, as CDR2 SEQ ID NO:21 and as CDR3 SEQ ID NO:32, or
[0264] n) as CDR1 SEQ ID NO:16, as CDR2 SEQ ID NO:21 and as CDR3
SEQ ID NO:28, or [0265] o) as CDR1 SEQ ID NO:17, as CDR2 SEQ ID
NO:26 and as CDR3 SEQ ID NO:31, or [0266] p) as CDR1 SEQ ID NO:14,
as CDR2 SEQ ID NO:24 and as CDR3 SEQ ID NO:28.
[0267] Even more preferred is a polypeptide according to the
present invention comprising: [0268] a) on the light chain as CDR1
SEQ ID NO:34, as CDR2 SEQ ID NO:38 and as CDR3 SEQ ID NO:44, and on
the heavy chain as CDR1 SEQ ID NO:13, as CDR2 SEQ ID NO:21 and as
CDR3 SEQ ID NO:28, or [0269] b) on the light chain as CDR1 SEQ ID
NO:33, as CDR2 SEQ ID NO:42 and as CDR3 SEQ ID NO:44, and on the
heavy chain as CDR1 SEQ ID NO:14, as CDR2 SEQ ID NO:27 and as CDR3
SEQ ID NO:30.
[0270] Even more preferred is a polypeptide according to the
present invention comprising a light variable chain having the
sequence of SEQ ID NO:53 and a variable heavy chain having the
sequence of SEQ ID NO:60, or a polypeptide comprising a light chain
having the CDR sequences of SEQ ID NO:33, SEQ ID NO: 41 and SEQ ID
NO:45 and a heavy chain having the CDR sequences of SEQ ID NO:15,
SEQ ID NO:23 and SEQ ID NO:29.
[0271] SEQ ID NOs: 47 to 55 and 145 to 147 denote variable regions
of the light chain and SEQ ID NOs: 56 to 71 and 148 denote variable
regions of the heavy chain of antibodies having the ability to bind
specifically to a C-terminal part of A.beta.(1-40), in particular
to A.beta.(21-37), as determined by the inventors. The variable
regions of the heavy and light chain are responsible for antigen
specificity. Therefore, in a further embodiment, the polypeptide
according to the present invention comprises a sequence selected
from SEQ ID NOs: 47 to 55 and 145 to 147 and/or a sequence selected
from SEQ ID NOs: 56 to 71 and 148. In a particularly preferred
embodiment the polypeptide according to the present invention
comprises SEQ ID NO:47 and SEQ ID NO:56 or SEQ ID NO:48 and SEQ ID
NO:57.
[0272] Each of the light chains of SEQ ID NOs: 47 to 55 and 145 to
147 can be combined with any of the heavy chains of SEQ ID NOs: 56
to 71 and 148. Accordingly, anti-.beta.-amyloid antibodies are
provided that comprise SEQ ID NO: 47 and SEQ ID NO:56; SEQ ID NO:
47 and SEQ ID NO:57; SEQ ID NO: 47 and SEQ ID NO: 58; SEQ ID NO: 47
and SEQ ID NO: 59; SEQ ID NO: 47 and SEQ ID NO:60; SEQ ID NO: 47
and SEQ ID NO: 61; SEQ ID NO: 47 and SEQ ID NO: 62; SEQ ID NO: 47
and SEQ ID NO: 63; SEQ ID NO: 47 and SEQ ID NO: 64; SEQ ID NO: 47
and SEQ ID NO: 65; SEQ ID NO: 47 and SEQ ID NO: 66; SEQ ID NO: 47
and SEQ ID NO: 67; SEQ ID NO: 47 and SEQ ID NO: 68; SEQ ID NO: 47
and SEQ ID NO: 69; SEQ ID NO: 47 and SEQ ID NO: 70; SEQ ID NO: 47
and SEQ ID NO: 71; SEQ ID NO: 47 and SEQ ID NO: 148; SEQ ID NO: 48
and SEQ ID NO:56; SEQ ID NO: 48 and SEQ ID NO:57; SEQ ID NO: 48 and
SEQ ID NO:58; SEQ ID NO: 48 and SEQ ID NO:59; SEQ ID NO: 48 and SEQ
ID NO:60; SEQ ID NO: 48 and SEQ ID NO:61; SEQ ID NO: 48 and SEQ ID
NO:62; SEQ ID NO: 48 and SEQ ID NO:63; SEQ ID NO: 48 and SEQ ID
NO:64; SEQ ID NO: 48 and SEQ ID NO:65; SEQ ID NO: 48 and SEQ ID
NO:66; SEQ ID NO: 48 and SEQ ID NO:67; SEQ ID NO: 48 and SEQ ID
NO:68; SEQ ID NO: 48 and SEQ ID NO:69; SEQ ID NO: 48 and SEQ ID
NO:70; SEQ ID NO: 48 and SEQ ID NO:71; SEQ ID NO: 48 and SEQ ID
NO:148; SEQ ID NO: 49 and SEQ ID NO:56; SEQ ID NO: 49 and SEQ ID
NO:57; SEQ ID NO: 49 and SEQ ID NO: 58; SEQ ID NO: 49 and SEQ ID
NO: 59; SEQ ID NO: 49 and SEQ ID NO: 60; SEQ ID NO: 49 and SEQ ID
NO: 61; SEQ ID NO: 49 and SEQ ID NO: 62; SEQ ID NO: 49 and SEQ ID
NO: 63; SEQ ID NO: 49 and SEQ ID NO: 64; SEQ ID NO: 49 and SEQ ID
NO: 65; SEQ ID NO: 49 and SEQ ID NO: 66; SEQ ID NO: 49 and SEQ ID
NO: 67; SEQ ID NO: 49 and SEQ ID NO: 68; SEQ ID NO: 49 and SEQ ID
NO: 69; SEQ ID NO: 49 and SEQ ID NO: 70; SEQ ID NO: 49 and SEQ ID
NO: 71; SEQ ID NO: 49 and SEQ ID NO: 148; SEQ ID NO: 50 and SEQ ID
NO: 56; SEQ ID NO: 50 and SEQ ID NO: 57; SEQ ID NO: 50 and SEQ ID
NO: 58; SEQ ID NO: 50 and SEQ ID NO: 59; SEQ ID NO: 50 and SEQ ID
NO: 60; SEQ ID NO: 50 and SEQ ID NO: 61; SEQ ID NO: 50 and SEQ ID
NO: 62; SEQ ID NO: 50 and SEQ ID NO: 63; SEQ ID NO: 50 and SEQ ID
NO: 64; SEQ ID NO: 50 and SEQ ID NO: 65; SEQ ID NO: 50 and SEQ ID
NO: 66; SEQ ID NO: 50 and SEQ ID NO: 67; SEQ ID NO: 50 and SEQ ID
NO: 68; SEQ ID NO: 50 and SEQ ID NO: 69; SEQ ID NO: 50 and SEQ ID
NO: 70; SEQ ID NO: 50 and SEQ ID NO: 71; SEQ ID NO: 50 and SEQ ID
NO: 148; SEQ ID NO: 51 and SEQ ID NO: 56; SEQ ID NO: 51 and SEQ ID
NO: 57; SEQ ID NO: 51 and SEQ ID NO: 58; SEQ ID NO: 51 and SEQ ID
NO: 59; SEQ ID NO: 51 and SEQ ID NO: 60; SEQ ID NO: 51 and SEQ ID
NO: 61; SEQ ID NO: 51 and SEQ ID NO: 62; SEQ ID NO: 51 and SEQ ID
NO: 63; SEQ ID NO: 51 and SEQ ID NO: 64; SEQ ID NO: 51 and SEQ ID
NO: 65; SEQ ID NO: 51 and SEQ ID NO: 66; SEQ ID NO: 51 and SEQ ID
NO: 67; SEQ ID NO: 51 and SEQ ID NO: 68; SEQ ID NO: 51 and SEQ ID
NO: 69; SEQ ID NO: 51 and SEQ ID NO: 70; SEQ ID NO: 51 and SEQ ID
NO: 71; SEQ ID NO: 51 and SEQ ID NO: 148; SEQ ID NO: 52 and SEQ ID
NO: 56; SEQ ID NO: 52 and SEQ ID NO: 57; SEQ ID NO: 52 and SEQ ID
NO: 58; SEQ ID NO: 52 and SEQ ID NO: 59; SEQ ID NO: 52 and SEQ ID
NO: 60; SEQ ID NO: 52 and SEQ ID NO: 61; SEQ ID NO: 52 and SEQ ID
NO: 62; SEQ ID NO: 52 and SEQ ID NO: 63; SEQ ID NO: 52 and SEQ ID
NO: 64; SEQ ID NO: 52 and SEQ ID NO: 65; SEQ ID NO: 52 and SEQ ID
NO:66; SEQ ID NO: 52 and SEQ ID NO: 67; SEQ ID NO: 52 and SEQ ID
NO: 68; SEQ ID NO: 52 and SEQ ID NO: 69; SEQ ID NO: 52 and SEQ ID
NO: 70; SEQ ID NO: 52 and SEQ ID NO: 71; SEQ ID NO: 52 and SEQ ID
NO:148; SEQ ID NO: 53 and SEQ ID NO: 56; SEQ ID NO: 53 and SEQ ID
NO: 57; SEQ ID NO: 53 and SEQ ID NO: 58; SEQ ID NO: 53 and SEQ ID
NO: 59; SEQ ID NO: 53 and SEQ ID NO: 60; SEQ ID NO: 53 and SEQ ID
NO: 61; SEQ ID NO: 53 and SEQ ID NO: 62; SEQ ID NO: 53 and SEQ ID
NO: 63; SEQ ID NO: 53 and SEQ ID NO: 64; SEQ ID NO: 53 and SEQ ID
NO: 65; SEQ ID NO: 53 and SEQ ID NO: 66; SEQ ID NO: 53 and SEQ ID
NO: 67; SEQ ID NO: 53 and SEQ ID NO: 68; SEQ ID NO: 53 and SEQ ID
NO: 69; SEQ ID NO: 53 and SEQ ID NO: 70; SEQ ID NO: 53 and SEQ ID
NO: 71; SEQ ID NO: 53 and SEQ ID NO: 148; SEQ ID NO: 54 and SEQ ID
NO: 56; SEQ ID NO: 54 and SEQ ID NO: 57; SEQ ID NO: 54 and SEQ ID
NO: 58; SEQ ID NO: 54 and SEQ ID NO: 59; SEQ ID NO: 54 and SEQ ID
NO: 60; SEQ ID NO: 54 and SEQ ID NO: 61; SEQ ID NO: 54 and SEQ ID
NO: 62; SEQ ID NO: 54 and SEQ ID NO: 63; SEQ ID NO: 54 and SEQ ID
NO: 64; SEQ ID NO: 54 and SEQ ID NO: 65; SEQ ID NO: 54 and SEQ ID
NO: 66; SEQ ID NO: 54 and SEQ ID NO: 67; SEQ ID NO: 54 and SEQ ID
NO: 68; SEQ ID NO: 54 and SEQ ID NO: 69; SEQ ID NO: 54 and SEQ ID
NO: 70; SEQ ID NO: 54 and SEQ ID NO: 71; SEQ ID NO: 54 and SEQ ID
NO: 148; SEQ ID NO: 55 and SEQ ID NO: 56; SEQ ID NO: 55 and SEQ ID
NO: 57; SEQ ID NO: 55 and SEQ ID NO: 58; SEQ ID NO: 55 and SEQ ID
NO: 59; SEQ ID NO: 55 and SEQ ID NO: 60; SEQ ID NO: 55 and SEQ ID
NO: 61; SEQ ID NO: 55 and SEQ ID NO: 62; SEQ ID NO: 55 and SEQ ID
NO: 63; SEQ ID NO: 55 and SEQ ID NO: 64; SEQ ID NO: 55 and SEQ ID
NO: 65; SEQ ID NO: 55 and SEQ ID NO: 66; SEQ ID NO: 55 and SEQ ID
NO: 67; SEQ ID NO: 55 and SEQ ID NO: 68; SEQ ID NO: 55 and SEQ ID
NO: 69; SEQ ID NO: 55 and SEQ ID NO: 70; SEQ ID NO: 55 and SEQ ID
NO: 71; SEQ ID NO: 55 and SEQ ID NO: 148; SEQ ID NO: 145 and SEQ ID
NO: 56; SEQ ID NO: 145 and SEQ ID NO: 57; SEQ ID NO: 145 and SEQ ID
NO: 58; SEQ ID NO: 145 and SEQ ID NO: 59; SEQ ID NO: 145 and SEQ ID
NO: 60; SEQ ID NO: 145 and SEQ ID NO: 61; SEQ ID NO: 145 and SEQ ID
NO: 62; SEQ ID NO: 145 and SEQ ID NO: 63; SEQ ID NO: 145 and SEQ ID
NO: 64; SEQ ID NO: 145 and SEQ ID NO: 65; SEQ ID NO: 145 and SEQ ID
NO: 66; SEQ ID NO: 145 and SEQ ID NO: 67; SEQ ID NO: 145 and SEQ ID
NO: 68; SEQ ID NO: 145 and SEQ ID NO: 69; SEQ ID NO: 145 and SEQ ID
NO: 70; SEQ ID NO: 145 and SEQ ID NO: 71; SEQ ID NO: 145 and SEQ ID
NO: 148; SEQ ID NO: 146 and SEQ ID NO: 56; SEQ ID NO: 146 and SEQ
ID NO: 57; SEQ ID NO: 146 and SEQ ID NO: 58; SEQ ID NO: 146 and SEQ
ID NO: 59; SEQ ID NO: 146 and SEQ ID NO: 60; SEQ ID NO: 146 and SEQ
ID NO: 61; SEQ ID NO: 146 and SEQ ID NO: 62; SEQ ID NO: 146 and SEQ
ID NO: 63; SEQ ID NO: 146 and SEQ ID NO: 64; SEQ ID NO: 146 and SEQ
ID NO: 65; SEQ ID NO: 146 and SEQ ID NO: 66; SEQ ID NO: 146 and SEQ
ID NO: 67; SEQ ID NO: 146 and SEQ ID NO: 68; SEQ ID NO: 146 and SEQ
ID NO: 69; SEQ ID NO: 146 and SEQ ID NO: 70; SEQ ID NO: 146 and SEQ
ID NO: 71; SEQ ID NO: 146 and SEQ ID NO: 148; SEQ ID NO: 147 and
SEQ ID NO: 56; SEQ ID NO: 147 and SEQ ID NO: 57; SEQ ID NO: 147 and
SEQ ID NO: 58; SEQ ID NO: 147 and SEQ ID NO: 59; SEQ ID NO: 147 and
SEQ ID NO: 60; SEQ ID NO: 147 and SEQ ID NO: 61; SEQ ID NO: 147 and
SEQ ID NO: 62; SEQ ID NO: 147 and SEQ ID NO: 63; SEQ ID NO: 147 and
SEQ ID NO: 64; SEQ ID NO: 147 and SEQ ID NO: 65; SEQ ID NO: 147 and
SEQ ID NO: 66; SEQ ID NO: 147 and SEQ ID NO: 67; SEQ ID NO: 147 and
SEQ ID NO: 68; SEQ ID NO: 147 and SEQ ID NO: 69; SEQ ID NO: 147 and
SEQ ID NO: 70; SEQ ID NO: 147 and SEQ ID NO: 71; or SEQ ID NO: 147
and SEQ ID NO: 148;
[0273] In preferred embodiments, the anti-.beta.-amyloid antibodies
comprise SEQ ID NO: 145 and SEQ ID NO:60; SEQ ID NO: 53 and SEQ ID
NO:60; SEQ ID NO: 51 and SEQ ID NO:60; SEQ ID NO: 52 and SEQ ID
NO:60; SEQ ID NO: 146 and SEQ ID NO:60; SEQ ID NO: 53 and SEQ ID
NO:148; SEQ ID NO: 55 and SEQ ID NO:61; SEQ ID NO: 145 and SEQ ID
NO:62; SEQ ID NO: 54 and SEQ ID NO:60; SEQ ID NO: 54 and SEQ ID
NO:61; SEQ ID NO: 54 and SEQ ID NO:148; SEQ ID NO: 145 and SEQ ID
NO:148; or SEQ ID NO: 146 and SEQ ID NO:61.
[0274] In addition, SEQ ID NOs 72 to 74 denote constant regions of
the light chain and SEQ ID NOs 75 to 77 denote constant regions of
the heavy chain of antibodies having the ability to bind
specifically to a C-terminal part of A.beta.(1-40), in particular
to A.beta.(21-37). Therefore, in a further embodiment, the
polypeptide according to the present invention can comprise a
sequence selected from SEQ ID NOs: 72 to 74 and/or a sequence
selected from SEQ ID NOs: 75 to 77. In particular, the polypeptide
according to the present invention can comprise a sequence selected
from SEQ ID NO:72 or 73 and a sequence selected from SEQ ID NOs 75
to 77.
[0275] The complete sequences, consisting of variable region and
constant region, for the light chain of antibodies having the
ability to bind specifically to a C-terminal part of A.beta.(1-40),
in particular to A.beta.(21-37), are denoted in SEQ ID NOs: 78 to
93.
[0276] Therefore, in another embodiment of the present invention,
the polypeptide according to the present invention comprises a
sequence selected from SEQ ID NOs: 78 to 93.
[0277] The complete sequences, consisting of variable region and
constant region, for the heavy chain of antibodies having the
ability to bind specifically to a C-terminal part of A.beta.(1-40),
in particular to A.beta.(21-37), are denoted in SEQ ID NOs: 94 to
137. Therefore, in another embodiment of the present invention, the
polypeptide according to the present invention comprises a sequence
selected from SEQ ID NOs: 94 to 137.
[0278] In an even more preferred embodiment, the polypeptide
according to the present invention comprises a sequence as denoted
in SEQ ID NO:78 and a sequence as denoted in SEQ ID NO:94 or a
sequence as denoted in SEQ ID NO:80 and a sequence as denoted in
SEQ ID NO:97. The same applies to the analogous isoforms of
respective chains and combinations thereof (as can be taken from
FIG. 23a).
[0279] Consensus sequences for framework regions also can be
identified. If the amino acid sequences of the variable regions of
the heavy and light chains are aligned respectively according to
the Kabat rules, the so-called framework regions N-terminal to
CDR1, between CDR1 and CDR2, between CDR2 and CDR3 and C-terminal
to CDR3 can be compared in a way analogous to that described above
for the CDR regions, and consensus sequences for the framework of
the antibodies of the invention can be derived.
[0280] It has to be noted that the inventors found that the
N-terminal sequence, about 18 amino acid residues, of kappa light
chain of antibodies having the ability to bind specifically to a
C-terminal part of A.beta.(1-40), in particular to A.beta.(21-37),
is well conserved. This applies for antibodies derived from IVIgG
preparations as well as for antibodies derived from patient serum.
In FIG. 31 said sequences are depicted, additionally also denoted
as SEQ ID NOs: 138 to 143. It is contemplated, that the
conservative nature of the N-terminus of kappa light chain of these
antibodies might contribute to antigen specificity and/or
prevention of plaque formation when bound to A.beta. peptide. Thus,
in a preferred embodiment, the polypeptide according to the present
invention comprises a sequence as denoted in the consensus sequence
of SEQ ID NO: 44, in particular a sequence as denoted in SEQ ID
NOs: 138 to 143.
[0281] In another embodiment, the inventive polypeptides bind
specifically to oligomeric forms of .beta.-amyloid polypeptide. By
way of non-limiting example, the polypeptides can bind oligomeric
forms of A.beta.(1-40) or oligomeric fauns of A.beta.(12-20) or
oligomeric forms of A.beta.(21-37). In one aspect, the inventive
polypeptides are capable of binding specifically to oligomeric
fauns of A.beta.(1-40) when incubated overnight at 10 mM sodium
phosphate, 150 mM NaCl, pH 7.4 at 4.degree. C.
[0282] In another aspect, the inventive autoantibodies bind with a
higher affinity to dimers of A.beta. than to corresponding monomers
of A.beta..
[0283] In general, binding affinity is a measure of
antibody-antigen combination and concerns the selectivity with
which a given antibody binds to an epitope when compared with
binding to any other epitope. This preferential or selective
binding can be quantified as a binding affinity or titer. Methods
of calculating antibody affinity are well-known in the field. See,
e.g. Practical Immunology Ch. 3, Frank C. Hay & Olwyn M. R.
Westwood, Blackwell Publishing (2002); Measuring Immunity: Basic
Biology and Clinical Assessment, Ch. 16, Michael T. Lotze &
Angus W. Thomson (eds.), Academic Press (2005), both which are
incorporated herein by reference.
[0284] In a preferred embodiment, where the polypeptide according
to the invention is an antibody, said antibody may be a monoclonal
antibody. Monoclonal antibodies have the advantage that they
exhibit less cross reactivity.
[0285] In another preferred embodiment the polypeptides according
to the present invention comprises derivatives and/or fragments of
antibodies binding to A.beta.(21-37) (SEQ ID NO:2). It is well
known in the art that antibodies can be treated enzymatically, e.g.
with proteases in order to obtain fragments of antibodies which
retain the antigen binding properties of the intact antibody, but
which lack the Fc-domain. Such fragments are for example Fab or
F(ab').sub.2 fragments, which are obtainable via enzymatic digest
of antibodies with papain or pepsin protease, respectively. Another
fragment of an antibody is a single chain variable fragment (scFv)
of an antibody, i.e. a fusion of the variable regions of the heavy
and light chains of an antibody via a short flexible linker
(usually serine, glycine). Normally, such a chimeric molecule
retains the specificity of the original antibody, despite removal
of the constant regions and the introduction of a linker peptide.
Single chain variable fragments can be obtained by genetic
engineering of a respective nucleic acid encoding for such a fusion
protein. The advantage of such a fragment is that it consists only
of a single polypeptide chain which is more easily expressed and
properly folded in artificial expression systems than the whole
antibody, which comprises at least 4 polypeptide chains which need
a correct assembly in order to function adequately.
[0286] If the polypeptide according the invention is an antibody, a
human antibody is preferred due to its low immunogenicity in
humans. However, the antibody or fragment thereof can be derived
from any species suitable for antibody production. Non-human
antibodies can be derived in particular from mouse, chicken,
rabbit, rat, donkey, camel, dromedary, shark and llamas. Antibodies
of camel, dromedary, shark and llamas have the advantage that these
animals have antibodies which consist only of a homodimer. Thus,
such polypeptides have similar advantages in expression and
assembly as described above for single chain variable
fragments.
[0287] In another preferred embodiment the antibody, derivatives or
fragments thereof is a humanized antibody, derivative or fragment
thereof, i.e. while the antigen binding domain or parts thereof
is/are of non-human origin, the rest of the antibody, derivative or
fragment thereof is of human origin. In another preferred
embodiment the antibody, derivative or fragment thereof is
chimeric, i.e. while the variable domain or parts thereof is/are of
non-human origin the constant domain is of human origin. Both
embodiments serve the purpose to reduce negative side effects due
to immunogenic properties of protein domains of non-human origin.
In an even more preferred embodiment, the polypeptide binding to
epitope A.beta.(21-37) (SEQ ID NO:2) of amyloid-beta peptide (1-40)
(SEQ ID NO:1) is an antibody derivative or fragment which comprises
only the paratope of an antibody binding to said epitope. Example
for such a paratope is, for example, a polypeptide comprising the
amino acid sequences for the respective CDR domains of heavy and
light chain connected via the intervening sequences or via
synthetic linkers.
[0288] In one aspect, the antibodies of the present invention
embrace allelic variants, conservatively modified variants, and
minor recombinant modifications to a specific anti-A.beta.(21-37)
autoantibody. Amino acid sequence variants of the antibody can be
prepared by introducing appropriate nucleotide changes into the
encoding DNA, or by peptide synthesis. Such variants include, for
example, deletions and/or insertions and/or substitutions of
residues within the amino acid sequences of the antibodies. Any
combination of deletion, insertion, and substitution is made to
arrive at the final construct, provided that the final construct
possesses the desired characteristics. The amino acid changes also
may alter post-translational processes of antibody, such as
changing the number or position of glycosylation sites.
[0289] Amino acid sequence insertions can include, for example,
amino- and/or carboxyl-terminal fusions ranging in length from one
residue to polypeptides containing a hundred or more residues, as
well as intra-sequence insertions of single or multiple amino acid
residues. Examples of terminal insertions include an antibody with
an N-terminal methionyl residue or the specific binding agent or
antibody (including antibody fragment) fused to an epitope tag or a
salvage receptor epitope. Other insertional variants include a
fusion to a polypeptide which increases the serum half-life of the
antibody, e.g. at the N-terminus or C-terminus.
[0290] Another type of variant is an amino acid substitution
variant. These variants have at least one amino acid residue in the
antibody molecule removed and a different residue inserted in its
place. Naturally occurring residues are divided into groups based
on common side-chain properties: [0291] (1) hydrophobic:
norleucine, met, ala, val, leu, ile; [0292] (2) neutral
hydrophilic: cys, ser, thr; [0293] (3) acidic: asp, glu; [0294] (4)
basic: asn, gin, his, lys, arg; [0295] (5) residues that influence
chain orientation: gly, pro; and [0296] (6) aromatic: trp, tyr,
phe.
[0297] Conservative substitutions involve replacing an amino acid
with another member of its class. Non-conservative substitutions
involve replacing a member of one of these classes with a member of
another class. Example of conservative substitutions include
replacing ala with val, leu or ile; arg with lys, gin or asn; asn
with gln, his, asp, lys, or gln; asp with glu or asn; cys with ser
or ala; gln with asn or glu; glu with asp or gin; gly with ala; his
with asn, gin, lys, or arg; ile with leu, val, met, ala or phe; leu
with norleucine, ile, val, met, ala, or phe; lys with arg, gly,
asn; met with leu, phe, or ile; phe with leu, val, ile, ala, or
tyr; pro with ala; ser with thr; thr with ser; trp with tyr or phe;
tyr with trp, phe, thr or ser; and val with ile, leu, met, phe,
ala, or norleucine.
[0298] Any cysteine residue not involved in maintaining the proper
conformation of the specific binding agent or humanized or variant
antibody also may be substituted, generally with serine, to improve
the oxidative stability of the molecule and prevent aberrant
crosslinking. Conversely, cysteine bond(s) may be added to the
specific binding agent or antibody to improve its stability
(particularly where the antibody is an antibody fragment such as an
Fv fragment).
[0299] Altered glycosylation variants also can be produced that
have a modified glycosylation pattern relative to the parent
antibody, for example, by deleting one or more carbohydrate
moieties found in the antibody, and/or adding one or more
glycosylation sites that are not present in the antibody.
Glycosylation of polypeptides including antibodies is typically
either N-linked or O-linked. N-linked refers to the attachment of
the carbohydrate moiety to the side chain of an asparagine residue.
The tripeptide sequences asparagine-X-serine and
asparagine-X-threonine, where X is any amino acid except proline,
are the recognition sequences for enzymatic attachment of the
carbohydrate moiety to the asparagine side chain. The presence of
either of these tripeptide sequences in a polypeptide creates a
potential glycosylation site. Thus, N-linked glycosylation sites
may be added to an antibody by altering the amino acid sequence
such that it contains one or more of these tripeptide sequences.
O-linked glycosylation refers to the attachment of one of the
sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino
acid, most commonly serine or threonine, although 5-hydroxyproline
or 5-hydroxylysine also may be used. O-linked glycosylation sites
may be added to a specific binding agent or antibody by inserting
or substituting one or more serine or threonine residues to the
sequence of the antibody.
[0300] Modifications to increase serum half-life also may
desirable, for example, by incorporation of or addition of a
salvage receptor binding epitope (e.g., by mutation of the
appropriate region or by incorporating the epitope into a peptide
tag that is then fused to the antibody at either end or in the
middle, e.g., by DNA or peptide synthesis; See, e.g., WO 96/32478)
or adding molecules such as PEG or other water soluble polymers,
including polysaccharide polymers.
Preparation of Antibodies
Polyclonal Antibodies
[0301] Polyclonal antibodies are preferably raised in animals by
multiple subcutaneous (sc) or intraperitoneal (ip) injections of
the relevant antigen and an adjuvant. Alternatively, antigen may be
injected directly into the animal's lymph node (see Kilpatrick et
al., Hybridoma, 16:381-389, 1997). An improved antibody response
may be obtained by conjugating the relevant antigen to a protein
that is immunogenic in the species to be immunized, e.g., keyhole
limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean
trypsin inhibitor using a bifunctional or derivatizing agent, for
example, maleimidobenzoyl sulfosuccinimide ester (conjugation
through cysteine residues), N-hydroxysuccinimide (through lysine
residues), glutaraldehyde, succinic anhydride or other agents known
in the art.
[0302] Animals are immunized against the antigen, immunogenic
conjugates or derivatives by combining, e.g., 100 .mu.g of the
protein or conjugate (for mice) with 3 volumes of Freund's complete
adjuvant and injecting the solution intradermally at multiple
sites. One month later, the animals are boosted with 1/5 to 1/10
the original amount of peptide or conjugate in Freund's complete
adjuvant by subcutaneous injection at multiple sites. At 7-14 days
post-booster injection, the animals are bled and the serum is
assayed for antibody titer Animals are boosted until the titer
plateaus. Preferably, the animal is boosted with the conjugate of
the same antigen, but conjugated through a different cross-linking
reagent. Conjugates also can be made in recombinant cell culture as
protein fusions. Also, aggregating agents such as alum are suitably
used to enhance the immune response.
Monoclonal Antibodies
[0303] Monoclonal antibodies can be made using the hybridoma method
first described by Kohler et al., Nature, 256:495 (1975), or by
recombinant DNA methods. In the hybridoma method, a mouse or other
appropriate host animal, such as rats, hamster or macaque monkey,
is immunized to elicit lymphocytes that produce or are capable of
producing antibodies that will specifically bind to the protein
used for immunization. Alternatively, lymphocytes may be immunized
in vitro. Lymphocytes then are fused with myeloma cells using a
suitable fusing agent, such as polyethylene glycol, to form a
hybridoma cell (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103 (Academic Press, 1986)). The hybridoma cells
thus prepared are seeded and grown in a suitable culture medium
that preferably contains one or more substances that inhibit the
growth or survival of the unfused, parental myeloma cells. For
example, if the parental myeloma cells lack the enzyme hypoxanthine
guanine phosphoribosyl transferase (HGPRT or HPRT), the culture
medium for the hybridomas typically will include hypoxanthine,
aminopterin, and thymidine (HAT medium), which substances prevent
the growth of HGPRT-deficient cells.
[0304] Preferred myeloma cells are those that fuse efficiently,
support stable high-level production of antibody by the selected
antibody-producing cells and are sensitive to a medium. Human
myeloma and mouse-human heteromyeloma cell lines also have been
described for the production of human monoclonal antibodies
(Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal
Antibody Production Techniques and Applications, pp. 51-63 (Marcel
Dekker, Inc., New York, 1987)). Exemplary murine myeloma lines
include those derived from MOP-21 and M.C.-11 mouse tumors
available from the Salk Institute Cell Distribution Center, San
Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the
American Type Culture Collection, Rockville, Md. USA. Culture
medium in which hybridoma cells are growing is assayed for
production of monoclonal antibodies directed against the antigen.
Preferably, the binding specificity of monoclonal antibodies
produced by hybridoma cells is determined by immunoprecipitation or
by an in vitro binding assay, such as radioimmunoassay (RIA) or
enzyme-linked immunoabsorbent assay (ELISA). The binding affinity
of the monoclonal antibody can be determined, for example, by
BIAcore or Scatchard analysis (Munson et al., Anal. Biochem.,
107:220 (1980)).
[0305] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the clones
can be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture
media for this purpose include, for example, D-MEMO or RPMI 1640
medium. In addition, the hybridoma cells can be grown in vivo as
ascites tumors in an animal. The monoclonal antibodies secreted by
the subclones are suitably separated from the culture medium,
ascites fluid, or serum by conventional immunoglobulin purification
procedures such as protein A-Sepharose, hydroxylapatite
chromatography, gel electrophoresis, dialysis, or affinity
chromatography.
Recombinant Production of Antibodies
[0306] The amino acid sequence of an immunoglobulin of interest can
be determined by direct protein sequencing, and suitable encoding
nucleotide sequences can be designed according to a universal codon
table.
[0307] Alternatively, DNA encoding the monoclonal antibodies can be
isolated and sequenced from the hybridoma cells using conventional
procedures (e.g., by using oligonucleotide probes that are capable
of binding specifically to genes encoding the heavy and light
chains of the monoclonal antibodies). Sequence determination will
generally require isolation of at least a portion of the gene or
cDNA of interest. Usually this requires cloning the DNA or mRNA
encoding the monoclonal antibodies. Cloning is carried out using
standard techniques (see, e.g., Sambrook et al. (1989) Molecular
Cloning: A Laboratory Guide, Vols 1-3, Cold Spring Harbor Press,
which is incorporated herein by reference). For example, a cDNA
library can be constructed by reverse transcription of polyA+ mRNA,
preferably membrane-associated mRNA, and the library screened using
probes specific for human immunoglobulin polypeptide gene
sequences. In a preferred embodiment, the polymerase chain reaction
(PCR) is used to amplify cDNAs (or portions of full-length cDNAs)
encoding an immunoglobulin gene segment of interest (e.g., a light
chain variable segment). The amplified sequences can be cloned
readily into any suitable vector, e.g., expression vectors,
minigene vectors, or phage display vectors. It will be appreciated
that the particular method of cloning used is not critical, so long
as it is possible to determine the sequence of some portion of the
immunoglobulin polypeptide of interest.
[0308] One source for RNA used for cloning and sequencing is a
hybridoma produced by obtaining a B cell from the transgenic mouse
and fusing the B cell to an immortal cell. An advantage of using
hybridomas is that they can be easily screened, and a hybridoma
that produces a human monoclonal antibody of interest selected.
Alternatively, RNA can be isolated from B cells (or whole spleen)
of the immunized animal. When sources other than hybridomas are
used, it may be desirable to screen for sequences encoding
immunoglobulins or immunoglobulin polypeptides with specific
binding characteristics. One method for such screening is the use
of phage display technology. Phage display is described in e.g.,
Dower et al., WO 91/17271, McCafferty et al., WO 92/01047, and
Caton and Koprowski, Proc. Natl. Acad. Sci. USA, 87:6450-6454
(1990), each of which is incorporated herein by reference. In one
embodiment using phage display technology, cDNA from an immunized
transgenic mouse (e.g., total spleen cDNA) is isolated, PCR is used
to amplify cDNA sequences that encode a portion of an
immunoglobulin polypeptide, e.g., CDR regions, and the amplified
sequences are inserted into a phage vector. cDNAs encoding peptides
of interest, e.g., variable region peptides with desired binding
characteristics, are identified by standard techniques such as
panning.
[0309] The sequence of the amplified or cloned nucleic acid is then
determined. Typically the sequence encoding an entire variable
region of the immunoglobulin polypeptide is determined, however,
sometimes only a portion of a variable region need be sequenced,
for example, the CDR-encoding portion. Typically the sequenced
portion will be at least 30 bases in length, and more often bases
coding for at least about one-third or at least about one-half of
the length of the variable region will be sequenced.
[0310] Sequencing can be carried out on clones isolated from a cDNA
library or, when PCR is used, after subcloning the amplified
sequence or by direct PCR sequencing of the amplified segment.
Sequencing is carried out using standard techniques (see, e.g.,
Sambrook et al. (1989) Molecular Cloning: A Laboratory Guide, Vols
1-3, Cold Spring Harbor Press, and Sanger, F. et al. (1977) Proc.
Natl. Acad. Sci. USA 74: 5463-5467, which is incorporated herein by
reference). By comparing the sequence of the cloned nucleic acid
with published sequences of human immunoglobulin genes and cDNAs,
an artisan can determine readily, depending on the region
sequenced, (i) the germline segment usage of the hybridoma
immunoglobulin polypeptide (including the isotype of the heavy
chain) and (ii) the sequence of the heavy and light chain variable
regions, including sequences resulting from N-region addition and
the process of somatic mutation. One source of immunoglobulin gene
sequence information is the National Center for Biotechnology
Information, National Library of Medicine, National Institutes of
Health, Bethesda, Md.
[0311] Once isolated, the DNA may be operably linked to expression
control sequences or placed into expression vectors, which are then
transfected into host cells such as E. coli cells, simian COS
cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do
not otherwise produce immunoglobulin protein, to direct the
synthesis of monoclonal antibodies in the recombinant host
cells.
[0312] Expression control sequences denote DNA sequences necessary
for the expression of an operably linked coding sequence in a
particular host organism. The control sequences that are suitable
for prokaryotes, for example, include a promoter, optionally an
operator sequence, and a ribosome-binding site. Eukaryotic cells
are known to utilize promoters, polyadenylation signals, and
enhancers.
[0313] Nucleic acid is operably linked when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome-binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, operably linked means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking can be accomplished
by ligation at convenient restriction sites. If such sites do not
exist, synthetic oligonucleotide adaptors or linkers can be used in
accordance with conventional practice.
[0314] Cell, cell line, and cell culture are often used
interchangeably and all such designations include progeny.
Transformants and transformed cells include the primary subject
cell and cultures derived therefrom without regard for the number
of transfers. It also is understood that all progeny may not be
precisely identical in DNA content, due to deliberate or
inadvertent mutations. Mutant progeny that have the same function
or biological activity as screened for in the originally
transformed cell are included.
[0315] Isolated nucleic acids also are provided that encode
specific antibodies, optionally operably linked to control
sequences recognized by a host cell, vectors and host cells
comprising the nucleic acids, and recombinant techniques for the
production of the antibodies, which may comprise culturing the host
cell so that the nucleic acid is expressed and, optionally,
recovering the antibody from the host cell culture or culture
medium.
[0316] A variety of vectors are known in the art. Vector components
can include one or more of the following: a signal sequence (that,
for example, can direct secretion of the antibody), an origin of
replication, one or more selective marker genes (that, for example,
can confer antibiotic or other drug resistance, complement
auxotrophic deficiencies, or supply critical nutrients not
available in the media), an enhancer element, a promoter, and a
transcription termination sequence, all of which are well known in
the art.
[0317] Suitable host cells include prokaryote, yeast, or higher
eukaryote cells. Suitable prokaryotes include eubacteria, such as
Gram-negative or Gram-positive organisms, for example,
Enterohacteriaceae such as Escherichia, e.g., E. coli,
Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g.,
Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B. subtilis and B.
licheniformis, Pseudomonas, and Streptomyces. In addition to
prokaryotes, eukaryotic microbes such as filamentous fungi or yeast
are suitable cloning or expression hosts for antibody-encoding
vectors. Saccharomyces cerevisiae, or common baker's yeast, is the
most commonly used among lower eukaryotic host microorganisms.
However, a number of other genera, species, and strains are
commonly available, such as Pichia, e.g. P. pastoris,
Schizosaccharomyces pombe; Kluyveromyces, Yarrowia; Candida;
Trichodemia reesia; Neurospora crassa; Schwanniomyces such as
Schwanniomyces occidentalis; and filamentous fungi such as, e.g.,
Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such
as A. nidulans and A. niger.
[0318] Suitable host cells for the expression of glycosylated
antibodies are derived from multicellular organisms. Examples of
invertebrate cells include plant and insect cells. Numerous
baculoviral strains and variants and corresponding permissive
insect host cells from hosts such as Spodoptera frugiperda
(caterpillar), Aedes aegypti (mosquito), Aedes albopictus
(mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori
have been identified. A variety of viral strains for transfection
of such cells are publicly available, e.g., the L-I variant of
Autographa californica NPV and the Bm-5 strain of Bombyx mori
NPV.
[0319] However, interest has been greatest in vertebrate cells, and
propagation of vertebrate cells in culture (tissue culture) has
become routine. Examples of useful mammalian host cell-lines are
Chinese hamster ovary cells, including CHOK1 cells (ATCC CCL61) and
Chinese hamster ovary cells/-DHFR (DXB-11, DG-44; Urlaub et al,
Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); monkey kidney CV1 line
transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney
line (293 or 293 cells subcloned for growth in suspension culture,
[Graham et al., J. Gen Virol. 36: 59 (1977)]; baby hamster kidney
cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol.
Reprod. 23: 243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70);
African green monkey kidney cells (VERO-76, ATCC CRL-1587); human
cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells
(MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL
1442); human lung cells (WI38, ATCC CCL 75); human hepatoma cells
(Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51);
TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383: 44-68
(1982)); MRC 5 cells and FS4 cells.
[0320] The host cells can be cultured in a variety of media.
Commercially available media such as Ham's F10 (Sigma), Minimal
Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's
Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing
the host cells. In addition, any of the media described in Ham et
al., Meth. Enz. 58: 44 (1979), Barnes et al., Anal. Biochem. 102:
255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;
4,560,655; or 5,122,469; WO90103430; WO 87/00195; or U.S. Pat. Re.
No. 30,985 can be used as culture media for the host cells. Any of
these media can be supplemented as necessary with hormones and/or
other growth factors (such as insulin, transferrin, or epidermal
growth factor), salts (such as sodium chloride, calcium, magnesium,
and phosphate), buffers (such as HEPES), nucleotides (such as
adenosine and thymidine), antibiotics (such as Gentamycin.TM.
drug), trace elements (defined as inorganic compounds usually
present at final concentrations in the micromolar range), and
glucose or an equivalent energy source. Any other necessary
supplements also can be included at appropriate concentrations that
would be known to those skilled in the art. The culture conditions,
such as temperature, pH, and the like, are those previously used
with the host cell selected for expression, and will be apparent to
the artisan.
[0321] The antibody composition can be purified using, for example,
hydroxylapatite chromatography, cation or anion exchange
chromatography, or preferably affinity chromatography, using the
antigen of interest or protein A or protein G as an affinity
ligand. Protein A can be used to purify antibodies that are based
on human .gamma.1, .gamma.2, or .gamma.4 heavy chains (Lindmark et
al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended
for all mouse isotypes and for human .gamma.3 (Guss et al., 20 EMBO
J. 5: 15671575 (1986)). The matrix to which the affinity ligand is
attached is most often agarose, but other matrices are available.
Mechanically stable matrices such as controlled pore glass or
poly(styrenedivinyl)benzene allow for faster flow rates and shorter
processing times than can be achieved with agarose. Where the
antibody comprises a CH3 domain, the Bakerbond ABX.TM. resin (J. T.
Baker, Phillipsburg, 25 NJ.) is useful for purification. Other
techniques for protein purification such as ethanol precipitation,
Reverse Phase HPLC, chromatofocusing, SDS-PAGE, and ammonium
sulfate precipitation are also possible depending on the specific
binding agent or antibody to be recovered.
[0322] A person skilled in the art with the information of this
patent application at hand will readily know how to perform the
methods according to the invention. One example would be to coat a
sepharose column with a polypeptide having the sequence of SEQ ID
NO: 2, followed by incubation with an immunoglobulin fraction
(antibody fraction) derived from the sample, performing a washing
step and subsequent elution of the A.beta.(21-37) specific
antibodies bound to the column. For the methods of separation
according to the invention the polypeptide, comprising an amino
acid sequence of an A.beta. polypeptide, wherein the sequence of
the A.beta. polypeptide has at least the sequence according to SEQ
ID NO:2 and at most the sequence according to SEQ ID NO:4,
additionally comprises moieties such as tags or markers, in
particular biotin, streptavidin, GST, HIS, STREP-tag, Myc, HA,
poly-L-lysine, poly-L-lysine-L-alanine copolymers, poly-Aib
(alpha-aminoisobutyric acid), poly-.beta.-alanine, poly-L-alanine,
poly-D-lysine, poly-D-lysine-D-alanine copolymers, poly-D-alanine,
or combinations of poly-L- and D-amino acids. These markers can
provide for a facilitated binding of this polypeptide to a carrier.
Elution, i.e. separation of polypeptides according to the present
invention from the carrier can, for instance, be achieved by
applying a short-term pH change to pH 2, by adding excess of a
polypeptide comprising the sequence according to SEQ ID NO:2 or by
increasing the salt content of the elution buffer. Further,
non-limiting examples for isolation of polypeptides from the serum
of a subject or from commercially available IVIgG preparations are
given in more detail in the examples section of this
application.
[0323] Antibodies can not only be derived from a human subject but
also from an animal. Besides isolating pre-existing
anti-A.beta.(21-37) autoantibodies from the blood of such an animal
in analogy to the methods described above, such animals can be
additionally immunized with a polypeptide comprising an amino acid
sequence of an A.beta. polypeptide, wherein the sequence of the
A.beta. polypeptide has at least the sequence according to SEQ ID
NO:2 and at most the sequence according to SEQ ID NO:4. After
several rounds of immunization the corresponding A.beta.-specific
antibody producing B-cells can be obtained from the blood of the
animal by routine methods.
[0324] If desired, such B cell clones (of human or animal origin)
can be converted into a cell line, for example by isolating cells
from the spleen of an animal and immortalizing them by transfection
with Epstein Barr Virus or by fusing the cells with myeloma cells
(hybridoma technology). The latter is especially useful for
producing antibodies in large quantities.
[0325] Alternatively, the polypeptides of the present invention can
be, if suitable, directly synthesized, either by conventional
polypeptide synthesis, by in vitro translation or by any other
means for synthesizing polypeptides and proteins. A person skilled
in the art will be familiar with a multitude of appropriate
techniques and will be readily able to apply them to the
subject-matter of the present invention.
[0326] Additionally, the polypeptide binding to the epitope
A.beta.(21-37) (SEQ ID NO:2) of amyloid-beta (1-40) (SEQ ID NO: 1)
can originate from other proteins than antibodies. As an example,
anticalins can be engineered to bind to certain epitopes i.e. to
the epitope as denoted in SEQ ID NO: 2. Anticalins are a class of
engineered ligand-binding proteins that are based on the lipocalin
scaffold. Using targeted mutagenesis of the loop region and
biochemical selection techniques, variants with novel ligand
specificities, both for low-molecular weight substances and for
macromolecular protein targets, can be generated (see DE 199 26
068; Schlehuber et al., J. Mol. Biol. (2000), 297 (5) p. 1105-1120;
Expert Opin Biol Ther. 2005, 5(11), p. 1453-62). Binding of such a
polypeptide to an A.beta. C-terminal part, in particular to A.beta.
(21-37) can also provide for an inhibition of polymerization of AD
peptide and thus provide an efficient means for treatment and or
prophylaxis of Alzheimer's Disease and other neurodementing
diseases.
[0327] The polypeptides according to the invention can also be
obtained via a recombinant expression system. In order to express a
polypeptide according to the invention, a respective nucleic acid
expression construct has to be generated. Therefore, the present
invention relates also to a nucleic acid having a sequence encoding
for a polypeptide according to the present invention, in particular
encoding the light chain or the heavy chain of one of the above
mentioned antibodies, derivatives or fragments thereof or encoding
for another protein according to the invention such as an anticalin
binding to A.beta.(21-37). Such a nucleic acid can be for instance
obtained by identifying the amino acid sequence of one of the
peptides mentioned above, for instance via mass spectrometry means,
via Edman sequencing or any other method for protein sequencing
known to the skilled artisan. Following the genetic code a nucleic
acid sequence can be derived from the amino acid sequence.
Preferably, the generated nucleic acid sequence is optimized in
regard to the codon usage of the respective expression system of
interest. Alternatively, cells expressing such an antibody can be
isolated (see above) and the genomic loci or mRNA encoding for the
heavy and light chain of the antibody specific for the C-Terminus
of A.beta. are sequenced. For certain expression systems this
nucleic acid sequence might need to be adapted in order to provide
for an optimal codon usage. A person skilled in the art with the
above mentioned nucleic acid sequences at hand will be readily
capable of generating expression constructs for use in a suitable
expression system. Therefore, the present invention also relates to
an expression construct providing for the expression of the
polypeptides of the invention and to an isolated cell, which
expresses a polypeptide or a fragment thereof according to the
invention. Expression constructs, i.e. vectors include plasmids,
cosmids, viruses (bacteriophage, animal viruses, and plant
viruses), episomes and artificial chromosomes (e.g., YACs). One of
skill in the art would be able to construct a vector by standard
recombinant techniques. Said cell can be for example a myeloma
cell, a Chinese hamster ovary cell, a Syrian hamster ovary cell, a
human embryonic kidney cell, insect cell (baculovirus system), or a
transgenic plant cell, which is transformed with an expression
vector according to the invention (see Schillberg et al., Cell Mol
Life Sci. 2003 60(3): p. 433-445) or a cell, which endogenously
expresses a polypeptide according to the invention (hybridoma). The
expression of recombinant polypeptides of the invention is not
restricted to eukaryotic cells but can also be expressed in
prokaryotes. Recombinant polypeptides according to the invention
can be obtained from any such cell by purification means which are
well known in the art.
[0328] In a preferred embodiment, the polypeptide according to the
invention is an antibody or a fragment thereof and is optionally
encoded by two expression vectors, i.e. one expression vector for
the light chain and one for the heavy chain.
[0329] In order to bind within the polypeptide fragment
A.beta.(21-37), an antibody or a fragment thereof requires
primarily an intact antigen binding domain (variable domain). The
constant region of such an antibody is usually not critical for
antigen binding. Thus, it is clear for a person skilled in the art,
that if the polypeptide according to the invention is an antibody
or a fragment thereof, this antibody/fragment may have any given
isotype selected from the group consisting of IgG, IgM, IgA, IgD
and IgE, including all respective subclasses of these isotypes. If
the polypeptide according to the present invention is expressed as
an antibody in an immune cell or hybridoma cell, the isotype can be
switched by additional expression or administration of activation
induced cytidine deaminase and administration of stimulating
factors known to the skilled artisan.
[0330] In one embodiment, the polypeptides according to the
invention are chemically coupled or fused with substances which
prevent plaque aggregation and/or lead to the disintegration of
toxic A.beta. oligomers. Such a substance could be for example a
protease cleaving Amyloid beta polypeptide as used for the
experimental characterization of the epitope according to the
invention, i.e. Serine-proteases like Trypsin, Chymotrypsin; or
Lys-C protease, Arg-C protease, Asp-N-proteases, also unspecific
proteases such as proteinase-K, thermolysine, subtilisin.
[0331] The present invention also relates to a method of isolation
and separation of a polypeptide according to the invention from a
sample, the method comprising the following steps: [0332] a)
incubating a polypeptide comprising an amino acid sequence of an
A.beta. polypeptide, wherein the sequence of the A.beta.
polypeptide has at least the sequence according to SEQ ID NO:2,
i.e. A.beta.(21-37) and at most the sequence according to SEQ ID
NO:4, i.e. A.beta.(12-40), which polypeptide is immobilized on a
carrier, with a sample, [0333] b) separating said sample from the
carrier. [0334] c) separating polypeptides according to the
invention bound to the polypeptide of step a) from the carrier.
[0335] In an alternative approach, a polypeptide according to the
invention can be obtained by incubating the sample with the
polypeptide comprising an amino acid sequence of an A.beta.
polypeptide, wherein the sequence of the A.beta. polypeptide has at
least the sequence according to SEQ ID NO:2 and at most the
sequence according to SEQ ID NO:4 prior to incubation with the
carrier. Thus, the present invention also relates to a method of
separation of a polypeptide according to the invention from a
sample, the method comprising the following steps: [0336] a)
incubating a polypeptide, comprising an amino acid sequence of an
A.beta. polypeptide, wherein the sequence of the A.beta.
polypeptide has at least the sequence according to SEQ ID NO:2 and
at most the sequence according to SEQ ID NO:4, with a sample and
subsequently [0337] b) incubating the sample with a carrier having
a binding affinity for the polypeptide of step a), and [0338] c)
separating said sample from the carrier, and, [0339] d) separating
polypeptides according to the invention bound to the polypeptide of
step a) from the carrier.
[0340] Strategies and techniques are well known in the art to
obtain the above mentioned polypeptides, i.e. via genetic
engineering and expression of the respective polypeptide in a cell
line of interest. Antibody fragments according to the invention do
not have to be physically derived from an intact antibody but can
also be genetically engineered by conventional means.
[0341] The above mentioned polypeptides can be obtained for example
by screening antibodies derived from the blood of a human subject
for the capacity of binding to amino acid 21 to 37 of SEQ ID NO 1
(i.e. SEQ ID NO: 2).
Methods of Treatment
[0342] In another aspect, the present invention relates to the use
of the above mentioned polypeptides according to the present
invention for use in human and veterinary medicine.
[0343] In particular, the polypeptides according to the present
invention can be used for the manufacture of a medicament in order
to treat and/or prevent the progression of Alzheimer's disease,
Down's syndrome, Dementia with Lewy bodies, fronto-temporal
dementia, cerebral amyloid angiopathy, and/or amyloidoses.
[0344] Furthermore, the present invention relates to the use of a
polypeptide comprising an amino acid sequence of an A.beta.
peptide, wherein the A.beta. peptide has at least the sequence
according to SEQ ID NO:2 and at most the sequence according to SEQ
ID NO:4 for use in human and veterinary medicine, in particular for
the manufacture of a medicament for the treatment and/or prevention
of Alzheimer's disease, Down's syndrome, Dementia with Lewy bodies,
fronto-temporal dementia, cerebral amyloid angiopathy, and
amyloidoses.
[0345] In this invention, the inventors identified two A.beta.
epitope sequences recognized by A.beta. autoantibodies isolated
from serum of AD patients as well as of healthy control
individuals. The A.beta. autoantibodies of healthy control
individuals were found to specifically recognize a C-terminal part
of the A.beta. sequence, namely A.beta.(21-37) (SEQ ID NO:2).
Furthermore, AD patients have an increased fraction of antibodies
recognizing the A.beta.(4-10) part of the A.beta. polypeptide (SEQ
ID NO:3), while having a decreased fraction of the antibodies
recognizing the A.beta.(21-37) part of A.beta. polypeptide.
Additionally, the inventors found that in healthy individuals not
suffering from AD, the ratio of the amount of AD autoantibodies
directed against a specific C-terminal epitope of A.beta., namely
A.beta.(21-37) compared to the amount of AD autoantibodies binding
to A.beta.(4-10), is much higher than in AD patients. The binding
to this epitope seems to inhibit the formation of plaques and
therefore delay the onset or progression of AD. This provides the
basis for a new, therapeutic approach by administration of agents
to a human subject or animal, which agents bind to said epitope and
thereby prevents the aggregation of A.beta. peptide. This delays
the onset and/or the progression of Alzheimer's disease and
consequently provides a valuable therapeutic/prophylactic
pharmaceutical.
[0346] The inventors believe to have identified a class of natural
human antibodies selected over time by evolution to target the
toxic oligomers of A.beta. as a natural way the human body prevents
neurodementing diseases. Such antibodies would be expected not to
have pathological effects caused by the binding to A.beta. or
related peptides to the brain vessels. Recently, it has been shown
that passive immunization with antibodies directed against
A.beta.'s N-terminal part causes bleeding in an animal transgenic
mouse model. In these experiments, following 5 months of passive
immunization, a significant amyloid reduction was found in the
neocortex of the immunized mice compared to sham-treated controls.
Immunized mice, however, exhibited a more than twofold increase in
the frequency of CAA-associated cerebral hemorrhage in addition to
an increase in hemorrhage severity over controls. These adverse
events are believed to be caused by A.beta. antibodies binding to
A.beta. deposited in brain vessels (Pfeifer M, et al. 2003. Herzig
M C, et al 2004).
[0347] Thus, the present invention also relates to the use of the
above mentioned polypeptides for the manufacture of a medicament in
order to treat and/or prevent the progression of a neurodementing
disease, Alzheimer's disease, Down's syndrome, Dementia with Lewy
bodies, fronto-temporal dementia, cerebral amyloid angiopathy, and
amyloidoses. The treatment/prevention of the above mentioned
diseases is provided by prevention of A.beta. plaque formation.
Depending on the stage of the respective disease this leads to a
prevention of the disease (no onset yet) or to a treatment (after
onset of the disease), e.g. by preventing further formation of
plaques. In a preferred embodiment the medicament is formulated for
the treatment and/or prevention of plaques in the brain of a
patient.
[0348] Alternatively, as already indicated above, the present
invention relates also to the use of a polypeptide comprising an
amino acid sequence of an A.beta. polypeptide, wherein the A.beta.
polypeptide has at least the sequence according to SEQ ID NO:2 and
at most the sequence according to SEQ ID NO:4, for the manufacture
of a medicament for the treatment of Alzheimer's disease, Down's
syndrome, Dementia with Lewy bodies, fronto-temporal dementia,
cerebral amyloid angiopathy, and/or amyloidoses. In such a
scenario, this epitope serves for the active immunization of a
human or animal subject in order to enhance endogenous antibody
production against A.beta.(21-37). Such immunization approaches can
also utilize DNA vaccines, which have the benefit of avoiding the
administration of AD protein fragments. Therefore, the present
invention also relates to a nucleic acid molecule encoding for a
polypeptide comprising an amino acid sequence of an A.beta.
polypeptide, wherein the A.beta. polypeptide has at least the
sequence according to SEQ ID NO:2 and at most the sequence
according to SEQ ID NO:4. Consequently the present invention also
relates to the use of this nucleic acid sequence for the
manufacture of a medicament for the treatment and/or prevention of
Alzheimer's disease, Down's syndrome, Dementia with Lewy bodies,
fronto-temporal dementia, cerebral amyloid angiopathy or
amyloidoses.
Administration and Preparation of Pharmaceutical Formulations
[0349] The anti-A.beta. antibodies can be formulated into
pharmaceutical compositions comprising a carrier suitable for the
desired delivery method. Suitable carriers include any material
which, when combined with the antibody, retains the high-affinity
binding of A.beta. and is nonreactive with the subject's immune
system. Examples include, but are not limited to, any of a number
of standard pharmaceutical carriers such as sterile phosphate
buffered saline solutions, bacteriostatic water, and the like. A
variety of aqueous carriers may be used, e.g., water, buffered
water, 0.4% saline, 0.3% glycine and the like, and can include
other proteins for enhanced stability, such as albumin,
lipoprotein, globulin, etc., subjected to mild chemical
modifications or the like.
[0350] Exemplary antibody concentrations in the formulation can
range from about 0.1 mg/ml to about 180 mg/ml or from about 0.1
mg/mL to about 50 mg/mL, or from about 0.5 mg/mL to about 25 mg/mL,
or alternatively from about 2 mg/mL to about 10 mg/mL. An aqueous
formulation of the antibody can be prepared in a pH-buffered
solution, for example, at pH ranging from about 4.5 to about 6.5,
or from about 4.8 to about 5.5, or alternatively about 5.0.
Examples of buffers that are suitable for a pH within this range
include, for example, acetate (e.g. sodium acetate), succinate
(such as sodium succinate), gluconate, histidine, citrate and other
organic acid buffers. The buffer concentration can be from about 1
mM to about 200 mM, or from about 10 mM to about 60 mM, depending,
for example, on the buffer and the desired isotonicity of the
formulation.
[0351] A tonicity agent, which also can stabilize the antibody, can
be included in the formulation. Exemplary tonicity agents include
sugar alcohols, such as mannitol, sucrose or trehalose. Preferably
the aqueous formulation is isotonic, although hypertonic or
hypotonic solutions may be suitable. Exemplary concentrations of
the sugar alcohol in the formulation may range from about 1% to
about 15% w/v.
[0352] A surfactant also may be added to the antibody formulation
to reduce aggregation of the formulated antibody and/or minimize
the formation of particulates in the formulation and/or reduce
adsorption. Exemplary surfactants include nonionic surfactants such
as polysorbates (e.g. polysorbate 20, or polysorbate 80) or
poloxamers (e.g. poloxamer 188). Exemplary concentrations of
surfactant may range from about 0.001% to about 0.5%, or from about
0.005% to about 0.2%, or alternatively from about 0.004% to about
0.01% w/v.
[0353] In one embodiment, the formulation contains the
above-identified agents (i.e. antibody, buffer, polyol and
surfactant) and is essentially free of one or more preservatives,
such as benzyl alcohol, phenol, m-cresol, chlorobutanol and
benzethonium Cl. In another embodiment, a preservative may be
included in the formulation, e.g., at concentrations ranging from
about 0.1% to about 2%, or alternatively from about 0.5% to about
1%. One or more other pharmaceutically acceptable carriers,
excipients or stabilizers such as those described in Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) may be
included in the formulation provided that they do not adversely
affect the desired characteristics of the formulation. Acceptable
carriers, excipients or stabilizers are nontoxic to recipients at
the dosages and concentrations employed and include: additional
buffering agents; co-solvents; antioxidants including ascorbic acid
and methionine; chelating agents such as EDTA; metal complexes
(e.g. Zn-protein complexes); biodegradable polymers such as
polyesters; and/or salt-forming counter-ions such as sodium.
[0354] Therapeutic formulations of the antibody are prepared for
storage by mixing the antibody having the desired degree of purity
with optional physiologically acceptable carriers, excipients or
stabilizers (Remington's Pharmaceutical Sciences 16th edition,
Osol, A. Ed. (1980)), in the form of lyophilized formulations or
aqueous solutions. Acceptable carriers, excipients, or stabilizers
are nontoxic to recipients at the dosages and concentrations
employed, and include buffers such as phosphate, citrate, and other
organic acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
maltose, or dextrins; chelating agents such as EDTA; sugars such as
sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions
such as sodium; metal complexes (e.g., Zn-protein complexes);
and/or non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
[0355] In one embodiment, a suitable formulation contains an
isotonic buffer such as a phosphate, acetate or TRIS buffer in
combination with a tonicity agent such as a sugar alcohol,
Sorbitol, sucrose or sodium chloride which tonicities and
stabilizes. One example of such a tonicity agent is 5% Sorbitol or
sucrose. In addition, the formulation optionally can include a
surfactant such as to prevent aggregation and for stabilization at
0.01 to 0.02% w/v. The pH of the formulation can range from 4.5-6.5
or 4.5 to 5.5. Other exemplary descriptions of pharmaceutical
formulations for antibodies can be found in US 2003/0113316 and
U.S. Pat. No. 6,171,586, each incorporated herein by reference in
its entirety.
[0356] The formulation herein also can contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect each other. For example, it may be desirable to
further provide an immunosuppressive agent. Such molecules are
suitably present in combination in amounts that are effective for
the purpose intended.
[0357] The active ingredients also can be entrapped in microcapsule
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsule and poly-(methylmethacrylate) microcapsule,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions or minicells. Such techniques
are disclosed in Remington's Pharmaceutical Sciences 16th edition,
Osol, A. Ed. (1980).
[0358] Suspensions and crystal forms of antibodies are also
contemplated. Methods to make suspensions and crystal forms are
known to one of skill in the art.
[0359] The formulations to be used for in vivo administration must
be sterile. The compositions of the invention can be sterilized by
conventional, well known sterilization techniques. For example,
sterilization is accomplished readily by filtration through sterile
filtration membranes. The resulting solutions may be packaged for
use or filtered under aseptic conditions and lyophilized, the
lyophilized preparation being combined with a sterile solution
prior to administration. The process of freeze-drying is often
employed to stabilize polypeptides for long-term storage,
particularly when the polypeptide is relatively unstable in liquid
compositions. A lyophilization cycle is usually composed of three
steps: freezing, primary drying, and secondary drying; Williams and
Polli, Journal of Parenteral Science and Technology, Volume 38,
Number 2, pages 48-59 (1984). In the freezing step, the solution is
cooled until it is adequately frozen. Bulk water in the solution
forms ice at this stage. The ice sublimes in the primary drying
stage, which is conducted by reducing chamber pressure below the
vapor pressure of the ice, using a vacuum. Finally, sorbed or bound
water is removed at the secondary drying stage under reduced
chamber pressure and an elevated shelf temperature. The process
produces a material known as a lyophilized cake. Thereafter the
cake can be reconstituted prior to use.
[0360] The standard reconstitution practice for lyophilized
material is to add back a volume of pure water (typically
equivalent to the volume removed during lyophilization), although
dilute solutions of antibacterial agents are sometimes used in the
production of pharmaceuticals for parenteral administration; Chen,
Drug Development and Industrial Pharmacy, Volume 18, Numbers 11 and
12, pages 1311-1354 (1992).
[0361] Excipients have been noted in some cases to act as
stabilizers for freeze-dried products; Carpenter et al.,
Developments in Biological Standardization, Volume 74, pages
225-239 (1991). For example, known excipients include sugar
alcohols (including mannitol, sorbitol and glycerol); sugars
(including glucose and sucrose); and amino acids (including
alanine, glycine and glutamic acid).
[0362] In addition, sugar alcohols and sugars are often used to
protect polypeptides from freezing and drying-induced damage and to
enhance the stability during storage in the dried state. In
general, sugars, in particular disaccharides are effective in both
the freeze-drying process and during storage. Other classes of
molecules, including mono- and di-saccharides and polymers such as
PVP, also have been reported as stabilizers of lyophilized
products.
[0363] For injection, the pharmaceutical formulation and/or
medicament can be a powder suitable for reconstitution with an
appropriate solution as described above. Examples of these include,
but are not limited to, freeze-dried, rotary-dried or spray-dried
powders, amorphous powders, granules, precipitates, or
particulates. For injection, the formulations may optionally
contain stabilizers, pH modifiers, surfactants, bioavailability
modifiers and combinations of these.
[0364] Sustained-release preparations can be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g., films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the Lupron Depot.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated antibodies remain in
the body for a long time, they can denature or aggregate as a
result of exposure to moisture at 3TC, resulting in a loss of
biological activity and possible changes in immunogenicity or other
functional properties. Rational strategies can be devised for
stabilization depending on the mechanism involved. For example, if
the aggregation mechanism is discovered to be intermolecular S--S
bond formation through thio-disulfide interchange, stabilization
may be achieved by modifying sulfhydryl residues, lyophilizing from
acidic solutions, controlling moisture content, using appropriate
additives, and developing specific polymer matrix compositions.
[0365] The formulations of the invention can be designed to be
short-acting, fast-releasing, long-acting, or sustained-releasing
as described herein. Thus, the pharmaceutical formulations also can
be formulated for controlled release or for slow release.
[0366] Specific dosages can be adjusted depending on conditions of
disease, age, body weight, general health conditions, sex, and diet
of the subject, dose intervals, administration routes, excretion
rate, and combinations of drugs. Any of the above dosage forms
containing effective amounts are well within the bounds of routine
experimentation and therefore, well within the scope of the instant
invention.
[0367] The specific binding agent or antibody is administered by
any suitable means, including parenteral, subcutaneous,
intraperitoneal, intrapulmonary, and intranasal, and, if desired
for local treatment, intralesional administration. Parenteral
infusions include intravenous, intraarterial, intraperitoneal,
intramuscular, intradermal or subcutaneous administration. In
addition, the specific binding agent or antibody is suitably
administered by pulse infusion, particularly with declining doses
of the specific binding agent or antibody. Preferably, the dosing
is given by injections, most preferably intravenous or subcutaneous
injections, depending in part on whether the administration is
brief or chronic. Other administration methods are contemplated,
including topical, particularly transdermal, transmucosal, rectal,
oral or local administration e.g. through a catheter placed close
to the desired site. Most preferably, the antibody is administered
intravenously in a physiological solution at a dose ranging between
0.01 mg/kg to 100 mg/kg at a frequency ranging from daily to weekly
to monthly (e.g. every day, every other day, every third day, or 2,
3, 4, 5, or 6 times per week), preferably a dose ranging from 0.1
to 45 mg/kg, 0.1 to 15 mg/kg or 0.1 to 10 mg/kg at a frequency of 2
or 3 times per week, or up to 45 mg/kg once a month.
Administration to Brain
[0368] A variety of approaches are known in the art to effect
administration of compounds to the brain. For example, a compound
can be administered by direct intraventricular or intrathecal
injection, preferably via slow infusion to minimize impact on brain
parenchyma. The desired drug also can be delivered using a slow
release implant in the brain, or (where the drug is a polypeptide)
implanted recombinant cells that produce the drug. The blood brain
barrier (BBB) can be permeabilized concomitant with drug
administration, to permit movement of the drug across the BBB.
Permeablizing agents include osmotic agents, such as hypertonic
mannitol, or another permeabilizing agent such as bradykinin, an
alkylglycerol, ultrasound, electromagnetic radiation or
parasympathetic innervation.
[0369] Alternatively, receptor-mediated transport can be utilized
to administer drug to the brain. It is known in the art that
peptides and proteins that directly cross the BBB may serve as
carriers for selective therapeutic agents that allow the
therapeutic agents to cross the BBB after delivery into the
bloodstream (Pan et al., Brain Research Reviews, 46:32-43, 2004;
Misra et al., J. Pharm. Pharmaceut. Sci., 6:252-273, 2003; Begley,
Pharmacol Ther. 2004 October; 104(1):29-45; Poduslo, US App. Pub.
No. 2003/0082191; Poduslo et al., Biochem., 43:6064-6075, 2004).
For example, Poduslo, WO 03/020212 describes conjugation of
antibodies to amyloid-beta protein fragments which are then taken
up by low-density lipoprotein receptor related protein-1, a
transporter at the BBB. Other examples of peptides which cross the
BBB include transferrin which binds to the transferrin receptor, a
transporter at the BBB; monoclonal antibodies to the transferrin
receptor such as OX26; cell-penetrating peptides such as TAT
transduction domain, penetratin, or Syn BI; and RAP which binds to
low-density lipoprotein receptor related protein-2, another
transporter at the BBB (see Pan et al., J Cell Sci. 2004 Oct. 1;
117(Pt 21):5071-8).
[0370] Receptor-mediated drug delivery to the brain can employ
chimeric peptide technology, wherein a non-transportable drug is
conjugated to a BBB transport vector. The latter can be a modified
protein or receptor-specific monoclonal antibody that undergoes
receptor-mediated transcytosis through the BBB in vivo. Conjugation
of drug to transport vector is facilitated with chemical linkers,
avidin-biotin technology, polyethylene glycol linkers, or
liposomes. Multiple classes of therapeutics have been delivered to
the brain with the chimeric peptide technology, including
peptide-based pharmaceuticals, anti-sense therapeutics including
peptide nucleic acids (PNAs), and small molecules incorporated
within liposomes. Alternatively, the drug can be encapsulated in a
liposome or nanoparticle which is then linked to the BBB transport
vector.
Administration with Other Agents
[0371] The antibodies can be concurrently administered with other
anti-amyloidgenic therapeutic agents. Concurrent administration
includes administration of the two different therapeutic agents at
different times and at different routes, as long as there is some
overlap in the time during which the agents are exerting their
therapeutic effects.
[0372] Exemplary anti-amyloidgenic agents known in the art include
other anti-amyloid-beta antibodies, anti-inflammatories known in
the art (e.g., NSAIDs and Cox-2 inhibitors) that reduce the
pathogenic effects of amyloid accumulation, cholesterol lowering
drugs, .beta.-secretase inhibitors, or anti-inflammatories that
reduce the inflammatory response due to the administration of
A.beta. antibody or that allow monitoring of the side effects of
the anti-A.beta. antibody.
[0373] Administration of medicaments according to the present
invention can be achieved via any common route. Although the
intravenous route is a preferred embodiment, other routes of
administration are contemplated. This includes oral, nasal, buccal,
rectal, vaginal or topical. Alternatively, administration may be by
orthotopic, intradermal, subcutaneous, intramuscular or
intraperitoneal.
[0374] In another preferred embodiment, the medicament according to
the present invention comprising a polypeptide according to the
invention is formulated for a combined administration with a second
medicament for the respective disease. Examples for such therapies
would be inhibitors of acetylcholine esterase or NMDA
(N-methyl-D-aspartate) receptor antagonists. The administration of
the medicament according to the invention can be prior to,
simultaneously with or after administration of the second
medicament.
[0375] In a further aspect the present invention relates to a
method of treatment and/or prevention of Alzheimer's disease,
Down's syndrome, Dementia with Lewy bodies, fronto-temporal
dementia, cerebral amyloid angiopathy or amyloidoses comprising
administering one or more of the above mentioned polypeptides of
the invention to a subject in need thereof.
[0376] The present invention also relates to the use of a
polypeptide comprising an amino acid sequence of an A.beta.
polypeptide, wherein the sequence of the AD polypeptide has at
least the sequence according to SEQ ID NO:2, i.e. A.beta.(21-37)
and at most the sequence according to SEQ ID NO:4, i.e.
A.beta.(12-40) for isolation and separation of a polypeptide
according to the present invention from a sample.
Detecting and Measuring the Progression of Disease
Peptides
[0377] In another aspect, A.beta. peptides are provided that are
useful for detecting and/or measuring the progression of
Alzheimer's and other neurodementing diseases.
[0378] The inventors identified two A.beta. epitope sequences
recognized by A.beta. autoantibodies isolated from serum of AD
patients as well as of healthy control individuals. The A.beta.
autoantibodies of healthy control individuals were found to
specifically recognize the C-terminal part of the A.beta. sequence,
namely A.beta.(21-37) (SEQ ID NO: 2) or any other sequence
comprising A.beta.(21-37). Furthermore A.beta. patients have an
increased fraction of antibodies recognizing the N-terminus, in
particular the A.beta.(4-10) epitope of the A.beta. polypeptide
(SEQ ID NO: 3), while having a decreased fraction of the antibodies
recognizing the C-Terminus, in particular the A.beta.(21-37)
epitope or any other sequence comprising A.beta.(21-37) of the
A.beta. polypeptide. This provides the basis for a new, early
A.beta. diagnostic method by determination of AD antibodies,
wherein an elevated level of A.beta. autoantibody (A.beta.21-37) is
a positive indicator, i.e. "healthy", while an elevated level of
"plaque specific" A.beta.(4-10)-antibody is a negative indicator,
i.e. "sick", with respect to the prognosis for disease progression
in a subject.
[0379] In one aspect, the present invention relates therefore to a
polypeptide comprising an amino acid sequence of an A.beta.
peptide, wherein the A.beta. peptide has at least the sequence
according to SEQ ID NO: 2 and at most the sequence according to SEQ
ID NO: 4.
[0380] A polypeptide according to the invention having a sequence
of A.beta. as set forth above may be for instance the
A.beta.(21-37) fragment of amyloid beta itself. Other possible
embodiments could comprise for example A.beta.(20-37),
A.beta.(12-37), A.beta.(12-40) (SEQ ID NO: 4), A.beta.(20-40),
A.beta.(21-40) and so forth. This polypeptide fragment can be
joined to other moieties. This means that a polypeptide according
to the invention can comprise besides the A.beta.(21-37) portion
other polypeptide sequences or non-polypeptide structures or
portions. The moieties attached to the A.beta. polypeptide
fragments may facilitate the performance of the methods according
to the present invention. In other aspects, A.beta. polypeptides
can form oligomers. Examples of such oligomers include, but are not
limited to, oligomeric forms of A.beta.(1-40) or oligomeric forms
of A.beta.(12-20) or oligomeric forms of A.beta.(21-37).
[0381] Thus, in a preferred embodiment the polypeptide according to
the invention additionally comprises other moieties such as tags or
markers, which facilitate in particular the attachment of the
polypeptide to a carrier. In particular such tags can provide for
the immobilisation of the polypeptide on a carrier coated with the
respective antagonist. Examples for such moieties are biotin,
streptavidin, GST, HIS, STREP-tag, Myc, HA, poly-L-lysine,
poly-L-lysine-L-alanine copolymers, poly-Aib (alpha-aminoisobutyric
acid), poly-.beta.-alanine, poly-L-alanine, poly-D-lysine,
poly-D-lysine-D-alanine copolymers, poly-D-alanine, or combinations
of poly-L- and -D-amino acids. The polypeptide comprising a
sequence according to SEQ ID NO 3 and at most the sequence
according to SEQ ID NO: 5, as used in some of the methods of the
invention, can also comprise such additional moieties.
[0382] The above mentioned peptide markers can be directly fused to
the polypeptides having e.g. the sequence of SEQ ID NO 2 or 3,
respectively. If a higher flexibility between the marker/tag and
the e.g. A.beta.(21-37) peptide is desired, linkers can be
introduced. Such linkers can be for instance polyglycine or
-alanine linkers. Biotin, a non-peptidic substance, can be
covalently linked to a polypeptide of the present invention. A
multitude of possible combinations of markers and carriers for the
immobilization of a polypeptide of the present invention is known
from the prior art.
[0383] In a preferred embodiment the region of the above mentioned
polypeptide having e.g. the sequence according to SEQ ID NO: 2 is
not in .beta.-sheet conformation. The .beta.-pleated sheet
conformation of A.beta. has been shown to be responsible for
neurotoxicity. Thus, A.beta.(21-37) antibodies in a healthy
individual recognize in particular the A.beta.(21-37) region or any
other sequence comprising A.beta.(21-37), if it is in random coil
or alpha-helix conformation. In another preferred embodiment,
therefore, the region of the above mentioned polypeptide having the
sequence according to SEQ ID NO: 2 or any other sequence comprising
A.beta.(21-37) is flanked by amino acid sequences, which prevent or
reduce .beta.-sheet formation of the polypeptide region having the
sequence according to SEQ ID NO: 2. Preferably said flanking amino
acid sequences are located in close proximity to the N- and/or
C-terminal ends of the A.beta. sequence stretch, e.g. having the
sequence according to SEQ ID NO: 2 and/or which flanking amino acid
sequences are composed of oligomeric peptides comprising, for
example, L-alanine, D-alanine, Aib (alpha-aminoisobutyric acid),
.beta.-alanine, D-valine, L-glycine, D-glycine and/or related
hydrophobic amino acids. Particularly preferred as flanking amino
acid sequences are oligomeric peptides such as -(L-alanine)n-,
-(D-Alanine)n-, -(Aib)n-, -(.beta.-alanine)n-, -(D-valine)n-,
-(L-glycine)n-, -(D-glycine)n- wherein n ranges preferably from
about 2 to about 6. Such flanking regions also ensure, that the
epitope A.beta.(21-37) of the polypeptides of the invention is not
present in .beta.-sheet conformation and thus accessible to the
A.beta.(21-37) autoantibodies in the samples. In some embodiments,
the .beta.-amyloid polypeptide is in oligomeric form.
[0384] However, in a particular embodiment, the polypeptide
according to the present invention is a minimal polypeptide that
has no further sequences than the sequence of A.beta.(20-37),
A.beta.(12-37), A.beta.(12-40), A.beta.(20-40) or A.beta.(21-40).
In particular oligomeric forms of said polypeptides are preferred
embodiments of the invention.
[0385] However, it has to be understood, that other polypeptides
than the ones mentioned above, which bind specifically to a first
monoclonal antibody, which first antibody is capable of binding
specifically to a sequence as denoted in SEQ ID NO 2 or any other
sequence comprising A.beta.(21-37), but which polypeptides do not
bind specifically to a second monoclonal antibody, which second
monoclonal antibody is capable of binding specifically to a
sequence as denoted in SEQ ID NO 3, can also be used for practicing
the methods of the present invention.
[0386] In a preferred embodiment the polypeptide according to the
invention is directly synthesized by conventional polypeptide
synthesis methods (see also Example 5). Similar approaches are
suitable for generation of a polypeptide comprising an amino acid
sequence of an A.beta. peptide, wherein the A.beta. peptide has at
least the sequence according to SEQ ID NO: 3 and at most the
sequence according to SEQ ID NO: 5.
[0387] Alternatively, the polypeptide according to the invention
can also be obtained by in vitro translation or via a recombinant
expression system. In order to express a polypeptide according to
the invention a respective nucleic acid expression construct has to
be generated. A person skilled in the art will clearly see several
ways to construct appropriate expression system harboring a nucleic
acid sequence encoding for a polypeptide according to the
invention. The construct is subsequently expressed in a suitable
host cell and the polypeptide is isolated. For this purification
step the above mentioned markers and/or tags can be used as
well.
[0388] The inventors found in serum of AD patients an additional
fraction of antibodies directed against a N-terminal epitope of AD
peptide, which is not present, or only in low abundance, in healthy
individuals. Thus, the detection of such antibodies is indicative
for the diagnosis, stage and/or progression of AD. To distinguish
between A.beta.(21-37) on one hand and A.beta.(4-10) or other
antibodies directed against the N-terminal part of A.beta., for
such diagnosis methods and assays a polypeptide can be used
comprising an amino acid sequence of an A.beta. peptide, wherein
the A.beta. peptide has at least the sequence according to SEQ ID
NO: 3 (A.beta.(4-10)) and at most the sequence according to SEQ ID
NO: 5 (A.beta.(1-20)).
[0389] In one embodiment, the invention relates to a polypeptide
comprising an amino acid sequence of an A.beta. peptide, wherein
the A.beta. peptide has at least the sequence according to SEQ ID
NO: 2 and at most the sequence according to SEQ ID NO: 4.
[0390] The polypeptide according to the invention thus comprises at
least a sequence stretch identical to the AD peptide amino acid
sequence ranging from amino acid 21 to amino acid 37 of SEQ ID NO:
1. This peptide sequence is denoted herein as SEQ ID NO: 2 or
A.beta.(21-37), respectively. A polypeptide according to the
present invention can exhibit also longer sequence stretches of the
A.beta. peptide sequence, going beyond the A.beta.(21-37) sequence.
However, the length of the A.beta. peptide sequence stretch
comprised by the polypeptide according to the invention should not
range further than from amino acid 12 to amino acid 40 of the AD
peptide, also denoted herein as SEQ ID NO:4 or A.beta.(12-40). This
ensures, that the A.beta. sequence stretch of the polypeptide of
the invention does not comprise amino acids relevant for the
binding of the plaque specific A.beta. antibody found in patients
with AD. The polypeptide according to the present invention can
also comprise other amino acid sequences. For example, tags,
markers, binding domains, activation domains or similar functional
moieties can be fused to the A.beta. sequence stretch, for
instance, to provide for a better binding of the polypeptide
according to the invention to certain surfaces. Analogous, the
polypeptide according to the present invention can be modified for
certain purposes, such as covalent coupling to a fluorophor or
chromophor, biotin, or the like. In certain cases the non-A.beta.
sequence stretches of the polypeptide according to the invention
provide for a structural stabilisation of the a A.beta.(21-37)
sequence stretch, preventing or reducing the formation of
.beta.-sheet conformation in this region.
[0391] In another embodiment, the inventive polypeptides bind
specifically to oligomeric forms of .beta.-amyloid polypeptide. By
way of non-limiting example, the polypeptides can bind oligomeric
forms of A.beta.(1-40) or oligomeric forms of A.beta.(12-40) or
oligomeric forms of A.beta.(21-37). In one aspect, the inventive
polypeptides are capable of binding specifically to oligomeric
forms of A.beta.(1-40) when incubated overnight with stirring in 10
mM sodium phosphate, 150 mM NaCl, pH 7.4 at 4.degree. C.
[0392] In a preferred embodiment the above mentioned polypeptide
additionally comprises one or more moieties such as tags or
markers, in particular biotin, streptavidin, GST, HIS, STREP-tag,
Myc, HA, poly-L-Iysine, poly-L-lysine-L-alanine copolymers,
poly-Aib (alpha-aminoisobutyric acid), poly-.beta.-alanine,
poly-L-alanine, poly-D-lysine, poly-D-lysine-D-alanine copolymers,
poly-D-alanine, or combinations of poly-L- and -D-amino acids.
[0393] In a preferred embodiment the region of the above mentioned
polypeptide, which has the sequence according to SEQ ID NO: 2 or
any other sequence comprising A.beta.(21-37) is not in a
.beta.-sheet conformation. In an even more preferred embodiment
this region is in random coil or exhibits .alpha.-helix
conformation.
[0394] In another preferred embodiment the region of the above
mentioned polypeptide having e.g. the sequence according to SEQ ID
NO: 2 or any other sequence comprising A.beta.(21-37) is flanked by
amino acid sequences, which prevent or reduce .beta.-sheet
formation of the A.beta. polypeptide region having e.g. the
sequence according to SEQ ID NO: 2 or any other sequence comprising
A.beta.(21-37), in particular wherein said flanking amino acid
sequences are located in close proximity to the N- and/or
C-terminal ends of the A.beta. sequence and which flanking amino
acid sequences are composed of oligomeric peptides comprising, for
example, L-alanine, D-alanine, Aib (alpha-aminoisobutyric acid),
.beta.-alanine, D-valine, L-glycine, D-glycine and/or related
hydrophobic amino acids. Particularly preferred as flanking amino
acid sequences are oligomeric peptides such as -(L-alanine).sub.n-,
-(D-Alanine).sub.n-, -(Aib).sub.n-, -(.beta.-alanine).sub.n-,
-(D-valine).sub.n-, -(L-glycine).sub.n-, -(D-glycine).sub.n-
wherein n ranges preferably from about 2 to about 6. In some
embodiments, the .beta.-amyloid polypeptide is in oligomeric
form.
[0395] Furthermore, the present invention relates to the use of a
polypeptide according to the present invention and/or of a
polypeptide comprising an amino acid sequence of an A.beta.
peptide, wherein the A.beta. peptide has at least the sequence
according to SEQ ID NO: 3 and at most the sequence according to SEQ
ID NO: 5, for diagnostic assays. Methods for such diagnostic assays
are exemplified below.
Methods
[0396] In another aspect the present invention relates to a method
for diagnosing a neurodementing disease, the method comprising the
following steps: [0397] a) incubating a polypeptide according to
the present invention immobilized on a carrier with a sample
derived from a subject, subsequently [0398] b) separating said
sample from the carrier, and [0399] c) detecting polypeptides bound
to the immobilized polypeptide of step a).
[0400] The present invention also relates to a method for
diagnosing a neurodementing disease, the method comprising the
following steps: [0401] a) incubating a polypeptide according to
the present invention with a sample derived from a subject, and
subsequently [0402] b) incubating the sample with a carrier having
a binding affinity for the polypeptide according to the present
invention, [0403] c) separating said sample from the carrier, and
[0404] d) detecting polypeptides bound to the polypeptide according
to the present invention, said polypeptide being bound to the
carrier.
[0405] In another aspect the present invention relates to methods
of diagnosis of a neurodementing disease, which utilize a
polypeptide or protein which comprises at least a sequence stretch
identical to the A.beta. peptide amino acid sequence ranging from
amino acid 4 to amino acid 10 of SEQ ID NO: 1. This peptide
sequence is denoted herein as SEQ ID NO: 3 or A.beta.(4-10) or the
N-terminal peptide, respectively. Such a polypeptide can exhibit
also longer sequence stretches of the A.beta. peptide sequence,
going beyond the A.beta.(4-10) sequence. However, the length of the
A.beta. peptide sequence stretch comprised by such a polypeptide
should preferably not range further than from amino acid 1 to amino
acid 20 of the A.beta. peptide, also denoted herein as SEQ ID NO:5
or A.beta. (1-20). This ensures, that the A.beta. sequence stretch
of the polypeptide of the invention does not comprise amino acids
relevant for the binding of the A.beta. autoantibody found in
healthy individuals and directed to A.beta.(21-37). The N-terminal
polypeptide can comprise besides the A.beta. sequence stretch other
amino acid sequences, moieties and modifications as well, as
already mentioned for the polypeptide according to the
invention.
[0406] Thus, the present invention also relates to a method for
diagnosing a neurodementing disease, the method comprising the
following steps: [0407] a) incubating a polypeptide immobilized on
a carrier with a sample derived from a subject, wherein the
polypeptide comprises an amino acid sequence of an A.beta. peptide,
wherein the AD peptide has at least the sequence according to SEQ
ID NO: 3 and at most the sequence according to SEQ ID NO: 5,
subsequently [0408] b) separating said sample from the carrier, and
[0409] c) detecting polypeptides bound to the immobilized
polypeptide of step a).
[0410] The present invention also relates to a method for
diagnosing a neurodementing disease, the method comprising the
following steps: [0411] a) incubating a polypeptide with a sample
derived from a subject, wherein the polypeptide comprises an amino
acid sequence of an A.beta. peptide, wherein the A.beta. peptide
has at least the sequence according to SEQ ID NO: 3 and at most the
sequence according to SEQ ID NO: 5, and subsequently [0412] b)
incubating the sample with a carrier having a binding affinity for
the polypeptide comprising an amino acid sequence of an A.beta.
peptide, wherein the A.beta. peptide has at least the sequence
according to SEQ ID NO: 3 and at most the sequence according to SEQ
ID NO: 5, [0413] c) separating said sample from the carrier, and
[0414] d) detecting polypeptides bound to the polypeptide, which
comprises an amino acid sequence of an A.beta. peptide, wherein the
AD peptide has at least the sequence according to SEQ ID NO: 3 and
at most the sequence according to SEQ ID NO: 5, said polypeptide
being bound to the carrier.
[0415] The present invention also relates to a method for
diagnosing a neurodementing disease, the method comprising the
following steps: [0416] a) incubating a polypeptide according to
the present invention immobilized on a carrier with a cell
containing sample derived from a subject, [0417] b) separating said
sample from the carrier, and [0418] c) detecting cells bound to the
immobilized polypeptide of step a).
[0419] The present invention also relates to a method for
diagnosing a neurodementing disease, the method comprising the
following steps: [0420] a) incubating a polypeptide according to
the present invention with a cell containing sample derived from a
subject, and subsequently [0421] b) incubating the sample with a
carrier having a binding affinity for a polypeptide of the present
invention, [0422] c) separating said sample from the carrier, and
[0423] d) detecting cells bound to the polypeptide according to the
present invention, said polypeptide being bound to the carrier.
[0424] The present invention also relates to a method for
diagnosing a neurodementing disease, the method comprising the
following steps: [0425] a) incubating a polypeptide immobilized on
a carrier with a cell containing sample derived from a subject,
wherein the polypeptide comprises an amino acid sequence of an
A.beta. peptide, wherein the A.beta. peptide has at least the
sequence according to SEQ ID NO: 3 and at most the sequence
according to SEQ ID NO: 5, [0426] b) separating said sample from
the carrier, and [0427] c) detecting cells bound to the immobilized
polypeptide of step a).
[0428] The present invention also relates to a method for
diagnosing a neurodementing disease, the method comprising the
following steps: [0429] a) incubating a polypeptide with a cell
containing sample derived from a subject, wherein the polypeptide
comprises an amino acid sequence of an A.beta. peptide, wherein the
A.beta. peptide has at least the sequence according to SEQ ID NO: 3
and at most the sequence according to SEQ ID NO: 5, and
subsequently [0430] b) incubating the sample with a carrier having
a binding affinity for the polypeptide comprising an amino acid
sequence of an A.beta. peptide, wherein the A.beta. peptide has at
least the sequence according to SEQ ID NO: 3 and at most the
sequence according to SEQ ID NO: 5, [0431] c) separating said
sample from the carrier, and [0432] d) detecting cells bound to the
polypeptide, which comprises an amino acid sequence of an A.beta.
peptide, wherein the AD peptide has at least the sequence according
to SEQ ID NO: 3 and at most the sequence according to SEQ ID NO: 5,
said polypeptide being bound to the carrier.
[0433] In a further embodiment of the invention the polypeptide,
which comprises an amino acid sequence of an A.beta. peptide,
wherein the A.beta. peptide has the sequence according to SEQ ID
NO: 3 and at most the sequence according to SEQ ID NO: 5, can
comprise, as mentioned above, additional moieties such as tags or
markers, in particular biotin, streptavidin, GST, HIS, STREP-tag,
Myc, HA, poly-L-lysine, poly-L-lysine-L-alanine copolymers,
poly-Aib (alpha-aminoisobutyric acid), poly-.beta.-alanine,
poly-L-alanine, poly-D-lysine, poly-D-lysine-D-alanine copolymers,
poly-D-alanine, or combinations of poly-L- and -D-amino acids. In a
preferred embodiment, a polypeptide of the invention is identical
to the polypeptide, which comprises an amino acid sequence of an
A.beta. peptide, wherein the A.beta. peptide has the sequence
according to SEQ ID NO: 3 and at most the sequence according to SEQ
ID NO: 5, except for the sequence stretch covering the
A.beta.-sequence.
[0434] In a preferred embodiment of the methods of the invention
employing a polypeptide according to the invention a solvent is
present in the incubation step(s), which prevents or reduces
.beta.-sheet formation of the polypeptide region having e.g. the
sequence according to SEQ ID NO: 2. Preferred solvents may be
trifluoroethanol (TFE), hexafluoro-isopropanol or similar solvents
to stabilize peptide conformations and to prevent or reduce
.beta.-sheet formation. Preferably, the solvent is present in an
aqueous solution comprising for example 5-10 mM phosphate buffer
and 150 mM NaCl. In an even more preferred embodiment the
concentration of TFE, hexafluoro-isopropanol and the like in the
aqueous solution ranges from about 1 to about 5%, preferably from
about 1% to about 2%.
[0435] In a preferred embodiment the methods of the invention
comprise an additional step, wherein at least one washing step is
performed before the detecting step.
[0436] In a preferred embodiment of the methods according to the
present invention, relating to the detection of cells, the methods
are carried out in form of affinity chromatography, in particular
immunoaffinity chromatography.
[0437] The amount of polypeptides, e.g. antibodies, binding to a
polypeptide according to the present invention, e.g. A.beta.
(21-37) or any other sequence comprising A.beta. (21-37), in a
sample of a subject is an indicator for the status of the subject
with regard to the development and/or progression of AD. The higher
the concentration of polypeptides directed against the A.beta.
(21-37) sequence stretch, the higher the protective capacity and
the lower the risk of development and/or progression of AD. The
amount of polypeptides, e.g. antibodies, directed against the
A.beta.(4-10) epitope, in a sample of a subject, is an indicator
for the status of the subject with regard to the development and/or
progression of AD as well. However, in this case the situation is
vice versa. The higher the concentration of polypeptides directed
against the A.beta. (4-10) sequence stretch, the higher the risk of
development and/or progression of AD. For diagnostic reasons, the
methods of detection according to the present invention can thus be
performed individually, but also in combination in order to provide
for a more specific diagnosis.
[0438] Thus, in a further embodiment of the invention a method
according to the invention employing a polypeptide according to the
invention is carried out in combination with a method of the
present invention employing a polypeptide comprising an amino acid
sequence of an A.beta. peptide, wherein the A.beta. peptide has at
least the sequence according to SEQ ID NO: 3 and at most the
sequence according to SEQ ID NO: 5. The method employing a
polypeptide of the present invention can be performed
simultaneously, prior to or after the second method.
[0439] It is also possible to diagnose the neurodementing disease
by way of an indirect approach. A method according to the present
invention--utilizing either a polypeptide according to the present
invention or a polypeptide comprising an amino acid sequence of an
A.beta. peptide, wherein the A.beta. peptide has at least the
sequence according to SEQ ID NO: 3 and at most the sequence
according to SEQ ID NO: 5--is combined with a similar method, which
only differs from the methods of the present invention by utilizing
polypeptide comprising a polypeptide having the length of
A.beta.(1-40) or A.beta.(1-42), i.e. full length A.beta. peptide
instead of the shorter A.beta. sequence stretches utilized by the
methods of the present invention. In such a scenario, the two
methods are carried out independently from each other and the
results of the methods are compared. To illustrate this concept,
the following example is given: [0440] 1) The method utilizing the
A.beta. full length polypeptide sequence yields the amount of all
polypeptides (or cells producing such polypeptides, respectively)
in the sample directed against full length A.beta. (Result A).
[0441] 2) On the other hand a method of the present invention
utilizing a polypeptide of the present invention yields the amount
of all polypeptides (or cells producing such polypeptides,
respectively) in the sample directed against a polypeptide of the
present invention such as A.beta.(21-37) or any other sequence
comprising A.beta.(21-37 (Result B). [0442] 3) A person skilled in
the art can now easily deduce the amount of polypeptides in the
sample directed against an epitope at the N-terminus of A.beta., in
particular against epitope A.beta.(4-10), by subtracting Result B
from Result A.
[0443] Analogously, the amount of polypeptides (or cells producing
such polypeptides, respectively) in the sample directed against a
polypeptide of the present invention such as A.beta.(21-37) or any
other sequence comprising A.beta.(21-37) can be obtained by
subtracting the results obtained with a polypeptide comprising an
amino acid sequence of an A.beta. peptide, wherein the A.beta.
peptide has at least the sequence according to SEQ ID NO: 3 and at
most the sequence according to SEQ ID NO: 5, e.g. A.beta.(4-10),
from the results obtained with A.beta. full length.
[0444] Thus, the present invention also refers to a method for
diagnosing a neurodementing disease, wherein the methods according
to the present invention comprise the following steps: [0445] i)
performing a first method according to the present invention as set
forth above, [0446] ii) performing a second method according to the
present invention proviso that the polypeptide to be incubated in
step a) of said second method comprises the full length amino acid
sequence of A.beta. peptide, and [0447] iii) comparing the result
obtained from step i) with the result of step ii).
[0448] A person skilled in the art will understand that it does not
matter for the above method whether step i) is carried out prior
to, simultaneously with or after step ii).
[0449] In one embodiment the polypeptides to be detected in the
methods of the invention are antibodies, in particular an
A.beta.(21-37) autoantibody or an A.beta.(4-10) autoantibody.
[0450] In a preferred embodiment the methods according to the
invention are carried out for diagnosing Alzheimer's disease,
Down's syndrome, Dementia with Lewy bodies, fronto-temporal
dementia, cerebral amyloid angiopathy, and/or amyloidoses.
[0451] In a further aspect the present inventions relates to a
carrier comprising a polypeptide according to the invention. In a
preferred embodiment the carrier additionally comprises a second
polypeptide comprising an amino acid sequence of an A.beta.
peptide, wherein the A.beta. peptide of the second polypeptide has
at least the sequence according to SEQ ID NO: 3 and at most the
sequence according to SEQ ID NO: 5.
[0452] The carriers according to the invention and used in the
methods of the invention can be of any suitable material capable of
binding polypeptides such as beads, in particular magnetic beads or
sepharose beads, membranes, in particular polyvinylidene fluoride
or nitrocellulose membranes, glass, sepharose matrices, gold
surfaces, synthetic surfaces, in particular microtiter plates. For
certain embodiments, the surface of the carriers can be coated with
agents, which are, for instance, capable of binding to the tags and
markers mentioned above.
Detection
[0453] A variety of assays can be employed in the inventive methods
to detect or measure antibody titer against the A.beta. peptides of
interest. Exemplary assays include, but are not limited to, ELISA,
ELISPOT, Western-Blot, Dot Blot, Protein-Chip, surface plasmon
resonance assay, immunoprecipitation or co-immunoprecipitation, or
affinity chromatography, in particular immunoaffinity
chromatography.
[0454] In a preferred embodiment, the methods according to the
invention comprising the detection of polypeptides in step c) or
d), respectively, represent an ELISA. In this case the carrier is
for example a microtiter plate. The separation of sample and
carrier is achieved by removing the sample liquid from the
microtiter plate and/or by washing the microtiter plate after the
incubation. Preferably, the bound polypeptide is in this scenario
an antibody and this antibody can be detected for example via a
secondary antibody, coupled with e.g. alkaline phosphatase (AP),
and the addition of a substrate for AP resulting in the turn over
of the substrate into, for example, a colored compound detectable
by an optical device. A person skilled in the art and familiar with
ELISA techniques will know several variations of the ELISA concept,
which can be applied to the methods of the present invention as
well.
[0455] Alternatively, in another preferred embodiment, the methods
according to the invention comprising the detection of polypeptides
in step c) or d), respectively, represent an ELISPOT assay. In this
case the carrier is for example a nitrocellulose plate. The
incubation step of the carrier with the sample provides in this
scenario enough time for a cell in the sample to produce sufficient
amounts of antibody. The separation of sample and carrier is
achieved by removing the sample liquid from the nitrocellulose
plate and/or by washing the nitrocellulose plate after the
incubation. Preferably, the bound polypeptide is an antibody and
this antibody can be detected, for example via a secondary
antibody, coupled with e.g. alkaline phosphatase (AP), and the
addition of a substrate for AP, such as BCIP/NBT
(Bromo-chloro-indoryl phosphate/Nitro Blue Tetrazolium) resulting
in the turn over of the substrate into, for example, a deep purple
stain detectable visually or by an optical device. A person skilled
in the art and familiar with ELISPOT techniques will know several
variations of the ELISPOT concept, which can be applied to the
methods of the present invention as well.
[0456] In a further embodiment, the methods according to the
invention comprising the detection of polypeptides in step c) or
d), respectively, represent a Western Blot or Dot Blot assay. In
this case the carrier is for example a nitrocellulose membrane. For
the Western Blot, on the nitrocellulose membrane is either
immobilized a polypeptide as used in step a) of the methods
according to the present invention or a substance with binding
affinity for a polypeptide as used in step a) of the methods
according to the present invention. The nitrocellulose or similar
membrane (PVDF etc.) itself can provide for the binding affinity to
the polypeptide as used in step a) of the methods according to the
present invention. The separation of sample and carrier is achieved
by removing the sample liquid from the nitrocellulose membrane
and/or by washing the nitrocellulose membrane after the incubation.
Preferably, the bound polypeptide is an antibody and this antibody
is for example detected via a labeled secondary antibody, e.g.
coupled with horseradish peroxidase, and subsequent luminescent
reaction and detection. A person skilled in the art and familiar
with Western Blot/Dot blot techniques will know several variations
of the Western Blot/Dot blot concept, which can be applied to the
methods of the present invention as well.
[0457] In a further embodiment, the methods according to the
invention comprising the detection of polypeptides in step c) or
d), respectively, represent a Protein chip, i.e. protein
microarray. In this case the carrier is for example a glass surface
functionalized for binding proteins. The separation of sample and
carrier is achieved by removing the sample liquid from the glass
carrier and/or by washing the glass carrier after the incubation.
Preferably, the bound polypeptide is an antibody and this antibody
is detected via a labelled secondary antibody, coupled with e.g. a
fluorescent dye, which can be detected by an optical device. A
person skilled in the art and familiar with protein chip techniques
will know several variations of the protein chip concept, which can
be applied to the methods of the present invention as well.
[0458] In a further embodiment, the methods according to the
invention comprising the detection of polypeptides in step c) or
d), respectively, represent a surface plasmon resonance analysis.
In this case the carrier is for example a metal surface such as a
gold surface. The separation of sample and carrier is achieved by
removing the sample liquid from the metal carrier and/or by washing
the metal carrier after the incubation. Preferably, the bound
polypeptide is an antibody and the binding of the antibody is
detected via measuring the intensity of the reflected light at a
specific incident angle with an optical device. A person skilled in
the art and familiar with plasmon resonance analysis techniques
will know several variations of the surface plasmon resonance
concept, which can be applied to the methods of the present
invention as well.
[0459] In a further embodiment, the methods according to the
invention comprising the detection of polypeptides in step c) or
d), respectively, represent a pull down or immunoprecipitation
experiment. In this case the carrier may consist of sepharose
beads. These sepharose beads are coated for example with
glutathione (for pull down assays) or with an antibody
(immunoprecipitation assays). Separation of sample and carrier is
achieved by removing the sample liquid from the sepharose beads
and/or by washing the sepharose beads after the incubation.
Preferably, the bound polypeptide is an antibody and this antibody
is detected via a subsequent Western Blot analysis of the
precipitated protein complexes. A person skilled in the art and
familiar with Pull down/Immunoprecipitation techniques will know
several variations of these concepts, which can be applied to the
methods of the present invention as well. One such variation would
be the application of a co-immunoprecipitation approach, wherein
the carrier has only an indirect binding affinity for the
polypeptide as used in step a) of the methods according to the
present invention.
[0460] In a further embodiment, the methods according to the
invention comprising the detection of polypeptides in step c) or
d), respectively, are carried out in form of an affinity
chromatography. In this case the carrier is the matrix, e.g.
sepharose within a conventional chromatography column, to which is
either linked a polypeptide as used in step a) of the methods
according to the present invention or a substance with binding
affinity for a polypeptide as used in step a) of the methods
according to the present invention. The incubation of the sample
with the carrier comprises the time frame the sample needs to pass
through the column. The separation of sample and carrier is
achieved by eluting the sample liquid from the column and/or by
washing the column after the incubation. Preferably, the bound
polypeptide is an antibody and this antibody is for example
detected by elution of the bound antibody, for instance with a
buffer having a high salt content, and subsequent detection of
eluted polypeptide, for example by direct optical determination or
by subsequent Western blot or similar analyses. A person skilled in
the art and familiar with affinity chromatography techniques will
know several variations of the affinity chromatography concept,
which can be applied to the methods of the present invention as
well. One such variation would be the application of an
immunoaffinity chromatography approach, wherein the carrier is
coated with antibodies directed against a polypeptide as used in
step a) of the methods according to the present invention.
[0461] In one embodiment, the methods according to the invention
comprising the detection of cells in step c) or d), respectively,
are carried out in form of an affinity chromatography. In this
case, for example, magnetic beads coated with sepharose represent
the carrier, to which either a polypeptide as used in step a) of
the methods according to the present invention is linked or a
substance with binding affinity for a polypeptide as used in step
a) of the methods according to the present invention. The
separation of sample and carrier is achieved by eluting the sample
liquid from the column and/or by washing the column after the
incubation. The cells bound to the matrix are for example B- or
T-cells, which can be detected, after elution of the cells from the
matrix, for example by way of flow cytometry. A person skilled in
the art and familiar with affinity chromatography and flow
cytometry techniques will know several variations of these
concepts, which can be applied to the methods of the present
invention as well.
[0462] Thus, the methods according to the present invention can be
carried out in particularly preferred embodiments as ELISA,
ELISPOT, Western-Blot, Protein-Chip, surface plasmon resonance
assay, immunoprecipitation or co-immunoprecipitation, or affinity
chromatography, in particular immunoaffinity chromatography. These
are all exemplifications of diagnostic assays. Examples for ELISA's
or affinity chromatography can be found in the examples section. In
diagnostic procedures based on surface plasmon resonance (SPR) the
detection, and quantification and analysis of binding kinetics of
A.gamma.-epitope specific antibodies can be performed by binding of
(i) a biotinylated A.beta.(21-37)- or A.beta.(4-10)-peptide to a
avidin/streptavidin-coated SPR chip surface, or by binding (ii)
A.beta.(21-37)- or A.beta.(4-10)-peptides with an N-terminal
Thiol-group containing carboxylic acid spacer to a gold-chip
surface; followed by binding and determination of the
A.beta.-autoantibodies. A person skilled in the art will readily
know how to incorporate the methods according to the present
invention into one of these standard techniques and procedures
mentioned above.
[0463] It has to be understood, that although the methods according
to the present invention can be carried out in form of one of the
above mentioned detection techniques per se (ELISA, ELISPOT,
Western-Blot, Dot Blot, Protein-Chip, surface plasmon resonance
assay, immunoprecipitation, affinity chromatography, etc.), it is
also possible to combine these detection techniques or to apply
them only for the detection step according to the invention, i.e.
step c) or d), respectively, while the other steps are carried out
in other formats. It is also obvious to a person skilled in the
art, that the information/signals obtained in the detection steps
in the methods of the present invention can provide the basis for
quantification of this information/signals.
[0464] In some cases it might be of higher diagnostic value, if,
instead of or in addition to the detection of polypeptides, e.g.
antibodies, cells producing said polypeptides are detected. For
example, it could be of importance, if in an AD patient the overall
number of cells producing an A.beta.(21-37) autoantibody is lower
than in healthy individuals, or if the amount of antibody secreted
by the respective antibody producing cells is reduced. Depending on
the result this can lead to different therapeutic approaches. Thus,
the present invention also relates to the detection of cells
producing polypeptides binding to a polypeptide according to the
invention or binding to a polypeptide comprising an amino acid
sequence of an A.beta. peptide, wherein the A.beta. peptide has at
least the sequence according to SEQ ID NO: 3 and at most the
sequence according to SEQ ID NO: 5.
[0465] As used in this invention, an immobilized polypeptide refers
in this regard to a polypeptide, which is coupled to a carrier. The
coupling can be covalently or non-covalently, it can be directly to
the carrier or via a linker/linking substance. If the
immobilization occurs non-covalently, then the carrier or the
linking substance exhibits a specific binding affinity for the
polypeptide according to the invention and vice versa. Binding
affinity refers to a property of a substance, in particular a
polypeptide, to associate with (an) other substance(s) and to form
a stable specific dimeric or multimeric complex. Such associations
rely usually on van der Waals- or hydrogen-bonds.
[0466] The incubating step(s) serves the purpose whereby two
partners of a binding pair, i.e. having a binding affinity for each
other, can associate and four a stable complex. The temperature of
the incubation step may vary, but is usually from about 0.degree.
C. to about 40.degree. C., preferably from about 4 to about
37.degree. C., even more preferred about 4.degree. C., about
16.degree. C., about 21.degree. C. or about 37.degree. C. The
higher the temperature, the shorter the time of incubation might
be. For example, if the incubation temperature is 4.degree. C. it
should last for at least 12 h or over night, while 1 h is usually
enough for an incubation at 37.degree. C. If suitable, the carrier
can be blocked prior to the method with a suitable blocking agent,
reducing the likelihood of unspecific binding events. Blocking
agents can be for example milk powder, BSA, fetal calf sera, or any
other blocking reagent.
[0467] The detection of polypeptides bound to an immobilized
polypeptide of the invention or to a polypeptide comprising an
amino acid sequence of an A.beta. peptide, wherein the A.beta.
peptide has at least the sequence according to SEQ ID NO: 3 and at
most the sequence according to SEQ ID NO: 5, can be performed by
several means. One possibility would be for example the
identification via mass spectrometrical means, for example
MALDI-TOF, ESI-MS, MS-FTICR. To this purpose, immunoglobulins are
first isolated from, for example, a serum sample of an AD patient
by protein G affinity chromatography, and subsequently
A.beta.-autoantibodies and A.beta.-plaque specific antibodies are
e.g. isolated by A.beta.-epitope-chromatography, respectively. The
antibodies are then immobilized, for example, on a sepharose
carrier as described in the examples. The specific
A.beta.-epitopes, A.beta.(21-37) and A.beta.(4-10), are then
identified after binding of full-length-A.beta.-polypeptide (for
example A.beta.(1-40) or A.beta.(1-42)), followed by proteolytic
epitope-excision mass spectrometric analysis using one or several
of the proteases, trypsin, chymotrypsin, Glu-C protease,
Asp-N-protease. After washing the affinity-bound A.beta.-epitope(s)
until no signal is detected in the supernatant, the specific
A.beta.-epitope is eluted from the column by treatment with,
typically, 0.1% trifluoroacetic acid, and identified by accurate
determination of its protonated molecular ions; the latter
molecular ion mass accuracy is entirely sufficient for
identification, but can be further ascertained by collision-induced
fragmentation and tandem-MS analysis of fragment ions.
[0468] For certain embodiments, secondary antibodies labeled with a
fluorescent dye or moiety (e.g. GFP) or labeled with an
enzymatically active substance such as horseradish peroxidase,
alkaline phosphatase, .beta.-galactosidase or other related enzymes
able to convert a colorless substrate to a suitable dye or
fluorescent product can be applied. The detection can also be
accomplished by detecting the amount of occupied binding sites,
i.e. utilizing a labeled A.beta.(21-37) antibody, which is
incubated with the carrier after the removal of the sample. The
amount of bound A.beta.(21-37) is in this scenario an indicator for
the amount of prior bound polypeptide. The lower the amount of
subsequently bound A.beta.(21-37) antibody is, the more
A.beta.(21-37) binding polypeptides contained in the sample. The
mentioned examples of detection are not to be considered limiting,
as a person skilled in the art will readily know a plurality of
methods of detection which can be used in the present
invention.
[0469] Usually, the polypeptides bound to the immobilized
polypeptide, which are detected in step c) or d), respectively, in
the methods of the present invention will be antibodies, in
particular an A.beta.(21-37) autoantibody or an A.beta.(4-10)
autoantibody, respectively. However, other substances in the human
body may also bind for instance to A.beta.(21-37) or A.beta.(4-10)
polypeptide.
[0470] Likewise, the detection of cells producing a polypeptide
binding to a polypeptide of the present invention or binding to a
polypeptide comprising an amino acid sequence of an A.beta.
peptide, wherein the A.beta. peptide has at least the sequence
according to SEQ ID NO: 3 and at most the sequence according to SEQ
ID NO: 5, is accomplished by standard techniques known to a person
skilled in the art. One example would be the analysis via flow
cytometry. Another approach would be the lysis of the cells,
DNA/RNA isolation and subsequent PCR amplification of specific
nucleotide sequences. Besides this, in the prior art there are
plurality of further possibilities published, which can be employed
to detect the cells in the methods of the present invention.
[0471] In a preferred embodiment the sample or the cell containing
sample, respectively, used for the methods of the present invention
is derived from blood, plasma, urine or cerebrospinal fluid (CSF)
of a subject. In an even more preferred embodiment the cell
containing sample is derived from blood and the cells are of the
B-cell lineage. The sample, i.e. the subject can be of human,
rodent, bovine, porcine, canine or avian origin. In particular the
sample or the cell containing sample can be derived from human,
mouse, rat, rabbit, cow, pig, dog, chicken and so forth.
[0472] A sample derived from a subject is derived from tissue or
body fluid of a subject. The subject can be a healthy individual,
i.e. not suffering from AD, or a "patient" suffering from a
neurodementing disorder. In a preferred embodiment the sample or
the cell containing sample, respectively, used for the methods of
the present invention is obtained from blood, plasma, urine or
cerebrospinal fluid (CSF) of a subject. In an even more preferred
embodiment the cell containing sample is obtained from blood and
the cells are of the B-cell lineage. The sample, i.e. the subject
can be of human, rodent, bovine, porcine, canine or avian origin.
In particular the sample or the cell containing sample can be
derived from human, mouse, rat, rabbit, cow, pig, dog, chicken and
so forth. Possible preparation procedures of such samples are well
known from the prior art.
[0473] In a preferred embodiment of the methods of the present
invention employing a polypeptide of the present invention a
solvent is present in the incubation step(s), which prevents or
reduces .beta.-sheet formation of the polypeptide region having the
sequence according to SEQ ID NO: 2. As mentioned above, the
.beta.-pleated sheet conformation of A.beta. has been shown to be
responsible for neurotoxicity. Thus, A.beta.(21-37) antibodies in a
healthy individual recognize in particular the A.beta.(21-37)
region or any other sequence comprising A.beta.(21-37), if it is in
random coil or .alpha.-helix conformation. Analogous to flanking
amino acid sequences, solvents can influence the conformational
state of the polypeptides of the invention. In particular, TFE,
hexafluoro-isopropanol and so forth can be used in the methods of
the present invention, for example in the incubation step, to
prevent or reduce a .beta.-sheet conformation of the important
epitope A.beta.(21-37), thus keeping it accessible to the
A.beta.(21-37) autoantibodies in healthy individuals. Preferably,
TFE is present in a concentration ranging from about 1% to about
5%, preferably about 1 to about 2%.
[0474] In a preferred embodiment the methods of the invention
comprise an additional step, wherein at least one washing step with
a washing solution is performed before the detecting step. A
washing step can increase the specificity of the later detection
signal and reduces background signals. Preferably, the washing
solution is water. More preferably buffers like PBS or TBS are used
to ensure a constant pH. The washing solution can contain small
amounts of detergent to increase the specificity of the signal. If
the specificity of the signal is low, the salt concentration or the
concentration of the detergent can be increased in the washing
solution.
[0475] In a further embodiment of the invention a method according
to the invention employing a polypeptide of the invention, i.e.
A.beta.(21-37) polypeptide, is carried out in combination with a
method of the present invention employing a polypeptide comprising
an amino acid sequence of an A.beta. peptide, wherein the A.beta.
peptide has at least the sequence according to SEQ ID NO: 3 and at
most the sequence according to SEQ ID NO: 5, i.e. A.alpha. (4-10).
The method employing a polypeptide of the present invention can be
performed simultaneously, prior to or after the second method. The
comparison of the abundance of A.beta.(21-37) specific polypeptides
with the abundance of A.beta.(4-10) specific polypeptides will
provide for a more detailed assessment of the stage and progression
of AD.
[0476] The detecting step in the methods of the present invention
provides for the possibility to quantify the amount of polypeptides
bound to A.beta.(21-37) or A.beta.(4-10). The determined values are
a measure for the stage and progression of AD. For instance, a
human subject can be considered healthy in regard to AD, if its
serum contains about 1 to 100 ng/.mu.l of A.beta.(21-37) specific
polypeptides and/or about 0 ng/.mu.l. (i.e. below the detection
limit) of the A.beta.(4-10) specific polypeptides. As reference for
a healthy individual might serve the average concentrations of the
respective polypeptides in the serum of people in the age of 20 to
35. In contrast, a subject might suffer from a neurodementing
disease or be endangered to develop a neurodementing disease, for
instance, if its serum contains about 0.01 to 5 ng/.mu.l of
A.beta.(4-10) specific polypeptides, preferably about 0.05 to 1
ng/.mu.l or even more preferably about 0.01 ng/.mu.l or if the
ratio of the concentration of the plaque specific polypeptide vs.
the concentration of the A.beta.(21-37) specific polypeptides in
the serum raises above 0, preferably if it is higher than 0.001,
0.002, 0.003, 0.004, 0.005, 0.010, 0.015, 0.020, 0.030 or even
higher than 0.050. With aging the amount of immunoglobulin produced
in a human body decreases naturally. Therefore, in particular cases
it might be necessary to consider the age of the subject before the
results obtained with the methods according to the present
invention are evaluated. In particular a person about 20 to about
35 years of age might be considered healthy, if its, for instance,
serum contains about 30 to 100 ng/.mu.l or more of A.beta.(21-37)
specific polypeptide, while a subject about 70 to about 80 years of
age can still be considered equally healthy with regard to AD with
"only" 2 to 5 ng/.mu.l of A.beta.(21-37) specific polypeptide in
its serum.
[0477] In a preferred embodiment the methods according to the
invention are carried out for diagnosing a neurodementing disease,
Alzheimer's disease, Down's syndrome, Dementia with Lewy bodies,
fronto-temporal dementia, cerebral amyloid angiopathy, and/or
amyloidoses. All diseases have in common, that the concentration of
A.beta.-autoantibody and A.beta.-plaque specific antibody is
affected by the respective disease as given above for AD.
[0478] In a further aspect the present invention relates to a
carrier comprising a polypeptide according to the invention. In a
preferred embodiment the carrier additionally comprises a second
polypeptide comprising an amino acid sequence of an A.beta.
peptide, wherein the A.beta. peptide of the second polypeptide has
at least the sequence according to SEQ ID NO: 3 and at most the
sequence according to SEQ ID NO: 5.
[0479] The carriers according to the invention and used in the
methods of the invention can be of any suitable material capable of
binding polypeptides such as beads, in particular magnetic beads or
sepharose beads, membranes, in particular polyvinylidene fluoride
or nitrocellulose membranes, glass, sepharose matrices, gold
surfaces, synthetic surfaces, in particular microtiter plates. For
certain embodiments, the surface of the carriers can be coated with
agents, which are, for instance, capable of binding to the tags and
markers mentioned above. A person skilled in the art will readily
know a broad variety of different carriers and possible coatings,
which can be applied for the methods according to the
invention.
Kits
[0480] In another aspect the present invention relates to a kit for
the diagnosis of a neurodementing disease, wherein the kit
comprises a polypeptide according to the invention. In one
embodiment, a kit comprises a second polypeptide comprising an
amino acid sequence of an A.beta. peptide, wherein the AD peptide
of the second polypeptide has at least the sequence according to
SEQ ID NO: 3 and at most the sequence according to SEQ ID NO: 5. In
another embodiment the kit comprises a carrier, in particular a
carrier as mentioned above. In another embodiment, a kit comprises
a first A.beta. peptide comprising at least the sequence according
to A.beta.(30-37) and at most the sequence according to
A.beta.(12-40), and a second A.beta. peptide wherein the second
A.beta. peptide comprising at least the sequence according to
A.beta.(4-10) and at most the sequence according to A.beta.(1-20).
Such kits can be used for example for routine diagnostics in
hospitals and nursing homes, for example to monitor the progression
of AD or to monitor the effectiveness of an AD therapy.
[0481] In another aspect the present invention relates to a kit for
the diagnosis of a neurodementing disease, wherein the kit
comprises a polypeptide according to the invention.
[0482] In a preferred embodiment the above mentioned kit comprises
a second polypeptide comprising an amino acid sequence of an
A.beta. peptide, wherein the A.beta. peptide of the second
polypeptide has at least the sequence according to SEQ ID NO: 3 and
at most the sequence according to SEQ ID NO: 5. In an even more
preferred embodiment the kit comprises a carrier, in particular a
carrier as mentioned above.
[0483] The kit can include one or more containers for the AD
peptides. In some embodiments, the kit contains separate
containers, dividers or compartments for the A.beta. peptides and
informational material. For example, each A.beta. peptide can be
contained in a bottle, vial, or syringe, and the informational
material can be contained in a plastic sleeve or packet. In other
embodiments, the separate elements of the kit are contained within
a single, undivided container. For example, each peptide is
contained in a bottle, vial, or syringe that has attached thereto
the informational material in the form of a label.
[0484] The following examples explain the invention but are not
considered to be limiting. Unless indicated differently, molecular
biological standard methods were used, as e.g., described by
Sambrock and Russel, 2001, Molecular cloning: A Laboratory Manual,
3. edition, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.
EXAMPLES
Example 1
Isolation of A.beta.-Antibody from an AD Patient
[0485] Immuno-isolation of the serum A.beta.-antibody from an AD
patient by epitope-specific affinity-chromatography was performed
on a Sepharose-G5A.beta.(4-10) affinity matrix column. The
Sepharose-G5A.beta.(4-10) affinity matrix was washed with 10 ml of
PBS (5 mmol L.sup.-1Na.sub.2HPO.sub.4, 150 mmol L.sup.-1 NaCl, pH
7.5) and transferred into a 1.7 ml vial using 300 .mu.l of PBS. 800
.mu.l (1 .mu.g/.mu.l) of two different A.beta. autoantibodies
(isolated from the sera of Alzheimer patients) were added and the
sample was slowly rotated overnight at 4.degree. C. The suspension
was transferred to a 0.8 ml micro-column (Mobitec, Gottingen,
Germany) providing the possibility of extensive washing without
significant Toss of material. The first 2 ml were collected as flow
through fraction. The column was washed with 20 ml of PBS and the
last 1 ml was collected for one-dimensional electrophoresis. The
affinity bound IgG was eluted with 6.times.0.5 ml 0.1% TFA; the
column was shaken gently for 15' and the released antibody
molecules collected in a microreaction cup. The samples were
lyophilized and stored until 1D-SDS-PAGE analysis (shown in FIG.
5).
[0486] Serum samples from healthy controls from all age ranges
investigated were also tested for the presence of plaque-antibodies
(N-terminal epitope), using the A.beta.(4-10) epitope column. In
all investigated samples, non-AD control samples were devoid of
detectable plaque-specific antibody.
Example 2
Isolation of Anti-A.beta.(21-37)-Autoantibodies from Healthy
Individuals
A. Affinity Isolation and Purification of
Anti-A.beta.(21-37)-Autoantibodies
[0487] The anti-A.beta.(21-37)-autoantibodies were isolated from
(i), commercially obtainable serum immunoglobulin and (ii) from
serum of healthy individuals (HI). Isolation of antibodies was
performed by A.beta.-epitope-specific affinity chromatography by a
procedure that employed a N-cysteinyl-A.beta.(12-40) column which
was immobilized on Ultralink-iodoacetyl-solid phase carrier as
described below.
[0488] N-Cysteinyl-A.beta.(12-40)
(H-CVHHQKLVFFAEDVGSNKGAIIGLMVGGVV-COOH) was synthesized by solid
phase peptide synthesis using 9-fluorenylmethoxycarbonyl/t-butyl
(Fmoc/tBu) chemistry on a NovaSyn TGR resin (0.23 mmole/g coupling
capacity) on a semi-automated Peptide Synthesizer EPS-221 (INTAVIS,
Langenfeld, Germany). The following side-chain protected amino acid
derivatives were used: Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH,
Fmoc-Gln(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Cys(Trt)-OH. The synthesis
was performed according to the following protocol: (i) DMF washing;
(ii) Fmoc deprotection with 2% DBU, 2% piperidine in DMF (5+10
min), (iii) DMF washing, (iv) coupling of 5 equiv of Fmoc amino
acid:PyBOP:NMM in DMF (40 min), (v) coupling of 5 mol-equivalents
of Fmoc amino acid:PyBOP:NMM in DMF (40 min) (vi) DMF washing
(3.times.1 min). Due to the hydrophobic character of the C-terminal
sequence of A.beta., double coupling of each amino acid was
employed throughout the synthesis. After completion of the
synthesis cycles, the peptide was cleaved from the resin for 3 h
using a mixture containing 95% TFA, 2.5% triisopropylsilan and 2.5%
deionized water. The crude product was precipitated with cold
tert-butylmethylether, washed three times with diethyl ether and
solubilized in 10% acetic acid (aqueous solution) prior to
freeze-drying. Purification of the peptides was performed by
semipreparative HPLC; subsequent characterization by HPLC and
MALDI-TOF mass spectrometric analysis ensured molecular homogeneity
of the peptide.
i) Immobilisation of CysA.beta.(12-40) on Ultralink Iodoacetyl
Gel
[0489] Since the A.beta.(12-40) sequence contains two internal
lysine residues which might lead to side reactions in
immobilization procedures using amino groups, a specific affinity
column was prepared using a cysteine residue attached to the
A.beta.-N-terminus, to ensure homogeneous orientation of peptide
molecules on the column support by immobilization through
cysteinyl-S-thioether linkage. The azlactone-activated support
contains an iodoacetyl group (UltraLink; Perbio, Bonn, Germany) at
the end of a hexadecyl-spacer group, which was reacted with the
cysteinyl-sulfhydryl group to yield a stable thioether linkage, in
order to reduce steric hindrance and provide maximum binding
capacity of the antibodies. For covalent attachment of the
Cys-A.beta.(12-40), 3.7 mg of peptide were dissolved in 50 mM Tris,
5 mM EDTA-Na coupling buffer (pH 8.5) to a final concentration of
0.37 mg/ml. The solution was added to 1 ml of drained
Ultralink-Iodoacetyl gel and the coupling reaction was performed
for 1 hr at 25.degree. C. under gentle mixing, followed by 30 min
reaction time without mixing. An aliquot of 0.5 ml of the
Cys-A.beta.(12-40) coupled support was packed into a column (2.5
ml, MoBiTec, Gottingen, Germany) allowing the solution to drain.
The column was washed with 3 ml of coupling buffer, and
non-specific binding sites on the gel were blocked for 2.times.45
mM by reaction with 1 ml of 50 mM L-Cysteine.HCl in coupling
buffer. Subsequently the column was washed with 5 ml of 1 M NaCl
and 5 ml of 0.1 M Na-phosphate, 0.15 M NaCl (pH 7.2) and stored at
4.degree. C. The gel support (0.5 ml) was transferred into a 15 ml
Falcon vial using 5 ml PBS and mixed with 5 ml IVIgG. After gentle
shaking overnight at 4.degree. C., the suspension was transferred
to the column using the effluent to completely rinse the matrix
back into the column. The column was washed eight times with 10 ml
of PBS followed by 2 wash cycles with 10 ml ultrapure water. The
affinity-bound antibodies were eluted from the column with
10.times.0.5 ml 0.1% trifluoroacetic acid (TFA). Subsequent
isolation and preparation of IgG for structural characterization
and affinity studies was performed using two different
protocols:
(a) The first procedure involved adjustment to neutral pH for each
fraction collected using 0.5 M NaH.sub.2PO.sub.4 (pH 8) in order to
maintain integrity of the antibodies for use in affinity studies.
The bound antibodies were eluted from the column with 10.times.0.5
ml 0.1 M glycine buffer, pH 2.8. Each fraction was collected in a
microreaction tube containing 35 .mu.l 1 M Tris-HCl, pH 9. To
maintain integrity of the antibodies neutral pH was adjusted
immediately after elution by adding the appropriate amount of
Tris-HCl or glycine buffer. To regenerate the column for further
use, the column was washed once with 10 ml 10 mM sodium-phosphate
buffer pH 6.8, followed by two wash cycles with 10 ml of PBS
containing 1M sodium chloride and finally two wash cycles with 10
ml PBS. Protein concentrations were determined by the BCA method
(Pierce; Perbio, Bonn, Germany). This procedure yielded the elution
of single, defined antibody. (b) The bound antibodies were eluted
from the column with 10.times.0.5 ml 0.1 M glycine buffer, pH 2.8.
For separation by gel electrophoresis the elution of affinity-bound
antibodies was not performed by subsequent pH adjustment, in order
to reduce the salt content of the samples subjected to isoelectric
focusing. Gel electrophoretic separations provided a set of defined
bands of antibodies (see numbering in FIG. 12).
ii) Antibody Quantification
[0490] Antibody concentrations in the elution fractions were
determined by the Micro BCA.TM. Protein Assay Kit method (Pierce;
Perbio, Bonn, Germany). The stock solution of 2 mg/ml of bovine
albumin supplied within the Micro BCA.TM. Kit was used to prepare
fresh standard dilutions within the range 40-0.5 .mu.g/ml. The
antibodies eluted between fractions 1 to 6, with highest
concentrations in fractions 1 and 3. For quantification of each set
of 10 elution fractions, fresh albumin standard dilutions were
prepared. Results were read at 562 nm with the ELISA reader.
B. Determination of the Epitope Recognized by the
Anti-A.beta.(21-37)-Autoantibodies Via Epitope Excision
[0491] Autoantibodies isolated as described in Example 2A from the
serum of healthy individuals were immobilized using a solution of
100 .mu.g A.beta.(21-37) autoantibodies in 500 .mu.l 0.2 M
NaHCO.sub.3/0.5 M NaCl (pH 8.3), which was added to
n-hydroxysuccinimidyl (NHS)-activated 6-aminohexanoic acid-coupled
sepharose (Sigma, St Louis, USA) and allowed to bind for 60 min at
20.degree. C. before transferring onto a microcapillary (MoBiTec,
Goettingen, Germany). The column was washed five times with 6 ml
blocking buffer (0.1M ethanolamine, 0.5 NaCl-pH=8.3) and between
the blocking steps with 6 ml washing buffer (0.2M NaOAc, 0.5M
NaCl-pH=4) each with one drop per second. Then the column was
incubated for 1 h in blocking buffer, followed by another wash
step: washed seven times alternatively with 6 ml washing buffer
(0.2M NaOAc, 0.5M NaCl-- pH=4) and with 6 ml blocking buffer (0.1M
ethanolamine, 0.5 NaCl-- pH=8.3). Finally, the column was washed
with 20 ml PBS (5 mM Na2HPO4, 150 mM NaCl, pH=7.5). Then the
peptides to be analyzed were applied in a molar ratio of about two
peptides per coupled antibody. Then the columns were washed with 10
ml PBS wash and then 10 ml double desalted H.sub.2O (MilliQ) to
remove unspecifically bound peptides. Then elution was done by
applying 500 .mu.l 0.1% TFA and incubating it for 15 minutes under
gentle agitation. Then the TFA solution containing specifically
bound peptides is eluted and TFA solution is applied again 2 to 4
times. These eluates are pooled and lyophilized then measured by
MS.
[0492] Epitope excision was performed by application of 2-5 .mu.g
A.beta.-antigen in PBS in a molar ratio of about two peptides per
coupled antibody to the antibody micro column produced as described
in the paragraph above for 60 min at room temperature
(20-25.degree. C.). After washing, digestion was performed on the
column for 2 h at 37.degree. C. with 0.2 .mu.g protease in 200
.mu.l PBS. Unbound peptides were removed and the epitope was
dissociated from the antibody using 500 .mu.l 0.1% trifluoroacetic
acid. After incubation for 15 min at 20.degree. C., this step was
repeated 5 times, the epitope eluate was lyophilized and
reconstituted in 10 .mu.l 0.1% TFA or MALDI solvent (3:2 AcCN: 0.1%
TFA-better) for mass spectrometric analysis.
[0493] Epitope extraction was performed in an analogous manner,
however, proteolytic digestion was performed first with the unbound
antigen and the proteolytic digest was applied directly to the
antibody column. As shown in FIG. 6, the carboxy-terminal
A.beta.(21-37) sequence was found to be specifically recognized
(proteolytically shielded), while N-terminal residues of A.beta.
were accessible for cleavage. The extracted Epitope bound by the
A.beta.-autoantibody isolated from the healthy individuals
exhibited thus the amino acid sequence of A.beta.(21-37).
Therefore, the antibodies of the invention were called
A.beta.(21-37) autoantibodies.
[0494] FIG. 3 shows a similar experiment in which it was shown that
the plaque specific antibodies directed against A.beta.(4-10) were
able to shield the amino acids F4, R5, D7 from proteolytic
digestion.
[0495] The specificity of the inventive antibodies was further
investigated using A.beta.(21-37) autoantibodies purified as
described in Example 2A from IVIgG. The anti-A.beta. antibody ACA
(based on U.S. Pat. No. 7,195,761 see example 5) also was
evaluated. The antibodies were incubated with different partial
A.beta. peptides as specified below and washed as described above
in Example 2B. The elution profiles were analyzed via MS as
above.
[0496] FIGS. 34 (a-d) show that antibodies of the invention
specifically bind in this experimental setting to A.beta.(12-40)
and A.beta.(20-37) but do not bind A.beta.(25-35), A.beta.(17-28)
or A.beta.(31-40).
[0497] FIG. 34 (b) shows that the A.beta.(21-37) autoantibodies
specifically bound the A.beta.(12-40) polypeptide
[0498] FIG. 34(c) shows that A.beta.(21-37) antibodies did not bind
to A.beta.-polypeptides A.beta.(25-35), A.beta.(17-28) or
A.beta.(31-40).
[0499] FIGS. 34(d) to 34(l) show that both the immobilized ACA
antibody and the immobilized A.beta.(21-37) autoantibodies bind to
A.beta.(1-40) and to A.beta.(12-40) but that only the immobilized
A.beta.(21-37) autoantibodies specifically bind to A.beta.(20-37)
and the ACA antibody does not. Neither immobilized antibody bound
A.beta.(17-28). In addition the immobilized antibody ACA did not
bind to A.beta.(4-10).
[0500] Therefore the antibodies of the invention are unique in that
they are characterized by specifically binding to A.beta.(12-40)
and A.beta.(20-37), whereas they do not bind to A.beta.(25-35),
A.beta.(17-28) or A.beta.(31-40) under the experimental conditions
specified above.
[0501] The structure and conformational properties, binding
affinity and specificity of the A.beta.-autoantibody epitope were
further characterized by investigation of synthetic peptides
comprising the A.beta.(21-37) epitope sequence, and by
fine-structure mapping using Alanine sequence mutations, H-D
exchange and high resolution mass spectrometry, ELISA studies and
CD spectroscopic conformational analysis in different solvents.
Biotinylated A.beta.(21-37) peptides and peptides derivative
flanked with oligo-Glycine and -(D-Ala) spacer groups were
synthesized by solid-phase peptide synthesis according to
previously described procedures for A.beta.-peptides, and were
purified by reversed-phase HPLC and characterized by MALDI- and
ESI-mass spectrometry for molecular homogeneity (Manea et al.,
2004; Mezo et al., 2004). Comparative binding studies were
performed with A.beta.-epitope peptides comprising different
C-terminal sequence lengths, using an ELISA system (see below, 3).
These results established an essential function for antibody
affinity of the carboxyterminal sequence end of A.beta., comprising
residues 30-37; this partial sequence is critically involved in
.beta.-sheet formation and aggregation of AD. Thus, full binding
affinity is obtained in A.beta.(12-40). In contrast the shortened
A.beta.(20-30) peptide showed almost completely abolished affinity.
Mass spectrometric studies of peptides upon H-D equilibrium
exchange showed rapid deuterium incorporation of peptide backbone
hydrogens only for the A.beta. sequence (20-30), but only little
backbone deuteration for residues (30-37), suggesting increased
shielding in this part due to conformational or aggregation
effects. Control binding studies of the epitope peptide
A.beta.(20-37) with antibodies that recognized the N-terminal,
A.beta.(1-16) peptide (plaque-specific mono- and polyclonal
antibodies) did not show any binding affinity.
C. Affinity Evaluation of Purified
Anti-A.beta.(21-37)-Autoantibodies
[0502] To assess the quality of the affinity purified antibodies,
ELISA was performed using the flow through of the
affinity-purification column (immobilized A.beta.12-40) as a
control. The 96-well plate was incubated with 200 ng/well of
A.beta.(1-40) in PBS buffer for 2 hrs at 20.degree. C. The plate
was washed 4 times with 200 .mu.l of PBS containing 0.05% Tween-20
and blocked for 2 hours with 5% BSA containing 0.05% Tween-20 in
PBS buffer. The plate was then incubated for 2 hrs at 20.degree. C.
under gentle shaking with the affinity-purified antibodies obtained
as described in example 2A in 5% BSA, 0.05% Tween-20 using the
IVIgG flow through as a control. After washing, anti-human
horse-radish peroxidase (HRP) conjugated antibodies were added to
the wells and incubated for 1 hr. After adding the substrate OPD
the optical density was determined at 450 nm. Affinities were
determined by competitive ELISA, with K.sub.d-values ranging
between about 8 to about 15.times.10.sup.-9 M.
D. Electrophoretic Separation for Sequence Determinations of
Anti-A.beta.(21-37)-Autoantibodies
[0503] Electrophoretic separation and isolation by isoelectric
focusing of the anti-A.beta.(21-37)-autoantibodies obtained as
described in example 2A was carried out by 1D- and 2D-SDS-PAGE.
Samples were equilibrated for 30 min in 6 M urea, 30% glycerol, 2%
w/v SDS, 0.05 M Tris-HCl (pH 8.8), 1% DTT and a trace of
bromophenol blue, then for 30 min in the same solution except that
DTT was replaced by 4.5% (w/v) iodoacetamide. Isoelectric focusing
(IEF) was carried out with a Multiphor II horizontal
electrophoresis system (Amersham Pharmacia Biotech) using 17 cm
immobilized pH gradient (IPG) strips (pH range 3-10 linear). The
second-dimensional separation was carried out with a Bio-Rad
Protean II xi cell vertical electrophoresis system using 10%
SDS-PAGE gels of 1.5 mm thickness. The IPG strips were rehydrated
overnight in a solution containing about 100 .mu.g lyophilized
anti-A.beta.(21-37)-autoantibody for Coomassie and 30 .mu.g for
silver staining solubilised in 7 M urea, 2 M thiourea, 4% CHAPS,
0.3% DTT, 2% Servalyt pH 3-10 and a trace of bromophenol blue. The
samples were applied using the in-gel rehydration method.
Rehydrated strips containing the sample were run in the first
dimension for about 30 kVh at 20.degree. C.
[0504] Strips placed on the vertical gels were overlayed with 1%
agarose in SDS running buffer (25 mM Tris-HCl, 192 mM glycine and
0.1% w/v SDS) and subjected to electrophoresis at 25 mA/gel for 30
min and 40 mA/gel until the tacking dye reached the anodic end of
gels. After separation in SDS-PAGE gels, the proteins were
visualized by silver staining or by sensitive colloidal Coomassie
staining and scanned using a GS-710 Calibrated Imaging Densitometer
(Bio-Rad) (see FIGS. 12 and 13).
Heavy and Light Chain Isolation
[0505] Isolation of light chains and heavy chains of antibodies was
made using 1D gel electrophoresis. The samples (50-200 .mu.g) were
dissolved in sample buffer (4% SDS, 25% glycerol, 50 mM
Tris-buffer, 0.02% Coomassie-blue, 6 M urea, pH 6.8) with repeated
agitation, sonication and centrifugation to ensure maximum
solubilization of the antibodies. Reduction of the disulfide
bridges was performed by reaction with dithiothreitol (DTT) at a
1000-x molar excess for 90 min at 20.degree. C. Subsequently,
alkylation of reduced cysteinyl-sulfhydryl groups was performed by
reaction with a 3-x molar excess of iodoacetamide (IAA)/DTT
concentration for 60 min at 20.degree. C. 1D-SDS-PAGE isolation of
heavy and light chain bands was performed on a 12% acrylamide gel,
using a BIO-RAD Protean-(II) Electrophoresis cell, by application
of approximately 20 .mu.g antibody per band. The PDQuest software
from Bio-Rad was employed for imaging and analyzing 1-D and 2-D
gels. After the gels had been stained and scanned, separate
algorithms of the PDQuest software were used to reduce background
noise levels, gel artifacts, and horizontal or vertical streaking
from the image. The PDQuest software was then used to automatically
detect the protein spots separated on 2-D gels, and for comparison
of different gels. Approximately 20 bands were detected as
discerned spots, of which 16 heavy chain and 15 light chain spots
were analysed and identified by mass spectrometric analysis.
E. Overview of Analytical Strategy and Methods for Sequence
Determination of Anti-A.beta.(21-37)-Autoantibodies
[0506] The primary structure determinations of antibodies,
encompassing amino acid sequences of heavy and light chains,
determination and multiplicity of CDR sequences, disulfide
linkages, and N-terminal sequences and their variations were
performed by a combination of the following complementary and
partially overlapping methods (overview in FIG. 10):
(i) Direct Edman N-terminal sequence determinations of intact
proteins, heavy and light chains following 1D-electrophoretic
isolation and blotting of antibody bands (N-terminus and CDR1
variable domains); (ii) 1D-Electrophoretic separation of heavy and
light chains, followed by tryptic digestion of isolated protein
bands, HPLC isolation of tryptic peptides, and Edman sequence
determinations of peptide fragments (variable sequences and CDR
regions); (iii) LC-ESI-MS/MS sequence determinations of proteolytic
peptides isolated by HPLC, using a combination of de-novo
sequencing and sequence determination from NCBI data base search
procedures (variable sequences and CDR regions); (iv) Mass
spectrometric sequence assignments of proteolytic digest peptides
from 2D-gel electrophoretic separations of antibody-isoforms, using
high resolution MALDI-FTICR-MS in conjunction with database search;
by performing two or more data base search procedures (e.g.,
Mascot, Profound search engines); constant domains and partially
variable sequence domains; (v) Mass spectrometric analysis of
HPLC-isolated proteolytic peptides, using MALDI-TOF-MS (constant
region sequences); in addition, MALDI-MS (MALDI-TOF and
MALDI-FTICR-MS) of proteolytic peptide fragments without and with
reduction of disulfide bridges was used for assignment and
confirmation of correct disulfide linkages; (vi) assignments of the
heavy and light chain connectivities of antibody-isoforms were
performed by MALDI-MS (MALDI-TOF- and MALDI-FTICR-MS) of
proteolytic peptide mixtures following 2D-gel electrophoretic
separation, in which the dithiothreitol (DTT) reduction step was
initially omitted, providing intact disulfide-linked antibodies
during the isoelectric focusing step. Disulfide reduction and
alkylation was then performed in the second electrophoresis
step.
F. Detailed Description of Sequences of
Anti-A.beta.(21-37)-Autoantibodies
[0507] Amino acid sequences were determined for
anti-A.beta.(21-37)-autoantibodies isolated by
affinity-purification from a) serum-IVIgG, and b) serum-IgGs
isolated from two healthy adult individuals (m, 30 yrs). Sequence
determinations for complete antibodies, heavy and light chains,
(identified with disulfide-linkages as described above) are shown
with assignments of structural details in FIGS. 29, 30 and 32. In
the experimental details depicted in FIGS. 25 to 30, the different
complementary methods used for completing the sequence
determinations by complementary and overlapping partial sequences
are shown by different underlining codes for (i), Edman N-terminal
protein sequence determinations; and (ii-v), sequence
determinations of proteolytic peptides isolated by HPLC using Edman
sequencing, LC-MS/MS, MALDI-TOF-MS and high resolution
MALDI-FTICR-MS, and MALDI-FTICR-MS of proteolytic peptide mixtures
upon 2D-electrophoretic isolation. Furthermore, intra-disulfide
linkages of cystinyl residues and heavy chain-light chain-disulfide
linkages identified at Cys-224 (HC-LC), Cys-230 and Cys-233 (HC),
have been annotated in the heavy chain sequence. In addition to
direct assignment of sequence positions of cysteine residues,
disulfide linkages were confirmed by mass spectrometric molecular
weight determinations from tryptic, non-reduced peptide mixtures
(not shown), and subsequent DTT-reduced peptides, showing full
agreement with homology comparison from predicted assignment of
disulfide linkages from crystallographic data of a reference IgG1
structure (PDB 1IG, Brookhaven protein structure data base).
[0508] From the serum-IVIgG antibodies, sequences were identified
for 12 heavy chain isoforms and 5 light chain isoforms of the
respective variable regions; the light chain sequences comprised 4
kappa and 1 lambda chain sequences. For a major portion of the
amino acid sequences, sequence data were corresponding with, and
ascertaining each other by at least two complementary, overlapping
partial sequence determinations. All antibody sequences were
identified as IgG.sub.1 subclass molecules.
[0509] Noteworthy results in the heavy and light chain sequences
are the identification of several single amino acid variations in
the constant domains, each of which was ascertained by several
complementing mass spectrometric methods and by Edman sequencing of
HLC-isolated tryptic peptides. The N-glycosylation site at N-301,
was ascertained by Edman sequence determination of the tryptic
peptide (297-304) (EEQYNSTYR); this peptide provided a blank in the
sequencing cycle-5. After N-deglycosylation with PNGaseF, this
peptide yielded the sequence determination, N-301; furthermore
MALDI-FTICR-MS analysis of the deglycosylated peptide provided
identification of the correct molecular mass. In addition a
non-glycosylated Fc sequence variation was identified by mutation
of N301A and by the presence of partially non-glycosylated N.
[0510] Variable sequences determined for light and heavy chains are
summarized in FIGS. 25 and 26, comprising 12 sequence variants
(heavy chain) and 5 (light chain). Sequence variations in the heavy
chains were considerably more frequent than for light chains,
except for the N-terminal sequences which showed several sequence
variations for the light chains but complete homogeneity of the
heavy chains. N-Terminal sequences were determined by a combination
of direct Edman sequence analysis of proteins, Edman sequencing of
tryptic N-terminal peptides, and LC-MS/MS sequencing of
HPLC-isolated N-terminal peptides. A characteristic feature is the
single uniform heavy chain N-terminal heavy chain (1-20), in
contrast to the light chain N-terminal mutations. The N-terminal
mutations of the light chains were confirmed by a blast homology
search using the NCBI data base.
[0511] CDR Sequence domains determined for both heavy and light
chain regions are summarized in FIG. 24 a to d. In agreement with
mutations identified within the variable domains, a higher
multiplicity was found for the heavy chain sequences with
identifications of up to 7 CDR1, CDR2 and CDR3 sequences; while 4
and 5 sequences were determined for light chain CDR1 and CDR2
domains, and two CDR3 sequence variants. Using a stepwise
examination of heavy and light chain CDRs, all CDR sequences were
in agreement matching the Kabat rules (Kabat, E. A., Wu, T. T.,
Perry, H. M., Gottesman, K. S.& Foeller, C. (1991) Sequences of
Proteins of Immunological Interest (Department of Health and Human
Services, Public Health Service, National Institutes of Health,
Bethesda, Md.) NIH Publ. No 91-3442 5th Ed and R. Kontermann, S.
Dubel (eds.), Antibody Engineering; Springer Lab Manual Series;
Springer, Heidelberg 2001;. The CDR sequences identified enabled
the derivation of consensus sequences.
G. Detailed Description of Experimental Procedures for Sequence
Determinations
i) Protein Sample Preparation for Sequence Determinations
[0512] Anti-A.beta.(21-37)-autoantibodies isolated from serum-IVIgG
obtained as described in Example 2A were lyophilized and
solubilized in denaturation buffer (6 M Urea; 50 mM Tris, pH=7.5)
at a concentration of 1 .mu.g/.mu.L. Reduction of disulfide bridges
was performed with DTT at a 1000.times. molar excess, for 2 hrs at
30.degree. C. Subsequent alkylation of free thiol groups was
carried out with iodoacetamide at a 3000.times. molar excess by
reaction for 1 hr at 20.degree. C. in subdued light. The samples
were subsequently lyophilized before separation of heavy and light
chains by 1D-gel electrophoresis.
ii) N-Terminal Edman Protein Sequence Analysis
[0513] Automated amino acid sequence analyses were performed with
an Applied Biosystems 494 HT Procise Sequencer attached to a 140C
Microgradient HPLC system, a 785A Programmable Absorbance Detector
and a 610A data analysis system. All solvents and reagents used
were of analytical ultragrade purity (supplied by Applied
Biosystems Europe, Darmstadt, Germany).
[0514] The following reagents and materials (Applied Biosystems)
were employed in all analyses: Blotting buffer: 25 mM Tris-HCl, 192
mM glycine, 0.1% SDS, 20% methanol; PVDF membrane: ProBlott,
Applied Biosystems; Filter papers: GB005 (Schleicher &
Schuell); Blotter: PeqLab PerfectBlue Tank-Elektroblotter Web M;
staining solution: 0.1% Coomassie Blue R-250 in 50% methanol;
destaining solution: 50% aqueous methanol.
[0515] The antibodies were reduced, alkylated and separated by
1D-SDS-PAGE into heavy and light chain components as described in
4). Immediately after electrophoretic separation the fresh gel was
equilibrated in transfer buffer for 10 min. The PVDF membrane
(10.times.10 cm, the size of the gel) was wetted in methanol
(analytical grade, Nomiapur) for 1 min and then equilibrated for 20
min in the transfer buffer. Two sheets of filter paper were cut to
the dimensions of the gel (10.times.10 cm), and filter papers were
soaked in the transfer buffer.
[0516] The blotting sandwich was assembled as follows: A wet filter
paper was placed onto the anode (+) side of the blotting cassette.
The equilibrated PVDF membrane was placed on top of the filter
paper. The equilibrated gel was placed on top of the transfer
membrane. The other wet filter paper was placed on top of the gel.
Care was taken not to include air bubbles between the sandwich
components. The cassette was then closed and immersed into the
blotting tank.
[0517] The blotting was carried out at constant current 1
mA/cm.sup.2 for 4 hours. After the protein transfer was completed,
the PVDF membrane was washed twice for 15 min with MilliQ water to
remove the SDS and glycine. The PVDF membrane was then washed with
methanol for 1 min and then stained for 1 min. The stained membrane
was washed with destaining solution until the protein spots were
clearly visible from the background. The membrane was then allowed
to dry in air. The protein spots were excised, placed in Eppendorf
tubes and stored at 4.degree. C. Before sequence analysis the spots
were washed with 100% methanol until complete destaining. The
protein spots were placed in the sequencer cartridge and sequenced
using the standard PL PVDF protein method (pulsed liquid sequencing
method for PVDF blotted proteins, Applied Biosystems).
iii) Proteolytic Digestion Of Antibodies Following Gel
Electrophoretic Separation
[0518] Heavy and light chains were separated by 1D-gel
electrophoresis with 12% separating gel and 5% stacking gel and
stained with colloidal Coomassie Blue as described in 4).
[0519] For in-gel proteolytic digestion and subsequent HPLC
isolation and mass spectrometric analysis of the tryptic peptides,
the gel bands were cut out and destained by addition of 60%
acetonitrile in MilliQ water for 20 min at 25.degree. C. After
removal of the supernatant and lyophilization of the gel spot to
dryness, 1 ml of a solution of 50 mM NH.sub.4HCO.sub.3 was added
for rehydration and incubated for 20 min at 25.degree. C. This
procedure was repeated two times and the final rehydration was
performed with the protease solution (12.5 ng/.mu.l trypsin in 50
mM NH.sub.4HCO.sub.3) at 4.degree. C. for 45 min. The gel spots
were then incubated for 12 hrs at 37.degree. C. in 1 ml 50 mM
NH.sub.4HCO.sub.3 and protein fragments were eluted three times
with 1 ml 60% acetonitrile in water for 1 hr. The eluates were
lyophilised to dryness and solubilised immediately prior to HPLC
and MS analysis.
[0520] For protein identification/sequence and data base analyses
following 2D-electrophoresis, sequence determinations by LC-MS/MS
and Edman N-terminal sequence determinations of proteolytic
peptides, the gel spots were excised, subjected to dehydration in
acetonitrile, and following removal of acetonitrile in vacuo dried
in a vacuum centrifuge. Sample preparation for proteolytic
digestion was performed as described above, by reduction with a
volume of 10 mM dithiotreitol (DTT) in 50 mM NH.sub.4HCO.sub.3
sufficient to cover the gel pieces, and protein was performed for 1
hr at 56.degree. C. After cooling to room temperature, the
DTT-containing solution was replaced with the same volume of a
solution of 55 mM iodoacetamide in 50 mM NH.sub.4HCO.sub.3. After
45 min incubation at room temperature in the dark with occasional
shaking (vortexing), the gel pieces were washed for 10 min with
50-100 .mu.L of 50 mM NH.sub.4HCO.sub.3, and dehydrated again by
addition of the same volume of acetonitrile. The liquid phase was
then removed and the gel pieces were completely dried in a vacuum
centrifuge.
[0521] Excised gel pieces were digested with trypsin either
manually or automatically using a DigestPro 96 robot (Intavis
Bioanalytical Instruments, Langenfeld, Germany) according to
literature procedures. For manual in-gel-digestion and subsequent
mass spectrometric analysis, the spots were excised and destained
by addition of 60% acetonitrile in MilliQ water for 20 min at
25.degree. C. After removal of supernatant and lyophilization of
the gel spot, a solution of 50 mM NH.sub.4HCO.sub.3 was added for
rehydration and incubated for 20 min at 25.degree. C. This
procedure was repeated two times, and final rehydration was then
performed for 45 min with the protease solution (12.5 ng/.mu.l
trypsin in 50 mM NH.sub.4HCO.sub.3) at 4.degree. C. The gel spots
were incubated for 12 h at 37.degree. C. in 50 mM NH.sub.4HCO.sub.3
and proteolytic peptides were eluted for 3-4 hrs with 60%
acetonitrile in water. The eluates were lyophilized to dryness and
dissolved immediately before MALDI-MS analysis in 5 .mu.l
acetonitrile/0.1% trifluoroacetic acid in water (2:1).
[0522] Automated in-gel-digestion for subsequent mass spectrometric
analysis was performed with a DigestPro 96 robot (Intavis
Bionalytical Instruments). The DigestPro 96 is a commercial digest
robot system consisting of a Gilson 221XL robot, equipped with a
module containing a temperature-regulated aluminium reactor block.
The block can hold up to 96 protein samples and is mounted on rails
so that it can be moved by the robot arm to either a washing or a
sample collection position. Protein gel pieces were excised from
the 2D-PAGE gels and loaded into a clean 96 well PCR plate which
contained small holes pierced into the well bottoms. The plate was
covered by a silicone membrane held in place by a lid and four
mounting screws. The holes in the silicone membrane allow for
reagent delivery by a specially designed dispensing needle. This
needle has a second, outer channel which delivers nitrogen pressure
to the reaction wells. Needle positioning allows either the
delivery of liquid to the vial or the ejection from the reactor by
2.6-bar nitrogen pressure. The entire in-gel digestion process, as
described below, was implemented on the robot platform, and was
controlled by the DigestPro 96 software (version 4.02; INTAVIS).
Briefly, the gel pieces were washed four times with 50 .mu.l of 50
mM NH.sub.4HCO.sub.3 and after each step dehydrated with 100 .mu.l
acetonitrile. After the last shrinking step, 50 .mu.l of enzyme
buffer (12.5 ng/.mu.l trypsin in 50 mM NH.sub.4HCO.sub.3, pH
.about.8) were added to the tubes. The enzyme was drawn into the
gel pieces for 30 min. Subsequently, 50 .mu.l solution of 50 mM
NH.sub.4HCO.sub.3 were added to cover the gel pieces, and after 6
hrs at 37.degree. C. the peptides were extracted. The first
extraction was performed with 50 .mu.l NH.sub.4HCO.sub.3 followed
by three extractions with 50 .mu.l of 10% formic acid; between
extractions, the gel pieces were dehydrated with acetonitrile as
described above. The collected extracts were then dried in a vacuum
centrifuge and redissolved immediately before MS analysis, in
either 5 .mu.l MALDI-MS solution (acetonitrile:0.1% trifluoroacetic
acid in water, 2:1) or 5 .mu.l ESI-MS solution
(methanol:water:acetic acid, 50:48:2 (v/v/v)). For in-gel
deglycosylation, the gel pieces were swollen in deglycosylation
buffer, which was prepared by mixing 100 .mu.L of a commercial
N-Glycosidase F (PNGase F) preparation (Roche, Mannheim, Germany)
with 100 .mu.L of 0.1 M ammonium bicarbonate buffer to provide a
final enzyme concentration of 100 units mL.sup.-1. If all liquid
was taken up by the gel pieces, further digestion buffer (but
without PNGase F) was added to the sample to keep the sample wet
during overnight incubation at 37.degree. C. To avoid possible
interference from PNGase F-related peptides in MALDI-MS analyses,
the glycosidase was removed prior to proteolysis, by washing the
gel pieces with 0.1% SDS in 0.1 M ammonium bicarbonate (four times
250 .mu.L for 1 hr each). All washing solutions were discarded and
SDS was removed by incubation with 50:45:5 (v/v/v)
methanol:water:acetic acid (30 min) and three times washing using
50% acetonitrile in 0.1 M ammonium bicarbonate (30 min each). All
washings were discarded, and the gel plugs then dried in a vacuum
centrifuge. The same procedure was used for in-gel deglycosylation
with EndoH glycosidase.
[0523] A ZipTip-cleanup procedure was then performed using
ZipTip.RTM..sub.C18 pipette tips from Millipore (Eschborn,
Germany). A ZipTip pipette tip is a microcolumn with a resin
prepacked into the narrow end of a 10 .mu.l pipette tip. ZipTip
pipette tips contain C.sub.18 or C.sub.4 reversed-phase material
for concentrating and purifying peptide and protein samples.
ZipTip.sub.C18 pipette tips were applied for peptides and low
molecular weight proteins, while ZipTip.sub.C4 pipette tips were
applied for higher molecular weight proteins. The complete ZipTip
procedure was carried out according to the instructions of the
manufacturer. Briefly, it consists essentially of five steps:
wetting; equilibration of the ZipTip pipette tip; binding of
peptides and proteins to the pipette tip; washing; and elution.
iv) HPLC Separation and Isolation of Proteolytic Peptides
[0524] All analytical HPLC separations were performed with a
BIO-RAD (Muenchen, Germany) 2700 HPLC system using a Vydac C.sub.4
column (250.times.4.6 mm I.D.) with 5 .mu.m silica (300 .ANG. pore
size). Linear gradient elution (0 min 0% solvent B; 5 min 0%
solvent B; 135 min 65% solvent B, 150 min 100% solvent B, 160 min
100% B), with eluant A consisting of 0.1% trifluoroacetic acid
(TFA) in water, and eluant B of 0.1% TFA in acetonitrile:water
(80:20, v/v) at a flow rate of 1 mL/min. The proteolytic peptide
samples, typically 50 .mu.g-aliquots were dissolved in 200 .mu.L of
eluant A. Detection of peptides was generally performed at 220 nm
using a BIO-RAD variable wavelength absorbance detector.
v) N-terminal Edman Sequence Determinations of Proteolytic
Peptides
[0525] Tryptic peptides were isolated by HPLC as described above,
and lyophilized and stored at -20.degree. C. prior to sequence
analysis. The sample support used for sequence determinations
consisted of a glass fibre filter (Applied Biosystems) which was
treated with a 30 .mu.l., BioBrene Plus (Applied Biosystems)
solution (100 .mu.g/.mu.L Biobrene and 6.66 .mu.g/.mu.l NaCl in
water), and precycled (3 cycles) using the standard filter precycle
method. For sequence analyses the lyophilized HPLC fractions were
reconstituted in 15 .mu.L 0.1% TFA, containing 20% (v/v)
acetonitrile in water. The reconstituted peptide solution was
applied on the precycled glass fiber filter in aliquots of 5 .mu.l
to ensure a distribution as close to the centre of the glass fiber
filter as possible, each application followed by drying under a
stream of Ar for 1 min.
[0526] All sequence analyses were performed on an Applied
Biosystems 494 HT Procise Sequencer/140C Microgradient System with
785A Programmable Absorbance Detector and 610A Data Analysis System
as described above. All solvents and reagents used were from
Applied Biosystems. The general method used for the analysis of
proteolytic peptides was the standard pulse-liquid method.
vi) Sequence Determinations by ESI-LC-MS/MS of Proteolytic
Peptides
[0527] All sequence determinations of tryptic peptides isolated by
HPLC (see above) were performed with a Broker Esquire-3000+
ion-trap LC-MS/MS system equipped with nano-ESI/LC ion source
systems (Broker Daltonics, Bremen, Germany). HPLC fractions of
proteolytic peptides were collected in 1 ml Eppendorf cups and
lyophilized to dryness and stored at -20.degree. C. until LC/MS
analysis. The HPLC fractions were dissolved in 16 .mu.l of a
solvent mixture containing 1% formic acid in water:acetonitrile
(9:1, v/v). The samples were sonicated for 5 min at 20.degree. C.
and centrifuged at 13 000 rpm/min for 3 min. The content of a
sample was transferred into a 2 ml screw cap vial equipped with an
internal microvial (0.1 ml) and placed in the LC/MS tray. A 3 .mu.l
aliquot of the sample was injected on the C-18 microcolumn by means
of the automatic injection system, and an elution gradient listed
in the table below (LC-MS gradient) was employed. The sample flows
from the injection loop into the column and is then directed into
the electrospray interface and through the ion optics into the ion
trap. The total ion current (TIC) was recorded as a function of
time, and is converted into the mass spectrum using the Data
Analysis software (Bruker Daltonics). The most intensive ions to be
used for MS/MS analysis were selected from the mass spectrum
resulting from the first LC/MS run. For each precursor ion
identified, a separate LC-MS/MS run was performed at identical
gradient conditions (see Table). Following start of the pumping
system, the isolation and fragmentation of parent ions was switched
on in the Esquire Control window, using the specification of
precursor mass, isolation width and fragmentation amplitude. The
total ion current (TIC) corresponding to the ion fragments was
recorded as a function of time; if a single parent ion was
subjected to fragmentation during the run, the TIC contains a
single peak. The MS/MS spectrum of the precursor ion was generated
by the Data Analysis software by averaging the pulses at half peak
width. The m/z values of the fragments contained in the MS/MS
spectrum and their intensities were exported into a data analysis
file type (wearing the extension *mgf). The file was uploaded into
the MS/MS Mascot search engine for performing the NCBInr data base
search, using the following search parameters: taxonomy, Homo
sapiens; allowed missed cleavages, -1; peptide search tolerance, 2
Da; MS/MS tolerance, 0.8 Da; fixed modification, carbamidomethyl
(cysteine); variable modification, Met-oxidation. The results
displayed contain the Mowse probability score in form of a chart,
providing the peptide sequence and the protein originating for each
hit result. If the result for the fragment ions of a given
precursor led to direct identification score of a peptide from
immunoglobulin heavy- or light-chain, the peptide sequence obtained
was taken to be a correct one. If no identification score was
directly obtained for a given precursor and its MS/MS spectrum, the
peptide sequence was ascertained by de novo sequencing, using the
assignment function from the Data Analysis software. This function
assigns the mass difference between two fragments into the mass for
a specific amino acid. If the peptide sequence data obtained by the
de novo procedure was identical with the sequence obtained by the
NCBI database search, the sequence result was taken as correct. If
the database search performed for a certain precursor ion did not
provide any immunoglobulin peptide fragment, the corresponding
precursor ion was assigned as unidentified.
TABLE-US-00001 Table of LC-MS/MS gradient elution parameters Time
(min) Solvent A Solvent B 0 80 20 3 80 20 6 50 50 16 20 80 18 2 98
20 2 98 22 98 2 24 98 2
vii) MALDI-TOF Mass Spectrometry Of Proteolytic Peptides
[0528] MALDI-TOF MS analysis was carried out with a Bruker Biflex
linear TOF mass spectrometer (Bruker Daltonics, Bremen, Germany)
equipped with a nitrogen UV laser (.lamda.=337 nm), a 26-sample
SCOUT source, a video system and a XMASS data system for spectra
acquisition and instrument control. A saturated solution of
.alpha.-cyano-4-hydroxy-cinnamic acid (HCCA) in acetonitrile: 0.1%
TFA in water (2:1 v/v) was used as the matrix. For all MALDI-MS
analyses, 0.8 .mu.L of matrix solution and 0.8 .mu.L of the sample
solution (proteolytic peptide mixture or tryptic peptides separated
by HPLC) were mixed on the stainless steel MALDI target and allowed
to dry. Acquisition of spectra was carried out at an acceleration
voltage (V.sub.acc) of 20 kV and a detector voltage of 1.5 kV.
External calibration was carried out using the average masses of
singly protonated ion signals of bovine insulin (5734.5 Da), bovine
insulin B-chain oxidized (3496.9), human neurotensin (1673.9 Da),
human angiotensin I (1297.5 Da), human bradykinin (1061.2) and
human angiotensin II (1047.2 Da).
viii) MALDI-FT-ICR-MS of Proteolytic Peptides
[0529] MALDI-FTICR mass spectrometric analyses were performed with
a Bruker APEX II FTICR instrument (Bruker Daltonics, Bremen,
Germany) equipped with an actively shielded 7T superconducting
magnet (Magnex, Oxford, UK), a cylindrical infinity ICR analyzer
cell, and an external Scout 100 fully automated X-Y target stage
MALDI source with pulsed collision gas. The pulsed nitrogen laser
was operated at 337 nm.
[0530] Analyses of peptide samples were performed with a 100 mg/mL
solution of 2,5-dihydroxybenzoic acid (DHB) in acetonitrile/0.1%
TFA in water (2:1 v/v) used as the matrix. An aliquot of 0.5 .mu.L
of matrix solution and 0.5 .mu.L of sample solution (tryptic
peptide or peptide mixture) were mixed on the stainless steel MALDI
target and allowed to dry. External calibration was carried out
using the monoisotopic masses of singly protonated ion signals of
bovine insulin (5730.609 Da), bovine insulin B-chain oxidized
(3494.651), human neurotensin (1672.917 Da), human angiotensin I
(1296.685 Da), human bradykinin (1060.569) and human angiotensin II
(1046.542 Da). Acquisition and processing of spectra were performed
with XMASS software (Bruker Daltonics, Bremen, Germany).
[0531] MALDI-FTICR-MS/MS analyses were performed with the Bruker
ApexII FTICR-MS instrument equipped with SORI-CID
(sustained-off-resonance-collision-induced--dissociation)
dissociation, IRMPD (Infrared Multiphoton Photodissociation)
instrumentation for fragmentation of peptide and protein ions
(Damoc et al., 2003). Ions formed by MALDI ionization were trapped
in the analyzer cell, and isolation of a precursor ion was
performed by ejecting from the ICR cell all ions of higher and
lower masses through the application of suitable excitation pulses,
using the appropriate frequencies and amplitudes. The following
experimental conditions were employed: correlated sweep
attenuation: 8-10 dB, ejection safety belt: 500-1000 Hz. For
SORI-CID, a low-amplitude rf-excitation was applied for 250 msec to
the precursor ion at a frequency that is slightly off-resonance
(500-1000 Hz) from the cyclotron frequency. The amplitude of the
excitation was kept low so that the ion never went too far from the
center of the cell. While this excitation was applied, the pressure
was raised in the analyzer cell (10.sup.-8 mbar) by admitting a
collision gas (argon) through a pulse valve for 20-80 msec. Under
these conditions, the precursor ion underwent many low-energy
collisions, which slowly activated the ion until it reached its
threshold for dissociation.
[0532] For IRMPD (infrared-multiphoton-dissociation) experiments
the mass-selected ions were photodissociated using a 25 W
continuous wave CO.sub.2 laser (10.6 .mu.m, Synard, Mukilteo,
Wash., USA). The laser power was set to 50% threshold and the laser
irradiation time to 50-200 msec.
[0533] For protein identifications and sequence determinations
(constant region sequences) of proteolytic peptide mixtures
following 2D-gel electrophoresis, the following (publicly
available) data base search engines were employed:
Mascot--Peptide mass fingerprint and MS/MS ion search from Matrix
Science Ltd., London. ProFound--Peptide mass fingerprint from
Rockefeller and New York Universities. MS-Fit--Peptide mass
fingerprint from University of California, San Francisco (UCSF).
MS-Tag--MS/MS ion search from University of California, San
Francisco (UCSF).
Example 3
Determination of Dissociation Constants of
Antigen-Antibody-Complexes by ELISA
[0534] The K.sub.d values were determined by a modification of the
method of Kim et al. (1990). For the determinations, the antibody
concentrations employed were first derived from an initial
calibration curve obtained by an indirect ELISA as described in
example 9B.
1) For the indirect ELISA, microtiter plates were coated with 150
.mu.L/well of streptavidin at 20.degree. C. for 2 hrs. Wells were
washed one time with 0.05% (v/v) Tween-20 detergent in
phosphate-buffered saline (PBS) (Na.sub.2HPO.sub.4 5 mM, NaCl 150
mM, pH 7.5). Biotinylated-(G).sub.5-A.beta. (12-40) peptide at
concentrations between 1.times.10.sup.-6 and 10.sup.-8 M were
prepared in PBS and deposited in the wells at a volume of 100
.mu.L/well. The wells were incubated for 2 hours at 20.degree. C.
temperature followed by a 4 times washing step and blocking with
blocking buffer (BSA 5% w/v, 0.05% Tween-20 v/v in PBS) for 2
hours. Anti-A.beta.(12-40) antibody was diluted to concentrations
between 1.4.times.10.sup.-7 and 10.sup.-9 M with blocking buffer
and added at 100 .mu.L/well. The microplate was incubated at
20.degree. C. for 2 hours and then washed with Covabuffer (0.15 M
PBS, pH 7.2 containing 2M NaCl, 0.083 M MgSO.sub.4 and 0.05%
Tween-20). The wells were incubated with peroxidase-conjugated
mouse anti-human IgG (1:5.000) for 45 min at 20.degree. C. Antibody
binding was detected with a freshly prepared solution of
1,2-Phenylendiamine (OPD) containing 0.1 M citrate-phosphate, 0.1%
OPD and 0.006% hydrogen peroxide. The enzyme reaction was monitored
as a function of time at 450 nm, using an ELISA plate reader
(Victor.sup.2, Perkin Elmer Life/Analytical Sciences, Boston,
Mass.). For each antibody and antigen concentration triplicate
wells were prepared and measured. Direct proportionality was
observed between absorbance and antibody concentration over a wide
concentration range. This concentration range was used to select
the initial concentration for K.sub.d determinations. The initial
concentration was selected to be within the linear region of the
plot of optical density vs. antibody concentration. 2) For the
determination of the K.sub.d values the following conditions were
applied. The antigen, A.beta.(12-40) peptide at various
concentrations (1.times.10.sup.-6 M to 4.8.times.10.sup.-10 M) was
mixed with a constant concentration of antibody derived from the
preliminary ELISA calibration. The incubation was performed in 5%
BSA, 0.05% Tween-20 in PBS using polypropylene test tubes to
minimize antibody loss by adsorption on the microreaction tube
walls. After 2 hrs, 100 .mu.l of each mixture was transferred and
incubated for 30 min into the wells of a microtiter plate
previously coated with biotinylated-(G).sub.5-A.beta.(12-40) (1
.mu.M) and blocked. The concentration of free antibody was then
measured by indirect ELISA as described above. The K.sub.d values
were obtained by plotting the experimental data using the Sips
coordinates. The following mean K.sub.d values were determined for
the A.beta.-antibody complexes of A.beta.(12-40) peptide: a)
Affinity purified IVIgG antibodies from commercially available
IVIgG preparation: [0535] 8.times.10.sup.-9M b) Affinity purified
IVIgG antibodies from Serum-A (healthy human individual; age above
30): 14.times.10.sup.-9 M c) Affinity purified IVIgG antibodies
from Serum-B (healthy human individual; age above 30):
18.times.10.sup.-9 M
[0536] Since the range of binding/dissociation constants for the
formation of A.beta.-fibrils/aggregates has been estimated in the
literature to be in the range of 10.sup.-6 M (determined), the
binding of antibodies is determined to be specific. For IgG
antibodies, typical K.sub.d-values in the range of 10.sup.-8 to
10.sup.-9 M have been determined for a large variety of oligo- and
polypeptide antigens and epitopes.
Example 4
Inhibition of Plaque Formation by Affinity Purified IVIgG
Antibodies
[0537] Human neuroblastoma cells (SH-Sy5y) were grown in RPMI
1640-Medium supplemented with 10% fetal calf serum, 10 mM Hepes, 4
mM glutamine and penicillin (200 units/ml), streptomycin (200
.mu.g/ml). Cells were incubated at a density of 30,000 cells/well
over night in a 96-well microtiter plate. After removal of medium,
cells were washed with PBS, and toxic A.beta.-oligomers (2 .mu.M
final concentration) were added at a volume of 100 .mu.l fresh
medium to 7.5 .mu.M or 15 .mu.M of anti-A.beta.(21-37)-autoantibody
or without anti-A.beta.(21-37)-autoantibody. The affinity purified
IVIgG antibodies were obtained by purifying antibodies from
commercially available IVIgG by affinity chromatography using
A.beta.(1-40) coupled to a gel using the coupling chemistry
described in Example 2A. MTT test was performed after 4-hrs
incubation. FIG. 4 shows that the A.beta.-mediated toxicity (grey
bars) is almost completely antagonized by affinity purified IVIgGs
(black bars).
[0538] The experiment was repeated with affinity purified IVIgG
(purified as described above, mab CSL Clone 7 (see Example 5). As a
negative control the antibodies CSL360 (see Example 10) or no
antibody was used. A positive control antibody used was ACA (see
Example 10). Results as shown in FIG. 49 clearly show a dose
dependent effectiveness of protecting cells from the neurotoxic
effects of A.beta. oligomers for both the affinity purified mab CSL
Clone 7 and affinity purified IVIgG.
[0539] Soluble toxic A.beta. oligomers as used in Examples 4 can be
prepared by dissolving 1.0 mg A.beta. in 400 .mu.L HFIP for 10-20
min at room temperature. 100 .mu.l of the resulting seedless
A.beta. solution are then added to 900 .mu.L DD H2O in a
siliconized Eppendorf tube. After 10-20 min incubation at room
temperature, the samples are centrifuged for 15 min. at
14,000.times.G and the supernatant fraction (pH 2.8-3.5) is
transferred to a new siliconized tube and subjected to a gentle
stream of N2 for 5-10 min to evaporate the HFIP. The samples are
then stirred at 500 RPM using a Teflon coated micro stir bar for
24-48 hr at 22.degree. C. Aliquots (10 .mu.l) are taken at 6-12 hr
intervals for observation by atomic force microscopy or electron
microscopy.
Example 5
Recombinant Expression of an Anti-A.beta.(21-37) Autoantibodies
A. Mammalian Expression Vector Construction for Transient
Expression
[0540] Amino acid sequences for both the light chain variable
region of CSL Clone 7 (SEQ ID NO: 53) and heavy chain variable
region of CSL Clone 7 (SEQ ID NO: 60) were used to synthesize cDNA
constructs encoding these sequences by GENEART AG (Regensburg,
Germany). The light and heavy chain cDNA constructs were also
designed to contain unique flanking restriction enzyme sites to
allow cloning into a mammalian expression vector upstream of the
human light and heavy chain constant regions respectively. The
constructs were also engineered with a Kozak translation initiation
sequence, an ATG start codon and signal peptides
(MESQTQVLMSLLFWVSGTCG--light chains and MGWSWIFLFLVSGTGGVLS--heavy
chains).
[0541] Using standard molecular biology techniques, the heavy chain
variable region was cloned into the mammalian expression vector
pcDNA3.1(+)-hIgG1, which is based on the pcDNA3.1(+) expression
vector (Invitrogen) modified to include the human IgG1 constant
region and a terminal stop codon downstream of the variable region
insertion site. The light chain variable region was cloned into the
expression vector pcDNA3.1(+)-h.kappa., which is based on the
pcDNA3.1(+) expression vector modified to include the human kappa
constant region and a stop codon downstream of the variable region
insertion site.
[0542] CSL Clone 7 also was engineered as a "murinized" version to
facilitate repetitive use in murine animal models. The heavy chain
variable region was cloned into the mammalian expression vector
pcDNA3.1(+)-mIgG2a, which is based on the pcDNA3.1(+) expression
vector (Invitrogen) modified to include the murine IgG2a constant
region and a terminal stop codon downstream of the variable region
insertion site. The light chain variable region was cloned into the
expression vector pcDNA3.1(+)-m.kappa., which is based on the
pcDNA3.1(+) expression vector modified to include the murine kappa
constant region and a stop codon downstream of the variable region
insertion site. Murinized CSL Clone 7 was expressed and purified as
described below.
B. Cell Culture
[0543] Serum-free suspension adapted 293-T cells were obtained from
Genechoice Inc. Cells were cultured in FreeStyle.TM. Expression
Medium (Invitrogen) supplemented with
penicillin/streptomycin/fungizone reagent (Invitrogen). Prior to
transfection the cells were maintained at 37.degree. C. in
humidified incubators with an atmosphere of 8% CO.sub.2.
C. Transient Transfection
[0544] Transient transfection of the clone 7 expression plasmids
using 293-T cells was performed using 293 fectin transfection
reagent (Invitrogen) according to the manufacturer's instructions.
The light and heavy chain expression vectors were combined and
co-transfected with the 293-T cells. Cells (1000 ml) were
transfected at a final concentration of 1.times.10.sup.6 viable
cells/ml and incubated in a Cellbag 2L (Wave Biotech/GE Healthcare)
for 5 days at 37.degree. C. with an atmosphere of 8% CO.sub.2 on a
2/10 Wave Bioreactor system 2/10 or 20/50 (Wave Biotech/GE
Healthcare). The culture conditions were 35 rocks per minute with
an angle of 8.degree.. Pluronic.RTM. F-68 (Invitrogen), to a final
concentration of 0.1% v/v, was added 4 hours post-transfection. 24
hours post-transfection the cell cultures were supplemented with
Tryptone N1 (Organotechnie, France) to a final concentration of
0.5% v/v. The cell culture supernatants were harvested by
centrifugation at 2500 rpm and were then passed through a 0.45
.mu.M filter (Nalgene) prior to purification.
D. Analysis of Protein Expression
[0545] After 5 days 20 .mu.l of culture supernatant was
electrophoresed on a 4-20% Tris-Glycine SDS polyacrylamide gel and
the antibody was visualized by staining with Coomassie Blue
reagent.
E. Antibody Purification
[0546] The CSL Clone 7 monoclonal antibody was purified using
protein A affinity chromatography at 4.degree. C., where MabSelect
resin (5 ml, GE Healthcare, UK) was packed into a 30 ml Poly-Prep
empty column (Bio-Rad, CA). The resin was first washed with 10
column volumes of pyrogen free GIBCO Distilled Water (Invitrogen,
CA) to remove storage ethanol and then equilibrated with 5 column
volumes of pyrogen free phosphate buffered saline (PBS) (GIBCO PBS,
Invitrogen, CA). The filtered conditioned cell culture media (1 L)
was loaded onto the resin by gravity feed. The resin was then
washed with 5 column volumes of pyrogen free PBS to remove
non-specific proteins. The bound antibody was eluted with 2 column
volumes of 0.1M glycine pH 2.8 (Sigma, Mo.) into a fraction
containing 0.2 column volumes of 2M Tris-HCl pH 8.0 (Sigma, Mo.) to
neutralize the low pH. The eluted antibody was dialysed for 18 hrs
at 4.degree. C. in a 12 ml Slide-A-Lyzer cassette MW cutoff 3.5 kD
(Pierce, Ill.) against 5 L PBS. The antibody concentration was
determined by measuring the absorbance at 280 nm using an Ultraspec
3000 (GE Healthcare, UK) spectrophotometer. The purity of the
antibody was analysed by SDS-PAGE, where 2 .mu.g protein in
reducing Sample Buffer (Invitrogen, CA) was loaded onto a Novex
10-20% Tris Glycine Gel (Invitrogen, CA) and a constant voltage of
150V was applied for 90 minutes in an XCell SureLock Mini-Cell
(Invitrogen, CA) with Tris Glycine SDS running buffer before being
visualized using Coomassie Stain, as per the manufacturer's
instructions.
[0547] The above-described techniques can be used to express and
purify any of the inventive antibodies. In subsequent experiments
light chain SEQ ID NOs: 47, 48, 50 to 55, and 145 to 147 were
cloned into the expression vector pcDNA3.1(+)-h.kappa. and
co-transfected with heavy chain SEQ ID NOs: 56 to 71 and 148 which
were cloned into the expression vector pcDNA3.1(.+-.)-hIgG1. A
total of 187 transient transfections were performed covering all
possible light and heavy chain antibody pairs. The following 42
light and heavy chain antibody pairs (SEQ ID NOs) expressed
sufficient antibody for purification and analysis: 47/56, 50/60,
50/61, 50/62, 50/67, 50/68, 50/69, 50/148, 51/60, 51/61, 51/62,
51/68, 51/148, 52/60, 52/148, 53/60, 53/68, 53/148, 54/60, 54/61,
54/62, 54/67, 54/68, 54/69, 54/148, 55/60, 55/61, 55/62, 55/67,
55/68, 55/69, 55/148, 145/60, 145/61, 145/62, 145/68, 145/148,
146/60, 146/61, 146/62, 146/68 and 146/148.
[0548] Such methods can also be employed to produce fully human
anti-.beta. amyloid antibodies comprising selected individual
sequences based on the consensus sequences of the respective CDRs,
such as SEQ ID NOs: 6 to 11 and SEQ ID NOs: 153 to 161, or the
single sequences of the respective CDRs, such as (a) for CDR I of
the heavy chain SEQ ID Nos: 13 to 20, b) for CDR2 of the heavy
chain SEQ ID NOs: 21 to 27, c) for CDR3 of the heavy chain SEQ ID
NOs: 28 to 32, d) for CDR1 of the light chain SEQ ID NOs: 33 to 37,
e) for CDR2 of the light chain SEQ ID NOs: 38 to 43 and SEQ ID NO:
53, f) for CDR3 of the light chain SEQ ID Nos: 44 to 46, g) for the
variable heavy chain SEQ ID NOs: 56 to 71 and h) for the variable
light chain SEQ ID NOs: 47 to 55.
[0549] For use as a control antibody (ACA) in our studies we also
cloned the light and heavy chain variable region sequences of the
humanized 266 antibody which is known to bind an epitope contained
within position 13-28 of the amyloid beta peptide. These sequences
were obtained from the U.S. Pat. No. 7,195,761 B2. Specifically the
genes for the humanized light chain variable region of 266 (U.S.
Pat. No. 7,195,761 B2, SEQ ID NO: 11) and the humanized heavy chain
variable region of 266 (U.S. Pat. No. 7,195,761 B2, SEQ ID No:12)
were synthesized, cloned into expression vectors, transiently
expressed and purified using the above-described methods.
Example 6
Binding of a Recombinantly Expressed
Anti-A.gamma.(21-37)-Autoantibody CSL Clone 7 and Affinity Purified
IVIgG to Oligomeric Forms of A.beta.
[0550] A synthetic amyloid beta 1-40 peptide (PSL GmbH Heidelberg)
containing an additional cysteine at the amino terminal
(A.beta.1-40.Cys) was analyzed in an immunoprecipitation assay
against anti-A.beta.(21-37) monoclonal antibody (mab) CSL Clone 7
and against affinity purified IVIgG as described in example 4. PBS
was employed as a negative control.
[0551] Specifically, it was evaluated whether mab CSL Clone 7 would
immunoprecipate the synthetic peptide in a monomer or an oligomer
form. The peptide, resuspended (1 mg/ml) in phosphate buffered
saline (PBS: 10 mM sodium phosphate, 150 mM NaCl, pH 7.4) was used
immediately (0 h) or subjected to oligomerisation (15 h) at
37.degree. C., 900 rpm and stored at -80.degree. C. in small
aliquots until use. For the immunoprecipitation, aliquots of 30
.mu.l of Protein-G beads (GE Healthcare) were incubated with 5
.mu.g antibody mab CSL Clone 7 or 5 .mu.g anti-A.beta.(21-37)
autoantibodies purified according to example 2 (A.beta.1-40 column,
2 .mu.g A.beta.1-40.Cys and 1.5 ml PBS over night at 4.degree. C.
Immobilized antibody/peptide were collected. After washing (five
times) with PBS, the peptide was eluted by adding 1.times.
non-reducing NuPAGE LDS Sample Buffer (Invitrogen) for 10 min at
95.degree. C. Protein separation was done by electrophoresis on
NuPAGE 4-12% Bis-Tris Gels (Invitrogen) and western transfer on
nitrocellulose membranes by wet blot according to the supplier
(Invitrogen). Membranes were blocked with 1.times. Roti-Block
(Roth) and then successively incubated with the first antibody,
1:6000 Bam90.1 (anti-A.beta.) (Sigma) and secondary antibody, goat
anti-mouse HRP conjugated (Pierce). A SuperSignal West Dura
Extended Duration Substrate (Thermo Scientific/Pierce) was used as
chemiluminescent substrate.
[0552] The results show that mab CSL Clone 7 (see FIG. 33) and
affinity purified IVIgG (see FIG. 37) bind oligomeric forms of
A.beta.1-40. In particular, mab CSL Clone 7 or affinity purified
IVIgG co-incubated either with monomeric (0 h) or oligomeric (15 h)
forms of A.beta.1-40,Cys precipitated oligomeric forms of the
peptide. More results on binding to oligomeric forms of A.beta. can
be found in Example 12D.
Example 7
Peptide Synthesis
[0553] Peptides Biotin-G.sub.5-FAEDVGSNKGA-NH.sub.2
(Biotin-G.sub.5-A.beta.20-30) and
Biotin-G.sub.5-FAEDVGSNKGAIIGLMVG-NH.sub.2
(Biotin-G.sub.5-A.beta.20-37) were synthesized by solid-phase
peptide synthesis (SPPS) on a NovaSyn TGR resin, containing a
polystyrene-polyethyleneglycol resin and Rink-amide-linker
cleavable under acidic conditions, according to commercially
available material and published literature procedures.
9-Fluorenylmethoxycarbonyl/t-butyl (Fmoc/tBu) chemistry was used
throughout for synthesis using a semi-automated Economy Peptide
Synthesizer EPS-221 (ABIMED, Germany). The following side-chain
protected amino acid derivatives were used: Fmoc-Lys(Boc)-OH,
Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(OtBu)-OH,
Fmoc-Glu(OtBu)-OH. The synthesis was performed according to the
following general protocol: (i) DMF washing (3.times.1 min), (ii)
Fmoc deprotection with 2% DBU, 2% piperidine in DMF (15 min), (iii)
DMF washing (6.times.1 min), (iv) coupling of 5 equiv of Fmoc amino
acid:PyBOP:NMM in DMF (40-60 min), (v) DMF washing (3.times.1 min).
For the synthesis of A.beta. (20-37) which has a hydrophobic
C-terminal part, double coupling of each amino acid was employed.
The biotinylation of the N-terminus was carried out on the resin
using D-(+)-Biotin. After completion of the syntheses, the peptides
were cleaved from the resin using a TFA, triethylsilane and
deionized water mixture (95:2.5:2.5, V/V/V) for 3 h at room
temperature. The synthetic peptides were used for example in
ELISAs.
Example 8
Amino Acid Sequencing of A.beta.(21-37)
[0554] Sequence determination of all epitopes either identified
(isolated A.beta. epitopes A.beta.(4-10) and A.beta.(21-37)) or
used (e.g. synthetic A.beta.(21-37)) was carried out by means
of
a) Edman sequencing;
b) ESI-Tandem MS/MS-sequencing, and
[0555] c) FTICR-MS analysis and fragmentation by means of
IRMPD-Fragmentation.
[0556] Automated amino acid sequence analysis was performed on an
Applied Biosystems Model 494 Procise Sequencer attached to a Model
140C Microgradient System, a 785A Programmable Absorbance Detector
and a 610A Data Analysis System.
[0557] All solvents and reagents used were of highest analytical
grade purity (Applied Biosystems). The sequencing method used was
pulsed liquid. Lyophilized samples were dissolved in 10 .mu.L 0.1%
TFA. To assure a distribution as close to the centre of the glass
fiber filter as possible, the sample was applied in aliquots of 2
.mu.L, each application followed by drying under a stream of
argon.
Example 9
ELISAs
[0558] ELISAs were used for the determination of [0559] (a)
plaque-specific anti-A.beta.(4-10) antibodies in the anti-A.beta.
autoantibodies mixture separated from IVIgG [0560] (b) binding of
anti-A.beta. autoantibodies from human serum to A.beta.(21-37)
peptide, A.beta.(12-40) peptide, and A.beta.(1-40) peptide [0561]
(c) binding of anti-A.beta. autoantibodies separated from IVIgG and
from individual human sera (AD serum and healthy individuals serum)
to A.beta.(1-40) peptide and to A.beta.(21-37) epitope peptide
[0562] (d) binding of a recombinantly expressed A.beta.(21-37)
autoantibody (CSL Clone 7) to A.beta. partial sequences
A. ELISA for A.beta.(4-10) Antibodies
[0563] In this experiment a standard dilution of the antibody
(anti-A.beta. antibodies isolated from IVIgG using a
Cys-A.beta.(1-40) antigen column) was used in combination with 12
serial dilutions of Biotin-G5A.beta.(4-10) peptide, used as coating
antigen. 96-well ELISA plates were coated with 150 .mu.L/well
streptavidin solution (c=5 .mu.g/mL in PBS) for 2 hours at room
temperature. After washing the wells four times with PBS-T (0.05%
Tween-20 v/v in PBS, pH=7.5), 100 .mu.L/well of biotinylated
epitope peptides (12 serial dilutions from 50 .mu.M to 0.024 .mu.M
in PBS, pH=7.5) was added and incubated for 2 hours at room
temperature. After that, the plates were washed four times with 200
.mu.L/well PBS-T and the non-specific adsorption sites were blocked
with 5% BSA, 0.05% Tween-20 in PBS (200 .mu.L/well, 2 h incubation
at RT). Then, 100 .mu.L/well of the anti-A.beta. autoantibodies
isolated from IVIgG (1:150 dilution prepared in 5% BSA, 0.05%
Tween-20 in PBS) was added to each well. Thereafter, the plates
were incubated at room temperature for two hours and subsequently
washed six times with PBS-T. 100 .mu.L of peroxidase goat
anti-human IgG diluted 5000 times in 5% BSA, 0.05% Tween-20 were
added to each well and the plates were incubated at room
temperature for one hour, then they were washed three times with
PBS-T and once with 0.05 M sodium phosphate-citrate buffer, pH=5.
100 .mu.L of o-phenylenediamine dihydrochloride (OPD) in substrate
buffer (phosphate-citrate) at c=1 mg/mL with 2 .mu.L of 30%
hydrogen-peroxide per 10 mL of substrate buffer were added. The
absorbance at 450 nm was measured on a Wallac 1420 Victor2 ELISA
Plate.
B. Antibody Determination in Human Serum by Indirect ELISA
[0564] 96-well ELISA plates were coated with 100 .mu.L/well of
A.beta.(1-40) peptide (c=2.5 .mu.g/mL in PBS buffer, pH 7.5) for 2
h at room temperature. Thereafter, the plates were washed four
times with 200 .mu.L/well of washing buffer (PBS-T; PBS with 0.05%
Tween-20) and blocked for 2 h at room temperature with blocking
buffer (5% BSA, 0.1% Tween-20 in PBS). After two times washing with
PBS-T, the sera were added at an initial dilution of 1:33.3, then
diluted 3 fold serially in blocking buffer and incubated for 2 h at
room temperature. Then, the plates were washed eight times with
PBS-T and goat anti-human IgG conjugated with horseradish
peroxidase diluted 1:5000 in blocking buffer was added to the
plates and incubated for 1 h at RT. The plates were washed four
times with PBS-T and two times with 0.05 M sodium phosphate-citrate
buffer, pH=5. 100 .mu.L of o-phenylenediamine dihydrochloride (OPD)
in substrate buffer (phosphate-citrate) at c=1 mg/mL with 2 .mu.L
of 30% hydrogen-peroxide per 10 mL of substrate buffer were added.
The absorbance at 450 nm was measured on a Wallac 1420 Victor2
ELISA Plate. A.beta.-antibody quantifications were performed with a
1 .mu.g/.mu.l stock solution, using a BSA reference curve for
calibration using the commercial protein quantification kit Pierce
micro-BCA. The results obtained are illustrated in FIG. 8. The
percentage given illustrates the A.beta.-antibody concentrations in
IVIgG from two separate ELISA determinations. Similar results were
obtained with the A.beta.(21-37) affinity chromatography. To the
contrary, affinity chromatography with A.beta.(4-10) peptide
yielded no detectable amounts of polypeptides binding to the
N-terminal epitope. Consequently, the results obtained with
A.beta.(1-40) are equivalent to those obtained with A.beta.(21-37)
for healthy individuals.
C. Binding of Anti-A13 Autoantibodies Isolated from IVIgG and from
Individual Human serum to A.beta.1-40 peptide
[0565] 96-well ELISA plates were coated with 100 .mu.L/well of
A.beta.1-40 peptide (c=2.5 .mu.g/mL in PBS buffer, pH 7.5) for 2 h
at room temperature. Thereafter, the plates were washed four times
with 200 .mu.L/well of washing buffer (PBS-T; PBS with 0.05%
Tween-20) and blocked for 2 h at room temperature with blocking
buffer (5% BSA in PBS). After washing the plates two times with 200
.mu.L/well of PBS-T, 100 .mu.L/well of the 1.sup.st antibody
(polyclonal anti-A.beta. autoantibodies isolated from IVIgG or from
individual human serum) (8 serial dilutions prepared in blocking
buffer; dilutions from 1:250 to 1:32000) was added and incubated
for 2 h at room temperature. Then, the plates were washed four
times with 200 .mu.L/well of PBS-T and the 2.sup.nd antibody
(HRP-goat anti-human IgG; c=1 .mu.g/.mu.L) diluted 2000 times in
blocking buffer was added (100 .mu.L/well; 2 h incubation at room
temperature). After washing the plates three times with 200
.mu.L/well of PBS-T and once with 200 .mu.L/well of
citrate-phosphate buffer, pH=5, 100 .mu.L of o-phenylenediamine
dihydrochloride (OPD) in substrate buffer (phosphate-citrate) at
c=1 mg/mL with 2 .mu.L of 30% hydrogen-peroxide per 10 mL of
substrate buffer were added. The absorbance at 450 nm was measured
on a Wallac 1420 Victor.sup.2 ELISA Plate.
D. Binding of a Recombinantly Expressed A.beta.(21-37) Autoantibody
(CSL Clone 7) to A.beta. Partial Sequences
[0566] Peptides Biotin-G.sub.5-A.beta.(1-40),
Biotin-G.sub.5-A.beta.(12-40) and Biotin-G.sub.5-A.beta.(4-10) were
compared for binding to a recombinantly expressed A.beta.(21-37)
autoantibody (CSL Clone 7) by the following indirect ELISA: 96-well
ELISA plates were coated with 150 .mu.L/well streptavidin solution
(c=2.5 .mu.g/mL in PBS pH7.4) for 2 hours at room temperature.
After washing the wells four times with 200 .mu.L/well PBS-T (0.05%
Tween-20 v/v in PBS, pH=7.4), 100 .mu.L/well of biotinylated
epitope peptides 1 .mu.M in PBS, pH=7.5) were added and incubated
for 2 hours at room temperature. After that, the plates were washed
four times with 200 .mu.L/well PBS-T and the non-specific
adsorption sites were blocked with 5% BSA, 0.1% Tween-20 in PBS
(200 .mu.L/well, over night at RT). Then the plates were washed
once with 200 .mu.L/well with PBS-T. Then, 8 serial dilutions of
CSL Clone 7 prepared in 5% BSA, 0.1% Tween-20, 1% DMSO in PBS) were
added to the wells. Thereafter, the plates were incubated at room
temperature for two hours and subsequently washed six times with
PBS-T. 100 .mu.l of peroxidase goat anti-human IgG diluted 5000
times in 5% BSA, 0.1% Tween-20 were added to each well and the
plates were incubated at room temperature for one hour, then they
were washed three times with 200 .mu.L/well PBS-T and once with
0.05 M sodium phosphate-citrate buffer, pH=5. 100 .mu.L of
o-phenylenediamine dihydrochloride (OPD) in substrate buffer
(phosphate-citrate) at c=1 mg/mL with 2 .mu.L of 30%
hydrogen-peroxide per 10 mL of substrate buffer were added. The
absorbance at 450 nm was measured on a Wallac 1420 Victor.sup.2
ELISA Plate. Background signals of the assay were measured with
antibody dilutions incubated in wells lacking the biotinylated
peptides.
[0567] The recombinantly expressed anti-A.beta.(21-37) autoantibody
CSL clone 7 (see Example 5) was evaluated as described above using
Biotin-G.sub.5-A.beta.(1-40), Biotin-G.sub.5-A.beta.(12-40) and
Biotin-G.sub.5-A.beta.(4-10). FIG. 39 shows that CSL clone 7 binds
to A.beta.(1-40) and A.beta.(12-40) but not to A.beta.(4-10).
Example 10
Prevention of Fibril Formation by the Antibodies of the
Invention
[0568] 1 mg of A.beta.1-40 (PSL Heidelberg) was dissolved in a
LoBind tube (Eppendorf) with 100 .mu.l trifluoroacetic acid 0.1%
(TFA) and incubated for 1 hour at room temperature. The solution
was diluted with PBS to 1 mM A.beta.1-40. To 100 .mu.l A.beta.
fiber formation sample, the antibodies were added to a final
concentration of 1.3 .mu.M.
[0569] The following antibodies were examined: [0570] affinity
purified IVIgG as described in Example 4 [0571] recombinant
antibody CSL Clone 7 (as described in example 5) [0572] antibody
ACA (U.S. Pat. No. 7,195,761 B2) [0573] negative control CSL360
(CSL360 is a chimeric antibody and shows no binding to
A.beta.(1-40) when tested using biosensor or ELISA analysis)
[0574] The incubation was carried out overnight at 37.degree. C. on
a heating block. A 2.5 mM Thioflavin T (THT) solution in Glycine
buffer pH 9.2 was prepared. The 100 .mu.l A.beta. fiber formation
sample was transferred into a black -96 well plate (Greiner), and
50 .mu.M THT was added. The fluorescence (excitation 450 nm
emission 490 nm) of the samples was measured after 24 hour with a
Tecan InfiniTE M200 plate reader. One measurement represents the
average of 25 flashes.
[0575] As shown in FIG. 40, all tested A.beta. antibodies showed an
inhibition of fibrilization by about 20% as compared to the
negative control.
Example 11
Assay Binding of Sera from an AD Patient and an Age Matched Healthy
Control
A: Dot Blot
[0576] Dot blots of A.beta..sub.1-15cys-and A.beta..sub.1-40Cys-,
(both peptides do have a cysteine do have at the N-terminus)
freshly resuspended in PBS buffer (sample-0 h) or subjected to
oligomerisation (sample-15 h) were applied on nitrocellulose
membrane (0.5 .mu.g/3 .mu.l spot).
[0577] The membranes were blocked with Roti-Block (Roth) for 1 h at
room temperature and then incubated with 10 .mu.g primary antibody
(6E10, Bam90.1, IVIG, CSL-7, ACA), an AD-serum (AD1) and a serum
(K4) from a healthy human individual) in 20 ml blocking reagent
(RotiBlock) over night at 4.degree. C. Incubation with secondary
antibodies (anti-human HRP: 1:100,000; anti-mouse HRP: 1:6,000) was
done for 1 h at room temperature. A SuperSignal West Dura Extended
Duration Substrate (Thermo Scientific/Pierce) was used as
chemiluminescence substrate according to the instructions of the
manufacturer. The signal on X-ray films was recorded for 10 s-5
min.
[0578] As shown in FIG. 41 all control antibodies showed
specificity to the expected epitope (6E10=A.beta.(1-17),
Bam90.1=A.beta.(13-28)). Purified antibodies CSL Clone7, anti
A.beta.(21-37) autoantibodies (purified according to example 4),
ACA (see example 5), but also antibodies from the AD serum and the
healthy individual bind to A.beta..sub.1-40 oligomers (sample 15
h). Only 6E10 and the AD-serum bound to A.beta. 1-15 but not the
control sera of the healthy patient.
[0579] Also, CSL Clone 7 and A.beta.(21-37) autoantibodies purified
from IVIgG as described in Example 4 exhibited preferential binding
to aggregated/oligomeric forms of A.beta. (A.beta.(1-40) 15 h as
opposed to A.beta. 1-40 0 h). By comparison, the control antibodies
6E10, Bam90.1 and ACA showed no such preference, binding instead
equally to both species of A.beta..
TABLE-US-00002 TABLE 1 Summary data of the Dot Blot analysis
Affinity se- se- CSL- purified rum rum Peptide 6E10 Bam90.1 Clone 7
IVIgG ACA AD1 K4 A.beta. 1-40, 0 h ++ ++ (trace) - ++ - - A.beta.
1-40, ++ ++ +++ ++ ++ +++ +++ 15 h A.beta. 1-15, 0 h ++ - - - - ++
- A.beta. 1-15, ++ - - - - ++ - 15 h
B: ELISA
[0580] IgG from serum samples (AD1 and an age matched healthy human
individual, see above in Example 11A) after purification on Protein
G (Pierce) according to the instructions of the manufacturer were
loaded on an A.beta.(1-16) affinity column (prepared according to
Example 1), washed with PBS and 10 mM sodium phosphate pH 6.8 and
eluted with 100 mM Glycine pH 2.8.
[0581] The eluate was analyzed in an ELISA on
Biotin-G.sub.5-A.beta.(4-10) coated plates as described in Example
9D.
[0582] The result shows a higher titer of A.beta.(4-10) antibodies
in the serum of the AD patient as compared to the signal detected
for the control sample from an age matched healthy human individual
(see FIG. 42).
[0583] Results represent an early experiment which suggests, at
least for the single AD1 serum tested, that A.beta.(4-10)
autoantibodies are present but either in low amount, of low
affinity, or possible both. If these results are verified, they
indicate that ultra-sensitive assay procedures will be required to
permit the procedure to become routine.
Example 12
Binding Characteristics of Recombinantly Expressed A.beta.(21-37)
Autoantibodies in ELISA, Biacore and Western Blot
TABLE-US-00003 [0584] TABLE 2 Binding characteristics of
recombinantly expressed A.beta.(21-37) autoantibodies in ELISA and
Biacore WESTERN BLOT ANTIBODY EIA BIACORE ANALYSIS ACA (# 80) ++++
++++ Binds to all species 53/60 (# 92) ++++ ++++ Dimer binding
53/60 (# 93) ++++ ++++ Dimer binding 50/60 + +++ nt 50/61 + ++ nt
50/62 - - nt 50/67 - - nt 50/68 - - nt 50/69 - nt nt 50/148 - + nt
47/56 - - nt 51/60 ++++ +++ Dimer binding 51/61 + + nt 51/62 - - nt
51/68 - + nt 51/148 + + nt 52/60 ++++ ++++ Dimer binding 52/148 +
++ nt 53/68 - - nt 53/148 ++++ ++++ nt 54/60 ++ +++ nt 54/61 ++ ++
Dimer binding 54/62 - - nt 54/67 - No capture nt 54/68 - - nt 54/69
- No capture nt 55/60 + ++ nt 55/61 +++ +++ Dimer binding 55/62 - -
nt 55/67 - No capture nt 55/68 - - nt 55/69 - - nt 55/148 + + nt
145/60 +++++ ++++ nt 145/61 - - nt 145/62 +++ + nt 145/68 - - nt
145/148 ++ ++ nt 146/60 ++++ ++++ nt 146/61 ++ ++ nt 146/62 + - nt
146/68 +/? - nt 146/148 + +++ nt 54/148 ++ + nt +/- qualitative
assessment of ELISA and biosensor binding where increasing (+)
indicates increase binding titre on ELISA and on biosensor
indicates an improvement on either off-rate or on rate that would
suggest an antibody with comparative higher affinity (-) indicates
either no binding by antibodies to immobilised antibodies Nt
denotes not tested
[0585] All transfectants that expressed immunoglobulin efficiently
were tested in an ELISA based on binding to amyloid .beta. peptide.
Biosensor data is ranked on a qualitative affinity binding
assessment of the antibody to the cys-dimer peptide as described
below.
[0586] Antibodies which are expressed in high quantities and show
binding in ELISA or Biacore or Western blot are preferred
embodiments of the invention. A failure to express in high quantity
or non-binding in any of the assays described below does not
necessarily mean that these antibodies would not be functional if
either expression would be improved or more sensitive detection
methods were be employed.
A: A.beta. Peptide Preparation
[0587] Lyophilized 1 mg A.beta.(1-40) peptide (Sigma) was
resuspended in 200 .mu.l 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)
(Sigma), aliquoted into 1.5 ml Eppendorf tubes and lyophilized
overnight. The aliquoted A.beta.(1-40) peptide was resuspended at 1
mg/ml with dimethyl sulphoxide (DMSO, ICN) and stored at 4.degree.
C. This material is referred to as A.beta. 1-40 monomer.
[0588] To oligomerize the A.beta.(1-40) peptide the A.beta.(1-40)
monomer was diluted to 0.1 mg/ml in 1.times.PBS (137 mM NaCl, 10 mM
phosphate, 2.7 mM KCl) and incubated for 3 to 6 days at 37.degree.
C. and then stored at 4.degree. C., this material is referred to as
A.beta.(1-40) oligomer.
[0589] A.beta.(1-40) peptide with an N-terminal cysteine residue
(A.beta.(1-40 Cys)) was resuspended from a lyophilized state in
ddH2O at 5.9 mg/ml and stored at -80.degree. C. The A.beta.(1-40
Cys) peptide was then diluted to 0.1 mg/ml in 1.times.PBS and
stored at 4.degree. C., this material is referred to as
A.beta.(1-40 Cys) oligomer.
B: Anti-Beta Amyloid Antibody ELISA Protocol
[0590] A.beta.(1-40) monomer, A.beta.(1-40) oligomer, A.beta.(1-40
Cys) oligomer, and control plate using bovine serum albumin (Sigma)
or Aprotinin (Sigma) were immobilized at 1 .mu.g/ml in PBS (50
.mu.l per well) on a Nunc Maxisorb 96-well plate overnight at
4.degree. C. Wells were washed once with 350 .mu.l Wash Buffer
(1.times.PBS, 0.05% (v/v) Tween-20 (Sigma)). Wells were then
blocked with Blocking Buffer (2% (w/v) Difco Skim milk (BD) in PBS,
50-150 .mu.l per well) for 2 hours at room-temperature and washed
once with 350 .mu.l Wash Buffer.
[0591] The primary antibody was serial diluted 1:2 in V-bottom well
96-well plates (Nunc) at starting concentration of 100 mg/ml in
Antibody Buffer (1% (w/v) Bovine Serum Albumin (Sigma) in PBS,
0.05% Tween-20 (Sigma)).
[0592] The control antibodies (eg. 6E10 mAb (Sigma) and ACA) were
analyzed at a starting concentration of 1 mg/ml
[0593] 100 .mu.l of primary antibody were transferred in serial
dilution to Nunc Maxisorb plates and incubated for 2-3 hours at
room-temperature. Wells were washed three times with 350 .mu.l Wash
Buffer rapidly.
[0594] The secondary antibodies sheep anti-human IgG-HRP and sheep
anti-mouse IgG-HRP (Chemicon) were added at 1:1000 in Antibody
Buffer (50 .mu.l per well) and incubated for 30 minutes at
room-temperature.
[0595] Wells were then washed three times with 350 .mu.l Wash
Buffer and the plates developed with TMB Substrate (Millipore,
Australia) (50 .mu.l per well) for 5 minutes. The reaction was
stopped with 2M phosphoric acid (25 .mu.l per well) and the plates
were read at 450 nm, 0.1 seconds in Wallac Victor 2 plate
reader.
C: Biosensor Analysis of A.beta.(1-40) Interaction with
Recombinantly Expressed A.beta.(21-37) Autoantibodies
Peptide Sample Preparation:
[0596] Preparation of Peptides for Analysis on Captured Monoclonal
Antibodies is as Described in section "A.beta. preparation of
peptides" above
Biosensor Immunoglobulin Capture Surface Preparation:
[0597] An anti-human immunoglobulin biosensor chip was prepared
using a human antibody capture kit (Biacore, Sweden) as per
manufacturer's instructions at flow rate of 5 .mu.l/min with a 6
minute contact time during NHS/ECD immobilisation. Approximately
10000 resonance units were achieved on all channels.
Capture Conditions:
[0598] All antibodies were captured (25 .mu.g/ml in 0.1 mg/ml BSA,
Hepes buffered saline) at a flow rate of 20 .mu.l/min for 2 mins.
In all experiments flow cell 1 served as a baseline subtracted
control channel using human IgG1 control antibody (Chemicon,
Australia).
[0599] The 3 remaining channels were used to capture either control
antibody (ACA) or human monoclonal antibodies to test for peptide
binding. Peptide binding (60 .mu.l) to the captured antibodies was
then analysed at a flow rate of 30 .mu.l/min over all channels
simultaneously and allowed to dissociate to for 300-600 seconds
prior to desorption from the sensor surface using a 5 .mu.l
injection of 3M MgCl.sub.2 as per instructions.
[0600] All samples were cooled prior to analysis at 12'C using a
Multitemp (GE, Sweden) attached to the biosensor 2000 (GE,
Sweden).
[0601] A second control experiment was performed for all monoclonal
antibodies where a 60 .mu.l injection of 0.1 mg/ml BSA, Hepes
buffered saline was directly injected post antibody capture to take
into account dissociation of human monoclonal antibody from
immobilised capture antibody. This result was manually subtracted
from data generated using peptides using BIAevaluation software
(GE, Sweden).
[0602] In all experiments A.beta.(1-40 Cys) oligomer was analyzed
at 20-50 .mu.g/ml in 0.1 mg/ml BSA, Hepes buffered saline.
A.beta.(1-40) monomer was diluted from a 1 mg/ml stock in 100% DMSO
to 10 .mu.g/ml in 0.1 mg/ml BSA, Hepes buffered saline prior to
analysis.
[0603] FIGS. 43 to 45 show the results obtained in this analysis.
Whereas the ACA control antibody show equal binding to both
A.beta.(1-40) monomer and A.beta.(1-40 Cys) oligomer most
antibodies of the invention show a preferential binding to
A.beta.(1-40 Cys) oligomer.
D: Anti-Beta Amyloid Antibody Tricine SDS-Page and Western Blot
Protocol
[0604] Novex Pre-cast 10-20% Tricine gels 10-well (Invitrogen) were
loaded with 0.5 .mu.g peptide per well in 20 .mu.l (0.5 .mu.g/20
.mu.l=25 .mu.g/ml).
[0605] Preparation of molecular weight marker: 20 .mu.l 2.times.
Tricine Sample Buffer--Non-reducing (TSB-NR) (300 mM Tris-HCl, pH
8.45, 24% Glycerol, 8% SDS, 0.005% Coomassie Blue G, 0.005% Phenol
Red) were added to 20 .mu.l Pre-stained markers (Bio-Rad), and 20
.mu.l per well were used.
Preparation of the Samples:
[0606] 100 .mu.l 1.times.TSB-NR loading samples were prepared as
follows
Peptides:
TABLE-US-00004 [0607] Stock Dilution 100 .mu.l A.beta. 1-40 1.0
mg/ml 1/40 2.5 .mu.l + 47.5 .mu.l ddH.sub.2O + 50 .mu.l 2X monomer
TSB-NR A.beta. 1-40 0.1 mg/ml 1/4 25 .mu.l + 25 .mu.l ddH.sub.2O +
50 .mu.l 2X oligomer TSB-NR A.beta. 1-40 Cys 0.1 mg/ml 1/4 25 .mu.l
+ 25 .mu.l ddH.sub.2O + 50 .mu.l 2X oligomer TSB-NR
[0608] Each gel was set up in a 10-lane format as below so that
each test antivody was flanked by a molecular weight marker lane
and a blank buffer lane. Molecular weight positions have been
marked for simplicity.
1) Pre-stained markers 2) A.beta.1-40 monomer 3) A.beta.1-40
oligomer 4) A.beta.1-40 Cys oligomer
5) 1.times.TSB-NR
[0609] 6) Pre-stained markers 7) A.beta.1-40 monomer 8) A.beta.1-40
oligomer 9) A.beta.1-40 Cys oligomer
10) 1.times.TSB-NR
[0610] A further gel was run as above and stained for protein by
coomassie and deep purple. The less sensitive Coomassie shows that
the peptide is essentially monomer in lanes 2 and 3 and dimer in
lane 4 (or lane 7, 8 and 9 respectively which is labeled 1, 2 and 3
in FIGS. 47 and 48). The more sensitive deep purple reveals a
ladder of oligomers in each lane, which is consistent with the
staining pattern for mabs 6E10 and ACA.
[0611] The Tricine gels were run in XCell SureLock Mini-Cell
(Invitrogen) with inner and outer buffer chambers containing
Tricine SDS Running Buffer (0.1 M Tris Base, 0.1M Tricine, 0.1%
SDS). The electrophoretic separation was completed applying 125V
for 90 minutes.
[0612] The gel was then transferred to nitrocellulose as per
Membrane Filter Paper Sandwich (Invitrogen) using the XCell II Blot
Module (Invitrogen) in the XCell SureLock Mini-Cell (Invitrogen).
The inner XCell II Blot Module chamber was filled with chilled
1.times. Tris-Glycine Transfer Buffer (12 mM Tris Base, 96 mM
glycine, 20% methanol)) and the outer chamber was filled with
chilled distilled water.
[0613] Transfer was achieved by applying 25V for 1.5 hours and the
nitrocellulose membrane was subsequently blocked overnight in 50 ml
Blocking Buffer (2% (w/v) Difco Skim milk (BD) in PBS) at 4.degree.
C.
[0614] The primary antibody, control antibodies 6E10 and ACA were
added to the membrane at 0.5 .mu.g/ml and the recombinant
A.beta.(21-37) antibodies at 10-20 .mu.g/ml in 10 ml, 1% (w/v)
Bovine Serum Albumin (Sigma) in PBS, 0.05% Tween-20 (Sigma)) and
were incubated with shaking for 2-3 hours. Membranes were washed 3
times for 10 minutes with 50 ml Wash Buffer (1.times.PBS, 0.05%
(v/v) Tween-20 (Sigma)). The secondary antibody, sheep anti-human
IgG-HRP and sheep anti-mouse IgG-HRP (Chemicon) was added at 0.5
.mu.g/ml in 10 ml buffer (as above) and incubated with the membrane
with shaking for 30 minutes.
[0615] The membranes were washed 3 times for 10 minutes with 50 ml
Wash Buffer and subsequently developed with ECL Plus (Perkin-Elmer)
on Amersham Hyperfilm ECL (GE Lifesciences) (see FIG. 47).
[0616] Where the signal was below detection threshold using the
above protocol, membranes were washed 3.times. with Wash buffer and
then additionally probed with biotinylated antihuman IgG1 (Sigma
Clone 8c/6-39, 1:2000) for 30-60 minute at RT. Membranes were then
washed as above. Streptavidin peroxidase (1:4000, Chemicon) was
then added for 30 mins and membranes washed again. This additional
step amplified the signal and resulted in detection using ECL
substrates as indicated above.
[0617] An equivalent gel as probed in the western blot analysis was
not transferred to nitrocellulose. One half was stained with
coomassie blue per manufacturers instructions (Novex, Invitrogen)
and the other half was subjected to deep purple high protein
sensitivity staining as per manufacturers instructions (GE,
Sweden)
Example 13
Plaque Deposition (Taconic Mice)
Antibody Treatment of Transgenic APP Mice
[0618] APP transgenic mice (Tg2576) at 10 month of age received
once a week an i.p. injection of 200 .mu.l containing 200 .mu.g (8
g/kg) antibodies for 8 weeks. One animal received an affinity
purified A.beta. autoantibodies from IVIgG, two animals received a
murinized monoclonal antibody against A.beta.(CSL Clone 7), and two
mice received a negative chimeric monoclonal control antibody
(CSL360). At the end of the experimental period, animals were
killed by decapitation. The brain was dissected and the hemispheres
separated along the midline. One hemisphere was fixed in 4%
buffered formaldehyde for 24 h followed by dehydration and paraffin
embedding. The other hemisphere was immediately snap-frozen in
liquid nitrogen and kept at -80.degree. C.
Plaque Evaluation
Materials:
[0619] 3 .mu.m thick slices of murine brain tissue mounted on
microscope slides from Mentzel Glas and immunostained with
6F3D-Antibody (see protocol: Immunohistochemistry).
[0620] Transmitting light microscope: Eclipse 80i, containing Plan
Apochromat objectives 2.times.-40.times. magnification; Nikon
Instruments Europe, Nikon GmbH, Duesseldorf, Germany
[0621] Imaging Software: NIS-Elements BR software version 2.3,
Nikon; Nikon Instruments Europe, Nikon GmbH, Duesseldorf,
Germany
[0622] Digital sight: 2 Megapixel digital camera, Nikon (Nikon
Instruments Europe, Nikon GmbH, Duesseldorf, Germany).
[0623] Excel Software (Microsoft Office 2003, Microsoft Corp.
Redmont, USA.)
Method:
[0624] Digital RGB-Pictures were taken using the above-mentioned
camera in a 40.times. magnification. A macro was created by
recording every step defining the threshold of intensity, minimum
and maximum diameter, excluding e.g. vessels ("objects with holes
in the middle") to ensure equal parameters for every analysis. The
area of interest was defined by applying a measurement frame.
Within this area, the aforementioned criteria were used to identify
"objects" (cluster of pixels) fitting into the scheme provided by
the macro. A binary picture was created by the software for
background subtraction. Five independent fields per location were
analyzed (Cortex and Hippocampal formation were evaluated
separately). Data of the five analyses were summarized by the
software and a small statistical analysis was provided, like number
of plaques per measured area, the quotient of plaque area and
measured area as percentage. Detailed data of every object were
transmitted into an excel file for further analysis.
TABLE-US-00005 TABLE 3 Number of Fields 1 Number of Objects 7
Objects per Field 7 Measured Area 33152.5 [.mu.m * .mu.m] Objects
per Area 0.000211146/[.mu.m * .mu.m] Area Fraction 0.152147 Feature
Mean St. Dev Minimum Maximum Area 720.58 1438.5 0.072697 4222.5
EqDiameter 19.112 23.499 0.30424 73.323 Perimeter 106.58 169.48
0.94227 514.37 Width 7.5773 5.9398 0.20097 17.626 Circularity
0.70335 0.24485 0.20055 1 MeasuredArea 33152 0 33152 33152
[0625] Of special importance is the "Area fraction" as it equals
the percentage of plaque area (the marked area) from the measured
area (total area).
Measured Features:
Area
[0626] Area is a principal size criterion. In a non-calibrated
system, it expresses the number of pixels; in a calibrated one, it
expresses the real area (given in .mu.m.sup.2).
Area Fraction
[0627] Area Fraction is the ratio of the segmented image area and
the Measured Area (defined as: square unit over selected
fields).
Area Fraction=Area/Measured Area
Circularity
[0628] Circularity equals "1" only for circles; all other shapes
are characterized by circularity values smaller than "1". It is a
derived shape measure, calculated from the area and perimeter. This
feature is useful for examining shape characteristics.
Circularity=4*.pi.*Area/Perimeter.sup.2
EqDiameter
[0629] The equivalent diameter (EqDiameter) is a size feature
derived from the area. It determines the diameter of a circle with
the same area as the measured object:
EqDiameter= (4*Area/.pi.)
Object Per Area
[0630] Number of objects per square unit over selected fields
(measured area),
Perimeter
[0631] Perimeter is the total boundary measure. It includes both
the outer and inner boundary (if there are holes within an object).
The perimeter is calculated from four projections in the directions
0, 45, 90 and 135 degrees using Crofton's formula.
Perimeter=.pi.*(Pr0+Pr45+Pr90+Pr135)/4
Width
[0632] Width is a derived feature appropriate for elongated or thin
structures. It is based on the rod model and is calculated
according to:
Width=Area/Length
Example 14
Evidence that the Antibodies of the Invention do not Show Vessel
Staining
Methods:
[0633] 3 .mu.m paraffin slices were cut from post-mortem brain
material of a patient suffering from Alzheimer's disease and
cerebral amyloid angiopathy (CAA) using the HM 355 S rotary
microtome from Microm (MICROM International GmbH, Walldorf,
Germany) and mounted on SuperFrost Plus microscope slides from
Menzel-Glaeser (Menzel-Glaeser GmbH & Co. KG Braunschweig,
Germany). All protocol steps were performed at room temperature if
not stated otherwise.
[0634] De-paraffining of the microscope slides was performed
according to the following protocol: xylene (4 changes), 96%
ethanol (3 changes), 70% ethanol (3 changes) followed by 2 changes
of de-ionised water. Each step was performed for 3 minutes.
[0635] As pre-treatment for the antigen retrieval, slides were
incubated in 70% (v/v) formic acid in PBS for 20 minutes, replaced
by de-ionised water and two changes of PBS as washing steps. The
endogenous peroxidase was blocked for 30 minutes using 1% (v/v)
H2O.sub.2 in Methanol. To prevent unspecific staining, slides were
incubated for 30 minutes with diluted goat serum (according to the
Vectastain.RTM. Elite ABC Kit instructions) or for one hour with
diluted mouse IgG (according to the Vectastain.RTM. M.O.M.-Kit
instructions), respectively. The blocking solutions were removed,
no washing step was used.
[0636] After the blocking step, primary antibodies were applied to
the slides in a dilution of 1:100 in Vectastain.RTM. Elite ABC Kit
diluent or 1:50 (6F3D, according to the manufacturer's
instructions) in Vectastain.RTM. M.O.M.-Kit diluent. Negative
controls were carried along consisting of one slide without any
antibody or detection system, two slides without primary antibody,
but with the Vectastain.RTM. Elite ABC Kit or the Vectastain.RTM.
M.O.M.-Kit secondary system, respectively, and two slides with
clone 53/60 recombinant human anti .beta.-amyloid immunoglobulin or
the 6F3D antibody, respectively, and without any detection
system.
[0637] The slides were incubated with the primary antibodies
overnight (18 hrs) at 4.degree. C. in a humid chamber. The day
after, the slides were washed twice for 2 minutes with PBS.
Afterwards, the slides were incubated for 30 minutes with
biotinylated anti-mouse antibody (6F3D antibody) or biotinylated
anti-human antibody (diluted according to the Vectastain.RTM. Elite
ABC Kit or the Vectastain.RTM. M.O.M.-Kit instructions). The slides
were washed twice for 2 minutes with PBS. Subsequently, the slides
were incubated for 30 minutes with the Vectastain.RTM. Elite ABC
reagent according to the instructions of the Vectastain.RTM. Elite
ABC Kit and the Vectastain.RTM. M.O.M.-Kit. This step was followed
by two washing steps with PBS for 2 minutes. Afterwards, the DAB
reagent was applied for 5 minutes (according to the manufacturer's
instructions) as chromogen. This reaction was stopped by a washing
step in de-ionised water for 5 minutes. A 10 second dip in Mayer's
acid haemalaun-solution followed by blueing for 5 minutes in
running tap-water served as a counterstain. Dehydration was
performed by putting the slides into a sequence of 70% (v/v)
ethanol (3 times), 96% ethanol (3 times), isopropanol (once),
xylene (4 times) for 30 seconds each. The slides were air-dried and
mounted with RotiHistokit.RTM. and coverslips from Menzel-Glas.
Images were taken using the Nikon Eclipse 80i microscope with a
Nikon digitalsight 2 Megapixel camera and the Nikon NIS-Elements BR
version 2.3 software.
[0638] The results shown in FIGS. 48a to 48f demonstrate that
neither the affinity purified autoantibodies (purified as in
Example 4) nor mab CSL Clone 7 (see Example 5) show staining of the
vessel walls, whereas the anti-A.beta. antibody ACA (see Example 5)
shows a staining of the vessel walls, comparable to that of the
antibody 6F3D which was used as a positive control. Such results
suggest that the antibodies of the invention will not trigger
adverse events which are believed to be caused by A.beta.
antibodies binding to A.beta. deposited brain vessels (Pfeifer M,
et al. Cerebral hemorrhage after passive anti-Abeta immunotherapy.
Science 298:1379. Herzig M C, et al (2004): Abeta is targeted to
the vasculature in a mouse model of hereditary cerebral hemorrhage
with amyloidosis. Nat Neurosci 7:954-960).
Sequence CWU 1
1
203140PRTHomo sapiens 1Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu
Val His His Gln Lys1 5 10 15Leu Val Phe Phe Ala Glu Asp Val Gly Ser
Asn Lys Gly Ala Ile Ile 20 25 30Gly Leu Met Val Gly Gly Val Val 35
40217PRTHomo sapiens 2Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile
Ile Gly Leu Met Val1 5 10 15Gly37PRTHomo sapiens 3Phe Arg His Asp
Ser Gly Tyr1 5429PRTHomo sapiens 4Val His His Gln Lys Leu Val Phe
Phe Ala Glu Asp Val Gly Ser Asn1 5 10 15Lys Gly Ala Ile Ile Gly Leu
Met Val Gly Gly Val Val 20 25520PRTHomo sapiens 5Asp Ala Glu Phe
Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys1 5 10 15Leu Val Phe
Phe 2065PRTHomo sapiensMOD_RES(1)..(1)Ser, Gly or Asn 6Xaa Tyr Xaa
Met Xaa1 5717PRTHomo sapiensMOD_RES(1)..(1)Ser, Arg or Glu 7Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa Lys1 5 10
15Xaa813PRTHomo sapiensMOD_RES(1)..(1)Asp or Gly 8Xaa Xaa Xaa Xaa
Trp Xaa Xaa Xaa Xaa Xaa Xaa Asp Xaa1 5 10911PRTHomo
sapiensMOD_RES(2)..(2)Glu or Ala 9Arg Xaa Ser Gln Xaa Xaa Xaa Xaa
Tyr Leu Ala1 5 10107PRTHomo sapiensMOD_RES(1)..(1)Gly, Trp, Ala or
Lys 10Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5119PRTHomo
sapiensMOD_RES(3)..(3)Ala or Tyr 11Gln Gln Xaa Xaa Ser Xaa Xaa Xaa
Thr1 5129PRTHomo sapiensMOD_RES(3)..(3)Tyr or Ala 12Gln Gln Xaa Xaa
Ser Xaa Xaa Xaa Thr1 5135PRTHomo sapiens 13Ser Tyr Trp Met Ser1
5145PRTHomo sapiens 14Gly Tyr Trp Met Ser1 5155PRTHomo sapiens
15Asn Tyr Asp Met His1 5165PRTHomo sapiens 16Ser Tyr Trp Met His1
5175PRTHomo sapiens 17Asn Tyr Trp Met Ser1 5185PRTHomo sapiens
18Ser Tyr Asp Met Ser1 5195PRTHomo sapiens 19Ser Tyr Asp Met Ser1
5205PRTHomo sapiens 20Ser Tyr Trp Met Ser1 52117PRTHomo sapiens
21Ser Val Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val Lys1
5 10 15Gly2216PRTHomo sapiens 22Arg Ile Gly Thr Ala Gly Asp Arg Tyr
Tyr Ala Gly Ser Val Lys Gly1 5 10 152316PRTHomo sapiens 23Arg Ile
Gly Thr Ala Gly Arg Thr Asn Tyr Asn Pro Ser Leu Lys Gly1 5 10
152417PRTHomo sapiens 24Ser Val Lys Gln Phe Phe Ser Gly Lys Tyr Tyr
Ala Gly Ser Val Lys1 5 10 15Gly2517PRTHomo sapiens 25Ser Val Lys
Gln Phe Phe Ser Gly Ser Ala Ala Thr Gly Ser Val Lys1 5 10
15Gly2617PRTHomo sapiens 26Ser Val Lys Gln Phe Phe Ser Gly Pro Leu
Ala Thr Gly Ser Val Lys1 5 10 15Gly2717PRTHomo sapiens 27Ser Val
Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val Lys1 5 10
15Gly2812PRTHomo sapiens 28Asp Ala Ser Ser Trp Tyr Arg Asp Trp Phe
Asp Pro1 5 102913PRTHomo sapiens 29Gly Ala Gly Arg Trp Ala Pro Leu
Gly Ala Phe Asp Ile1 5 103012PRTHomo sapiens 30Asp Gly Ser Ser Trp
Tyr Arg Asp Trp Phe Asp Pro1 5 103112PRTHomo sapiens 31Asp Gly Ser
Ser Trp Tyr Arg Asp Trp Phe Asp Pro1 5 103212PRTHomo sapiens 32Asp
Ala Gly Arg Trp Ala Asp Leu Ala Phe Asp Ile1 5 103311PRTHomo
sapiens 33Arg Glu Ser Gln Gly Ile Arg Asn Tyr Leu Ala1 5
103411PRTHomo sapiens 34Arg Ala Ser Gln Ser Val Asn Ser Tyr Leu
Ala1 5 103511PRTHomo sapiens 35Arg Glu Ser Gln Gly Ile Arg Asn Tyr
Leu Ala1 5 103611PRTHomo sapiens 36Arg Ala Ser Gln Ser Val Ser Ser
Tyr Leu Ala1 5 103712PRTHomo sapiens 37Thr Leu Ser Ser Glu His Ser
Thr Tyr Thr Ile Glu1 5 10387PRTHomo sapiens 38Gly Ala Ser Thr Arg
Ala Thr1 5397PRTHomo sapiens 39Ala Ala Ser Ile Arg Ala Thr1
5407PRTHomo sapiens 40Gly Ala Ala Ser Arg Ala Thr1 5417PRTHomo
sapiens 41Lys Ala Ser Ser Leu Gln Ser1 5427PRTHomo sapiens 42Ala
Ala Ser Ser Arg Ala Thr1 5437PRTHomo sapiens 43Val Lys Ser Asp Gly
Ser His1 5449PRTHomo sapiens 44Gln Gln Tyr Gly Ser Ser Gln Gly Thr1
5459PRTHomo sapiens 45Gln Gln Ala Asn Ser Phe Pro Leu Thr1
54610PRTHomo sapiens 46Gly Glu Ser His Thr Ile Asp Gly Gln Cys1 5
1047108PRTHomo sapiens 47Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Val Asn Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Gly Ala Ser Thr Arg Ala
Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75 80Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Gln Gly 85 90 95Thr Phe Gly
Pro Gly Thr Lys Val Asp Ile Lys Arg 100 10548108PRTHomo sapiens
48Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1
5 10 15Glu Arg Val Thr Ile Thr Cys Arg Glu Ser Gln Gly Ile Arg Asn
Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile 35 40 45Tyr Ala Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Arg Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Tyr Gly Ser Ser Gln Gly 85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp
Ile Lys Arg 100 10549111PRTHomo sapiens 49Leu Pro Val Leu Thr Gln
Pro Pro Ser Ala Ser Ala Leu Leu Gly Ala1 5 10 15Ser Ile Lys Leu Thr
Cys Thr Leu Ser Ser Glu His Ser Thr Tyr Thr 20 25 30Ile Glu Trp Tyr
Gln Gln Arg Pro Gly Arg Ser Pro Gln Tyr Ile Met 35 40 45Lys Val Lys
Ser Asp Gly Ser His Ser Lys Gly Asp Gly Ile Pro Asp 50 55 60Arg Phe
Met Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Thr Phe Ser65 70 75
80Asn Leu Gln Ser Asp Asp Glu Ala Glu Tyr His Cys Gly Glu Ser His
85 90 95Thr Ile Asp Gly Gln Cys Trp Val Phe Gly Gly Gly Thr Lys Leu
100 105 11050108PRTHomo sapiens 50Asp Ile Gln Met Thr Gln Ser Pro
Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Ala Leu Ser Cys
Arg Ala Ser Gln Ser Val Asn Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Gly Ala Ala Ser
Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75 80Glu Asp
Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Gln Gly 85 90 95Thr
Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg 100 10551108PRTHomo
sapiens 51Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser
Pro Gly1 5 10 15Glu Arg Val Thr Ile Thr Cys Arg Glu Ser Gln Gly Ile
Arg Asn Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Arg Leu Leu Ile 35 40 45Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro
Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Arg Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Tyr Gly Ser Ser Gln Gly 85 90 95Thr Phe Gly Pro Gly Thr Lys
Val Asp Ile Lys Arg 100 10552108PRTHomo sapiens 52Glu Ile Val Met
Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Val
Thr Ile Thr Cys Arg Glu Ser Gln Gly Ile Arg Asn Tyr 20 25 30Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr
Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro65
70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Gln
Gly 85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg 100
10553108PRTHomo sapiens 53Asp Val Val Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Glu
Ser Gln Gly Ile Arg Asn Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Lys Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Leu Leu Gly Gly Lys
Ala Ala Leu Thr Leu Ser Gly Val Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Leu 85 90 95Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys Arg 100 10554108PRTHomo sapiens
54Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1
5 10 15Glu Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asn Ser
Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile 35 40 45Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Arg Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Tyr Gly Ser Ser Gln Gly 85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp
Ile Lys Arg 100 10555108PRTHomo sapiens 55Asp Ile Gln Met Thr Gln
Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Ala Leu
Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Ala Ala
Ser Ile Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75
80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Gln Gly
85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg 100
10556120PRTHomo sapiens 56Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Arg Ser Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Val Lys Gln Asp Gly
Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Thr Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Ala Ser Ser Trp Tyr Arg Asp Trp Phe Asp Pro Trp Gly 100 105 110Gln
Gly Thr Leu Val Thr Val Ser 115 12057120PRTHomo sapiens 57Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Gly Tyr 20 25
30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Ser Val Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ser Ser Trp Tyr Arg Asp Trp
Phe Asp Pro Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser 115
12058120PRTHomo sapiens 58Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Arg Ser Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Val Lys Gln Phe Phe
Ser Gly Pro Leu Ala Thr Gly Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Ala Ser Ser Trp Tyr Arg Asp Trp Phe Asp Pro Trp Gly 100 105 110Gln
Gly Thr Leu Val Thr Val Ser 115 12059120PRTHomo sapiens 59Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Gly Tyr 20 25
30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Ser Val Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser
Val 50 55 60Lys Gly Arg Pro Thr Ile Ser Arg Asp Asn Ala Lys Asn Gln
Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ser Ser Trp Tyr Arg Asp Trp
Phe Asp Pro Trp Gly 100 105 110Gln Gly Thr Leu Val Ser Val Ser 115
12060120PRTHomo sapiens 60Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Arg Asn Tyr 20 25 30Asp Met His Trp Val Arg Gln Gly
Ile Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Arg Ile Gly Thr Ala Gly
Arg Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Glu Asn Ala Lys Asp Ser Leu Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Arg Val Gly Asp Ala Ala Val Tyr Tyr Cys Ala 85 90 95Arg Gly Ala
Gly Arg Trp Ala Pro Leu Gly Ala Phe Asp Ile Trp Gly 100 105 110Gln
Gly Thr Leu Val Ile Val Ser 115 12061120PRTHomo sapiens 61Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn Tyr 20 25
30Asp Met His Trp Val Arg Gln Gly Ile Gly Lys Gly Leu Val Trp Val
35 40 45Ser Arg Ile Gly Thr Ala Gly Asp Arg Tyr Tyr Ala Gly Ser Val
Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asp Ser Leu
Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Val Gly Asp Ala Ala Val
Tyr Tyr Cys Ala 85 90 95Arg Gly Ala Gly Arg Trp Ala Pro Leu Gly Ala
Phe Asp Ile Trp Gly 100 105 110Gln Gly Thr Leu Val Ile Val Ser 115
12062120PRTHomo sapiens 62Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Val Lys Gln Asp Gly
Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile
Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Gly Ser Ser Trp Tyr Arg Asp Trp Phe Asp Pro Trp Gly 100 105 110Gln
Gly Thr Leu Val Thr Val Ser 115 12063120PRTHomo sapiens 63Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr 20 25
30Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Ser Val Lys Gln Phe Phe Ser Gly Ser Ala Ala Thr Gly Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ser Ser Trp Tyr Arg Asp Trp
Phe Asp Pro Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser 115
12064120PRTHomo sapiens 64Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Arg Ser Tyr 20 25 30Asp Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Val Lys Gln Asp Gly
Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Pro Thr Ile
Ser Arg Asp Asn Ala Lys Asn Gln Leu Ser65 70 75 80Leu Lys Leu Ser
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Gly Ser Ser Trp Tyr Arg Asp Trp Phe Asp Pro Trp Gly 100 105 110Gln
Gly Thr Leu Val Thr Val Ser 115 12065120PRTHomo sapiens 65Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Gly Tyr 20 25
30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Ser Val Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser
Val 50 55 60Lys Gly Arg Leu Thr Leu Ser Val Asp Thr Ser Lys Asn Gln
Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ser Ser Trp Tyr Arg Asp Trp
Phe Asp Pro Trp Gly 100 105 110Gln Gly Thr Leu Val Ser Val Ser 115
12066120PRTHomo sapiens 66Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Arg Gly Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Val Lys Gln Asp Gly
Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Val Thr Ile
Ser Val Glu Thr Ser Lys Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Gly Ser Ser Trp Tyr Arg Asp Trp Phe Asp Pro Trp Gly 100 105 110Gln
Gly Thr Leu Val Ser Val Ser 115 12067120PRTHomo sapiens 67Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr 20 25
30Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Ser Val Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser
Val 50 55 60Lys Gly Arg Val Thr Ile Ser Leu Asp Thr Ser Lys Asn Gln
Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ser Ser Trp Tyr Arg Asp Trp
Phe Asp Pro Trp Gly 100 105 110Gln Gly Thr Leu Val Ser Val Ser 115
12068120PRTHomo sapiens 68Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Ile Thr Phe Arg Ser Tyr 20 25 30Asp Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Val Lys Gln Asp Gly
Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Val
Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Ala Gly Arg Trp Ala Asp Leu Ala Phe Asp Ile Trp Gly 100 105 110Gln
Gly Thr Leu Val Thr Val Ser 115 12069120PRTHomo sapiens 69Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr 20 25
30Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Ser Val Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser
Val 50 55 60Lys Gly Arg Val Thr Ile Thr Ala Asp Arg Ala Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ser Ser Trp Tyr Arg Asp Trp
Phe Asp Pro Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser 115
12070120PRTHomo sapiens 70Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Arg Asn Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Val Lys Gln Phe Phe
Ser Gly Pro Leu Ala Thr Gly Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Thr Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Gly Ser Ser Trp Tyr Arg Asp Trp Phe Asp Pro Trp Gly 100 105 110Gln
Gly Thr Leu Val Thr Val Ser 115 12071120PRTHomo sapiens 71Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Gly Tyr 20 25
30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Ser Val Lys Gln Phe Phe Ser Gly Lys Tyr Tyr Ala Gly Ser
Val 50 55 60Lys Gly Arg Val Thr Ile Ser Val Glu Thr Ser Lys Asn Gln
Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Asp Ala Ser Ser Trp Tyr Arg Asp Trp
Phe Asp Pro Trp Gly 100 105 110Gln Gly Thr Leu Val Ser Val Ser 115
12072106PRTHomo sapiens 72Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln1 5 10 15Leu Lys Ser Gly Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr 20 25 30Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser 35 40 45Gly Asn Ser Gln Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60Tyr Ser Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys65 70 75 80His Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 100 10573106PRTHomo sapiens 73Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln1 5 10 15Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 20 25
30Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr 50 55 60Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys65 70 75 80His Lys Leu Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro 85 90 95Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100
10574109PRTHomo sapiens 74Thr Val Leu Gly Gln Pro Lys Ala Ala Pro
Ser Val Thr Leu Phe Pro1 5 10 15Pro Ser Ser Glu Glu Leu Gln Ala Asn
Lys Ala Thr Leu Val Cys Leu 20 25 30Ile Ser Asp Phe Tyr Pro Gly Ala
Val Thr Val Ala Trp Lys Ala Asp 35 40 45Ser Ser Pro Val Lys Ala Gly
Val Glu Thr Thr Thr Pro Ser Lys Gln 50 55 60Ser Asn Asn Lys Tyr Ala
Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu65 70 75 80Gln Trp Lys Ser
His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly 85 90 95Ser Thr Val
Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 10575331PRTHomo sapiens
75Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser1
5 10 15Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp 20 25 30Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr 35 40 45Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr 50 55 60Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Gln65 70 75 80Thr Tyr Ile Cys Asn Val Asn His Lys Pro
Ser Asn Thr Lys Val Asp 85 90 95Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr His Thr Cys Pro Pro 100 105 110Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro 115 120 125Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 130 135 140Cys Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn145 150 155
160Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
165 170 175Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu
Thr Val 180 185 190Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser 195 200 205Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys 210 215 220Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Asp225 230 235 240Glu Leu Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 245 250 255Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 260 265 270Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 275 280
285Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
290 295 300Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr305 310 315 320Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 33076331PRTHomo sapiens 76Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Cys Ser1 5 10 15Arg Ser Thr Ser Glu Ser Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp 20 25 30Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr 35 40 45Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 50 55 60Ser Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln65 70 75 80Thr Tyr Thr
Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp 85 90 95Lys Lys
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 100 105
110Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
115 120 125Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr 130 135 140Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn145 150 155 160Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg 165 170 175Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val 180 185 190Val His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 195 200 205Asn Lys Gly
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 210 215 220Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu225 230
235 240Glu Met Thr Lys Asn Gln Val Thr Leu Thr Cys Leu Val Lys Gly
Phe 245 250 255Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu 260 265 270Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp
Ser Asp Gly Ser Phe 275 280 285Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly 290 295 300Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr305 310 315 320Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 325 33077331PRTHomo sapiens 77Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser1 5 10 15Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 20 25
30Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
35 40 45Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr 50 55 60Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln65 70 75 80Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp 85 90 95Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro 100 105 110Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro 115 120 125Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr 130 135 140Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn145 150 155 160Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 165 170
175Glu Glu Gln Phe Ala Ser Thr Phe Arg Val Val Ser Val Leu Thr Val
180 185 190Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser 195 200 205Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys 210 215 220Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp225 230 235 240Glu Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe 245 250 255Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 260 265 270Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 275 280 285Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 290 295
300Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr305
310 315 320Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
33078214PRTHomo sapiens 78Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Val Asn Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Gly Ala Ser Thr Arg Ala
Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75 80Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Gln Gly 85 90 95Thr Phe Gly
Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala 100 105 110Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu
Cys 21079214PRTHomo sapiens 79Glu Ile Val Leu Thr Gln Ser Pro Ala
Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Val Asn Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Gly Ala Ser Thr Arg
Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75 80Glu Asp Phe
Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Gln Gly 85 90 95Thr Phe
Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala 100 105
110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Leu Tyr 180 185 190Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg
Gly Glu Cys 21080214PRTHomo sapiens 80Glu Ile Val Leu Thr Gln Ser
Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Val Thr Ile Thr
Cys Arg Glu Ser Gln Gly Ile Arg Asn Tyr 20 25 30Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Ala Ala Ser
Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75 80Glu
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Gln Gly 85 90
95Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala
100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe
Asn Arg Gly Glu Cys 21081214PRTHomo sapiens 81Glu Ile Val Leu Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Val Thr
Ile Thr Cys Arg Glu Ser Gln Gly Ile Arg Asn Tyr 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Ala
Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75
80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Gln Gly
85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Leu Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 21082220PRTHomo sapiens 82Leu Pro Val
Leu Thr Gln Pro Pro Ser Ala Ser Ala Leu Leu Gly Ala1 5 10 15Ser Ile
Lys Leu Thr Cys Thr Leu Ser Ser Glu His Ser Thr Tyr Thr 20 25 30Ile
Glu Trp Tyr Gln Gln Arg Pro Gly Arg Ser Pro Gln Tyr Ile Met 35 40
45Lys Val Lys Ser Asp Gly Ser His Ser Lys Gly Asp Gly Ile Pro Asp
50 55 60Arg Phe Met Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Thr Phe
Ser65 70 75 80Asn Leu Gln Ser Asp Asp Glu Ala Glu Tyr His Cys Gly
Glu Ser His 85 90 95Thr Ile Asp Gly Gln Cys Trp Val Phe Gly Gly Gly
Thr Lys Leu Thr 100 105 110Val Leu Gly Gln Pro Lys Ala Ala Pro Ser
Val Thr Leu Phe Pro Pro 115 120 125Ser Ser Glu Glu Leu Gln Ala Asn
Lys Ala Thr Leu Val Cys Leu Ile 130 135 140Ser Asp Phe Tyr Pro Gly
Ala Val Thr Val Ala Trp Lys Ala Asp Ser145 150 155 160Ser Pro Val
Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser 165 170 175Asn
Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln 180 185
190Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser
195 200 205Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 210 215
22083214PRTHomo sapiens 83Asp Ile Gln Met Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Ala Leu Ser Cys Arg Ala
Ser Gln Ser Val Asn Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Gly Ala Ala Ser Arg Ala
Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75 80Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Gln Gly 85 90 95Thr Phe Gly
Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala 100 105 110Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu
Cys 21084214PRTHomo sapiens 84Asp Ile Gln Met Thr Gln Ser Pro Ala
Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Ala Leu Ser Cys Arg
Ala Ser Gln Ser Val Asn Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Gly Ala Ala Ser Arg
Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75 80Glu Asp Phe
Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Gln Gly 85 90 95Thr Phe
Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala 100 105
110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Leu Tyr 180 185 190Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg
Gly Glu Cys 21085214PRTHomo sapiens 85Glu Ile Val Leu Thr Gln Ser
Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Val Thr Ile Thr
Cys Arg Glu Ser Gln Gly Ile Arg Asn Tyr 20 25 30Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Gly Ala Ser
Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75 80Glu
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Gln Gly 85 90
95Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala
100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe
Asn Arg Gly Glu Cys 21086214PRTHomo sapiens 86Glu Ile Val Leu Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Val Thr
Ile Thr Cys Arg Glu Ser Gln Gly Ile Arg Asn Tyr 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Gly
Ala Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75
80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Gln Gly
85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Leu Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 21087214PRTHomo sapiens 87Glu Ile Val
Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg
Val Thr Ile Thr Cys Arg Glu Ser Gln Gly Ile Arg Asn Tyr 20 25 30Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser
Ser Gln Gly 85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg
Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185
190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205Phe Asn Arg Gly Glu Cys 21088214PRTHomo sapiens 88Glu
Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10
15Glu Arg Val Thr Ile Thr Cys Arg Glu Ser Gln Gly Ile Arg Asn Tyr
20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
Ile 35 40 45Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr
Gly Ser Ser Gln Gly 85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp Ile
Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Leu Tyr
180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 21089214PRTHomo sapiens
89Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Glu Ser Gln Gly Ile Arg Asn
Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Lys Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser
Gly Val Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ala
Asn Ser Phe Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 21090214PRTHomo sapiens
90Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Glu Ser Gln Gly Ile Arg Asn
Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Lys Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser
Gly Val Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ala Asn Ser Phe Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155
160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Leu Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 21091214PRTHomo
sapiens 91Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser
Pro Gly1 5 10 15Glu Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val
Asn Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Arg Leu Leu Ile 35 40 45Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro
Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Arg Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Tyr Gly Ser Ser Gln Gly 85 90 95Thr Phe Gly Pro Gly Thr Lys
Val Asp Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150
155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
21092214PRTHomo sapiens 92Asp Ile Gln Met Thr Gln Ser Pro Gly Thr
Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Ala Leu Ser Cys Arg Ala
Ser Gln Ser Val Ser Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ile Arg Ala
Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75 80Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Gln Gly 85 90 95Thr Phe Gly
Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala 100 105 110Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu
Cys 21093214PRTHomo sapiens 93Asp Ile Gln Met Thr Gln Ser Pro Gly
Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Ala Leu Ser Cys Arg
Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ile Arg
Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75 80Glu Asp Phe
Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Gln Gly 85 90 95Thr Phe
Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala 100 105
110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Leu Tyr 180 185 190Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg
Gly Glu Cys 21094451PRTHomo sapiens 94Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr 20 25 30Trp Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Val Lys
Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg
Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Leu Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Asp Ala Ser Ser Trp Tyr Arg Asp Trp Phe Asp Pro Trp Gly
100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215
220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe 290 295 300Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330
335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro
Gly Lys 45095451PRTHomo sapiens 95Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Arg Ser Tyr 20 25 30Trp Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Val Lys Gln
Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Phe
Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Asp Ala Ser Ser Trp Tyr Arg Asp Trp Phe Asp Pro Trp Gly 100 105
110Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser
Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His 195 200 205Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230
235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr
Val Val His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile 325 330 335Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345
350Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Thr
355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro385 390 395 400Met Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Lys
45096451PRTHomo sapiens 96Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Arg Ser Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Val Lys Gln Asp Gly
Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Thr Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Ala Ser Ser Trp Tyr Arg Asp Trp Phe Asp Pro Trp Gly 100 105 110Gln
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230 235
240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Ala Ser Thr Phe 290 295 300Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360
365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Lys
45097451PRTHomo sapiens 97Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Arg Gly Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Val Lys Gln Asp Gly
Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Thr Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Gly Ser Ser Trp Tyr Arg Asp Trp Phe Asp Pro Trp Gly 100 105 110Gln
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185
190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310
315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Thr 355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Met Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425
430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445Pro Gly Lys 45098451PRTHomo sapiens 98Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Gly Tyr 20 25 30Trp Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala
Ser Val Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Asp Gly Ser Ser Trp Tyr Arg Asp Trp Phe Asp Pro
Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Cys Ser Arg Ser
Thr Ser Glu Ser Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val
Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly305 310 315
320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile
325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Thr 355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Met Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440
445Pro Gly Lys 45099451PRTHomo sapiens 99Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Arg Gly Tyr 20 25 30Trp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Val
Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asp Gly Ser Ser Trp Tyr Arg Asp Trp Phe Asp Pro Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Phe Ala Ser Thr Phe 290 295 300Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315
320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile
325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440
445Pro Gly Lys 450100451PRTHomo sapiens 100Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr 20 25 30Trp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Val
Lys Gln Phe Phe Ser Gly Pro Leu Ala Thr Gly Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asp Ala Ser Ser Trp Tyr Arg Asp Trp Phe Asp Pro Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe 290 295 300Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315
320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440
445Pro Gly Lys 450101451PRTHomo sapiens 101Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr 20 25 30Trp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Val
Lys Gln Phe Phe Ser Gly Pro Leu Ala Thr Gly Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asp Ala Ser Ser Trp Tyr Arg Asp Trp Phe Asp Pro Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr
Ser Glu Ser Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val
Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly305 310 315
320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile
325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Thr 355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Met Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440
445Pro Gly Lys 450102451PRTHomo sapiens 102Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr 20 25 30Trp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Val
Lys Gln Phe Phe Ser Gly Pro Leu Ala Thr Gly Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asp Ala Ser Ser Trp Tyr Arg Asp Trp Phe Asp Pro Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Phe Ala Ser Thr Phe 290 295 300Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315
320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile
325 330 335Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390
395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 435 440 445Pro Gly Lys 450103451PRTHomo sapiens
103Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Gly
Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Ser Val Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val
Asp Ser Val 50 55 60Lys Gly Arg Pro Thr Ile Ser Arg Asp Asn Ala Lys
Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ser Ser Trp Tyr Arg
Asp Trp Phe Asp Pro Trp Gly 100 105 110Gln Gly Thr Leu Val Ser Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280
285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe
290 295 300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395
400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 435 440 445Pro Gly Lys 450104451PRTHomo sapiens
104Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Gly
Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Ser Val Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val
Asp Ser Val 50 55 60Lys Gly Arg Pro Thr Ile Ser Arg Asp Asn Ala Lys
Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ser Ser Trp Tyr Arg
Asp Trp Phe Asp Pro Trp Gly 100 105 110Gln Gly Thr Leu Val Ser Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala
Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala 130 135 140Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys
Asn Val Asp His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280
285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu
Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Thr 355 360 365Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395
400Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 435 440 445Pro Gly Lys 450105451PRTHomo sapiens
105Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Gly
Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Ser Val Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val
Asp Ser Val 50 55 60Lys Gly Arg Pro Thr Ile Ser Arg Asp Asn Ala Lys
Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ser Ser Trp Tyr Arg
Asp Trp Phe Asp Pro Trp Gly 100 105 110Gln Gly Thr Leu Val Ser Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280
285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Ala Ser Thr Phe
290 295 300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395
400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 435 440 445Pro Gly Lys 450106451PRTHomo sapiens
106Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn
Tyr 20 25 30Asp Met His Trp Val Arg Gln Gly Ile Gly Lys Gly Leu Glu
Trp Val 35 40 45Gly Arg Ile Gly Thr Ala Gly Arg Thr Asn Tyr Asn Pro
Ser Leu Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asp
Ser Leu Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Val Gly Asp Ala
Ala Val Tyr Tyr Cys Ala 85 90 95Arg Gly Ala Gly Arg Trp Ala Pro Leu
Gly Ala Phe Asp Ile Trp Gly 100 105 110Gln Gly Thr Leu Val Ile Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280
285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe
290 295 300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395
400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 435 440 445Pro Gly Lys 450107451PRTHomo sapiens
107Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn
Tyr 20 25 30Asp Met His Trp Val Arg Gln Gly Ile Gly Lys Gly Leu Glu
Trp Val 35 40 45Gly Arg Ile Gly Thr Ala Gly Arg Thr Asn Tyr Asn Pro
Ser Leu Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asp
Ser Leu Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Val Gly Asp Ala
Ala Val Tyr Tyr Cys Ala 85 90 95Arg Gly Ala Gly Arg Trp Ala Pro Leu
Gly Ala Phe Asp Ile Trp Gly 100 105 110Gln Gly Thr Leu Val Ile Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala
Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala 130 135 140Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys
Asn Val Asp His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280
285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu
Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Thr 355 360 365Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395
400Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 435 440 445Pro Gly Lys 450108451PRTHomo sapiens
108Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn
Tyr 20 25 30Asp Met His Trp Val Arg Gln Gly Ile Gly Lys Gly Leu Glu
Trp Val 35 40 45Gly Arg Ile Gly Thr Ala Gly Arg Thr Asn Tyr Asn Pro
Ser Leu Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asp
Ser Leu Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Val Gly Asp Ala
Ala Val Tyr Tyr Cys Ala 85 90
95Arg Gly Ala Gly Arg Trp Ala Pro Leu Gly Ala Phe Asp Ile Trp Gly
100 105 110Gln Gly Thr Leu Val Ile Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215
220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Phe Ala Ser Thr Phe 290 295 300Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile 325 330
335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro
Gly Lys 450109451PRTHomo sapiens 109Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Arg Asn Tyr 20 25 30Asp Met His Trp Val Arg
Gln Gly Ile Gly Lys Gly Leu Val Trp Val 35 40 45Ser Arg Ile Gly Thr
Ala Gly Asp Arg Tyr Tyr Ala Gly Ser Val Lys 50 55 60Gly Arg Phe Thr
Ile Ser Arg Glu Asn Ala Lys Asp Ser Leu Tyr Leu65 70 75 80Gln Met
Asn Ser Leu Arg Val Gly Asp Ala Ala Val Tyr Tyr Cys Ala 85 90 95Arg
Gly Ala Gly Arg Trp Ala Pro Leu Gly Ala Phe Asp Ile Trp Gly 100 105
110Gln Gly Thr Leu Val Ile Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230
235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Phe Asn Ser Thr Phe 290 295 300Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345
350Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Lys
450110451PRTHomo sapiens 110Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Arg Asn Tyr 20 25 30Asp Met His Trp Val Arg Gln Gly
Ile Gly Lys Gly Leu Val Trp Val 35 40 45Ser Arg Ile Gly Thr Ala Gly
Asp Arg Tyr Tyr Ala Gly Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Glu Asn Ala Lys Asp Ser Leu Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Arg Val Gly Asp Ala Ala Val Tyr Tyr Cys Ala 85 90 95Arg Gly Ala
Gly Arg Trp Ala Pro Leu Gly Ala Phe Asp Ile Trp Gly 100 105 110Gln
Gly Thr Leu Val Ile Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Thr Cys Asn Val Asp His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230 235
240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val
Val His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Thr 355 360
365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro385 390 395 400Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Lys
450111451PRTHomo sapiens 111Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Arg Asn Tyr 20 25 30Asp Met His Trp Val Arg Gln Gly
Ile Gly Lys Gly Leu Val Trp Val 35 40 45Ser Arg Ile Gly Thr Ala Gly
Asp Arg Tyr Tyr Ala Gly Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Glu Asn Ala Lys Asp Ser Leu Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Arg Val Gly Asp Ala Ala Val Tyr Tyr Cys Ala 85 90 95Arg Gly Ala
Gly Arg Trp Ala Pro Leu Gly Ala Phe Asp Ile Trp Gly 100 105 110Gln
Gly Thr Leu Val Ile Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230 235
240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Ala Ser Thr Phe 290 295 300Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360
365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Lys
450112451PRTHomo sapiens 112Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Val Lys Gln Asp Gly
Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Gly Ser Ser Trp Tyr Arg Asp Trp Phe Asp Pro Trp Gly 100 105 110Gln
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230 235
240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr Phe 290 295 300Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360
365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Lys
450113451PRTHomo sapiens 113Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Val Lys Gln Asp Gly
Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Gly Ser Ser Trp Tyr Arg Asp Trp Phe Asp Pro Trp Gly 100 105 110Gln
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Thr Cys Asn Val Asp His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230 235
240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 290 295
300Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn
Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Thr 355 360 365Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Met
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410
415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 435 440 445Pro Gly Lys 450114451PRTHomo sapiens 114Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Ser Val Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ser Ser Trp Tyr Arg Asp Trp
Phe Asp Pro Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170
175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Ala Ser Thr Phe 290 295
300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410
415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 435 440 445Pro Gly Lys 450115451PRTHomo sapiens 115Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr 20 25
30Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Ser Val Lys Gln Phe Phe Ser Gly Ser Ala Ala Thr Gly Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ser Ser Trp Tyr Arg Asp Trp
Phe Asp Pro Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170
175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe 290 295
300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410
415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 435 440 445Pro Gly Lys 450116451PRTHomo sapiens 116Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr 20 25
30Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Ser Val Lys Gln Phe Phe Ser Gly Ser Ala Ala Thr Gly Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ser Ser Trp Tyr Arg Asp Trp
Phe Asp Pro Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Cys
Ser Arg Ser Thr Ser Glu Ser Thr Ala 130 135 140Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170
175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys Asn Val
Asp His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295
300Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn
Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Thr 355 360 365Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Met
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410
415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 435 440 445Pro Gly Lys 450117451PRTHomo sapiens 117Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr 20 25
30Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Ser Val Lys Gln Phe Phe Ser Gly Ser Ala Ala Thr Gly Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ser Ser Trp Tyr Arg Asp Trp
Phe Asp Pro Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170
175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Ala Ser Thr Phe 290 295
300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410
415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 435 440 445Pro Gly Lys 450118451PRTHomo sapiens 118Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr 20 25
30Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Ser Val Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser
Val 50 55 60Lys Gly Arg Pro Thr Ile Ser Arg Asp Asn Ala Lys Asn Gln
Leu Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ser Ser Trp Tyr Arg Asp Trp
Phe Asp Pro Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170
175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe 290 295
300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410
415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 435 440 445Pro Gly Lys 450119451PRTHomo sapiens 119Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr 20 25
30Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Ser Val Lys Gln Asp Gly Ser Glu Lys Tyr Tyr
Val Asp Ser Val 50 55 60Lys Gly Arg Pro Thr Ile Ser Arg Asp Asn Ala
Lys Asn Gln Leu Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ser Ser Trp Tyr
Arg Asp Trp Phe Asp Pro Trp Gly 100 105 110Gln Gly Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala 130 135 140Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys
Asn Val Asp His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280
285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu
Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Thr 355 360 365Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395
400Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 435 440 445Pro Gly Lys 450120451PRTHomo sapiens
120Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser
Tyr 20 25 30Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Ser Val Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val
Asp Ser Val 50 55 60Lys Gly Arg Pro Thr Ile Ser Arg Asp Asn Ala Lys
Asn Gln Leu Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ser Ser Trp Tyr Arg
Asp Trp Phe Asp Pro Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280
285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Ala Ser Thr Phe
290 295 300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395
400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 435 440 445Pro Gly Lys 450121451PRTHomo sapiens
121Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Gly
Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Ser Val Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val
Asp Ser Val 50 55 60Lys Gly Arg Leu Thr Leu Ser Val Asp Thr Ser Lys
Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ser Ser Trp Tyr Arg
Asp Trp Phe Asp Pro Trp Gly 100 105 110Gln Gly Thr Leu Val Ser Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280
285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe
290 295 300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395
400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 435 440 445Pro Gly Lys 450122451PRTHomo sapiens
122Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Gly
Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Ser Val Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val
Asp Ser Val 50 55 60Lys Gly Arg Leu Thr Leu Ser Val Asp Thr Ser Lys
Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ser Ser Trp Tyr Arg
Asp Trp Phe Asp Pro Trp Gly 100 105 110Gln Gly Thr Leu Val Ser Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala
Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala 130 135 140Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys
Asn Val Asp His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280
285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu
Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Thr 355 360 365Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395
400Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 435 440 445Pro Gly Lys 450123451PRTHomo sapiens
123Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Gly
Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Ser Val Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val
Asp Ser Val 50 55 60Lys Gly Arg Leu Thr Leu Ser Val Asp Thr Ser Lys
Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ser Ser Trp Tyr Arg
Asp Trp Phe Asp Pro Trp Gly 100 105 110Gln Gly Thr Leu Val Ser Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280
285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Ala Ser Thr Phe
290 295 300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395
400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 435 440 445Pro Gly Lys 450124451PRTHomo sapiens
124Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Gly
Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Ser Val Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val
Asp Ser Val 50 55 60Lys Gly Arg Val Thr Ile Ser Val Glu Thr Ser Lys
Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ser Ser Trp Tyr Arg
Asp Trp Phe Asp Pro Trp Gly 100 105 110Gln Gly Thr Leu Val Ser Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg
Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe 290 295 300Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330
335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro
Gly Lys 450125451PRTHomo sapiens 125Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Arg Gly Tyr 20 25 30Trp Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Val Lys Gln
Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Val
Thr Ile Ser Val Glu Thr Ser Lys Asn Gln Phe Ser65 70 75 80Leu Lys
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Asp Gly Ser Ser Trp Tyr Arg Asp Trp Phe Asp Pro Trp Gly 100 105
110Gln Gly Thr Leu Val Ser Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser
Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His 195 200 205Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230
235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr
Val Val His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile 325 330 335Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345
350Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Thr
355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro385 390 395 400Met Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Lys
450126451PRTHomo sapiens 126Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Arg Gly Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Val Lys Gln Asp Gly
Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Val Thr Ile
Ser Val Glu Thr Ser Lys Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Gly Ser Ser Trp Tyr Arg Asp Trp Phe Asp Pro Trp Gly 100 105 110Gln
Gly Thr Leu Val Ser Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230 235
240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Ala Ser Thr Phe 290 295 300Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360
365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Lys
450127451PRTHomo sapiens 127Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Arg Ser Tyr 20 25 30Trp Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Val Lys Gln Asp Gly
Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Val Thr Ile
Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Gly Ser Ser Trp Tyr Arg Asp Trp Phe Asp Pro Trp Gly 100 105 110Gln
Gly Thr Leu Val Ser Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230 235
240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr Phe 290 295 300Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360
365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Lys
450128451PRTHomo sapiens 128Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Arg Ser Tyr 20 25 30Trp Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Val Lys Gln Asp Gly
Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Val Thr Ile
Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Gly Ser Ser Trp Tyr Arg Asp Trp Phe Asp Pro Trp Gly 100 105 110Gln
Gly Thr Leu Val Ser Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Thr Cys Asn Val Asp His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230 235
240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val
Val His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Thr 355 360
365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro385 390 395 400Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Lys
450129451PRTHomo sapiens 129Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Arg Ser Tyr 20 25 30Trp Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Val Lys Gln Asp Gly
Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Val Thr Ile
Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Gly Ser Ser Trp Tyr Arg Asp Trp Phe Asp Pro Trp Gly 100 105 110Gln
Gly Thr Leu Val Ser Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230 235
240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Ala Ser Thr Phe 290 295 300Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360
365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Lys
450130451PRTHomo sapiens 130Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Arg Ser Tyr
20 25 30Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Ser Val Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Ala Gly Arg Trp Ala Asp Leu
Ala Phe Asp Ile Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170
175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe 290 295
300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410
415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 435 440 445Pro Gly Lys 450131451PRTHomo sapiens 131Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Arg Ser Tyr 20 25
30Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Ser Val Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Asp Ala Gly Arg Trp Ala Asp Leu Ala
Phe Asp Ile Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Cys
Ser Arg Ser Thr Ser Glu Ser Thr Ala 130 135 140Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170
175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys Asn Val
Asp His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295
300Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn
Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Thr 355 360 365Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Met
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410
415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 435 440 445Pro Gly Lys 450132451PRTHomo sapiens 132Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr 20 25
30Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Ser Val Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser
Val 50 55 60Lys Gly Arg Val Thr Ile Thr Ala Asp Arg Ala Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ser Ser Trp Tyr Arg Asp Trp
Phe Asp Pro Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170
175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe 290 295
300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410
415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 435 440 445Pro Gly Lys 450133451PRTHomo sapiens 133Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr 20 25
30Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Ser Val Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser
Val 50 55 60Lys Gly Arg Val Thr Ile Thr Ala Asp Arg Ala Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ser Ser Trp Tyr Arg Asp Trp
Phe Asp Pro Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Cys
Ser Arg Ser Thr Ser Glu Ser Thr Ala 130 135 140Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170
175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys Asn Val
Asp His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295
300Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn
Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Thr 355 360 365Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Met
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410
415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 435 440 445Pro Gly Lys 450134451PRTHomo sapiens 134Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn Tyr 20 25
30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Ser Val Lys Gln Phe Phe Ser Gly Pro Leu Ala Thr Gly Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ser Ser Trp Tyr Arg Asp Trp
Phe Asp Pro Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170
175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295
300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Met
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410
415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 435 440 445Pro Gly Lys 450135451PRTHomo sapiens 135Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn Tyr 20 25
30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Ser Val Lys Gln Phe Phe Ser Gly Pro Leu Ala Thr Gly Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ser Ser Trp Tyr Arg Asp Trp
Phe Asp Pro Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170
175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys 210 215
220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Phe Ala Ser Thr Phe 290 295 300Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile 325 330
335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro
Gly Lys 450136451PRTHomo sapiens 136Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Arg Gly Tyr 20 25 30Trp Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Val Lys Gln
Phe Phe Ser Gly Lys Tyr Tyr Ala Gly Ser Val 50 55 60Lys Gly Arg Val
Thr Ile Ser Val Glu Thr Ser Lys Asn Gln Phe Ser65 70 75 80Leu Lys
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Asp Ala Ser Ser Trp Tyr Arg Asp Trp Phe Asp Pro Trp Gly 100 105
110Gln Gly Thr Leu Val Ser Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230
235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Phe Asn Ser Thr Phe 290 295 300Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345
350Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Lys
450137451PRTHomo sapiens 137Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Arg Gly Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Val Lys Gln Phe Phe
Ser Gly Lys Tyr Tyr Ala Gly Ser Val 50 55 60Lys Gly Arg Val Thr Ile
Ser Val Glu Thr Ser Lys Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Ala Ser Ser Trp Tyr Arg Asp Trp Phe Asp Pro Trp Gly 100 105 110Gln
Gly Thr Leu Val Ser Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230 235
240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Ala Ser Thr Phe 290 295 300Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360
365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Lys
45013818PRTHomo sapiens 138Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg13918PRTHomo sapiens 139Glu
Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10
15Glu Arg14018PRTHomo sapiens 140Asp Ile Gln Met Thr Gln Ser Pro
Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg14118PRTHomo sapiens
141Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg14218PRTHomo sapiens 142Asp Ile Val Leu Thr Gln Ser
Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg14318PRTHomo
sapiens 143Asp Ile Gln Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser
Pro Gly1 5 10 15Glu Arg14418PRTHomo sapiensMOD_RES(1)..(1)Glu or
Asp 144Xaa Xaa Xaa Xaa Thr Gln Ser Pro Xaa Xaa Leu Ser Xaa Ser Xaa
Gly1 5 10 15Xaa Arg145108PRTHomo sapiens 145Asp Val Val Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Glu Ser Gln Gly Ile Arg Asn Tyr 20 25 30Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala
Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Leu
Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Leu
85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100
105146108PRTHomo sapiens 146Asp Val Val Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Glu
Ser Gln Gly Ile Arg Asn Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Lys Val Ser Asn Arg Phe
Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Leu Leu Gly Gly Lys
Ala Ala Leu Thr Leu Ser Gly Val Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Leu 85 90 95Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105147108PRTHomo sapiens
147Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Glu Ser Gln Gly Ile Arg Asn
Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg
Phe Ser Gly 50 55 60Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser
Gly Val Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ala Asn Ser Phe Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys Arg 100 105148120PRTHomo sapiens 148Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn Tyr 20 25 30Asp Met His Trp
Val Arg Gln Gly Ile Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile
Asn Arg Ser Gly Ala Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg
Phe Thr Ile Ser Arg Glu Asn Ala Lys Asp Ser Leu Tyr Leu65 70 75
80Gln Met Asn Ser Leu Arg Val Gly Asp Ala Ala Val Tyr Tyr Cys Ala
85 90 95Arg Gly Ala Gly Arg Trp Ala Pro Leu Gly Ala Phe Asp Ile Trp
Gly 100 105 110Gln Gly Thr Leu Val Ile Val Ser 115 12014916PRTHomo
sapiens 149Glu Ile Asn Arg Ser Gly Ala Thr Asn Tyr Asn Pro Ser Leu
Lys Ser1 5 10 151507PRTHomo sapiens 150Ala Ala Ser Thr Leu Gln Ser1
51517PRTHomo sapiens 151Lys Val Ser Asn Arg Phe Ser1 51527PRTHomo
sapiens 152Trp Ala Ser Thr Arg Glu Ser1 51535PRTHomo
sapiensMOD_RES(1)..(1)Asn or Ser 153Xaa Tyr Xaa Met Xaa1
51545PRTHomo sapiens 154Asn Tyr Asp Met His1 51555PRTHomo
sapiensMOD_RES(3)..(3)Trp or Asp 155Ser Tyr Xaa Met Ser1
515617PRTHomo sapiensMOD_RES(1)..(1)Arg, Ser or Glu 156Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Ser Xaa Lys1 5 10
15Xaa15716PRTHomo sapiensMOD_RES(1)..(1)Arg or Glu 157Xaa Ile Xaa
Xaa Xaa Gly Xaa Xaa Xaa Tyr Xaa Xaa Ser Xaa Lys Xaa1 5 10
1515817PRTHomo sapiens 158Ser Val Lys Gln Asp Gly Ser Glu Lys Tyr
Tyr Val Asp Ser Val Lys1 5 10 15Gly15913PRTHomo
sapiensMOD_RES(1)..(1)Gly or Asp 159Xaa Xaa Xaa Xaa Trp Xaa Xaa Xaa
Xaa Xaa Xaa Asp Xaa1 5 1016013PRTHomo sapiens 160Gly Ala Gly Arg
Trp Ala Pro Leu Gly Ala Phe Asp Ile1 5 1016112PRTHomo
sapiensMOD_RES(2)..(2)Gly or Ala 161Asp Xaa Xaa Xaa Trp Xaa Xaa Xaa
Xaa Phe Asp Xaa1 5 1016211PRTHomo sapiens 162Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg1 5 1016330PRTHomo sapiens 163Cys Val His
His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser1 5 10 15Asn Lys
Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val 20 25 301649PRTHomo
sapiens 164Glu Glu Gln Tyr Asn Ser Thr Tyr Arg1 516520PRTHomo
sapiens 165Met Glu Ser Gln Thr Gln Val Leu Met Ser Leu Leu Phe Trp
Val Ser1 5 10 15Gly Thr Cys Gly 2016619PRTHomo sapiens 166Met Gly
Trp Ser Trp Ile Phe Leu Phe Leu Val Ser Gly Thr Gly Gly1 5 10 15Val
Leu Ser16711PRTHomo sapiens 167Phe Ala Glu Asp Val Gly Ser Asn Lys
Gly Ala1 5 1016818PRTHomo sapiens 168Phe Ala Glu Asp Val Gly Ser
Asn Lys Gly Ala Ile Ile Gly Leu Met1 5 10 15Val Gly16958PRTHomo
sapiens 169Glu Val Lys Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr
Glu Val1 5 10 15His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly
Ser Asn Lys 20 25 30Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val
Ile Ala Thr Val 35 40 45Ile Val Ile Thr Leu Val Met Leu Lys Lys 50
5517011PRTHomo sapiens 170Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Arg1 5 1017114PRTHomo sapiens 171Ser Thr Ser Glu Ser Thr Ala Ala
Leu Gly Cys Leu Val Lys1 5 1017222PRTHomo sapiens 172Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln1 5 10 15Pro Glu
Asn Asn Tyr Lys 2017314PRTHomo sapiens 173Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys1 5 1017416PRTHomo sapiens 174Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys1 5 10
1517514PRTHomo sapiens 175Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys1 5 1017619PRTHomo sapiens 176Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro1 5 10 15Glu Val
Lys17716PRTHomo sapiens 177Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys1 5 10 1517828PRTHomo sapiens 178Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly1 5 10 15Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 20 2517921PRTHomo sapiens
179Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp1
5 10 15Pro Glu Val Lys Phe 2018016PRTHomo sapiens 180Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr1 5 10
1518118PRTHomo sapiens 181Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly1 5 10 15Lys Glu18213PRTHomo sapiens 182Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp1 5 1018318PRTHomo
sapiens 183Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr1 5 10 15Lys Asn18424PRTHomo sapiens 184Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly1 5 10 15Gln Pro Glu Asn
Asn Tyr Lys Thr 2018525PRTHomo sapiens 185Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala1 5 10 15Leu His Asn His Tyr
Thr Gln Lys Ser 20 2518613PRTHomo sapiens 186Lys Asn Thr Leu Tyr
Leu
Gln Met Asn Ser Leu Arg Ala1 5 1018714PRTHomo sapiens 187Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser1 5 1018832PRTHomo
sapiens 188Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu1 5 10 15Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp 20 25 3018928PRTHomo sapiens 189Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly1 5 10 15Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp 20 2519021PRTHomo sapiens 190Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp1 5 10 15Pro
Glu Val Lys Phe 2019115PRTHomo sapiens 191Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val1 5 10 1519218PRTHomo sapiens
192Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly1
5 10 15Lys Glu19313PRTHomo sapiens 193Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu1 5 1019418PRTHomo sapiens 194Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr1 5 10 15Lys
Asn19512PRTHomo sapiens 195Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly1 5 1019624PRTHomo sapiens 196Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly1 5 10 15Gln Pro Glu Asn Asn Tyr
Lys Thr 2019725PRTHomo sapiens 197Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala1 5 10 15Leu His Asn His Tyr Thr Gln
Lys Ser 20 2519829PRTHomo sapiens 198Arg Lys Cys Cys Val Glu Cys
Pro Pro Cys Pro Ala Pro Pro Val Ala1 5 10 15Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp 20 2519928PRTHomo sapiens 199Lys Cys
Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly1 5 10 15Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 20 2520018PRTHomo
sapiens 200Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu
Asn Gly1 5 10 15Lys Glu20113PRTHomo sapiens 201Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu1 5 1020224PRTHomo sapiens 202Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly1 5 10
15Gln Pro Glu Asn Asn Tyr Lys Thr 2020325PRTHomo sapiens 203Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala1 5 10 15Leu
His Asn His Tyr Thr Gln Lys Ser 20 25
* * * * *