U.S. patent application number 15/785667 was filed with the patent office on 2018-04-05 for amyloid-beta binding proteins.
The applicant listed for this patent is AbbVie Deutschland GmbH & Co. KG, AbbVie Inc.. Invention is credited to Stefan Barghorn, Lorenzo Benatuil, Ulrich Ebert, Simone Giaisi, Heinz Hillen, Andreas Striebinger.
Application Number | 20180094049 15/785667 |
Document ID | / |
Family ID | 44583713 |
Filed Date | 2018-04-05 |
United States Patent
Application |
20180094049 |
Kind Code |
A1 |
Barghorn; Stefan ; et
al. |
April 5, 2018 |
AMYLOID-BETA BINDING PROTEINS
Abstract
The present invention relates to amyloid-beta (A.beta.) binding
proteins. Antibodies of the invention have high affinity to
A.beta.(20-42) globulomer or any A.beta. form that comprises the
globulomer epitope. Method of making and method of using the
antibodies of the invention are also provided.
Inventors: |
Barghorn; Stefan; (Mannheim,
DE) ; Hillen; Heinz; (Hassloch, DE) ;
Striebinger; Andreas; (Speyer, DE) ; Giaisi;
Simone; (Edlingen-Neckarhaus, DE) ; Ebert;
Ulrich; (Mannheim, DE) ; Benatuil; Lorenzo;
(Northborough, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AbbVie Deutschland GmbH & Co. KG
AbbVie Inc. |
Wiesbaden
North Chicago |
IL |
DE
US |
|
|
Family ID: |
44583713 |
Appl. No.: |
15/785667 |
Filed: |
October 17, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14618632 |
Feb 10, 2015 |
9822171 |
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15785667 |
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13085891 |
Apr 13, 2011 |
8987419 |
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14618632 |
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61446624 |
Feb 25, 2011 |
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61373825 |
Aug 14, 2010 |
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61324386 |
Apr 15, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 7/06 20180101; C07K
2317/56 20130101; A61P 25/00 20180101; A61P 25/14 20180101; A61K
39/3955 20130101; A61K 49/00 20130101; C07K 2317/24 20130101; C07K
2317/626 20130101; A61P 35/00 20180101; C07K 14/4711 20130101; A61P
7/00 20180101; A61P 43/00 20180101; A61P 25/16 20180101; A61P 13/08
20180101; A61P 3/10 20180101; C07K 2317/569 20130101; C07K 2317/21
20130101; C07K 2317/565 20130101; C07K 2317/31 20130101; C07K
2317/55 20130101; C07K 2317/92 20130101; C07K 16/18 20130101; C07K
2317/30 20130101; A61P 7/10 20180101; A61K 47/6835 20170801; A61P
25/28 20180101; C07K 2317/622 20130101; A61P 25/20 20180101; C07K
2317/624 20130101; C07K 2317/54 20130101; A61P 7/02 20180101 |
International
Class: |
C07K 16/18 20060101
C07K016/18; A61K 47/68 20060101 A61K047/68; C07K 14/47 20060101
C07K014/47; A61K 49/00 20060101 A61K049/00; A61K 39/395 20060101
A61K039/395 |
Claims
1-17. (canceled)
18. A method for treating a subject for a disease or a disorder
selected from the group consisting of Systemic AL amyloidosis,
Nodular AL amyloidosis, Systemic AA amyloidosis, Prostatic
amyloidosis, Hemodialysis amyloidosis, Familial visceral
amyloidosis, Senile systemic amyloidosis, Familial cardiac
amyloidosis and Down syndrome by administering to the subject an
effective amount of an anti-A.beta.(20-42) globulomer antibody
comprising: a first amino acid sequence which is at least 90%
identical to SEQ ID NO:2 or SEQ ID NO:3; and a second amino acid
sequence which is at least 90% identical to SEQ ID NO: 1, wherein
the first amino acid sequence comprises three complementary
determining regions consisting of amino acids 31-35, 50-65 and
98-101, respectively, of SEQ ID NO:2 or SEQ ID NO:3; and the second
amino acid sequence comprises three complementary determining
regions consisting of amino acids 24-39, 55-61 and 94-102,
respectively, of SEQ ID NO: 1.
19. The method of claim 18, wherein the first amino acid sequence
is at least 90% identical to an amino acid sequence selected from
the group consisting of SEQ ID NO:4, SEQ ID NO:5, 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.
20. The method of claim 18, wherein the second amino acid sequence
is at least 90% identical to an amino acid sequence selected from
the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,
SEQ ID NO:15, and SEQ ID NO:16.
21. The method of claim 18, wherein the first amino acid sequence
is at least 90% identical to an amino acid sequence selected from
the group consisting of SEQ ID NO:4, SEQ ID NO:5, 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;
and the second amino acid sequence is at least 90% identical to an
amino acid sequence selected from the group consisting of SEQ ID
NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID
NO:16.
22. The method of claim 18, wherein the first amino acid sequence
is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5,
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; and the second amino acid sequence is selected
from the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:14, SEQ ID NO:15, and SEQ ID NO:16.
23. The method of claim 18, wherein the binding protein is selected
from the group consisting of: a monoclonal antibody, a
multispecific antibody, a dual specific antibody, a DVD, and a
bispecific antibody.
24. The method of claim 18, wherein the binding protein further
comprises an immunoglobulin light chain constant region having an
amino acid sequence selected from the group consisting of SEQ ID
NO:27 and SEQ ID NO:28.
25. The method of claim 18, wherein the binding protein further
comprises an agent selected from the group consisting of: an
immunoadhesion molecule, an imaging agent, and a therapeutic
agent.
26. The method of claim 18, wherein the binding protein possesses a
human glycosylation pattern.
27. A method for treating a subject for a disease or a disorder
selected from the group consisting of Systemic AL amyloidosis,
Nodular AL amyloidosis, Systemic AA amyloidosis, Prostatic
amyloidosis, Hemodialysis amyloidosis, Familial visceral
amyloidosis, Senile systemic amyloidosis, Familial cardiac
amyloidosis and Down syndrome by administering to the subject an
effective amount of a pharmaceutical composition, the
pharmaceutical composition comprising: an anti-A.beta.(20-42)
globulomer antibody comprising: a first amino acid sequence which
is at least 90% identical to SEQ ID NO:2 or SEQ ID NO:3; and a
second amino acid sequence which is at least 90% identical to SEQ
ID NO: 1, wherein the first amino acid sequence comprises three
complementary determining regions consisting of amino acids 31-35,
50-65 and 98-101, respectively, of SEQ ID NO:2 or SEQ ID NO:3; and
the second amino acid sequence comprises three complementary
determining regions consisting of amino acids 24-39, 55-61 and
94-102, respectively, of SEQ ID NO: 1; and a pharmaceutically
acceptable carrier.
28. The method of claim 27, wherein the pharmaceutical composition
further comprises at least one additional therapeutic agent.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of U.S. patent application Ser. No.
14/618,632, filed Feb. 10, 2015, which is a divisional of U.S.
patent application Ser. No. 13/085,891, filed on Apr. 13, 2011, now
U.S. Pat. No. 8,987,419, which claims priority to U.S. Patent
Application No. 61/446,624, filed on Feb. 25, 2011, U.S. Patent
Application No. 61/373,825, filed on Aug. 14, 2010, and U.S. Patent
Application No. 61/324,386, filed on Apr. 15, 2010, the entire
contents of all of which are fully incorporated herein by
reference.
SEQUENCE LISTING
[0002] This instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Oct. 16, 2017, is named 2017_10_16_10419USC2_SEQ-LIST.txt, and
is 45,498 bytes in size.
FIELD OF THE INVENTION
[0003] The present invention relates to amyloid-beta (A.beta.)
binding proteins, nucleic acids encoding said proteins, methods of
producing said proteins, compositions comprising said proteins and
the use of said proteins in diagnosis, treatment and prevention of
conditions such as amyloidoses, e.g., Alzheimer's disease.
BACKGROUND OF THE INVENTION
[0004] Alzheimer's disease (AD) is a neurodegenerative disorder
characterized by a progressive loss of cognitive abilities and by
characteristic neuropathological features comprising deposits of
amyloid beta (A.beta.) peptide, neurofibrillary tangles and
neuronal loss in several regions of the brain (Hardy and Selkoe,
Science 297: 353, 2002; Mattson, Nature 431: 7004, 2004. Cerebral
amyloid deposits and cognitive impairments very similar to those
observed in Alzheimer's disease are also hallmarks of Down syndrome
(trisomy 21), which occurs at a frequency of about 1 in 800
births.
[0005] The A.beta. peptide arises from the amyloid precursor
protein (APP) by proteolytic processing. This processing is
effected by the cooperative activity of several proteases named
.alpha.-, .beta.- and .gamma.-secretase and leads to a number of
specific fragments of differing length. The amyloid desposits
consist mostly of peptides with a length of 40 or 42 amino acids
(A.beta.40, A.beta.42). This also includes, in addition to human
variants, isoforms of the amyloid .beta.(1-42) protein present in
organisms other than humans, in particular, other mammals,
especially rats. This protein, which tends to polymerize in an
aqueous environment, may be present in very different molecular
forms. A simple correlation of the deposition of insoluble protein
with the occurrence or progression of dementia disorders such as,
for example, Alzheimer's disease, has proved to be unconvincing
(Terry et al., Ann. Neurol. 30: 572-580, 1991; Dickson et al.,
Neurobiol. Aging 16: 285-298, 1995). In contrast, the loss of
synapses and cognitive perception seems to correlate better with
soluble forms of A.beta.(1-42) (Lue et al., Am. J. Pathol. 155:
853-862, 1999; McLean et al., Ann. Neurol. 46: 860-866, 1999).
[0006] None of the polyclonal and monoclonal antibodies which have
been raised in the past against monomeric A.beta.(1-42) have proven
to produce the desired therapeutic effect without also causing
serious side effects in animals and/or humans. For example, passive
immunization results from preclinical studies in very old APP23
mice which received a N-terminal directed anti-A.beta.(1-42)
antibody once weekly for 5 months indicate therapeutically relevant
side effects. In particular, these mice showed an increase in
number and severity of microhemorrhages compared to saline-treated
mice (Pfeifer et al., Science 298: 1379, 2002). A similar increase
in hemorrhages was also described for very old (>24 months)
Tg2576 and PDAPP mice (Wilcock et al., J Neuroscience 23: 3745-51,
2003; Racke et al., J Neuroscience 25: 629-636, 2005). In both
strains, injection of anti-A.beta.(1-42) resulted in a significant
increase of microhemorrhages.
[0007] WO 2004/067561 refers to globular oligomers ("globulomers")
of A.beta.(1-42) peptide and a process for preparing them. WO
2006/094724 relates to non-diffusible globular A.beta.(X-38 . . .
43) oligomers wherein X is selected from the group consisting of
numbers 1 . . . 24. WO 2004/067561 and WO 2006/094724 further
describes that limited proteolysis of the globulomers yields
truncated versions of said globulomers such as A.beta.(20-42) or
A.beta.(12-42) globulomers. WO 2007/064917 describes the cloning,
expression and isolation of recombinant forms of amyloid .beta.
peptide (referred to hereafter as N-Met A.beta.(1-42)) and
globulomeric forms thereof. The data suggest the existence of an
amyloid fibril independent pathway of A.beta. folding and assembly
into A.beta. oligomers which display one or more unique epitopes
(hereinafter referred to as the globulomer epitopes). Since
globulomer epitopes were detected in the brain of AD patients and
APP transgenic mice and the globulomer specifically binds to
neurons and blocks LTP, the globulomer represents a pathologically
relevant A.beta. conformer. It has been found that soluble A.beta.
globulomer exert its detrimental effects essentially by interaction
with the P/Q type presynaptic calcium channel, and that inhibitors
of this interaction are therefore useful for treatment of
amyloidoses such as Alzheimer's disease (WO 2008/104385).
[0008] Antibodies which selectively bind to such globulomeric forms
of A.beta. have been described in WO 2007/064972, WO 2007/062852,
WO 2008067464, WO 2008/150946 and WO 2008/150949. For instance,
several monoclonal antibodies known from WO 2007/062852 and WO
2008/150949 specifically recognize A.beta.(20-42) globulomer.
[0009] There exists a tremendous, unmet therapeutic need for the
development of biologics such as A.beta. binding proteins that
prevent or slow down the progression of the disease without
inducing negative and potentially lethal effects on the human body.
Such a need is particularly evident in view of the increasing
longevity of the general population and, with this increase, an
associated rise in the number of patients annually diagnosed with
Alzheimer's disease or related disorders. Further, such A.beta.
binding proteins will allow for proper diagnosis of Alzheimer's
disease in a patient experiencing symptoms thereof, a diagnosis
which can only be confirmed upon autopsy at the present time.
Additionally, the A.beta. binding proteins will allow for the
elucidation of the biological properties of the proteins and other
biological factors responsible for this debilitating disease.
SUMMARY OF THE INVENTION
[0010] The present invention provides a novel family of A.beta.
binding proteins (or simply "binding proteins"), CDR grafted
antibodies, humanized antibodies, and fragments thereof, capable of
binding to soluble A.beta. globulomers, for example, A.beta.(20-42)
globulomer as described herein. It is noted that the binding
proteins of the present invention may also be reactive with (i.e.
bind to) AP forms other than the AP globulomers described herein,
such A.beta. forms may be present in the brain of a patient having
an amyloidosis such as Alzheimer's disease. These A.beta. forms may
or may not be oligomeric or globulomeric. The A.beta. forms to
which the binding proteins of the present invention bind include
any A.beta. form that comprises the globulomer epitope with which
the murine/mouse monoclonal antibody m4D10 is reactive (hereinafter
referred to as "m4D10"). m4D10 and its properties are described in
WO 2007/062852, which is incorporated herein by reference. Such
A.beta. forms are hereinafter referred to as "targeted A.beta.
forms". Further, the present invention also provides a therapeutic
means with which to inhibit the activity of said targeted A.beta.
forms and provides compositions and methods for treating diseases
associated with said targeted A.beta. forms, particularly
amyloidosis such as Alzheimer's disease.
[0011] In one aspect, the invention provides a binding protein
comprising: a first amino acid sequence which is at least 90%
identical to
TABLE-US-00001 SEQ ID NO: 2:
EVQLVESGGGLX.sup.12QPGGSLRLSCAX.sup.24SGFTX.sup.29SSYGVHWVRQAPGK
GLEWX.sup.48X.sup.49VIWRGGRIDYNAAFMSRX.sup.67TISX.sup.71DNSKX.sup.76TX.sup-
.78YLQM NSLRAEDTAVYYCARNSDVWGQGTTVTVSS,
[0012] wherein X.sup.12 is I or V, X.sup.24 is A or V, X.sup.29 is
V or L, X.sup.48 is V or L, X.sup.49 is S or G, X.sup.67 is F or L,
X.sup.71 is R or K, X.sup.76 is N or S, and X.sup.78 is L or V;
or
TABLE-US-00002 SEQ ID NO: 3:
X.sup.1VQLQESGPGLVKPSETLSLTCTVSGX.sup.27SX.sup.29SSYGVHWX.sup.37RQPPG
KGLEWX.sup.48GVIWRGGRIDYNAAFMSRX.sup.67TISX.sup.71DTSKX.sup.76QX.sup.78SLK-
L SSVTAADTAVYYCARNSDVWGQGTTVTVSS,
[0013] wherein X.sup.1 is Q or E, X.sup.27 is G or F, X.sup.29 is I
or L, X.sup.37 is I or V, X.sup.48 is I or L, X.sup.67 is V or L,
X.sup.71 is V or K, X.sup.76 is N or S, and X.sup.78 is F or V;
[0014] and a second amino acid sequence which is at least 90%
identical to
TABLE-US-00003 SEQ ID NO: 1:
DVVMTQX.sup.7PLSLPVTX.sup.15GQPASISCKSSQSLLDIDGKTYLNWX.sup.41X.sup.42
QX.sup.44PGQSPX.sup.50RLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAE
DVGVYYCWQGTHFPYTFGQGTKLEIKR,
[0015] wherein X.sup.7 is S or T, X.sup.15 is L or P, X.sup.41 is F
or L, X.sup.42 is Q or L, X.sup.44 is R or K, and X.sup.50 is R or
Q.
[0016] In a further aspect of the invention, the binding protein
described above comprises a first amino acid sequence which is at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical
to an amino acid sequence selected from the group consisting of SEQ
ID NO:4, SEQ ID NO:5, 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. In still a further aspect of
the invention, the binding protein described above comprises a
first amino acid sequence selected from the group consisting of SEQ
ID NO:4, SEQ ID NO:5, 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.
[0017] In another aspect of the invention, the binding protein
described above comprises a second amino acid sequence which is at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical
to an amino acid sequence selected from the group consisting of SEQ
ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID
NO:16. In still another aspect of the invention, the binding
protein described above comprises a second amino acid sequence
selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13,
SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16.
[0018] In one aspect of the invention, the binding protein
described above comprises a first amino acid sequence which is at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical
to an amino acid sequence selected from the group consisting of SEQ
ID NO:4, SEQ ID NO:5, 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; and a second amino acid
sequence which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identical to an amino acid sequence selected from the
group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ
ID NO:15, and SEQ ID NO:16. In a further aspect of the invention,
the binding protein described above comprises a first amino acid
sequence selected from the group consisting of SEQ ID NO:4, SEQ ID
NO:5, 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; and a second amino acid sequence selected
from the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:14, SEQ ID NO:15, and SEQ ID NO:16.
[0019] In a particular aspect of the invention, the binding protein
described above comprises a first amino acid sequence which is at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical
to the amino acid sequence set forth in SEQ ID NO:6; and a second
amino acid sequence which is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% identical to an amino acid sequence set forth
in SEQ ID NO:14. In a further particular aspect of the invention,
the binding protein described above comprises a first amino acid
sequence set forth in SEQ ID NO:6; and a second amino acid sequence
set forth in SEQ ID NO:14.
[0020] In a particular aspect of the invention, the binding protein
described above comprises a first amino acid sequence which is at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical
to the amino acid sequence set forth in SEQ ID NO:10; and a second
amino acid sequence which is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% identical to an amino acid sequence set forth
in SEQ ID NO:14. In a further particular aspect of the invention,
the binding protein described above comprises a first amino acid
sequence set forth in SEQ ID NO:10; and a second amino acid
sequence set forth in SEQ ID NO:14.
[0021] In one aspect, the binding protein described herein is an
antibody. This antibody may be, for example, an immunoglobulin
molecule, a disulfide linked Fv, a monoclonal antibody (mab), a
single chain Fv (scFv), a chimeric antibody, a single domain
antibody, a CDR-grafted antibody, a diabody, a humanized antibody,
a multispecific antibody, a Fab, a dual specific antibody, a dual
variable domain (DVD) binding molecule, a Fab', a bispecific
antibody, a F(ab').sub.2, or a Fv.
[0022] When the binding protein described herein is an antibody, it
comprises at least one variable heavy chain that corresponds to the
first amino acid sequence as defined above, and at least one
variable light chain that corresponds to the second amino acid
sequence as defined above. For example, an antibody of the
invention comprises (i) at least one variable heavy chain
comprising an amino acid sequence which is at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino
acid sequence selected from the group consisting of SEQ ID NO:2,
SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, 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, and (ii)
at least one variable light chain comprising an amino acid sequence
which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%
or 100% identical to an amino acid sequence selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:14, SEQ ID NO:15, and SEQ ID NO:16. In a particular aspect of
the invention, the antibody of the invention comprises (i) at least
one variable heavy chain comprising an amino acid sequence which is
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
identical to an amino acid sequence set forth in SEQ ID NO:6 or SEQ
ID NO:10, and (ii) at least one variable light chain comprising an
amino acid sequence which is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or 100% identical to an amino acid sequence set
forth in SEQ ID NO:14.
[0023] The binding protein described herein may further (in
addition to the first and second amino acid sequence) comprise
another moiety which may be another amino acid sequence or other
chemical moiety. For instance, an antibody of the present invention
may comprise a heavy chain immunoglobulin constant domain. Said
heavy chain immunoglobulin constant domain may be selected from the
group consisting of a human IgM constant domain, a human IgG4
constant domain, a human IgG1 constant domain, a human IgE constant
domain, a human IgG2 constant domain, a human IgG3 constant domain,
and a human IgA constant domain. In another aspect, the binding
protein of the invention further comprises a heavy chain constant
region having an amino acid sequence selected from the group
consisting of SEQ ID NO:25 and SEQ ID NO:26, additionally a light
chain constant region having an amino acid sequence selected from
the group consisting of SEQ ID NO:27 and SEQ ID NO:28. In a
particular aspect of the invention, the binding protein described
herein comprises a variable heavy chain comprising an amino acid
sequence set forth in SEQ ID NO:6 or SEQ ID NO:10; a variable light
chain comprising an amino acid sequence set forth in SEQ ID NO:14;
a heavy chain constant region having an amino acid sequence set
forth in SEQ ID NO:25; and a light chain constant region having an
amino acid sequence set forth in SEQ ID NO:27. In a further
particular aspect of the invention, the binding protein described
herein comprises a first amino acid sequence set forth in SEQ ID
NO:46 or SEQ ID NO:47, and a second first amino acid sequence set
forth in SEQ ID NO:48.
[0024] The binding protein, e.g. the antibody, described herein may
further comprise a therapeutic agent, an imaging agent, residues
capable of facilitating formation of an immunoadhesion molecule
and/or another functional molecule (e.g. another peptide or
protein). The imaging agent can be a radiolabel including but not
limited to .sup.3H, .sup.14C, .sup.35S, .sup.90Y, .sup.99Tc,
.sup.111In, .sup.125I, .sup.131I, .sup.177Lu, .sup.166Ho, and
.sup.153Sm; an enzyme, a fluorescent label, a luminescent label, a
bioluminescent label, a magnetic label, or biotin.
[0025] The binding protein of the present invention can be
glycosylated. According to one aspect of the invention, the
glycosylation pattern is a human glycosylation pattern.
[0026] In one aspect of the invention, the above-described binding
protein binds to an A.beta. form that comprises the globulomer
epitope with which the murine monoclonal antibody m4D10 is reactive
(i.e. a targeted A.beta. form). In particular the above-described
binding proteins bind to amyloid-beta (20-42) globulomer as
described herein.
[0027] In one aspect of the invention, the binding protein
described herein is capable of modulating a biological function of
A.beta.(20-42) globulomer. In a further aspect of the invention,
the binding protein described herein is capable of neutralizing
A.beta.(20-42) globulomer activity.
[0028] The binding protein of the present invention may exist as a
crystal. In one aspect, the crystal is a carrier-free
pharmaceutical controlled release crystal. In another aspect, the
crystallized binding protein has a greater half life in vivo than
its soluble counterpart. In still another aspect, the crystallized
binding protein retains biological activity after
crystallization.
[0029] The present invention also provides an isolated nucleic acid
encoding any one of the binding proteins disclosed herein. A
further embodiment provides a vector comprising said nucleic acid.
Said vector may be selected from the group consisting of pcDNA, pTT
(Durocher et al., Nucleic Acids Research 30(2), 2002), pTT3 (pTT
with additional multiple cloning site), pEFBOS (Mizushima and
Nagata, Nucleic acids Research 18(17), 1990), pBV, pJV, and
pBJ.
[0030] In another aspect of the invention, a host cell is
transformed with the vector disclosed above. According to one
embodiment, the host cell is a prokaryotic cell including but not
limited to E. coli. In a related embodiment, the host cell is a
eukaryotic cell selected from the group comprising a protist cell,
animal cell, plant cell and fungal cell. The animal cell may be
selected from the group consisting of a mammalian cell, an avian
cell and an insect cell. According to one aspect of the invention,
said mammalian cell is selected from the group comprising CHO and
COS, said fungal cell is a yeast cell such as Saccharomyces
cerevisiae, and said insect cell is an insect Sf9 cell.
[0031] Further, the invention provides a method of producing a
binding protein as disclosed herein that comprises culturing any
one of the host cells disclosed herein in a culture medium under
conditions and for a time suitable to produce said binding protein.
Another embodiment provides a binding protein of the invention
produced according to the method disclosed herein. In another
embodiment, the invention provides a binding protein produced
according to the method disclosed above.
[0032] The invention also provides a pharmaceutical composition
comprising a binding protein, e.g. an antibody, as disclosed herein
and a pharmaceutically acceptable carrier.
[0033] One embodiment of the invention provides a composition for
the release of the binding protein described herein wherein the
composition comprises a formulation which in turn comprises a
crystallized binding protein, e.g. a crystallized antibody, as
disclosed above, and an ingredient; and at least one polymeric
carrier. In one aspect the polymeric carrier is a polymer selected
from one or more of the group consisting of: poly(acrylic acid),
poly(cyano-acrylates), poly(amino acids), poly(anhydrides),
poly(depsipeptides), poly(esters), poly(lactic acid),
poly(lactic-co-glycolic acid) or PLGA,
poly(.beta.-hydroxybutryate), poly(caprolactone), poly(dioxanone);
poly(ethylene glycol), poly((hydroxypropyl) methacrylamide),
poly((organo)phosphazene), poly(ortho esters), poly(vinyl alcohol),
poly(vinylpyrrolidone), maleic anhydride-alkyl vinyl ether
copolymers, pluronic polyols, albumin, alginate, cellulose and
cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid,
oligosaccharides, glycaminoglycans, sulfated polysaccharides,
blends and copolymers thereof. In another aspect the ingredient is
selected from the group consisting of: albumin, sucrose, trehalose,
lactitol, gelatin, hydroxypropyl-.beta.-cyclodextrin,
methoxypolyethylene glycol and polyethylene glycol.
[0034] The present invention also relates to a method of inhibiting
(i.e. reducing) the activity of A.beta.(20-42) globulomer (or any
other targeted A.beta. form) comprising contacting said targeted
A.beta. form with binding protein(s) of the invention such that the
activity of said targeted A.beta. form is inhibited (i.e. reduced).
In a particular embodiment, said activity is inhibited in vitro.
This method may comprise adding the binding protein of the
invention to a sample, e.g. a sample derived from a subject (e.g.,
whole blood, cerebrospinal fluid, serum, tissue, etc.) or a cell
culture which contains or is suspected to contain a targeted
A.beta. form, in order to inhibit (i.e. reduce) the activity of the
A.beta. form in the sample. Alternatively, the activity of said
targeted A.beta. form may be inhibited (i.e. reduced) in a subject
in vivo. Thus, the present invention further relates to the binding
protein described herein for use in inhibiting (i.e. reducing) the
activity of a targeted A.beta. form in a subject comprising
contacting said A.beta. form with binding protein(s) of the
invention such that the activity of the A.beta. form is inhibited
(i.e. reduced).
[0035] In a related aspect, the invention provides a method for
inhibiting (i.e. reducing) the activity of a targeted A.beta. form
in a subject suffering from a disease or disorder in which the
activity of said A.beta. form is detrimental. In one embodiment,
said method comprises administering to the subject at least one of
the binding proteins disclosed herein such that the activity of a
targeted A.beta. form in the subject is inhibited (i.e. reduced).
Thus, the invention provides the A.beta. binding proteins described
herein for use in inhibiting (i.e. reducing) a targeted A.beta.
form in a subject suffering from a disease or disorder as described
herein, wherein at least one of the binding proteins disclosed
herein is administered to the subject such that the activity of
said A.beta. form in the subject is inhibited (i.e. reduced).
[0036] In a related aspect, the invention provides a method for
treating (e.g., curing, suppressing, ameliorating, delaying or
preventing the onset of, or preventing recurrence or relapse of) or
preventing a disease or disorder selected from the group consisting
of Alpha1-antitrypsin-deficiency, C1-inhibitor deficiency
angioedema, Antithrombin deficiency thromboembolic disease, Kuru,
Creutzfeld-Jacob disease/scrapie, Bovine spongiform encephalopathy,
Gerstmann-Straussler-Scheinker disease, Fatal familial insomnia,
Huntington's disease, Spinocerebellar ataxia, Machado-Joseph
atrophy, Dentato-rubro-pallidoluysian atrophy, Frontotemporal
dementia, Sickle cell anemia, Unstable hemoglobin inclusion-body
hemolysis, Drug-induced inclusion body hemolysis, Parkinson's
disease, Systemic AL amyloidosis, Nodular AL amyloidosis, Systemic
AA amyloidosis, Prostatic amyloidosis, Hemodialysis amyloidosis,
Hereditary (Icelandic) cerebral angiopathy, Huntington's disease,
Familial visceral amyloidosis, Familial visceral polyneuropathy,
Familial visceral amyloidosis, Senile systemic amyloidosis,
Familial amyloid neurophathy, Familial cardiac amyloidosis,
Alzheimer's disease, Down syndrome, Medullary carcinoma thyroid and
Type 2 diabetes mellitus (T2DM). In a particular embodiment, said
disease or disorder is an amyloidosis such as Alzheimer's disease
or Down syndrome. In one embodiment, said method comprising the
step of administering any one of the A.beta. binding proteins
disclosed herein such that treatment is achieved. In another
embodiment, the invention provides a method of treating a subject
suffering from a disease or disorder disclosed herein comprising
the step of administering any one of the A.beta. binding proteins
disclosed herein, concurrent with or after the administration of
one or more additional therapeutic agent(s). Thus, the invention
provides the A.beta. binding proteins disclosed herein for use in
treating a subject suffering from a diseases or disorder disclosed
herein comprising the step of administering any one of the binding
proteins disclosed herein, concurrent with or after the
administration of one or more additional therapeutic agent(s). For
instance, the additional therapeutic agent is selected from the
group of therapeutic agents listed herein.
[0037] The binding proteins disclosed herein and the pharmaceutical
compositions comprising said binding proteins are administered to a
subject by at least one mode selected from parenteral,
subcutaneous, intramuscular, intravenous, intraarticular,
intrabronchial, intraabdominal, intracapsular, intracartilaginous,
intracavitary, intracelial, intracelebellar,
intracerebroventricular, intracolic, intracervical, intragastric,
intrahepatic, intramyocardial, intraosteal, intrapelvic,
intrapericardiac, intraperitoneal, intrapleural, intraprostatic,
intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,
intrasynovial, intrathoracic, intrauterine, intravesical, bolus,
vaginal, rectal, buccal, sublingual, intranasal, and
transdermal.
[0038] In another embodiment, the present invention provides a
method for detecting a targeted A.beta. form in a sample comprising
(i) contacting said sample with binding protein(s) of the invention
and (ii) detecting formation of a complex between said binding
protein(s) and elements of said sample, wherein formation or
increased formation of the complex in the sample relative to a
control sample indicates the presence of said A.beta. form in the
sample. The sample may be a biological sample obtained from a
subject which is suspected of having a disease or disorder as
disclosed herein (e.g., whole blood, cerebrospinal fluid, serum,
tissue, etc.) or a cell culture which contains or is suspected to
contain said A.beta. form. The control sample does not contain said
A.beta. form or is obtained from a patient not having a disease as
described above. The presence of a complex between said binding
protein(s) and elements of a sample obtained from a patient
suspected of having Alzheimer's disease indicates a diagnosis of
this disease in said patient.
[0039] In an alternative embodiment, the detection of the targeted
A.beta. form may be performed in vivo, e.g. by in vivo imaging in a
subject. For this purpose, the binding protein(s) of the invention
may be administered to a subject or a control subject under
conditions that allow binding of said protein(s) to the targeted
A.beta. form and detecting formation of a complex between said
binding protein(s) and said A.beta. form, wherein formation or
increased formation of the complex in the subject relative to the
control subject indicates the presence of said A.beta. form in the
subject. The subject may be a subject which is known or suspected
to suffer from a disorder or disease in which activity of a
targeted A.beta. form is detrimental.
BRIEF DESCRIPTION OF THE FIGURES
[0040] FIG. 1 illustrates amino acid sequences (SEQ ID NO:1) of the
variable light chain of humanized 4D10 antibodies comprising
J.kappa.2 and V.kappa. A17/2-30 framework regions. All CDR regions
are underlined.
[0041] FIG. 2 illustrates amino acid sequences (SEQ ID NO:2) of the
variable heavy chain of humanized 4D10 antibodies comprising human
JH6 (hJH6) and VH3_53 framework regions. All CDR regions are
underlined.
[0042] FIG. 3 illustrates amino acid sequences (SEQ ID NO:3) of the
variable heavy chain of humanized 4D10 antibodies comprising human
JH6 and VH4_59 framework regions. All CDR regions are
underlined.
[0043] FIG. 4 illustrates the amino acid sequence (SEQ ID NO:4) of
the variable heavy chain of humanized 4D10 antibodies comprising
human JH6 (hJH6) and VH3_53 framework regions. All CDR regions are
underlined.
[0044] FIG. 5 illustrates the amino acid sequence (SEQ ID NO:5) of
the variable heavy chain of humanized 4D10 antibodies comprising
human JH6 and VH3_53 framework regions with VH3 consensus change
I12V. All CDR regions are underlined.
[0045] FIG. 6 illustrates the amino acid sequence (SEQ ID NO:6) of
the variable heavy chain of humanized 4D10 antibodies comprising
human JH6 and VH3_53 framework regions with VH3 consensus change
I12V and framework backmutations A24V, V29L, V48L, S49G, F67L,
R71K, N76S and L78V. All CDR regions are underlined.
[0046] FIG. 7 illustrates the amino acid sequence (SEQ ID NO:7) of
the variable heavy chain of humanized 4D10 antibodies comprising
human JH6 and VH3_53 framework regions with framework backmutations
V29L and R71K. All CDR regions are underlined.
[0047] FIG. 8 illustrates the amino acid sequence (SEQ ID NO:8) of
the variable heavy chain of humanized 4D10 antibodies comprising
human JH6 and VH4_59 framework regions. All CDR regions are
underlined.
[0048] FIG. 9 illustrates the amino acid sequence (SEQ ID NO:9) of
the variable heavy chain of humanized 4D10 antibodies comprising
human JH6 and VH4_59 framework regions with a Q1E change to prevent
N-terminal pyroglutamate formation. All CDR regions are
underlined.
[0049] FIG. 10 illustrates the amino acid sequence (SEQ ID NO:10)
of the variable heavy chain of humanized 4D10 antibodies comprising
human JH6 and VH4_59 framework regions with a Q1E change to prevent
N-terminal pyroglutamate formation, and framework backmutations
G27F, I29L, I37V, I48L, V67L, V71K, N76S and F78V. All CDR regions
are underlined.
[0050] FIG. 11 illustrates the amino acid sequence (SEQ ID NO:11)
of the variable heavy chain of humanized 4D10 antibodies comprising
human JH6 and VH4_59 framework regions with a Q1E change to prevent
N-terminal pyroglutamate formation, and framework backmutations
G27F, I29L and V71K. All CDR regions are underlined.
[0051] FIG. 12 illustrate the amino acid sequence (SEQ ID NO:12) of
the variable light chain of humanized 4D10 antibodies comprising
J.kappa.2 and V.kappa. A17/2-30 framework regions. All CDR regions
are underlined.
[0052] FIG. 13 illustrates the amino acid sequence (SEQ ID NO:13)
of the variable light chain of humanized 4D10 antibodies comprising
J.kappa.2 and V.kappa. A17/2-30 framework regions with V.kappa.2
consensus changes S7T, LISP, Q37L, R39K and R45Q. All CDR regions
are underlined.
[0053] FIG. 14 illustrates the amino acid sequence (SEQ ID NO:14)
of the variable light chain of humanized 4D10 antibodies comprising
J.kappa.2 and V.kappa. A17/2-30 framework regions with V.kappa.2
consensus changes S7T, LISP, Q37L, R39K and R45Q, and framework
backmutation F36L. All CDR regions are underlined.
[0054] FIG. 15 illustrates the amino acid sequence (SEQ ID NO:15)
of the variable light chain of humanized 4D10 antibodies comprising
J.kappa.2 and V.kappa. A17/2-30 framework regions with V.kappa.2
consensus changes S7T and Q37L. All CDR regions are underlined.
[0055] FIG. 16 illustrates the amino acid sequence (SEQ ID NO:16)
of the variable light chain of humanized 4D10 antibodies comprising
J.kappa.2 and V.kappa. A17/2-30 framework regions with V.kappa.2
consensus changes S7T, Q37L and R39K. All CDR regions are
underlined.
[0056] FIG. 17 illustrates an amino acid sequence alignment of the
variable heavy chains of murine monoclonal antibody 4D10 (m4D19)
and humanized 4D10 antibodies (4D10hum) comprising human JH6 (hJH6)
and VH3_53 framework regions. All CDR regions are printed in bold
letters. X on position 12 is I or V, X on position 24 is A or V, X
on position 29 is V or L, X on position 48 is V or L, X on position
49 is S or G, X on position 67 is F or L, X on position 71 is R or
K, X on position 76 is N or S, and X on position 78 is L or V.
[0057] FIG. 18 illustrates an amino acid sequence alignment of the
variable heavy chains of murine monoclonal antibody 4D10 (m4D19)
and humanized 4D10 antibodies (4D10hum) comprising human JH6 and
VH4_59 framework regions. All CDR regions are printed in bold
letters. X on position 1 is Q or E, X on position 27 is G or F, X
on position 29 is I or L, X on position 37 is I or V, X on position
48 is I or L, X on position 67 is V or L, X on position 71 is V or
K, X on position 76 is N or S, and X on position 78 is F or V.
[0058] FIG. 19 illustrates an amino acid sequence alignment of the
variable light chains of murine monoclonal antibody 4D10 (m4D19)
and humanized 4D10 antibodies (4D10hum) comprising J.kappa.2 and
V.kappa. A17/2-30 framework regions. All CDR regions are printed in
bold letters. X on position 7 is S or T, X on position 15 is L or
P, X on position 41 is F or L, X on position 42 is Q or L, X on
position 44 is R or K, and X on position 50 is R or Q.
[0059] FIGS. 20A and B show platelet factor 4 (PF-4) cross-reaction
of humanized monoclonal antibodies 4D10hum#1 and 4D10hum#2,
human/mouse chimeric antibody h1G5 (positive control) and human
polyclonal antibody hIgG1 (negative control) in (A) Cynomolgus
monkey plasma and (B) human plasma, as determined by
sandwich-ELISA. Binding of PF-4 to the immobilized antibodies was
detected.
[0060] FIGS. 21A and B show platelet factor 4 (PF-4) cross-reaction
of humanized monoclonal antibodies 4D10hum#1 and 4D10hum#2,
human/mouse chimeric antibody h1G5 (positive control) and human
polyclonal antibody hIgG1 (negative control) in (A) Cynomolgus
monkey plasma and (B) human plasma, as determined by aligned
sandwich-ELISA. The antibodies were captured on the plate by
immobilized anti-mouse IgG. Binding of PF-4 to the captured
antibodies was detected.
[0061] FIGS. 22A and B show platelet factor 4 (PF-4) cross-reaction
of murine monoclonal antibodies m4D10 and m1G5, anti human PF-4
antibody (positive control) and IgG2a (negative control) in (A)
Cynomolgus monkey plasma and (B) human plasma, as determined by
sandwich-ELISA. Binding of PF-4 to the immobilized antibodies was
detected.
[0062] FIGS. 23A and B show platelet factor 4 (PF-4) cross-reaction
of murine monoclonal antibodies m4D10 and m1G5, anti human PF-4
antibody (positive control) and IgG2a (negative control) in (A)
Cynomolgus monkey plasma and (B) human plasma, as determined by
aligned sandwich-ELISA. The antibodies were captured on the plate
by immobilized anti-mouse IgG. Binding of PF-4 to the captured
antibodies was detected.
[0063] FIG. 24 illustrates the amino acid sequence (SEQ ID NO:46)
of the heavy chain of a humanized 4D10 antibody comprising human
JH6 and VH3_53 framework regions with VH3 consensus change I12V and
framework backmutations A24V, V29L, V48L, S49G, F67L, R71K, N76S
and L78V; and an Ig gamma-1 constant region. All CDR regions are
underlined.
[0064] FIG. 25 illustrates the amino acid sequence (SEQ ID NO:47)
of the heavy chain of a humanized 4D10 antibody comprising human
JH6 and VH4_59 framework regions with a Q1E change to prevent
N-terminal pyroglutamate formation, and framework backmutations
G27F, I29L, I37V, I48L, V67L, V71K, N76S and F78V; and an Ig
gamma-1 constant region. All CDR regions are underlined.
[0065] FIG. 26 illustrates the amino acid sequence (SEQ ID NO:48)
of the light chain of a humanized 4D10 antibody comprising
J.kappa.2 and V.kappa. A17/2-30 framework regions with V.kappa.2
consensus changes S7T, LISP, Q37L, R39K and R45Q, and framework
backmutation F36L; and an Ig kappa constant region. All CDR regions
are underlined.
DETAILED DESCRIPTION OF THE INVENTION
[0066] Unless otherwise defined herein, scientific and technical
terms used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. The meaning and scope of the terms should be clear,
however, in the event of any latent ambiguity, definitions provided
herein take precedent over any dictionary or extrinsic definition.
Further, unless otherwise required by context, singular terms shall
include pluralities and plural terms shall include the singular. In
this application, the use of "or" means "and/or" unless stated
otherwise. Furthermore, the use of the term "including", as well as
other forms, such as "includes" and "included", is not limiting.
Also, terms such as "element" or "component" encompass both
elements and components comprising one unit and elements and
components that comprise more than one subunit unless specifically
stated otherwise.
[0067] Generally, nomenclatures used in connection with, and
techniques of, cell and tissue culture, molecular biology,
immunology, microbiology, genetics, protein and nucleic acid
chemistry, and hybridization described herein are those well known
and commonly used in the art. The methods and techniques of the
present invention are generally performed according to conventional
methods well known in the art and as described in various general
and more specific references that are cited and discussed
throughout the present specification unless otherwise indicated.
Enzymatic reactions and purification techniques are performed
according to manufacturer's specifications, as commonly
accomplished in the art or as described herein. The nomenclatures
used in connection with, and the laboratory procedures and
techniques of, analytical chemistry, synthetic organic chemistry,
and medicinal and pharmaceutical chemistry described herein are
those well known and commonly used in the art. Standard techniques
are used for chemical syntheses, chemical analyses, pharmaceutical
preparation, formulation, and delivery, and treatment of
patients.
[0068] The present invention pertains to A.beta. binding proteins,
particularly anti-A.beta. antibodies or an A.beta. binding portion
thereof, particularly those binding to A.beta.(20-42) globulomer.
These A.beta. binding proteins are capable of discriminating not
only other forms of A.beta. peptides, particularly monomers and
fibrils, but also untruncated forms of A.beta. globulomers. Thus,
the present invention relates to an A.beta. binding protein having
a binding affinity to an A.beta.(20-42) globulomer that is greater
than the binding affinity of this A.beta. binding protein to an
A.beta.(1-42) globulomer.
[0069] The term "A.beta.(X-Y)" as used herein refers to the amino
acid sequence from amino acid position X to amino acid position Y
of the human amyloid beta (A.beta.) protein including both X and Y,
in particular to the amino acid sequence from amino acid position X
to amino acid position Y of the amino acid sequence DAEFRHDSGY
EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IAT (SEQ ID NO:29) (corresponding
to amino acid positions 1 to 43) or any of its naturally occurring
variants, in particular those with at least one mutation selected
from the group consisting of A2T, H6R, D7N, A21G ("Flemish"), E22G
("Arctic"), E22Q ("Dutch"), E22K ("Italian"), D23N ("Iowa"), A42T
and A42V wherein the numbers are relative to the start of the
A.beta. peptide, including both position X and position Y or a
sequence with up to three additional amino acid substitutions none
of which may prevent globulomer formation. According to one aspect,
there are no additional amino acid substitutions in the portion
from amino acid 12 or X, whichever number is higher, to amino acid
42 or Y, whichever number is lower. According to another aspect,
there are no additional amino acid substitutions in the portion
from amino acid 20 or X, whichever number is higher, to amino acid
42 or Y, whichever number is lower. According to another aspect,
there are no additional amino acid substitutions in the portion
from amino acid 20 or X, whichever number is higher, to amino acid
40 or Y, whichever number is lower. An "additional" amino acid
substitution herein is any deviation from the canonical sequence
that is not found in nature.
[0070] More specifically, the term "A.beta.(1-42)" as used herein
refers to the amino acid sequence from amino acid position 1 to
amino acid position 42 of the human A.beta. protein including both
1 and 42, in particular to the amino acid sequence DAEFRHDSGY
EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA (SEQ ID NO:30) or any of its
naturally occurring variants, in particular those with at least one
mutation selected from the group consisting of A2T, H6R, D7N, A21G
("Flemish"), E22G ("Arctic"), E22Q ("Dutch"), E22K ("Italian"),
D23N ("Iowa"), A42T and A42V wherein the numbers are relative to
the start of the A.beta. peptide, including both 1 and 42 or a
sequence with up to three additional amino acid substitutions none
of which may prevent globulomer formation. According to one aspect,
there are no additional amino acid substitutions in the portion
from amino acid 20 to amino acid 42. Likewise, the term
"A.beta.(1-40)" as used herein refers to the amino acid sequence
from amino acid position 1 to amino acid position 40 of the human
A.beta. protein including both 1 and 40, in particular to the amino
acid sequence DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV (SEQ ID
NO:31) or any of its naturally occurring variants, in particular
those with at least one mutation selected from the group consisting
of A2T, H6R, D7N, A21G ("Flemish"), E22G ("Arctic"), E22Q
("Dutch"), E22K ("Italian"), and D23N ("Iowa") wherein the numbers
are relative to the start of the A.beta. peptide, including both 1
and 40 or a sequence with up to three additional amino acid
substitutions none of which may prevent globulomer formation.
According to one aspect, there are no additional amino acid
substitutions in the portion from amino acid 20 to amino acid
40.
[0071] More specifically, the term "A.beta.(12-42)" as used herein
refers to the amino acid sequence from amino acid position 12 to
amino acid position 42 of the human A.beta. protein including both
12 and 42, in particular to the amino acid sequence VHHQKLVFF
AEDVGSNKGA IIGLMVGGVV IA (SEQ ID NO:32) or any of its naturally
occurring variants, in particular those with at least one mutation
selected from the group consisting of A21G ("Flemish"), E22G
("Arctic"), E22Q ("Dutch"), E22K ("Italian"), D23N ("Iowa"), A42T
and A42V wherein the numbers are relative to the start of the
A.beta. peptide, including both 12 and 42 or a sequence with up to
three additional amino acid substitutions none of which may prevent
globulomer formation. According to one aspect, there are no
additional amino acid substitutions in the portion from amino acid
20 to amino acid 42. Likewise, the term "A.beta.(20-42)" as used
herein refers to the amino acid sequence from amino acid position
20 to amino acid position 42 of the human amyloid .beta. protein
including both 20 and 42, in particular to the amino acid sequence
F AEDVGSNKGA IIGLMVGGVV IA (SEQ ID NO:33) or any of its naturally
occurring variants, in particular those with at least one mutation
selected from the group consisting of A21G ("Flemish"), E22G
("Arctic"), E22Q ("Dutch"), E22K ("Italian"), D23N ("Iowa"), A42T
and A42V wherein the numbers are relative to the start of the
A.beta. peptide, including both 20 and 42 or a sequence with up to
three additional amino acid substitutions none of which may prevent
globulomer formation. According to one aspect, there are any
additional amino acid substitutions.
[0072] The term "A.beta.(X-Y) globulomer" (A.beta.(X-Y) globular
oligomer) as used herein refers to a soluble, globular,
non-covalent association of A.beta.(X-Y) peptides as defined above,
possessing homogeneity and distinct physical characteristics.
According to one aspect, A.beta.(X-Y) globulomers are stable,
non-fibrillar, oligomeric assemblies of A.beta.(X-Y) peptides which
are obtainable by incubation with anionic detergents. In contrast
to monomer and fibrils, these globulomers are characterized by
defined assembly numbers of subunits (e.g. early assembly forms
with 4-6 subunits, "oligomers A"; and late assembly forms with
12-14 subunits, "oligomers B"; as described in WO2004/067561). The
globulomers have a 3-dimensional globular type structure ("molten
globule", see Barghorn et al., J Neurochem 95: 834-847, 2005). They
may be further characterized by one or more of the following
features: [0073] cleavability of N-terminal amino acids X-23 with
promiscuous proteases (such as thermolysin or endoproteinase GluC)
yielding truncated forms of globulomers; [0074] non-accessibility
of C-terminal amino acids 24-Y with promiscuous proteases and
antibodies; [0075] truncated forms of these globulomers maintain
the 3-dimensional core structure of said globulomers with a better
accessibility of the core epitope A.beta.(20-Y) in its globulomer
conformation.
[0076] According to the invention and in particular for the purpose
of assessing the binding affinities of the A.beta. binding proteins
of the present invention, the term "A.beta.(X-Y) globulomer" here
refers in particular to a product which is obtainable by a process
as described in WO2004/067561, which is incorporated herein by
reference. Said process comprises unfolding a natural, recombinant
or synthetic A.beta.(X-Y) peptide or a derivative thereof; exposing
the at least partially unfolded A.beta.(X-Y) peptide or derivative
thereof to a detergent, reducing the detergent action and
continuing incubation.
[0077] For the purpose of unfolding the peptide, hydrogen
bond-breaking agents such as, for example, hexafluoroisopropanol
(HFIP) may be allowed to act on the protein. Times of action of a
few minutes, for example about 10 to 60 minutes, are sufficient
when the temperature of action is from about 20 to 50.degree. C.
and in particular about 35 to 40.degree. C. Subsequent dissolution
of the residue evaporated to dryness, e.g. in concentrated form, in
suitable organic solvents miscible with aqueous buffers, such as,
for example, dimethyl sulfoxide (DMSO), results in a suspension of
the at least partially unfolded peptide or derivative thereof,
which can be used subsequently. If required, the stock suspension
may be stored at low temperature, for example at about 20.degree.
C., for an interim period. Alternatively, the peptide or the
derivative thereof may be taken up in slightly acidic, e.g.
aqueous, solution, for example, an about 10 mM aqueous HCl
solution. After an incubation time of usually a few minutes,
insoluble components are removed by centrifugation. A few minutes
at 10,000 g is expedient. These method steps can be carried out at
room temperature, i.e. a temperature in the range from 20 to
30.degree. C. The supernatant obtained after centrifugation
contains the A.beta.(X-Y) peptide or the derivative thereof and may
be stored at low temperature, for example at about -20.degree. C.,
for an interim period. The following exposure to a detergent
relates to the oligomerization of the peptide or the derivative
thereof to give an intermediate type of oligomers (in WO
2004/067561 referred to as oligomers A). For this purpose, a
detergent is allowed to act on the at least partially unfolded
peptide or derivative thereof until sufficient intermediate
oligomer has been produced. Preference is given to using ionic
detergents, in particular anionic detergents.
[0078] According to a particular embodiment, a detergent of the
formula (I):
R--X,
[0079] is used, in which the radical R is unbranched or branched
alkyl having from 6 to 20, e.g. 10 to 14, carbon atoms or
unbranched or branched alkenyl having from 6 to 20, e.g. 10 to 14,
carbon atoms, the radical X is an acidic group or salt thereof,
with X being selected, e.g., from among --COO-M.sup.+,
--SO.sub.3-M.sup.+, and especially --OSO.sub.3-M.sup.+ and M.sup.+
is a hydrogen cation or an inorganic or organic cation selected
from, e.g., alkali metal and alkaline earth metal cations and
ammonium cations. Advantageous are detergents of the formula (I),
in which R is unbranched alkyl of which alk-1-yl radicals must be
mentioned in particular. For example, sodium dodecyl sulfate (SDS),
lauric acid, the sodium salt of the detergent lauroylsarcosin (also
known as sarkosyl NL-30 or Gardol.RTM.) and oleic acid can be used
advantageously. The time of detergent action in particular depends
on whether (and if yes, to what extent) the peptide or the
derivative thereof subjected to oligomerization has unfolded. If,
according to the unfolding step, the peptide or derivative thereof
has been treated beforehand with a hydrogen bond-breaking agent,
i.e. in particular with hexafluoroisopropanol, times of action in
the range of a few hours, advantageously from about 1 to 20 and in
particular from about 2 to 10 hours, are sufficient when the
temperature of action is about 20 to 50.degree. C. and in
particular about 35 to 40.degree. C. If a less unfolded or an
essentially not unfolded peptide or derivative thereof is the
starting point, correspondingly longer times of action are
expedient. If the peptide or the derivative thereof has been
pretreated, for example, according to the procedure indicated above
as an alternative to the HFIP treatment or said peptide or
derivative thereof is directly subjected to oligomerization, times
of action in the range from about 5 to 30 hours and in particular
from about 10 to 20 hours are sufficient when the temperature of
action is about 20 to 50.degree. C. and in particular about 35 to
40.degree. C. After incubation, insoluble components are
advantageously removed by centrifugation. A few minutes at 10,000 g
is expedient. The detergent concentration to be chosen depends on
the detergent used. If SDS is used, a concentration in the range
from 0.01 to 1% by weight, e.g. from 0.05 to 0.5% by weight, for
example of about 0.2% by weight, proves expedient. If lauric acid
or oleic acid are used, somewhat higher concentrations are
expedient, for example in a range from 0.05 to 2% by weight, e.g.
from 0.1 to 0.5% by weight, for example of about 0.5% by weight.
The detergent action should take place at a salt concentration
approximately in the physiological range. Thus, in particular NaCl
concentrations in the range from 50 to 500 mM, e.g. from 100 to 200
mM or at about 140 mM are expedient. The subsequent reduction of
the detergent action and continuation of incubation relates to a
further oligomerization to give the A.beta.(X-Y) globulomer of the
invention (in WO2004/067561 referred to as oligomers B). Since the
composition obtained from the preceding step regularly contains
detergent and a salt concentration in the physiological range it is
then expedient to reduce detergent action and also the salt
concentration. This may be carried out by reducing the
concentration of detergent and salt, for example, by diluting,
expediently with water or a buffer of lower salt concentration, for
example Tris-HCl, pH 7.3. Dilution factors in the range from about
2 to 10, advantageously in the range from about 3 to 8 and in
particular of about 4, have proved suitable. The reduction in
detergent action may also be achieved by adding substances which
can neutralize said detergent action. Examples of these include
substances capable of complexing the detergents, like substances
capable of stabilizing cells in the course of purification and
extraction measures, for example particular EO/PO block copolymers,
in particular the block copolymer under the trade name
Pluronic.RTM. F 68. Alkoxylated and, in particular, ethoxylated
alkyl phenols such as the ethoxylated t-octylphenols of the
Triton.RTM. X series, in particular Triton.RTM. X100,
3-(3-cholamidopropyldimethylammonio)-1-propanesulfonate
(CHAPS.RTM.) or alkoxylated and, in particular, ethoxylated
sorbitan fatty esters such as those of the Tween.RTM. series, in
particular Tween.RTM. 20, in concentration ranges around or above
the particular critical micelle concentration, may be equally used.
Subsequently, the solution is incubated until sufficient
A.beta.(X-Y) globulomer of the invention has been produced. Times
of action in the range of several hours, e.g. in the range from
about 10 to 30 hours or in the range from about 15 to 25 hours, are
sufficient when the temperature of action is about 20 to 50.degree.
C. and in particular about 35 to 40.degree. C. The solution may
then be concentrated and possible residues may be removed by
centrifugation. Here too, a few minutes at 10,000 g proves
expedient. The supernatant obtained after centrifugation contains
an A.beta.(X-Y) globulomer of the invention. An A.beta.(X-Y)
globulomer of the invention can be finally recovered in a manner
known per se, e.g. by ultrafiltration, dialysis, precipitation or
centrifugation. For example, electrophoretic separation of the
A.beta.(X-Y) globulomers under denaturing conditions, e.g. by
SDS-PAGE, may produce a double band (e.g. with an apparent
molecular weight of 38/48 kDa for A.beta.(1-42)), and upon
glutardialdehyde treatment of the globulomers before separation
these two bands can merge into one. Size exclusion chromatography
of the globulomers may result in a single peak (e.g. corresponding
to a molecular weight of approximately 100 kDa for A.beta.(1-42)
globulomer or of approximately 60 kDa for glutardialdehyde
cross-linked A.beta.(1-42) globulomer), respectively. Starting out
from A.beta.(1-42) peptide, A.beta.(12-42) peptide, and
A.beta.(20-42) peptide said processes are in particular suitable
for obtaining A.beta.(1-42) globulomers, A.beta.(12-42)
globulomers, and A.beta.(20-42) globulomers.
[0080] In a particular embodiment of the invention, A.beta.(X-Y)
globulomers wherein X is selected from the group consisting of the
numbers 2 . . . 24 and Y is as defined above, are those which are
obtainable by truncating A.beta.(1-Y) globulomers into shorter
forms wherein X is selected from the group consisting of the
numbers 2 . . . 24, for example with X being 20 or 12, and Y is as
defined above, which can be achieved by treatment with appropriate
proteases. For instance, an A.beta.(20-42) globulomer can be
obtained by subjecting an A.beta.(1-42) globulomer to thermolysin
proteolysis, and an A.beta.(12-42) globulomer can be obtained by
subjecting an A.beta.(1-42) globulomer to endoproteinase GluC
proteolysis. When the desired degree of proteolysis is reached, the
protease is inactivated in a generally known manner. The resulting
globulomers may then be isolated following the procedures already
described herein and, if required, processed further by further
work-up and purification steps. A detailed description of said
processes is disclosed in WO2004/067561, which is incorporated
herein by reference.
[0081] For the purposes of the present invention, an A.beta.(1-42)
globulomer is, in particular, the A.beta.(1-42) globulomer as
described in Example 1a below; an A.beta.(20-42) globulomer is in
particular the A.beta.(20-42) globulomer as described in Example 1b
below, and an A.beta.(12-42) globulomer is in particular the
A.beta.(12-42) globulomer as described in Example 1c below.
According to one aspect of the invention, the globulomer shows
affinity to neuronal cells and/or exhibits neuromodulating
effects.
[0082] According to another aspect of the invention, the globulomer
consists of 11 to 16, e.g. of 12 to 14 A.beta.(X-Y) peptides.
According to another aspect of the invention, the term
"A.beta.(X-Y) globulomer" herein refers to a globulomer consisting
essentially of A.beta.(X-Y) subunits, where for example on average
at least 11 of 12 subunits are of the A.beta.(X-Y) type, or less
than 10% of the globulomers comprise any non-A.beta.(X-Y) peptides,
or the content of non-A.beta.(X-Y) peptides is below the detection
threshold. More specifically, the term "A.beta.(1-42) globulomer"
herein refers to a globulomer consisting essentially of
A.beta.(1-42) units as defined above; the term "A.beta.(12-42)
globulomer" herein refers to a globulomer consisting essentially of
A.beta.(12-42) units as defined above; and the term "A.beta.(20-42)
globulomer" herein refers to a globulomer consisting essentially of
A.beta.(20-42) units as defined above.
[0083] The term "cross-linked A.beta.(X-Y) globulomer" herein
refers to a molecule obtainable from an A.beta.(X-Y) globulomer as
described above by cross-linking, e.g. by chemically cross-linking,
aldehyde cross-linking, glutardialdehyde cross-linking, of the
constituent units of the globulomer. In another aspect of the
invention, a cross-linked globulomer is essentially a globulomer in
which the units are at least partially joined by covalent bonds,
rather than being held together by non-covalent interactions only.
For the purposes of the present invention, a cross-linked
A.beta.(1-42) globulomer is in particular the cross-linked
A.beta.(1-42) oligomer as described in Example 1d below.
[0084] The term "A.beta.(X-Y) globulomer derivative" herein refers
in particular to a globulomer that is labelled by being covalently
linked to a group that facilitates detection, for example a
fluorophore, e.g. fluorescein isothiocyanate, phycoerythrin,
Aequorea victoria fluorescent protein, Dictyosoma fluorescent
protein or any combination or fluorescence-active derivative
thereof; a chromophore; a chemoluminophore, e.g. luciferase, in
particular Photinus pyralis luciferase, Vibrio fischeri luciferase,
or any combination or chemoluminescence-active derivative thereof;
an enzymatically active group, e.g. peroxidase, e.g. horseradish
peroxidase, or any enzymatically active derivative thereof; an
electron-dense group, e.g. a heavy metal containing group, e.g. a
gold containing group; a hapten, e.g. a phenol derived hapten; a
strongly antigenic structure, e.g. peptide sequence predicted to be
antigenic, e.g. predicted to be antigenic by the algorithm of
Kolaskar and Tongaonkar; an aptamer for another molecule; a
chelating group, e.g. hexahistidinyl; a natural or nature-derived
protein structure mediating further specific protein-protein
interactions, e.g. a member of the fos/jun pair; a magnetic group,
e.g. a ferromagnetic group; or a radioactive group, e.g. a group
comprising 1H, 14C, 32P, 35S or 125I or any combination thereof; or
to a globulomer flagged by being covalently or by non-covalent
high-affinity interaction linked to a group that facilitates
inactivation, sequestration, degradation and/or precipitation, for
example flagged with a group that promotes in vivo degradation such
as ubiquitin, this flagged oligomer being, e.g., assembled in vivo;
or to a globulomer modified by any combination of the above. Such
labelling and flagging groups and methods for attaching them to
proteins are known in the art. Labelling and/or flagging may be
performed before, during or after globulomerisation. In another
aspect of the invention, a globulomer derivative is a molecule
obtainable from a globulomer by a labelling and/or flagging
reaction. Correspondingly, term "A.beta.(X-Y) monomer derivative"
here refers in particular to an A.beta. monomer that is labelled or
flagged as described for the globulomer.
[0085] In a further aspect of the invention, the binding proteins
described herein bind to the A.beta.(20-42) globulomer with a high
affinity, for instance with a dissociation constant (K.sub.D) of at
most about 10.sup.-6M; at most about 10.sup.-7 M; at most about
10.sup.-8 M; at most about 10.sup.-9 M; at most about 10.sup.-10 M;
at most about 10.sup.-11 M; at most about 10.sup.-12 M; and at most
10.sup.-13 M. In one aspect the on-rate constant (k.sub.on) of the
binding protein described herein to A.beta.(20-42) globulomer is
selected from the group consisting of: at least about 10.sup.2
M.sup.-1 s.sup.-1; at least about 10.sup.3 M.sup.-1 s.sup.-1; at
least about 10.sup.4 M.sup.-1 s.sup.-1; at least about 10.sup.5
M.sup.-1s.sup.-1; and at least about 10.sup.6 M.sup.-1s.sup.-1; as
measured by surface plasmon resonance. In another aspect, the
binding proteins have an off-rate constant (k.sub.off) to
A.beta.(20-42) globulomer selected from the group consisting of: at
most about 10.sup.-3 s.sup.-1; at most about 10.sup.-4 s.sup.-1; at
most about 10.sup.-5 s.sup.-1; and at most about 10.sup.-6
s.sup.-1, as measured by surface plasmon resonance. In a particular
aspect of the invention, the binding proteins described herein bind
to the A.beta.(20-42) globulomer with a dissociation constant from
1.times.10.sup.-9 to 1.times.10.sup.-10 M. In a further particular
aspect of the invention, the on-rate constant (k.sub.on) of the
binding protein described herein to A.beta.(20-42) globulomer is
from 1.times.10.sup.5 to 1.times.10.sup.6 M.sup.-1 s.sup.-1. In a
further particular aspect of the invention, the binding proteins
described herein have an off-rate constant (k.sub.off) to
A.beta.(20-42) globulomer from 8.times.10.sup.-5 to
8.times.10.sup.-4 s.sup.-1.
[0086] In another aspect of the invention, the binding affinity of
the binding proteins described herein to A.beta.(20-42) globulomer
is greater than to an A.beta.(1-42) globulomer.
[0087] The term "greater affinity" herein refers to a degree of
interaction where the equilibrium between unbound A.beta. binding
protein and unbound A.beta. globulomer on the one hand and A.beta.
binding protein-globulomer complex on the other is further in
favour of the A.beta. binding protein-globulomer complex. Likewise,
the term "smaller affinity" here refers to a degree of interaction
where the equilibrium between unbound A.beta. binding protein and
unbound A.beta. globulomer on the one hand and A.beta. binding
protein-globulomer complex on the other is further in favour of the
unbound A.beta. binding protein and unbound A.beta. globulomer. The
term "greater affinity" is synonymous with the term "higher
affinity" and term "smaller affinity" is synonymous with the term
"lower affinity".
[0088] In a related aspect of the invention, the binding affinity
of the binding proteins described herein to A.beta.(20-42)
globulomer is at least 2 times (e.g., at least 3 or at least 5
times), at least 10 times (e.g., at least 20 times, at least 30
times or at least 50 times), at least 100 times (e.g., at least 200
times, at least 300 times or at least 500 times), and at least
1,000 times (e.g., at least 2,000 times, at least 3,000 times or at
least 5000 times), at least 10,000 times (e.g., at least 20,000
times, at least 30,000 times or at least 50,000 times), or at least
100,000 times greater than the binding affinity of the binding
protein to the A.beta.(1-42) globulomer.
[0089] In still a further aspect of the invention, the binding
proteins described herein bind to the A.beta.(12-42) globulomer
with a relatively high affinity, for instance with a dissociation
constant (K.sub.D) of at most about 10.sup.-6M; at most about
10.sup.-7 M; at most about 10.sup.-8 M; at most about 10.sup.-9 M;
at most about 10.sup.-10 at most about 10.sup.-11M; at most about
10.sup.-12 M; and at most 10.sup.-13 M. In one aspect the on-rate
constant (k.sub.on) of the binding protein described herein to
A.beta.(12-42) globulomer is selected from the group consisting of:
at least about 10.sup.2 M.sup.-1 s.sup.-1; at least about 10.sup.3
M.sup.-1s.sup.-1; at least about 10.sup.4 M.sup.-1 s.sup.-1; at
least about 10.sup.5 M.sup.-1s.sup.-1; and at least about 10.sup.6
M.sup.-1 s.sup.-1; as measured by surface plasmon resonance. In
another aspect, the binding proteins have an off-rate constant
(k.sub.off) to A.beta.(12-42) globulomer selected from the group
consisting of: at most about 10.sup.-3 s.sup.-1; at most about
10.sup.4 s.sup.-1; at most about 10.sup.-5 s.sup.-1; and at most
about 10.sup.-6 s.sup.-1, as measured by surface plasmon
resonance.
[0090] In a related aspect of the invention, the binding affinity
of the binding proteins described herein to A.beta.(20-42)
globulomer is about 1.1 to 3 times greater than the binding
affinity of the binding proteins to A.beta.(12-42) globulomer.
[0091] According to one aspect, the A.beta. binding proteins of the
present invention bind to at least one A.beta. globulomer, as
defined above, and have a comparatively smaller affinity for at
least one non-globulomer form of A.beta.. A.beta. binding proteins
of the present invention with a comparatively smaller affinity for
at least one non-globulomer form of A.beta. than for at least one
A.beta. globulomer include A.beta. binding protein with a binding
affinity to the A.beta.(20-42) globulomer that is greater than to
an A.beta.(1-42) monomer. According to an alternative or additional
aspect of the invention, the binding affinity of the A.beta.
binding protein to the A.beta.(20-42) globulomer is greater than to
an A.beta.(1-40) monomer. In particular, the affinity of the
A.beta. binding proteins to the A.beta.(20-42) globulomer is
greater than its affinity to both the A.beta.(1-40) and the
A.beta.(1-42) monomer.
[0092] The term "A.beta.(X-Y) monomer" as used herein refers to the
isolated form of the A.beta.(X-Y) peptide, in particular to a form
of the A.beta.(X-Y) peptide which is not engaged in essentially
non-covalent interactions with other A.beta. peptides. Practically,
the A.beta.(X-Y) monomer is usually provided in the form of an
aqueous solution. In a particular embodiment of the invention, the
aqueous monomer solution contains 0.05% to 0.2%, e.g. about 0.1%
NH.sub.4OH. In another particular embodiment of the invention, the
aqueous monomer solution contains 0.05% to 0.2%, e.g. about 0.1%
NaOH. When used (for instance for determining the binding
affinities of the A.beta. binding proteins of the present
invention), it may be expedient to dilute said solution in an
appropriate manner. Further, it is usually expedient to use said
solution within 2 hours, in particular within 1 hour, and
especially within 30 minutes after its preparation.
[0093] More specifically, the term "A.beta.(1-40) monomer" here
refers to an A.beta.(1-40) monomer preparation as described herein,
and the term "A.beta.(1-42) monomer" here refers to an
A.beta.(1-42) preparation as described herein.
[0094] Expediently, the A.beta. binding proteins of the present
invention bind to one or both monomers with low affinity, for
example with a K.sub.D of 1.times.10.sup.-8 M or smaller affinity,
e.g. with a K.sub.D of 3.times.10.sup.-8 M or smaller affinity,
with a K.sub.D of 1.times.10.sup.-7 M or smaller affinity, e.g.
with a K.sub.D of 3.times.10.sup.-7 M or smaller affinity, or with
a K.sub.D of 1.times.10.sup.-6 M or smaller affinity, e.g. with a
K.sub.D of 3.times.10.sup.-5 M or smaller affinity, or with a
K.sub.D of 1.times.10.sup.-5 M or smaller affinity.
[0095] According to one aspect of the invention, the binding
affinity of the A.beta. binding proteins of the present invention
to the A.beta.(20-42) globulomer is at least 2 times, e.g. at least
3 times or at least 5 times, at least 10 times, e.g. at least 20
times, at least 30 times or at least 50 times, at least 100 times,
e.g. at least 200 times, at least 300 times or at least 500 times,
at least 1,000 times, e.g. at least 2,000 times, at least 3,000
times or at least 5,000 times, at least 10,000 times, e.g. at least
20,000 times, at least 30,000 or at least 50,000 times, or at least
100,000 times greater than the binding affinity of the A.beta.
binding proteins to one or both monomers.
[0096] A.beta. binding proteins of the present invention having a
comparatively smaller affinity for at least one non-globulomer form
of A.beta. than for at least one A.beta. globulomer further include
A.beta. binding proteins having a binding affinity to the
A.beta.(20-42) globulomer that is greater than to A.beta.(1-42)
fibrils. According to an alternative or additional aspect of the
invention, the binding affinity of the A.beta. binding proteins to
the A.beta.(20-42) globulomer is greater than to A.beta.(1-40)
fibrils. According to one particular embodiment, the invention
relates to A.beta. binding proteins having a binding affinity to
the A.beta.(20-42) globulomer which is greater than their binding
affinity to both A.beta.(1-40) and A.beta.(1-42) fibrils.
[0097] The term "fibril" herein refers to a molecular structure
that comprises assemblies of non-covalently associated, individual
A.beta.(X-Y) peptides, which show fibrillary structure in the
electron microscope, which bind Congo red and then exhibit
birefringence under polarized light and whose X-ray diffraction
pattern is a cross-.beta. structure. In another aspect of the
invention, a fibril is a molecular structure obtainable by a
process that comprises the self-induced polymeric aggregation of a
suitable A.beta. peptide in the absence of detergents, e.g. in 0.1
M HCl, leading to the formation of aggregates of more than 24 or
more than 100 units. This process is well known in the art.
Expediently, A.beta.(X-Y) fibrils are used in the form of an
aqueous solution. In a particular embodiment of the invention, the
aqueous fibril solution is made by dissolving the AO peptide in
0.1% NH.sub.4OH, diluting it 1:4 with 20 mM NaH.sub.2PO.sub.4, 140
mM NaCl, pH 7.4, followed by readjusting the pH to 7.4, incubating
the solution at 37.degree. C. for 20 h, followed by centrifugation
at 10,000 g for 10 min and resuspension in 20 mM NaH.sub.2PO.sub.4,
140 mM NaCl, pH 7.4. The term "A.beta.(X-Y) fibril" herein also
refers to a fibril comprising A.beta.(X-Y) subunits where, e.g., on
average, at least 90% of the subunits are of the A.beta.(X-Y) type,
at least 98% of the subunits are of the A.beta.(X-Y) type or the
content of non-A.beta.(X-Y) peptides is below the detection
threshold. More specifically, the term "A.beta.(1-42) fibril"
herein refers to a A.beta.(1-42) fibril preparation as described in
Example 3.
[0098] Expediently, the A.beta. binding proteins of the present
invention bind to one or both fibrils with low affinity, for
example with a K.sub.D of 1.times.10.sup.-8M or smaller affinity,
e.g. with a K.sub.D of 3.times.10.sup.-8 M or smaller affinity,
with a K.sub.D of 1.times.10.sup.-7 M or smaller affinity, e.g.
with a K.sub.D of 3.times.10.sup.-7 M or smaller affinity, or with
a K.sub.D of 1.times.10.sup.-6 M or smaller affinity, e.g. with a
K.sub.D of 3.times.10.sup.-5 M or smaller affinity, or with a
K.sub.D of 1.times.10.sup.-5 M or smaller affinity.
[0099] According to one aspect of the invention, the binding
affinity of the A.beta. binding proteins of the present invention
to the A.beta.(20-42) globulomer is at least 2 times, e.g. at least
3 times or at least 5 times, at least 10 times, e.g. at least 20
times, at least 30 times or at least 50 times, at least 100 times,
e.g. at least 200 times, at least 300 times or at least 500 times,
at least 1,000 times, e.g. at least 2,000 times, at least 3,000
times or at least 5,000 times, at least 10,000 times, e.g. at least
20,000 times, at least 30,000 or at least 50,000 times, or at least
100,000 times greater than the binding affinity of the A.beta.
binding proteins to one or both fibrils.
[0100] According to a particular embodiment, the present invention
relates to A.beta. binding proteins having a comparatively smaller
affinity for both the monomeric and fibrillary forms of A.beta.
than for at least one A.beta. globulomer, in particular
A.beta.(20-42) globulomer. These A.beta. binding proteins sometimes
are referred to as globulomer-specific A.beta. binding
proteins.
[0101] The binding proteins of the present invention, e.g.
humanized antibody 4D10 (4D10hum), include globulomer-specific
binding proteins recognizing predominantly A.beta.(20-42)
globulomer forms and not standard preparations of A.beta.(1-40)
monomers, A.beta.(1-42) monomers, A.beta.-fibrils or sAPP (i.e.
soluble A.beta. precursor) in contrast to, for example, competitor
antibodies such as m266 and 3D6. Such specificity for globulomers
is important because specifically targeting the globulomer form of
A.beta. with humanized 4D10 will: 1) avoid targeting insoluble
amyloid deposits, binding to which may account for inflammatory
side effects observed during immunizations with insoluble A.beta.;
2) spare A.beta. monomer and APP that are reported to have
precognitive physiological functions (Plan et al., J Neurosci 23:
5531-5535, 2003; and 3) increase the bioavailability of the
antibody, as it would not be shaded or inaccessible through
extensive binding to insoluble deposits.
[0102] PF-4 is a small, 70-amino acid cytokine that belongs to the
CXC chemokine family and is also known as chemokine (C-X-C motif)
ligand 4 (CXCL4). PF-4 is released from alpha-granules of activated
platelets during platelet aggregation, and promotes blood
coagulation by moderating the effects of heparin-like molecules.
Due to these functions, it is predicted to be involved in wound
repair and inflammation (Eismann et al., Blood 76(2): 336-44,
1990). PF-4 is usually found in a complex with proteoglycan and can
form complexes with the anticoagulant heparin which is in use as
pharmacological treatment of thrombosis. It has a well described
pathological function in heparin-induced thrombocytopenia (HIT), an
idiosyncratic autoimmune reaction to the administration of the
anticoagulant heparin (Warkentin, N. Engl. J. Med. 356(9): 891-3,
2007), wherein the heparin:PF4 complex is the antigen. PF4
autoantibodies have also been found in patients with thrombosis and
features resembling HIT but no prior administration of heparin
(Warkentin et al., Am. J. Med. 121(7): 632-6, 2008).
Heparin-induced thrombocytopenia is characterized by the
development of thrombocytopenia (a low platelet count), and in
addition HIT predisposes to thrombosis. In view of these functions
and involvement of PF-4 in pathological processes it can be
concluded that the administration of binding proteins (e.g.
antibodies) showing binding (e.g. cross-reactivity) to the PF-4
present in a subject may affect said PF-4 functions and thus result
in adverse (side) effects. The degree and nature of such adverse
effects may vary depending on parameters such as location and size
of the epitope on PF-4, binding strength and nature of the
respective binding protein.
[0103] According to one aspect of the invention, the binding
proteins of the present invention do show no or low binding to
platelet factor 4 (PF-4). Said cross-reaction to PF-4 may be
evaluated by using standardized in vitro immunoassays such as
ELISA, dot blot or BIAcore analyses.
[0104] According to a particular embodiment, the cross-reaction to
PF-4 of a binding protein defined herein refers to ratio of values
for said binding protein and a reference anti-PF-4 antibody
obtained by (i) performing a sandwich-ELISA with a .about.1:3
dilution series of human or cynomolgus plasma from about 1:3.16 to
about 1:3160 (final plasma dilution) (e.g. as described in examples
3.1 and 3.2), (ii) plotting detected signal (y-axis) against
log-transformed plasma dilutions (x-axis), and (iii) determining
the area under the curve (AUC, or total peak area) from these
non-curve fitted data in the measured range (final plasma dilutions
from about 1:3.16 to about 1:3160). According to a particular
embodiment of the invention, determining the cross-reaction to PF-4
by sandwich-ELISA comprises the following: a certain amount of the
binding protein under investigation or the reference anti-PF-4
antibody or, expediently, an appropriate dilution thereof, for
instance 100 .mu.l of a 10 .mu.g/ml binding protein or antibody
solution in 100 mM sodium hydrogen carbonate, pH 9.6, is used for
coating wells of a protein adsorbing microtiter plate; the plate is
then washed, blocked, and washed again; then contacted with a
.about.1:3 dilution series of cynomolgus or human plasma, e.g.
human plasma spiked with human PF-4, from about 1:3.16 to about
1:3160 (final plasma dilution) followed by detection of the PF-4
bound to each well, e.g. by means of a primary PF-4 specific
antibody, an enzyme-conjugated secondary antibody and a
colorimetric reaction.
[0105] A "reference anti-PF-4 antibody", as used herein, is an
antibody, in particular a monoclonal antibody, that is specifically
reactive with PF-4, in particular human (HPF4). Such an antibody is
obtainable by providing an antigen comprising human PF-4, for
instance human PF-4 having amino acid sequence
EAEEDGDLQCLCVKTTSQVRPRHITSLEVIKAGPHCPTAQLIATLKNGRKICLDLQAP
LYKKIIKKLLES (SEQ ID NO:70), exposing an antibody repertoire to
said antigen and selecting from said antigen repertoire an antibody
which binds specifically to human PF-4. The antibody may optionally
be affinity purified using the immunogen (human PF-4). Such
reference anti-PF4 antibodies are commercially available, for
example, monoclonal anti-HPF4 antibody, Abcam cat. no.:
ab49735.
[0106] According to another particular embodiment, the
cross-reaction to PF-4 of a binding protein defined herein refers
to ratio of AUC values for said binding protein and a reference
anti-PF-4 antibody obtained by (i) performing an aligned
sandwich-ELISA with human or cynomolgus plasma and .about.1:3
dilution series of binding protein and reference anti-PF-4 antibody
from about 10 ng/ml to about 10000 ng/ml (final concentration)
(e.g. as described in examples 3.3 and 3.4), (ii) plotting detected
signal (y-axis) against log-transformed concentrations of binding
protein or reference anti-PF-4 antibody (x-axis), and (iii)
determining the area under the curve (AUC, or total peak area) from
these non-curve fitted data in the measured range (concentrations
of binding protein or reference antibody from about 10 ng/ml to
about 10000 ng/ml). According to a particular embodiment of the
invention, determining the cross-reaction to PF-4 by aligned
sandwich-ELISA comprises the following: the wells of a protein
adsorbing microtiter plate are coated with a certain amount of an
aligning antibody suitable to capture the binding protein under
investigation and the reference anti-PF-4 antibody, for example 100
.mu.l/well of 50 .mu.g/ml Fc specific anti-mouse IgG, Sigma cat.
no.: M3534, in 100 mM sodium hydrogen carbonate, pH 9.6); the plate
is then washed, blocked, and washed again; then contacted with a
.about.1:3 dilution series of the binding protein under
investigation or of the reference anti-PF-4 antibody from about 10
ng/ml to about 10000 ng/ml (final concentration); after another
washing step the plate is contacted with, e.g. 1:10 diluted, human
or cynomolgus plasma, e.g. human plasma spiked with human PF-4,
followed by detection of the PF-4 bound to the plate, e.g. by means
of a primary PF-4 specific antibody, an enzyme-conjugated secondary
antibody and a colorimetric reaction.
[0107] According to one aspect of the invention, the cross-reaction
of A.beta. binding protein of the present invention to PF-4, when
analyzed via sandwich-ELISA with cynomolgus plasma as described
herein, is smaller than the corresponding cross-reaction of a
reference anti-PF-4 antibody, for example at least 2 times, at
least 5 times, at least 10 times, at least 20 times, or at least 30
times smaller; and/or, when analyzed via sandwich-ELISA with human
plasma as described herein, is smaller than the corresponding
cross-reaction of a reference anti-PF-4 antibody, for example or at
least 2 times, at least 5 times, at least 10 times, at least 15
times, or at least 20 times smaller.
[0108] According to another aspect of the invention, the
cross-reaction of A.beta. binding protein of the present invention
to PF-4, when analyzed via aligned sandwich-ELISA with cynomolgus
plasma as described herein, is smaller than the corresponding
cross-reaction of a reference anti-PF-4 antibody, for example at
least 2 times, at least 5 times, at least 10 times, at least 20
times, at least 30 times, at least 50 times, at least 80 times or
at least 115 times smaller; and/or, when analyzed via aligned
sandwich-ELISA with human plasma as described herein, is smaller
than the corresponding cross-reaction of a reference anti-PF-4
antibody, for example at least 2 times, at least 5 times, at least
10 times, at least 15 times, at least 20 times, at least 25 times
smaller.
[0109] According to another aspect of the invention, the
cross-reaction of A.beta. binding protein of the present invention
to PF-4, when analyzed via sandwich-ELISA and aligned
sandwich-ELISA with cynomolgus plasma as described herein, is
smaller than the corresponding cross-reaction of a reference
anti-PF-4 antibody, for example at least 2 times, at least 5 times,
at least 10 times, at least 20 times, or at least 30 times
smaller.
[0110] According to another aspect of the invention, the
cross-reaction of A.beta. binding protein of the present invention
to PF-4, when analyzed via sandwich-ELISA and aligned
sandwich-ELISA with human plasma as described herein, is smaller
than the corresponding cross-reaction of a reference anti-PF-4
antibody, for example at least 2 times, at least 5 times, at least
10 times, at least 20 times, or at least 30 times smaller.
[0111] According to another aspect of the invention, the
cross-reaction of A.beta. binding protein of the present invention
to PF-4, when analyzed via sandwich-ELISA and aligned
sandwich-ELISA with cynomolgus and human plasma as described
herein, is smaller than the corresponding cross-reaction of a
reference anti-PF-4 antibody, for example at least 2 times, at
least 5 times, at least 10 times, at least 20 times, or at least 30
times smaller.
[0112] The term "polypeptide" as used herein, refers to any
polymeric chain of amino acids. The terms "peptide" and "protein"
are used interchangeably with the term polypeptide and also refer
to a polymeric chain of amino acids. The term "polypeptide"
encompasses native or artificial proteins, protein fragments and
polypeptide analogs of a protein sequence. A polypeptide may be
monomeric or polymeric.
[0113] The term "isolated protein" or "isolated polypeptide" is a
protein or polypeptide that by virtue of its origin or source of
derivation is not associated with naturally associated components
that accompany it in its native state; is substantially free of
other proteins from the same species; is expressed by a cell from a
different species; or does not occur in nature. Thus, a polypeptide
that is chemically synthesized or synthesized in a cellular system
different from the cell from which it naturally originates will be
"isolated" from its naturally associated components. A protein may
also be rendered substantially free of naturally associated
components by isolation, using protein purification techniques well
known in the art.
[0114] The term "recovering", as used herein, refers to the process
of rendering a chemical species such as a polypeptide substantially
free of naturally associated components by isolation, e.g., using
protein purification techniques well known in the art.
[0115] The terms "specific binding" or "specifically binding", as
used herein, in reference to the interaction of an antibody, a
protein, or a peptide with a second chemical species, mean that the
interaction is dependent upon the presence of a particular
structure (e.g., an antigenic determinant or epitope) on the
chemical species; for example, an antibody recognizes and binds to
a specific protein structure rather than to proteins generally. If
an antibody is specific for epitope "A", the presence of a molecule
containing epitope A (or free, unlabeled A), in a reaction
containing labeled "A" and the antibody, will reduce the amount of
labeled A bound to the antibody.
[0116] The term "antibody", as used herein, broadly refers to any
immunoglobulin (Ig) molecule comprised of four polypeptide chains,
two heavy (H) chains and two light (L) chains, or any functional
fragment, mutant, variant, or derivation thereof, which retains the
essential epitope binding features of an Ig molecule. Such
functional fragment, mutant, variant, or derivative antibody
formats are known in the art. Nonlimiting embodiments of which are
discussed below. A "full-length antibody", as used herein, refers
to an Ig molecule comprising four polypeptide chains, two heavy
chains and two light chains. The chains are usually linked to one
another via disulfide bonds. Each heavy chain is comprised of a
heavy chain variable region (also referred to herein as "variable
heavy chain", or abbreviated herein as HCVR or VH) and a heavy
chain constant region. The heavy chain constant region is comprised
of three domains, CH1, CH2 and CH3. Each light chain is comprised
of a light chain variable region (also referred to herein as
"variable light chain", or abbreviated herein as LCVR or VL) and a
light chain constant region. The light chain constant region is
comprised of one domain, CL. The VH and VL regions can be further
subdivided into regions of hypervariability, termed complementarity
determining regions (CDR), interspersed with regions that are more
conserved, termed framework regions (FR). Each VH and VL is
composed of three CDRs and four FRs, arranged from amino-terminus
to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2,
FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (e.g.,
IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG2, IgG3,
IgG4, IgA1 and IgA2) or subclass.
[0117] The terms "antigen-binding portion" of an antibody (or
simply "antibody portion"), "antigen-binding moiety" of an antibody
(or simply "antibody moiety"), as used herein, refers to one or
more fragments of an antibody that retain the ability to
specifically bind to an antigen (e.g., A.beta.(20-42) globulomer),
i.e. are functional fragments of an antibody. It has been shown
that the antigen-binding function of an antibody can be performed
by one or more fragments of a full-length antibody. Such antibody
embodiments may also be bispecific, dual specific, or
multi-specific, specifically binding to two or more different
antigens. Examples of binding fragments encompassed within the term
"antigen-binding portion" of an antibody include (i) a Fab
fragment, a monovalent fragment consisting of the VL, VH, CL and
CH1 domains; (ii) a F(ab').sub.2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fd fragment consisting of the VH and CH1
domains; (iv) a Fv fragment consisting of the VL and VH domains of
a single arm of an antibody, (v) a dAb fragment (Ward et al.,
Nature 341: 544-546, 1989; Winter et al., WO 90/05144 A1, herein
incorporated by reference), which comprises a single variable
domain; and (vi) an isolated complementarity determining region
(CDR). Furthermore, although the two domains of the Fv fragment, VL
and VH, are coded for by separate genes, they can be joined, using
recombinant methods, by a synthetic linker that enables them to be
made as a single protein chain in which the VL and VH regions pair
to form monovalent molecules (known as single chain Fv (scFv); see
e.g., Bird et al., Science 242: 423-426, 1988; and Huston et al.,
Proc. Natl. Acad. Sci. USA 85: 5879-5883, 1988). Such single chain
antibodies are also encompassed within the term "antigen-binding
portion" of an antibody. Other forms of single chain antibodies,
such as diabodies, are also encompassed. Diabodies are bivalent,
bispecific antibodies in which VH and VL domains are expressed on a
single polypeptide chain, but using a linker that is too short to
allow for pairing between the two domains on the same chain,
thereby forcing the domains to pair with complementary domains of
another chain and creating two antigen binding sites (see e.g.,
Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448, 1993;
Poljak et al., Structure 2: 1121-1123, 1994). Such antibody binding
portions are known in the art (Kontermann and Dubel eds., Antibody
Engineering, Springer-Verlag. New York. 790 pp., 2001, ISBN
3-540-41354-5).
[0118] The term "antibody", as used herein, also comprises antibody
constructs. The term "antibody construct" as used herein refers to
a polypeptide comprising one or more of the antigen-binding
portions of the invention linked to a linker polypeptide or an
immunoglobulin constant domain. Linker polypeptides comprise two or
more amino acid residues joined by peptide bonds and are used to
link one or more antigen binding portions. Such linker polypeptides
are well known in the art (see e.g., Holliger et al., Proc. Natl.
Acad. Sci. USA 90: 6444-6448, 1993; Poljak et al., Structure 2:
1121-1123, 1994).
[0119] An immunoglobulin constant domain refers to a heavy or light
chain constant domain. Human IgG heavy chain and light chain
constant domain amino acid sequences are known in the art and
represented in Table 1.
TABLE-US-00004 TABLE 1 SEQUENCE OF HUMAN IgG HEAVY CHAIN CONSTANT
DOMAIN AND LIGHT CHAIN CONSTANT DOMAIN Sequence Sequence Protein
Identifier 123456789012345678901234567890 Ig SEQ ID
ASTKGPSVFFLAPSSKSTSGGTAALGCLVK gamma-1 NO: 25
DYFPEPVTVSWNSGALTSGVHTFPAVLQSS constant
GLYSLSSVVTVPSSSLGTQTYICNVNHKPS region
NTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKA
LPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK Ig
SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVK gamma-1 NO: 26
DYFPEPVTVSWNSGALTSGVHTFPAVLQSS constant
GLYSLSSVVTVPSSSLGTQTYICNVNHKPS region
NTKVDKKVEPKSCDKTHTCPPCPAPEAAGG mutant
PSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREE
MTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK Ig Kappa SEQ ID
TVAAPSVFIFPPSDEQLKSGTASVVCLLNN constant NO: 27
FYPREAKVQWKVDNALQSGNSQESVTEQDS region
KDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC Ig Lambda SEQ ID
QPKAAPSVTLFPPSSEELQANKATLVCLIS constant NO: 28
DFYPGAVTVAWKADSSPVKAGVETTTPSKQ region
SNNKYAASSYLSLTPEQWKSHRSYSCQVTH EGSTVEKTVAPTECS
[0120] Still further, a binding protein of the present invention
(e.g. an antibody) may be part of a larger immunoadhesion molecule,
formed by covalent or noncovalent association of the binding
protein of the invention with one or more other proteins or
peptides. Examples of such immunoadhesion molecules include the use
of the streptavidin core region to make a tetrameric scFv molecule
(Kipriyanov et al., Human Antibodies and Hybridomas 6: 93-101,
1995) and use of a cysteine residue, a marker peptide and a
C-terminal polyhistidine tag to make bivalent and biotinylated scFv
molecules (Kipriyanov et al., Mol. Immunol. 31: 1047-1058, 1994).
Antibody portions, such as Fab and F(ab').sub.2 fragments, can be
prepared from whole antibodies using conventional techniques, such
as papain or pepsin digestion, respectively, of whole antibodies.
Moreover, antibodies, antibody portions and immunoadhesion
molecules can be obtained using standard recombinant DNA
techniques, as described herein.
[0121] An "isolated antibody", as used herein, is intended to refer
to an antibody that is substantially free of other antibodies
having different antigenic specificities. An isolated antibody that
specifically binds A.beta.(20-42) globulomer may, however, have
cross-reactivity to other antigens, such as A.beta. globulomers,
e.g. A.beta.(12-42) globulomer or other A.beta. forms. Moreover, an
isolated antibody may be substantially free of other cellular
material and/or chemicals and/or any other targeted A.beta.
form.
[0122] The term "human antibody", as used herein, is intended to
include antibodies having variable and constant regions derived
from human germline immunoglobulin sequences. The human antibodies
of the invention may include amino acid residues not encoded by
human germline immunoglobulin sequences (e.g. mutations introduced
by random or site-specific mutagenesis in vitro or by somatic
mutation in vivo), for example in the CDRs and in particular in
CDR3. However, the term "human antibody", as used herein, is not
intended to include antibodies in which CDR sequences derived from
the germline of another mammalian species, such as a mouse, have
been grafted onto human framework sequences.
[0123] The term "recombinant human antibody", as used herein, is
intended to include all human antibodies that are prepared,
expressed, created or isolated by recombinant means, such as
antibodies expressed using a recombinant expression vector
transfected into a host cell (described further in Section B,
below), antibodies isolated from a recombinant, combinatorial human
antibody library (Hoogenboom, TIB Tech. 15: 62-70, 1997; Azzazy and
Highsmith, Clin. Biochem. 35: 425-445, 2002; Gavilondo J. V., and
Larrick J. W. (2002) BioTechniques 29:128-145; Hoogenboom H., and
Chames P. (2000) Immunology Today 21:371-378), antibodies isolated
from an animal (e.g. a mouse) that is transgenic for human
immunoglobulin genes (see e.g. Taylor, L. D., et al. (1992) Nucl.
Acids Res. 20:6287-6295; Kellermann S-A., and Green L. L. (2002)
Current Opinion in Biotechnology 13:593-597; Little M. et al (2000)
Immunology Today 21:364-370) or antibodies prepared, expressed,
created or isolated by any other means that involves splicing of
human immunoglobulin gene sequences to other DNA sequences. Such
recombinant human antibodies have variable and constant regions
derived from human germline immunoglobulin sequences. In certain
embodiments, however, such recombinant human antibodies are
subjected to in vitro mutagenesis (or, when an animal transgenic
for human Ig sequences is used, in vivo somatic mutagenesis) and
thus the amino acid sequences of the VH and VL regions of the
recombinant antibodies are sequences that, while derived from and
related to human germline VH and VL sequences, may not naturally
exist within the human antibody germline repertoire in vivo.
[0124] The term "chimeric antibody" refers to antibodies which
comprise heavy and light chain variable region sequences from one
species and constant region sequences from another species, such as
antibodies having murine heavy and light chain variable regions
linked to human constant regions.
[0125] The term "CDR-grafted antibody" refers to antibodies which
comprise heavy and light chain variable region sequences from one
species but in which the sequences of one or more of the CDR
regions of VH and/or VL are replaced with CDR sequences of another
species, such as antibodies having murine CDRs (e.g., CDR3) in
which one or more of the murine variable heavy and light chain
regions has been replaced with human variable heavy and light chain
sequences.
[0126] The terms "Kabat numbering", "Kabat definitions and "Kabat
labeling" are used interchangeably herein. These terms, which are
recognized in the art, refer to a system of numbering amino acid
residues which are more variable (i.e. hypervariable) than other
amino acid residues in the heavy and light chain variable regions
of an antibody, or an antigen binding portion thereof (Kabat et al.
(1971) Ann. NY Acad, Sci. 190:382-391 and, Kabat, E. A., et al.
(1991) Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, NIH
Publication No. 91-3242). For the heavy chain variable region, the
hypervariable region ranges from amino acid positions 31 to 35 for
CDR1, amino acid positions 50 to 65 for CDR2, and amino acid
positions 95 to 102 for CDR3. For the light chain variable region,
the hypervariable region ranges from amino acid positions 24 to 34
for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid
positions 89 to 97 for CDR3.
[0127] As used herein, the terms "acceptor" and "acceptor antibody"
refer to the antibody or nucleic acid sequence providing or
encoding at least 80%, at least 85%, at least 90%, at least 95%, at
least 98% or 100% of the amino acid sequences of one or more of the
framework regions. In some embodiments, the term "acceptor" refers
to the antibody amino acid or nucleic acid sequence providing or
encoding the constant region(s). In yet another embodiment, the
term "acceptor" refers to the antibody amino acid or nucleic acid
sequence providing or encoding one or more of the framework regions
and the constant region(s). In a specific embodiment, the term
"acceptor" refers to a human antibody amino acid or nucleic acid
sequence that provides or encodes at least 80%, for example at
least 85%, at least 90%, at least 95%, at least 98%, or 100% of the
amino acid sequences of one or more of the framework regions. In
accordance with this embodiment, an acceptor may contain at least
1, at least 2, at least 3, least 4, at least 5, or at least 10
amino acid residues that does (do) not occur at one or more
specific positions of a human antibody. An acceptor framework
region and/or acceptor constant region(s) may be, e.g., derived or
obtained from a germline antibody gene, a mature antibody gene, a
functional antibody (e.g., antibodies well-known in the art,
antibodies in development, or antibodies commercially
available).
[0128] As used herein, the term "CDR" refers to the complementarity
determining region within antibody variable sequences. There are
three CDRs in each of the variable regions of the heavy chain and
the light chain, which are designated CDR1, CDR2 and CDR3, for each
of the variable regions. The term "CDR set" as used herein refers
to a group of three CDRs that occur in a single variable region
capable of binding the antigen. The exact boundaries of these CDRs
have been defined differently according to different systems. The
system described by Kabat (Kabat et al., Sequences of Proteins of
Immunological Interest (National Institutes of Health, Bethesda,
Md. (1987) and (1991)) not only provides an unambiguous residue
numbering system applicable to any variable region of an antibody,
but also provides precise residue boundaries defining the three
CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and
coworkers (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987) and
Chothia et al., Nature 342:877-883 (1989)) found that certain
sub-portions within Kabat CDRs adopt nearly identical peptide
backbone conformations, despite having great diversity at the level
of amino acid sequence. These sub-portions were designated as L1,
L2 and L3 or H1, H2 and H3 where the "L" and the "H" designates the
light chain and the heavy chains regions, respectively. These
regions may be referred to as Chothia CDRs, which have boundaries
that overlap with Kabat CDRs. Other boundaries defining CDRs
overlapping with the Kabat CDRs have been described by Padlan
(FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45
(1996)). Still other CDR boundary definitions may not strictly
follow one of the above systems, but will nonetheless overlap with
the Kabat CDRs, although they may be shortened or lengthened in
light of prediction or experimental findings that particular
residues or groups of residues or even entire CDRs do not
significantly impact antigen binding. The methods used herein may
utilize CDRs defined according to any of these systems, particular
embodiments use Kabat or Chothia defined CDRs.
[0129] As used herein, the term "canonical" residue refers to a
residue in a CDR or framework that defines a particular canonical
CDR structure as defined by Chothia et al. (J. Mol. Biol.
196:901-907 (1987); Chothia et al., J. Mol. Biol. 227:799 (1992),
both are incorporated herein by reference). According to Chothia et
al., critical portions of the CDRs of many antibodies have nearly
identical peptide backbone confirmations despite great diversity at
the level of amino acid sequence. Each canonical structure
specifies primarily a set of peptide backbone torsion angles for a
contiguous segment of amino acid residues forming a loop.
[0130] As used herein, the terms "donor" and "donor antibody" refer
to an antibody providing one or more CDRs. In one embodiment, the
donor antibody is an antibody from a species different from the
antibody from which the framework regions are obtained or derived.
In the context of a humanized antibody, the term "donor antibody"
refers to a non-human antibody providing one or more CDRs.
[0131] As used herein, the term "framework" or "framework sequence"
refers to the remaining sequences of a variable region minus the
CDRs. Because the exact definition of a CDR sequence can be
determined by different systems, the meaning of a framework
sequence is subject to correspondingly different interpretations.
The six CDRs (CDR-L1, -L2, and -L3 of light chain and CDR-H1, -H2,
and -H3 of heavy chain) also divide the framework regions on the
light chain and the heavy chain into four sub-regions (FR1, FR2,
FR3 and FR4) on each chain, in which CDR1 is positioned between FR1
and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4.
Without specifying the particular sub-regions as FR1, FR2, FR3 or
FR4, a framework region, as referred by others, represents the
combined FR's within the variable region of a single, naturally
occurring immunoglobulin chain. As used herein, a FR represents one
of the four sub-regions, and FRs represents two or more of the four
sub-regions constituting a framework region.
[0132] Human heavy chain and light chain acceptor sequences are
known in the art. In one embodiment of the invention, the human
heavy chain and light chain acceptor sequences are selected from
the sequences described in Table 2 and Table 3. In another
embodiment, the human heavy chain and light chain acceptor
sequences are selected from sequences which are at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identical
to the sequences described in Table 2 and Table 3.
TABLE-US-00005 TABLE 2 HEAVY CHAIN ACCEPTOR SEQUENCES SEQ Protein
Sequence ID NO region 123456789012345678901234567890 34 VH3_53/
EVQLVESGGGLIQPGGSLRLSCAASGFTVS JH6 FR1 35 VH3_53/ WVRQAPGKGLEWVS
JH6 FR2 36 VH3_53/ RFTISRDNSKNTLYLQMNSLRAEDTAVYYC JH6 FR3 AR 37
VH3_53/ WGQGTTVTVSS JH6 FR4 38 VH4_59/
QVQLQESGPGLVKPSETLSLTCTVSGGSIS JH6 FR1 39 VH4_59/ WIRQPPGKGLEWIG
JH6 FR2 40 VH4_59/ RVTISVDTSKNQFSLKLSSVTAADTAVYYC JH6 FR3 AR 41
VH4_59/ WGQGTTVTVSS JH6 FR4
TABLE-US-00006 TABLE 3 LIGHT CHAIN ACCEPTOR SEQUENCES SEQ Protein
Sequence ID NO region 123456789012345678901234567890 42 A1/2-30/
DVVMTQSPLSLPVTLGQPASISC J.kappa.2 FR1 43 A1/2-30/ WFQQRPGQSPRRLIY
J.kappa.2 FR2 44 A1/2-30/ GVPDRFSGSGSGTDFTLKISRVEAEDVGVY J.kappa.2
FR3 YC 45 A1/2-30/ FGQGTKLEIKR J.kappa.2 FR4
[0133] As used herein, the term "germline antibody gene" or "gene
fragment" refers to an immunoglobulin sequence encoded by
non-lymphoid cells that have not undergone the maturation process
that leads to genetic rearrangement and mutation for expression of
a particular immunoglobulin. (See, e.g., Shapiro et al., Crit. Rev.
Immunol. 22(3): 183-200 (2002); Marchalonis et al., Adv Exp Med
Biol. 484:13-30 (2001)). One of the advantages provided by various
embodiments of the present invention stems from the recognition
that germline antibody genes are more likely than mature antibody
genes to conserve essential amino acid sequence structures
characteristic of individuals in the species, hence less likely to
be recognized as from a foreign source when used therapeutically in
that species.
[0134] As used herein, the term "key" residues refer to certain
residues within the variable region that have more impact on the
binding specificity and/or affinity of an antibody, in particular a
humanized antibody. A key residue includes, but is not limited to,
one or more of the following: a residue that is adjacent to a CDR,
a potential glycosylation site (can be either N- or O-glycosylation
site), a rare residue, a residue capable of interacting with the
antigen, a residue capable of interacting with a CDR, a canonical
residue, a contact residue between heavy chain variable region and
light chain variable region, a residue within the Vernier zone, and
a residue in the region that overlaps between the Chothia
definition of a variable heavy chain CDR1 and the Kabat definition
of the first heavy chain framework.
[0135] As used herein, the term "humanized antibody" is an antibody
or a variant, derivative, analog or portion thereof which
immunospecifically binds to an antigen of interest and which
comprises a framework (FR) region having substantially the amino
acid sequence of a human antibody and a complementary determining
region (CDR) having substantially the amino acid sequence of a
non-human antibody. As used herein, the term "substantially" in the
context of a CDR refers to a CDR having an amino acid sequence at
least 90%, at least 95%, at least 98% or at least 99% identical to
the amino acid sequence of a non-human antibody CDR. A humanized
antibody comprises substantially all of at least one, and typically
two, variable domains (Fab, Fab', F(ab').sub.2, FabC, Fv) in which
all or substantially all of the CDR regions correspond to those of
a non-human immunoglobulin (i.e., donor antibody) and all or
substantially all of the framework regions are those of a human
immunoglobulin consensus sequence. According to one aspect, a
humanized antibody also comprises at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. In some embodiments, a humanized antibody contains
both the light chain as well as at least the variable domain of a
heavy chain. The antibody also may include the CH1, hinge, CH2,
CH3, and CH4 regions of the heavy chain. In some embodiments, a
humanized antibody only contains a humanized light chain. In some
embodiments, a humanized antibody only contains a humanized heavy
chain. In specific embodiments, a humanized antibody only contains
a humanized variable domain of a light chain and/or of a heavy
chain.
[0136] The humanized antibody can be selected from any class of
immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any
isotype, including without limitation IgG 1, IgG2, IgG3 and IgG4.
The humanized antibody may comprise sequences from more than one
class or isotype, and particular constant domains may be selected
to optimize desired effector functions using techniques well-known
in the art.
[0137] The framework and CDR regions of a humanized antibody need
not correspond precisely to the parental sequences, e.g., the donor
antibody CDR or the consensus framework may be mutagenized by
substitution, insertion and/or deletion of at least one amino acid
residue so that the CDR or framework residue at that site does not
correspond to either the donor antibody or the consensus framework.
In one embodiment, such mutations, however, will not be extensive.
Usually, at least 90%, at least 95%, at least 98%, or at least 99%
of the humanized antibody residues will correspond to those of the
parental FR and CDR sequences. As used herein, the term "consensus
framework" refers to the framework region in the consensus
immunoglobulin sequence. As used herein, the term "consensus
immunoglobulin sequence" refers to the sequence formed from the
most frequently occurring amino acids (or nucleotides) in a family
of related immunoglobulin sequences (See e.g., Winnaker, From Genes
to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In a
family of immunoglobulins, each position in the consensus sequence
is occupied by the amino acid occurring most frequently at that
position in the family. If two amino acids occur equally
frequently, either can be included in the consensus sequence.
[0138] As used herein, "Vernier" zone refers to a subset of
framework residues that may adjust CDR structure and fine-tune the
fit to antigen as described by Foote and Winter (1992, J. Mol.
Biol. 224:487-499, which is incorporated herein by reference).
Vernier zone residues form a layer underlying the CDRs and may
impact on the structure of CDRs and the affinity of the
antibody.
[0139] The term "antibody", as used herein, also comprises
multivalent binding proteins. The term "multivalent binding
protein" is used in this specification to denote a binding protein
comprising two or more antigen binding sites. The multivalent
binding protein is engineered to have the three or more antigen
binding sites, and is generally not a naturally occurring antibody.
The term "multispecific binding protein" refers to a binding
protein capable of binding two or more related or unrelated
targets. Dual variable domain (DVD) binding proteins as used
herein, are binding proteins that comprise two or more antigen
binding sites and are tetravalent or multivalent binding proteins.
Such DVDs may be monospecific, i.e. capable of binding one antigen
or multispecific, i.e. capable of binding two or more antigens. DVD
binding proteins comprising two heavy chain DVD polypeptides and
two light chain DVD polypeptides are referred to a DVD Ig. Each
half of a DVD Ig comprises a heavy chain DVD polypeptide, and a
light chain DVD polypeptide, and two antigen binding sites. Each
binding site comprises a heavy chain variable domain and a light
chain variable domain with a total of 6 CDRs involved in antigen
binding per antigen binding site. DVD binding proteins and methods
of making DVD binding proteins are disclosed in U.S. patent
application Ser. No. 11/507,050 and incorporated herein by
reference.
[0140] The term "epitope" includes any polypeptide determinant
capable of specific binding to an immunoglobulin or T-cell
receptor. In certain embodiments, epitope determinants include
chemically active surface groupings of molecules such as amino
acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain
embodiments, may have specific three dimensional structural
characteristics, and/or specific charge characteristics. An epitope
is a region of an antigen that is bound by a binding protein, in
particular by an antibody. In certain embodiments, a binding
protein or an antibody is said to specifically bind an antigen when
it preferentially recognizes its target antigen in a complex
mixture of proteins and/or macromolecules.
[0141] The binding affinities of the antibodies of the invention
may be evaluated by using standardized in-vitro immunoassays such
as ELISA, dot blot or BIAcore analyses (Pharmacia Biosensor AB,
Uppsala, Sweden and Piscataway, N.J.). For further descriptions,
see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson,
U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., et al.
(1995) J. Mol. Recognit. 8:125-131; and Johnsson, B., et al. (1991)
Anal. Biochem. 198:268-277.
[0142] According to a particular embodiment, the affinities defined
herein refer to the values obtained by performing a dot blot and
evaluating it by densitometry. According to a particular embodiment
of the invention, determining the binding affinity by dot blot
comprises the following: a certain amount of the antigen (e.g. the
A.beta.(X-Y) globulomer, A.beta.(X-Y) monomer or A.beta.(X-Y)
fibrils, as defined above) or, expediently, an appropriate dilution
thereof, for instance in 20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH
7.4, 0.2 mg/ml BSA to an antigen concentration of, for example, 100
pmol/.mu.l, 10 pmol/.mu.l, 1 pmol/.mu.l, 0.1 pmol/.mu.l and 0.01
pmol/.mu.l, is dotted onto a nitrocellulose membrane, the membrane
is then blocked with milk to prevent unspecific binding and washed,
then contacted with the antibody of interest followed by detection
of the latter by means of an enzyme-conjugated secondary antibody
and a colorimetric reaction; at defined antibody concentrations,
the amount of antibody bound allows affinity determination. Thus
the relative affinity of two different antibodies to one target, or
of one antibody to two different targets, is here defined as the
relation of the respective amounts of target-bound antibody
observed with the two antibody-target combinations under otherwise
identical dot blot conditions. Unlike a similar approach based on
Western blotting, the dot blot approach will determine an
antibody's affinity to a given target in the latter's natural
conformation; unlike the ELISA approach, the dot blot approach does
not suffer from differences in the affinities between different
targets and the matrix, thereby allowing for more precise
comparisons between different targets.
[0143] The term "surface plasmon resonance", as used herein, refers
to an optical phenomenon that allows for the analysis of real-time
biospecific interactions by detection of alterations in protein
concentrations within a biosensor matrix, for example using the
BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and
Piscataway, N.J.). For further descriptions, see Jonsson, U., et
al. (1993) Ann. Biol. Clin., 51: 19-26; Jonsson et al., (1991)
BioTechniques, 11: 620-627; Johnsson et al., (1995) J. Mol.
Recognit., 8: 125-131; and Johnnson et al. (1991) Anal. Biochem.,
198: 268-277.
[0144] The term "k.sub.on" (also "Kon", "kon", "K.sub.on"), as used
herein, is intended to refer to the on-rate constant for
association of a binding protein (e.g., an antibody) to an antigen
to form an association complex, e.g., antibody/antigen complex, as
is known in the art. The "k.sub.on" also is known by the terms
"association rate constant", or "ka", as used interchangeably
herein. This value indicates the binding rate of a binding protein
(e.g., an antibody) to its target antigen or the rate of complex
formation between a binding protein (e.g., an antibody) and antigen
as is shown by the equation below:
Antibody ("Ab")+Antigen ("Ag").fwdarw.Ab-Ag.
[0145] The term "k.sub.off" (also "Koff", "koff", "K.sub.off"), as
used herein, is intended to refer to the off rate constant for
dissociation, or "dissociation rate constant", of a binding protein
(e.g., an antibody) from an association complex (e.g., an
antibody/antigen complex) as is known in the art. This value
indicates the dissociation rate of a binding protein (e.g., an
antibody) from its target antigen, or separation of the Ab-Ag
complex over time into free antibody and antigen as shown by the
equation below:
Ab+Ag.rarw.Ab-Ag.
[0146] The term "K.sub.D" (also "K.sub.a" or "KD"), as used herein,
is intended to refer to the "equilibrium dissociation constant",
and refers to the value obtained in a titration measurement at
equilibrium, or by dividing the dissociation rate constant
(k.sub.off) by the association rate constant (k.sub.on). The
association rate constant (k.sub.on), the dissociation rate
constant (k.sub.off), and the equilibrium dissociation constant
(K.sub.D) are used to represent the binding affinity of a binding
protein (e.g., an antibody) to an antigen. Methods for determining
association and dissociation rate constants are well known in the
art. Using fluorescence-based techniques offers high sensitivity
and the ability to examine samples in physiological buffers at
equilibrium. Other experimental approaches and instruments such as
a BIAcore.RTM. (biomolecular interaction analysis) assay can be
used (e.g., instrument available from BIAcore International
A.beta., a GE Healthcare company, Uppsala, Sweden). Additionally, a
KinExA.RTM. (Kinetic Exclusion Assay) assay, available from
Sapidyne Instruments (Boise, Id.) can also be used.
[0147] The term "labeled binding protein", as used herein, refers
to a binding protein with a label incorporated that provides for
the identification of the binding protein. Likewise, the term
"labeled antibody" as used herein, refers to an antibody with a
label incorporated that provides for the identification of the
antibody. In one aspect, the label is a detectable marker, e.g.,
incorporation of a radiolabeled amino acid or attachment to a
polypeptide of biotinyl moieties that can be detected by marked
avidin (e.g., streptavidin containing a fluorescent marker or
enzymatic activity that can be detected by optical or colorimetric
methods). Examples of labels for polypeptides include, but are not
limited to, the following: radioisotopes or radionuclides (e.g.,
.sup.3H, .sup.14C, .sup.35S, .sup.90Y, .sup.99Tc, .sup.111In,
.sup.125I, .sup.131I, .sup.177Lu, .sup.166Ho, or .sup.153Sm);
fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors),
enzymatic labels (e.g., horseradish peroxidase, luciferase,
alkaline phosphatase); chemiluminescent markers; biotinyl groups;
predetermined polypeptide epitopes recognized by a secondary
reporter (e.g., leucine zipper pair sequences, binding sites for
secondary antibodies, metal binding domains, epitope tags); and
magnetic agents, such as gadolinium chelates.
[0148] The term "antibody", as used herein, also comprises antibody
conjugates. The term "antibody conjugate" refers to a binding
protein, such as an antibody, chemically linked to a second
chemical moiety, such as a therapeutic agent.
[0149] The term "therapeutic agent" is used herein to denote a
chemical compound, a mixture of chemical compounds, a biological
macromolecule, or an extract made from biological materials that is
a "cognitive enhancing drug," which is a drug that improves
impaired human cognitive abilities of the brain (namely, thinking,
learning, and memory). Cognitive enhancing drugs work by altering
the availability of neurochemicals (e.g., neurotransmitters,
enzymes, and hormones), by improving oxygen supply, by stimulating
nerve growth, or by inhibiting nerve damage. Examples of cognitive
enhancing drugs include a compound that increases the activity of
acetylcholine such as, but not limited to, an acetylcholine
receptor agonist (e.g., a nicotinic .alpha.-7 receptor agonist or
allosteric modulator, an .alpha.4.beta.2 nicotinic receptor agonist
or allosteric modulators), an acetylcholinesterase inhibitor (e.g.,
donepezil, rivastigmine, and galantamine), a butyrylcholinesterase
inhibitor, an N-methyl-D-aspartate (NMDA) receptor antagonist
(e.g., memantine), an activity-dependent neuroprotective protein
(ADNP) agonist, a serotonin 5-HT1A receptor agonist (e.g.,
xaliproden), a 5-HT.sub.4 receptor agonist, a 5-HT.sub.6 receptor
antagonist, a serotonin 1A receptor antagonist, a histamine H.sub.3
receptor antagonist, a calpain inhibitor, a vascular endothelial
growth factor (VEGF) protein or agonist, a trophic growth factor,
an anti-apoptotic compound, an AMPA-type glutamate receptor
activator, a L-type or N-type calcium channel blocker or modulator,
a potassium channel blocker, a hypoxia inducible factor (HIF)
activator, a HIF prolyl 4-hydroxylase inhibitor, an
anti-inflammatory agent, an inhibitor of amyloid A.beta. peptide or
amyloid plaque, an inhibitor of tau hyperphosphorylation, a
phosphodiesterase 5 inhibitor (e.g., tadalafil, sildenafil), a
phosphodiesterase 4 inhibitor, a monoamine oxidase inhibitor, or
pharmaceutically acceptable salt thereof. Specific examples of such
cognitive enhancing drugs include, but are not limited to,
cholinesterase inhibitors such as donepezil (Aricept.RTM.),
rivastigmine (Exelon.RTM.), galanthamine (Reminyl.RTM.),
N-methyl-D-aspartate antagonists such as memantine
(Namenda.RTM.).
[0150] The terms "crystal" and "crystallized", as used herein,
refer to a binding protein (e.g., an antibody, or antigen binding
portion thereof), that exists in the form of a crystal. Crystals
are one form of the solid state of matter, which is distinct from
other forms such as the amorphous solid state or the liquid
crystalline state. Crystals are composed of regular, repeating,
three-dimensional arrays of atoms, ions, molecules (e.g., proteins
such as antibodies), or molecular assemblies (e.g.,
antigen/antibody complexes). These three-dimensional arrays are
arranged according to specific mathematical relationships that are
well-understood in the field. The fundamental unit, or building
block, that is repeated in a crystal is called the asymmetric unit.
Repetition of the asymmetric unit in an arrangement that conforms
to a given, well-defined crystallographic symmetry provides the
"unit cell" of the crystal. Repetition of the unit cell by regular
translations in all three dimensions provides the crystal. See
Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids
and Proteins, a Practical Approach, 2.sup.nd ed., pp. 20 1-16,
Oxford University Press, New York, N.Y., (1999)."
[0151] As used herein, the term "neutralizing" refers to
neutralization of biological activity of a targeted A.beta. form
when a binding protein specifically binds said A.beta. form. For
example, a neutralizing binding protein is a neutralizing antibody
whose binding to the A.beta.(20-42) amino acid region of the
globulomer (and/or any other targeted A.beta. form) results in
inhibition of a biological activity of the globulomer. According to
one aspect of the invention, the neutralizing binding protein binds
to the A.beta.(20-42) region of the globulomer (and/or any other
targeted A.beta. form), and reduces a biologically activity of the
targeted A.beta. form by at least about 20%, 40%, 60%, 80%, 85% or
more. Inhibition of a biological activity of the targeted A.beta.
form by a neutralizing binding protein can be assessed by measuring
one or more indicators of the targeted A.beta. form biological
activity well known in the art, for example interaction (e.g.
binding) of the targeted A.beta. form to a P/Q type voltage-gated
presynaptic calcium channel, inhibition of P/Q type voltage-gated
presynaptic calcium channel activity, Ca.sup.++ flux through P/Q
type voltage-gated presynaptic calcium channel, local (e.g.
intracellular) Ca.sup.++ concentration, synaptic activity.
[0152] The term "activity" includes activities such as the binding
specificity/affinity of a binding protein, in particular of an
antibody, for an antigen, for example an A.beta.(20-42) globulomer
(and any other targeted A.beta. form); and/or the neutralizing
potency of an antibody, for example an antibody whose binding to a
targeted A.beta. form inhibits the biological activity of the
targeted A.beta. form. Said biological activity of the targeted
A.beta. form comprises interaction of the A.beta. form to P/Q type
voltage-gated presynaptic calcium channels, which results in
inhibition of the activity of said calcium channels.
[0153] The subject invention also provides isolated nucleotide
sequences encoding the binding proteins of the present invention.
The present invention also provides those nucleotide sequences (or
fragments thereof) having sequences comprising, corresponding to,
identical to, hybridizable to, or complementary to, at least about
70% (e.g., 70% 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% or 79%), at
least about 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%
or 89%), or at least about 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100%) identity to these encoding nucleotide
sequences. (All integers (and portions thereof) between and
including 70% and 100% are considered to be within the scope of the
present invention with respect to percent identity.) Such sequences
may be derived from any source (e.g., either isolated from a
natural source, produced via a semi-synthetic route, or synthesized
de novo). In particular, such sequences may be isolated or derived
from sources other than described in the examples (e.g., bacteria,
fungus, algae, mouse or human).
[0154] For purposes of the present invention, a "fragment" of a
nucleotide sequence is defined as a contiguous sequence of
approximately at least 6, e.g. at least about 8, at least about 10
nucleotides, or at least about 15 nucleotides, corresponding to a
region of the specified nucleotide sequence.
[0155] The term "identity" refers to the relatedness of two
sequences on a nucleotide-by-nucleotide basis over a particular
comparison window or segment. Thus, identity is defined as the
degree of sameness, correspondence or equivalence between the same
strands (either sense or antisense) of two DNA segments (or two
amino acid sequences). "Percentage of sequence identity" is
calculated by comparing two optimally aligned sequences over a
particular region, determining the number of positions at which the
identical base or amino acid occurs in both sequences in order to
yield the number of matched positions, dividing the number of such
positions by the total number of positions in the segment being
compared and multiplying the result by 100. Optimal alignment of
sequences may be conducted by the algorithm of Smith &
Waterman, Appl. Math. 2: 482, 1981, by the algorithm of Needleman
& Wunsch, J. Mol. Biol. 48: 443, 1970, by the method of Pearson
& Lipman, Proc. Natl. Acad. Sci. (USA) 85: 2444, 1988, and by
computer programs which implement the relevant algorithms (e.g.,
Clustal Macaw Pileup (Higgins et al., CABIOS. 5L151-153, 1989),
FASTDB (Intelligenetics), BLAST (National Center for Biomedical
Information; Altschul et al., Nucleic Acids Research 25: 3389-3402,
1997), PILEUP (Genetics Computer Group, Madison, Wis.) or GAP,
BESTFIT, FASTA and TFASTA (Wisconsin Genetics Software Package
Release 7.0, Genetics Computer Group, Madison, Wis.)). (See U.S.
Pat. No. 5,912,120.)
[0156] For purposes of the present invention, "complementarity" is
defined as the degree of relatedness between two DNA segments. It
is determined by measuring the ability of the sense strand of one
DNA segment to hybridize with the anti-sense strand of the other
DNA segment, under appropriate conditions, to form a double helix.
A "complement" is defined as a sequence which pairs to a given
sequence based upon the canonic base-pairing rules. For example, a
sequence A-G-T in one nucleotide strand is "complementary" to T-C-A
in the other strand. In the double helix, adenine appears in one
strand, thymine appears in the other strand. Similarly, wherever
guanine is found in one strand, cytosine is found in the other. The
greater the relatedness between the nucleotide sequences of two DNA
segments, the greater the ability to form hybrid duplexes between
the strands of the two DNA segments.
[0157] "Similarity" between two amino acid sequences is defined as
the presence of a series of identical as well as conserved amino
acid residues in both sequences. The higher the degree of
similarity between two amino acid sequences, the higher the
correspondence, sameness or equivalence of the two sequences.
("Identity between two amino acid sequences is defined as the
presence of a series of exactly alike or invariant amino acid
residues in both sequences.) The definitions of "complementarity",
"identity" and "similarity" are well known to those of ordinary
skill in the art.
[0158] "Encoded by" refers to a nucleic acid sequence which codes
for a polypeptide sequence, wherein the polypeptide sequence or a
portion thereof contains an amino acid sequence of at least 3 amino
acids, e.g. at least 8 amino acids, or at least 15 amino acids,
from a polypeptide encoded by the nucleic acid sequence.
[0159] The term "polynucleotide" as referred to herein, means a
polymeric form of two or more nucleotides, either ribonucleotides
or deoxynucleotides or a modified form of either type of
nucleotide. The term includes single and double stranded forms of
DNA, but preferably is double-stranded DNA.
[0160] The term "isolated polynucleotide" as used herein shall mean
a polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or
some combination thereof) that, by virtue of its origin, the
"isolated polynucleotide": is not associated with all or a portion
of a polynucleotide with which the "isolated polynucleotide" is
found in nature; is operably linked to a polynucleotide that it is
not linked to in nature; or does not occur in nature as part of a
larger sequence.
[0161] The term "vector", as used herein, is intended to refer to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked. One type of vector is a "plasmid",
which refers to a circular double stranded DNA loop into which
additional DNA segments may be ligated. Another type of vector is a
viral vector, wherein additional DNA segments may be ligated into
the viral genome. Certain vectors are capable of autonomous
replication in a host cell into which they are introduced (e.g.,
bacterial vectors having a bacterial origin of replication and
episomal mammalian vectors). Other vectors (e.g., non-episomal
mammalian vectors) can be integrated into the genome of a host cell
upon introduction into the host cell, and thereby are replicated
along with the host genome. Moreover, certain vectors are capable
of directing the expression of genes to which they are operatively
linked. Such vectors are referred to herein as "recombinant
expression vectors" (or simply, "expression vectors"). In general,
expression vectors of utility in recombinant DNA techniques are
often in the form of plasmids. In the present specification,
"plasmid" and "vector" may be used interchangeably as the plasmid
is the most commonly used form of vector. However, the invention is
intended to include such other forms of expression vectors, such as
viral vectors (e.g., replication defective retroviruses,
adenoviruses and adeno-associated viruses), which serve equivalent
functions.
[0162] The term "operably linked" refers to a juxtaposition wherein
the components described are in a relationship permitting them to
function in their intended manner. A control sequence "operably
linked" to a coding sequence is ligated in such a way that
expression of the coding sequence is achieved under conditions
compatible with the control sequences. "Operably linked" sequences
include both expression control sequences that are contiguous with
the gene of interest and expression control sequences that act in
trans or at a distance to control the gene of interest. The term
"expression control sequence" as used herein refers to
polynucleotide sequences which are necessary to effect the
expression and processing of coding sequences to which they are
ligated. Expression control sequences include appropriate
transcription initiation, termination, promoter and enhancer
sequences; efficient RNA processing signals such as splicing and
polyadenylation signals; sequences that stabilize cytoplasmic mRNA;
sequences that enhance translation efficiency (i.e., Kozak
consensus sequence); sequences that enhance protein stability; and
when desired, sequences that enhance protein secretion. The nature
of such control sequences differs depending upon the host organism;
in prokaryotes, such control sequences generally include promoter,
ribosomal binding site, and transcription termination sequence; in
eukaryotes, generally, such control sequences include promoters and
transcription termination sequence. The term "control sequences" is
intended to include components whose presence is essential for
expression and processing, and can also include additional
components whose presence is advantageous, for example, leader
sequences and fusion partner sequences.
[0163] "Transformation", as defined herein, refers to any process
by which exogenous DNA enters a host cell. Transformation may occur
under natural or artificial conditions using various methods well
known in the art. Transformation may rely on any known method for
the insertion of foreign nucleic acid sequences into a prokaryotic
or eukaryotic host cell. The method is selected based on the host
cell being transformed and may include, but is not limited to,
viral infection, electroporation, lipofection, and particle
bombardment. Such "transformed" cells include stably transformed
cells in which the inserted DNA is capable of replication either as
an autonomously replicating plasmid or as part of the host
chromosome. They also include cells which transiently express the
inserted DNA or RNA for limited periods of time.
[0164] The term "recombinant host cell" (or simply "host cell"), as
used herein, is intended to refer to a cell into which exogenous
DNA has been introduced. It should be understood that such terms
are intended to refer not only to the particular subject cell, but,
to the progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term "host cell" as used herein. In one aspect, host
cells include prokaryotic and eukaryotic cells selected from any of
the kingdoms of life. Eukaryotic cells include protist, fungal,
plant and animal cells. In another aspect host cells include, but
are not limited to, the prokaryotic cell line E. coli; mammalian
cell lines CHO, HEK 293 and COS; the insect cell line Sf9; and the
fungal cell Saccharomyces cerevisiae.
[0165] Standard techniques may be used for recombinant DNA,
oligonucleotide synthesis, and tissue culture and transformation
(e.g., electroporation, lipofection). Enzymatic reactions and
purification techniques may be performed according to
manufacturer's specifications or as commonly accomplished in the
art or as described herein. The foregoing techniques and procedures
may be generally performed according to conventional methods well
known in the art and as described in various general and more
specific references that are cited and discussed throughout the
present specification. See e.g., Sambrook et al. Molecular Cloning:
A Laboratory Manual (2.sup.nd ed., Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated
herein by reference for any purpose.
[0166] "Transgenic organism", as known in the art and as used
herein, refers to an organism having cells that contain a
transgene, wherein the transgene introduced into the organism (or
an ancestor of the organism) expresses a polypeptide not naturally
expressed in the organism. A "transgene" is a DNA construct, which
is stably and operably integrated into the genome of a cell from
which a transgenic organism develops, directing the expression of
an encoded gene product in one or more cell types or tissues of the
transgenic organism.
[0167] The terms "regulate" and "modulate" are used
interchangeably, and, as used herein, refer to a change or an
alteration in the activity of a molecule of interest (e.g., the
biological activity of a targeted A.beta. form). Modulation may be
an increase or a decrease in the magnitude of a certain activity or
function of the molecule of interest. Exemplary activities and
functions of a molecule include, but are not limited to, binding
characteristics, enzymatic activity, cell receptor activation, and
signal transduction.
[0168] Correspondingly, the term "modulator," as used herein, is a
compound capable of changing or altering an activity or function of
a molecule of interest (e.g., the biological activity of a targeted
A.beta. form). For example, a modulator may cause an increase or
decrease in the magnitude of a certain activity or function of a
molecule compared to the magnitude of the activity or function
observed in the absence of the modulator. In certain embodiments, a
modulator is an inhibitor, which decreases the magnitude of at
least one activity or function of a molecule.
[0169] The term "agonist", as used herein, refers to a modulator
that, when contacted with a molecule of interest, causes an
increase in the magnitude of a certain activity or function of the
molecule compared to the magnitude of the activity or function
observed in the absence of the agonist.
[0170] The term "antagonist" or "inhibitor", as used herein, refer
to a modulator that, when contacted with a molecule of interest
causes a decrease in the magnitude of a certain activity or
function of the molecule compared to the magnitude of the activity
or function observed in the absence of the antagonist. Particular
antagonists of interest include those that block or modulate the
biological activity of a targeted A.beta. form. Antagonists and
inhibitors of a targeted A.beta. form may include, but are not
limited to, the binding proteins of the invention, which bind to
A.beta.(20-42) globulomer and any other targeted A.beta. form. An
antagonist or inhibitor of a targeted A.beta. form may, for
example, reduce the inhibitory effect of said A.beta. form on the
activity of a P/Q type voltage-gated presynaptic calcium
channel.
[0171] As used herein, the term "effective amount" refers to the
amount of a therapy which is sufficient to reduce or ameliorate the
severity and/or duration of a disorder or one or more symptoms
thereof, prevent the advancement of a disorder, cause regression of
a disorder, prevent the recurrence, development, onset or
progression of one or more symptoms associated with a disorder,
detect a disorder, or enhance or improve the prophylactic or
therapeutic effect(s) of another therapy (e.g., prophylactic or
therapeutic agent).
[0172] The term "sample", as used herein, is used in its broadest
sense. A "biological sample", as used herein, includes, but is not
limited to, any quantity of a substance from a living thing or
formerly living thing. Such living things include, but are not
limited to, humans, mice, rats, monkeys, dogs, rabbits and other
animals. Such substances include, but are not limited to, blood,
serum, urine, synovial fluid, cells, organs, tissues, bone marrow,
lymph nodes and spleen.
[0173] I. Antibodies of the Invention
[0174] A first particular aspect of the invention provides CDR
grafted antibodies, or antigen-binding portions thereof, that bind
A.beta.(20-42) globulomer and/or any other targeted A.beta. form. A
second particular aspect of the invention provides humanized
antibodies, or antigen-binding portions thereof, that bind
A.beta.(20-42) globulomer and/or any other targeted A.beta. form.
According to one particular aspect, the antibodies, or portions
thereof, are isolated antibodies. According to a further particular
aspect, the antibodies of the invention neutralize an activity of
A.beta.(20-42) globulomer and/or of any other targeted A.beta.
form.
[0175] A. Method of Making Anti-A.beta.(20-42) Globulomer
Antibodies
[0176] Antibodies of the present invention may be made by any of a
number of techniques known in the art.
[0177] 1. Anti-A.beta.(20-42) Globulomer Monoclonal Antibodies
Using Hybridoma Technology
[0178] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et
al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681
(Elsevier, N.Y., 1981) (said references incorporated by reference
in their entireties). The term "monoclonal antibody" as used herein
is not limited to antibodies produced through hybridoma technology.
The term "monoclonal antibody" refers to an antibody that is
derived from a single clone, including any eukaryotic, prokaryotic,
or phage clone, and not the method by which it is produced.
[0179] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art.
In one embodiment, the present invention provides methods of
generating monoclonal antibodies as well as antibodies produced by
the method comprising culturing a hybridoma cell secreting an
antibody of the invention wherein, e.g., the hybridoma is generated
by fusing splenocytes isolated from a mouse immunized with an
antigen of the invention with myeloma cells and then screening the
hybridomas resulting from the fusion for hybridoma clones that
secrete an antibody able to bind a polypeptide of the invention.
Briefly, mice can be immunized with an A.beta.(20-42) globulomer
antigen. In a particular embodiment, the antigen is administered
with a adjuvant to stimulate the immune response. Such adjuvants
include complete or incomplete Freund's adjuvant, RIBI (muramyl
dipeptides) or ISCOM (immunostimulating complexes). Such adjuvants
may protect the polypeptide from rapid dispersal by sequestering it
in a local deposit, or they may contain substances that stimulate
the host to secrete factors that are chemotactic for macrophages
and other components of the immune system. Preferably, if a
polypeptide is being administered, the immunization schedule will
involve two or more administrations of the polypeptide, spread out
over several weeks.
[0180] After immunization of an animal with an A.beta.(20-42)
globulomer antigen, antibodies and/or antibody-producing cells may
be obtained from the animal. An anti-A.beta.(20-42) globulomer
antibody-containing serum is obtained from the animal by bleeding
or sacrificing the animal. The serum may be used as it is obtained
from the animal, an immunoglobulin fraction may be obtained from
the serum, or the anti-A.beta.(20-42) globulomer antibodies may be
purified from the serum. Serum or immunoglobulins obtained in this
manner are polyclonal, thus having a heterogeneous array of
properties.
[0181] Once an immune response is detected, e.g., antibodies
specific for the antigen A.beta.(20-42) globulomer are detected in
the mouse serum, the mouse spleen is harvested and splenocytes
isolated. The splenocytes are then fused by well-known techniques
to any suitable myeloma cells, for example cells from cell line
SP20 available from the ATCC. Hybridomas are selected and cloned by
limited dilution. The hybridoma clones are then assayed by methods
known in the art for cells that secrete antibodies capable of
binding A.beta.(20-42) globulomer. Ascites fluid, which generally
contains high levels of antibodies, can be generated by immunizing
mice with positive hybridoma clones.
[0182] In another embodiment, antibody-producing immortalized
hybridomas may be prepared from the immunized animal. After
immunization, the animal is sacrificed and the splenic B cells are
fused to immortalized myeloma cells as is well known in the art
(See, e.g., Harlow and Lane, supra). In a particular embodiment,
the myeloma cells do not secrete immunoglobulin polypeptides (a
non-secretory cell line). After fusion and antibiotic selection,
the hybridomas are screened using A.beta.(20-42) globulomer, or a
portion thereof, or a cell expressing A.beta.(20-42) globulomer. In
a particular embodiment, the initial screening is performed using
an enzyme-linked immunoassay (ELISA) or a radioimmunoassay (RIA).
An example of ELISA screening is provided in WO 00/37504, herein
incorporated by reference.
[0183] Anti-A.beta.(20-42) globulomer antibody-producing hybridomas
are selected, cloned and further screened for desirable
characteristics, including robust hybridoma growth, high antibody
production and desirable antibody characteristics, as discussed
further below. Hybridomas may be cultured and expanded in vivo in
syngeneic animals, in animals that lack an immune system, e.g.,
nude mice, or in cell culture in vitro. Methods of selecting,
cloning and expanding hybridomas are well known to those of
ordinary skill in the art.
[0184] In a particular embodiment, the hybridomas are mouse
hybridomas, as described above. In another particular embodiment,
the hybridomas are produced in a non-human, non-mouse species such
as rats, sheep, pigs, goats, cattle or horses. In another
embodiment, the hybridomas are human hybridomas, in which a human
non-secretory myeloma is fused with a human cell expressing an
anti-A.beta.(20-42) globulomer antibody.
[0185] Antibody fragments that recognize specific epitopes may be
generated by known techniques. For example, Fab and F(ab')2
fragments of the invention may be produced by proteolytic cleavage
of immunoglobulin molecules, using enzymes such as papain (to
produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
F(ab')2 fragments contain the variable region, the light chain
constant region and the CH1 domain of the heavy chain.
[0186] 2. Anti-A.beta.(20-42) Globulomer Monoclonal Antibodies
Using Slam
[0187] In another aspect of the invention, recombinant antibodies
are generated from single, isolated lymphocytes using a procedure
referred to in the art as the selected lymphocyte antibody method
(SLAM), as described in U.S. Pat. No. 5,627,052, PCT Publication
WO92/02551 and Babcock, J. S. et al. (1996) Proc. Natl. Acad. Sci.
USA 93:7843-7848. In this method, single cells secreting antibodies
of interest, e.g., lymphocytes derived from any one of the
immunized animals described in Section 1, are screened using an
antigen-specific hemolytic plaque assay, wherein the antigen
A.beta.(20-42) globulomer, or a subunit thereof, is coupled to
sheep red blood cells using a linker, such as biotin, and used to
identify single cells that secrete antibodies with specificity for
A.beta.(20-42) globulomer. Following identification of
antibody-secreting cells of interest, heavy- and light-chain
variable region cDNAs are rescued from the cells by reverse
transcriptase-PCR and these variable regions can then be expressed,
in the context of appropriate immunoglobulin constant regions
(e.g., human constant regions), in mammalian host cells, such as
COS or CHO cells. The host cells transfected with the amplified
immunoglobulin sequences, derived from in vivo selected
lymphocytes, can then undergo further analysis and selection in
vitro, for example by panning the transfected cells to isolate
cells expressing antibodies to A.beta.(20-42) globulomer. The
amplified immunoglobulin sequences further can be manipulated in
vitro, such as by in vitro affinity maturation methods such as
those described in PCT Publication WO 97/29131 and PCT Publication
WO 00/56772.
[0188] 3. Anti-A.beta.(20-42) Globulomer Monoclonal Antibodies
Using Transgenic Animals
[0189] In another embodiment of the instant invention, antibodies
are produced by immunizing a non-human animal comprising some, or
all, of the human immunoglobulin locus with an A.beta.(20-42)
globulomer antigen. In a particular embodiment, the non-human
animal is a XENOMOUSE transgenic mouse, an engineered mouse strain
that comprises large fragments of the human immunoglobulin loci and
is deficient in mouse antibody production. See, e.g., Green et al.
Nature Genetics 7:13-21 (1994) and U.S. Pat. Nos. 5,916,771,
5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598
and 6,130,364. See also WO 91/10741, published Jul. 25, 1991, WO
94/02602, published Feb. 3, 1994, WO 96/34096 and WO 96/33735, both
published Oct. 31, 1996, WO 98/16654, published Apr. 23, 1998, WO
98/24893, published Jun. 11, 1998, WO 98/50433, published Nov. 12,
1998, WO 99/45031, published Sep. 10, 1999, WO 99/53049, published
Oct. 21, 1999, WO 00 09560, published Feb. 24, 2000 and WO
00/037504, published Jun. 29, 2000. The XENOMOUSE transgenic mouse
produces an adult-like human repertoire of fully human antibodies,
and generates antigen-specific human monoclonal antibodies. The
XENOMOUSE transgenic mouse contains approximately 80% of the human
antibody repertoire through introduction of megabase sized,
germline configuration YAC fragments of the human heavy chain loci
and x light chain loci. See Mendez et al., Nature Genetics
15:146-156 (1997), Green and Jakobovits J. Exp. Med. 188:483-495
(1998), the disclosures of which are hereby incorporated by
reference.
[0190] 4. Anti-A.beta.(20-42) Globulomer Monoclonal Antibodies
Using Recombinant Antibody Libraries
[0191] In vitro methods also can be used to make the antibodies of
the invention, wherein an antibody library is screened to identify
an antibody having the desired binding specificity. Methods for
such screening of recombinant antibody libraries are well known in
the art and include methods described in, for example, Ladner et
al. U.S. Pat. No. 5,223,409; Kang et al. PCT Publication No.
WO92/18619; Dower et al. PCT Publication No. WO91/17271; Winter et
al. PCT Publication No. WO92/20791; Markland et al. PCT Publication
No. WO92/15679; Breitling et al. PCT Publication No. WO93/01288;
McCafferty et al. PCT Publication No. WO92/01047; Garrard et al.
PCT Publication No. WO92/09690; Fuchs et al. (1991) Bio/Technology
9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse
et al. (1989) Science 246:1275-1281; McCafferty et al., Nature
(1990) 348:552-554; Griffiths et al. (1993) EMBO J 12:725-734;
Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al.
(1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580;
Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al.
(1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS
88:7978-7982, US patent application publication 20030186374, and
PCT Publication No. WO97/29131, the contents of each of which are
incorporated herein by reference.
[0192] The recombinant antibody library may be from a subject
immunized with A.beta.(20-42) globulomer, or a portion of
A.beta.(20-42) globulomer. Alternatively, the recombinant antibody
library may be from a naive subject, i.e., one who has not been
immunized with A.beta.(20-42) globulomer, such as a human antibody
library from a human subject who has not been immunized with human
A.beta.(20-42) globulomer. Antibodies of the invention are selected
by screening the recombinant antibody library with the peptide
comprising human A.beta.(20-42) globulomer to thereby select those
antibodies that recognize A.beta.(20-42) globulomer and
discriminate A.beta.(1-42)globulomer, A.beta.(1-40) and
A.beta.(1-42)monomer, A.beta.-fibrils and sAPP.alpha.. Methods for
conducting such screening and selection are well known in the art,
such as described in the references in the preceding paragraph. To
select antibodies of the invention having particular binding
affinities for A.beta.(20-42) globulomer and discriminate
A.beta.(1-42) globulomer, A.beta.(1-40) and A.beta.(1-42) monomer,
A.beta.-fibrils and sAPP.alpha., such as those that dissociate from
human A.beta.(20-42) globulomer with a particular koff rate
constant, the art-known method of dot blot can be used to select
antibodies having the desired koff rate constant. To select
antibodies of the invention having a particular neutralizing
activity for A.beta.(20-42) globulomer and discriminate
A.beta.(1-42) globulomer, A.beta.(1-40) and A.beta.(1-42) monomer,
A.beta.-fibrils and sAPP.alpha., such as those with a particular an
IC50 standard methods known in the art for assessing the inhibition
of A.beta.(20-42) globulomer activity may be used.
[0193] In one aspect, the invention pertains to an isolated
antibody, or an antigen-binding portion thereof, that binds human
A.beta.(20-42) globulomer and discriminates A.beta.(1-42)
globulomer, A.beta.(1-40) and A.beta.(1-42) monomer,
A.beta.-fibrils and sAPP.alpha.. According to one aspect, the
antibody is a neutralizing antibody. In various embodiments, the
antibody is a recombinant antibody or a monoclonal antibody.
[0194] For example, the antibodies of the present invention can
also be generated using various phage display methods known in the
art. In phage display methods, functional antibody domains are
displayed on the surface of phage particles which carry the
polynucleotide sequences encoding them. In a particular, such phage
can be utilized to display antigen-binding domains expressed from a
repertoire or combinatorial antibody library (e.g., human or
murine). Phage expressing an antigen binding domain that binds the
antigen of interest can be selected or identified with antigen,
e.g., using labeled antigen or antigen bound or captured to a solid
surface or bead. Phage used in these methods are typically
filamentous phage including fd and M13 binding domains expressed
from phage with Fab, Fv or disulfide stabilized Fv antibody domains
recombinantly fused to either the phage gene III or gene VIII
protein. Examples of phage display methods that can be used to make
the antibodies of the present invention include those disclosed in
Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al.,
J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur.
J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997);
Burton et al., Advances in Immunology 57:191-280 (1994); PCT
application No. PCT/GB91/01134; PCT publications WO90/02809;
WO91/10737; WO92/01047; WO92/18619; WO93/11236; WO95/15982;
WO95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484;
5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908;
5,516,637; 5,780, 225; 5,658,727; 5,733,743 and 5,969,108; each of
which is incorporated herein by reference in its entirety.
[0195] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies including human antibodies or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described in detail below. For
example, techniques to recombinantly produce Fab, Fab' and F(ab')2
fragments can also be employed using methods known in the art such
as those disclosed in PCT publication WO92/22324; Mullinax et al.,
BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34
(1995); and Better et al., Science 240:1041-1043 (1988) (said
references incorporated by reference in their entireties). Examples
of techniques which can be used to produce single-chain Fvs and
antibodies include those described in U.S. Pat. Nos. 4,946,778 and
5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991);
Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science
240:1038-1040 (1988).
[0196] Alternative to screening of recombinant antibody libraries
by phage display, other methodologies known in the art for
screening large combinatorial libraries can be applied to the
identification of dual specificity antibodies of the invention. One
type of alternative expression system is one in which the
recombinant antibody library is expressed as RNA-protein fusions,
as described in PCT Publication No. WO 98/31700 by Szostak and
Roberts, and in Roberts, R. W. and Szostak, J. W. (1997) Proc.
Natl. Acad. Sci. USA 94:12297-12302. In this system, a covalent
fusion is created between an mRNA and the peptide or protein that
it encodes by in vitro translation of synthetic mRNAs that carry
puromycin, a peptidyl acceptor antibiotic, at their 3' end. Thus, a
specific mRNA can be enriched from a complex mixture of mRNAs
(e.g., a combinatorial library) based on the properties of the
encoded peptide or protein, e.g., antibody, or portion thereof,
such as binding of the antibody, or portion thereof, to the dual
specificity antigen. Nucleic acid sequences encoding antibodies, or
portions thereof, recovered from screening of such libraries can be
expressed by recombinant means as described above (e.g., in
mammalian host cells) and, moreover, can be subjected to further
affinity maturation by either additional rounds of screening of
mRNA-peptide fusions in which mutations have been introduced into
the originally selected sequence(s), or by other methods for
affinity maturation in vitro of recombinant antibodies, as
described above.
[0197] In another approach the antibodies of the present invention
can also be generated using yeast display methods known in the art.
In yeast display methods, genetic methods are used to tether
antibody domains to the yeast cell wall and display them on the
surface of yeast. In particular, such yeast can be utilized to
display antigen-binding domains expressed from a repertoire or
combinatorial antibody library (e.g., human or murine). Examples of
yeast display methods that can be used to make the antibodies of
the present invention include those disclosed Wittrup, et al. U.S.
Pat. No. 6,699,658 incorporated herein by reference.
[0198] B. Production of Recombinant A.beta.(20-42) Globulomer
Antibodies
[0199] Antibodies of the present invention may be produced by any
of a number of techniques known in the art. For example, expression
from host cells, wherein expression vector(s) encoding the heavy
and light chains is (are) transfected into a host cell by standard
techniques. The various forms of the term "transfection" are
intended to encompass a wide variety of techniques commonly used
for the introduction of exogenous DNA into a prokaryotic or
eukaryotic host cell, e.g., electroporation, calcium-phosphate
precipitation, DEAE-dextran transfection and the like. It is
possible to express the antibodies of the invention in either
prokaryotic or eukaryotic host cells. According to a particular
aspect of the invention, expression of antibodies is performed
using eukaryotic cells, for example mammalian host cells, because
such eukaryotic cells (and in particular mammalian cells) are more
likely than prokaryotic cells to assemble and secrete a properly
folded and immunologically active antibody.
[0200] According to one aspect, mammalian host cells for expressing
the recombinant antibodies of the invention include Chinese Hamster
Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub
and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used
with a DHFR selectable marker, e.g., as described in R. J. Kaufman
and P. A. Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells,
COS cells and SP2 cells. When recombinant expression vectors
encoding antibodies genes are introduced into mammalian host cells,
the antibodies are produced by culturing the host cells for a
period of time sufficient to allow for expression of the antibodies
in the host cells or secretion of the antibodies into the culture
medium in which the host cells are grown. Antibodies can be
recovered from the culture medium using standard protein
purification methods.
[0201] Host cells can also be used to produce functional antibody
fragments, such as Fab fragments or scFv molecules. It will be
understood that variations on the above procedure are within the
scope of the present invention. For example, it may be desirable to
transfect a host cell with DNA encoding functional fragments of
either the light chain and/or the heavy chain of an antibody of
this invention. Recombinant DNA technology may also be used to
remove some, or all, of the DNA encoding either or both of the
light and heavy chains that is not necessary for binding to the
antigens of interest. The molecules expressed from such truncated
DNA molecules are also encompassed by the antibodies of the
invention. In addition, bifunctional antibodies may be produced in
which one heavy and one light chain are an antibody of the
invention and the other heavy and light chain are specific for an
antigen other than the antigens of interest by crosslinking an
antibody of the invention to a second antibody by standard chemical
crosslinking methods.
[0202] In a particular system for recombinant expression of an
antibody, or antigen-binding portion thereof, of the invention, a
recombinant expression vector encoding both the antibody heavy
chain and the antibody light chain is introduced into dhfr- CHO
cells by calcium phosphate-mediated transfection. Within the
recombinant expression vector, the antibody heavy and light chain
genes are each operatively linked to CMV enhancer/AdMLP promoter
regulatory elements to drive high levels of transcription of the
genes. The recombinant expression vector also carries a DHFR gene,
which allows for selection of CHO cells that have been transfected
with the vector using methotrexate selection/amplification. The
selected transformant host cells are cultured to allow for
expression of the antibody heavy and light chains and intact
antibody is recovered from the culture medium. Standard molecular
biology techniques are used to prepare the recombinant expression
vector, transfect the host cells, select for transformants, culture
the host cells and recover the antibody from the culture medium.
Still further the invention provides a method of synthesizing a
recombinant antibody of the invention by culturing a host cell of
the invention in a suitable culture medium until a recombinant
antibody of the invention is synthesized. The method can further
comprise isolating the recombinant antibody from the culture
medium.
[0203] 1. Anti-A.beta.(20-42) Globulomer Murine Antibodies
[0204] Table 4 is a list of amino acid sequences of VH and VL
regions of murine 4D10.
TABLE-US-00007 TABLE 4 LIST OF AMINO ACID SEQUENCES OF VH AND VL
REGIONS SEQ PROTEIN SEQUENCE ID NO REGION
123456789012345678901234567890 23 m4D10_VH
QVQLKQSGPSLIQPSQSLSITCTVSGFSLT SYGVHWVRQSPGKGLEWLGVIWRGGRIDYN
AAFMSRLSITKDNSKSQVFFKMNSLQADDT AIYYCARNSDVWGTGTTVTVSS 24 m4D10_VL
DVVMTQTPLTLSVTIGQPASISCKSSQSLL DIDGKTYLNWLLQRPGQSPKRLIYLVSKLD
SGVPDRFTGSGSGTDFTLKISRVEAEDLGV YYCWQGTHEPYTEGGGTKLEIKR *CDRs are
underlined in murine light and heavy chains.
[0205] 2. Anti-A.beta.(20-42) Globulomer Chimeric Antibodies
[0206] A chimeric antibody is a molecule in which different
portions of the antibody are derived from different animal species,
such as antibodies having a variable region derived from a murine
monoclonal antibody and a human immunoglobulin constant region.
Methods for producing chimeric antibodies are known in the art and
discussed in detail herein. See e.g., Morrison, Science 229:1202
(1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al.,
(1989) J. Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715;
4,816,567; and 4,816,397, which are incorporated herein by
reference in their entireties. In addition, techniques developed
for the production of "chimeric antibodies" (Morrison et al., 1984,
Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature
312:604-608; Takeda et al., 1985, Nature 314:452-454 which are
incorporated herein by reference in their entireties) by splicing
genes from a mouse antibody molecule of appropriate antigen
specificity together with genes from a human antibody molecule of
appropriate biological activity can be used.
[0207] In one embodiment, the chimeric antibodies of the invention
are produced by replacing the heavy chain constant region of the
murine monoclonal anti-A.beta.(20-42) globulomer antibody 4D10
described in WO2007/062852 with a human IgG1 constant region.
[0208] 3. Anti-A.beta.(20-42) Globulomer CDR Grafted Antibodies
[0209] CDR-grafted antibodies of the invention comprise heavy and
light chain variable region sequences from a human antibody wherein
one or more of the CDR regions of VH and/or VL are replaced with
CDR sequences of the murine antibodies of the invention. A
framework sequence from any human antibody may serve as the
template for CDR grafting. However, straight chain replacement onto
such a framework often leads to some loss of binding affinity to
the antigen. The more homologous a human antibody is to the
original murine antibody, the less likely the possibility that
combining the murine CDRs with the human framework will introduce
distortions in the CDRs that could reduce affinity. Therefore, the
human variable framework chosen to replace the murine variable
framework apart from the CDRs have for example at least a 65%
sequence identity with the murine antibody variable region
framework. The human and murine variable regions apart from the
CDRs have for example at least 70%, least 75% sequence identity, or
at least 80% sequence identity. Methods for producing chimeric
antibodies are known in the art and discussed in detail herein.
(also see EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos.
5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP
592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498
(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);
Roguska et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S.
Pat. No. 5,565,352).
[0210] Table 5 below illustrates the sequences of CDR grafted
antibodies of the present invention (4D10hum antibodies) and the
CDRs contained therein.
TABLE-US-00008 TABLE 5 LIST OF AMINO ACID SEQUENCES OF VH AND VL
REGIONS OF CDR GRAFTED ANTIBODIES SEQ SEQUENCE ID NO PROTEIN REGION
123456789012345678901234567890 4 4D10hum_VH.1z
EVQLVESGGGLIQPGGSLRLSCAASGFTVS SYGVHWVRQAPGKGLEWVSVIWRGGRIDYN
AAFMSRFTISRDNSKNTLYLQMNSLRAEDT AVYYCARNSDVWGQGTTVTVSS 8
4D10hum_VH.2z QVQLQESGPGLVKPSETLSLTCTVSGGSIS
SYGVHWIRQPPGKGLEWIGVIWRGGRIDYN AAFMSRVTISVDTSKNQFSLKLSSVTAADT
AVYYCARNSDVWGQGTTVTVSS 17 VH 4D10hum Residues 31-35 of SYGVH CDR-H1
SEQ ID NOs: 4, 8 18 VH 4D10hum Residues 50-65 of VIWRGGRIDYNAAFMS
CDR-H2 SEQ ID NOs: 4, 8 19 VH 4D10hum Residues 98-101 of NSDV
CDR-H3 SEQ ID NOs: 4, 8 12 4D10hum_V.kappa..1z
DVVMTQSPLSLPVTLGQPASISCKSSQSLL DIDGKTYLNWFQQRPGQSPRRLIYLVSKLD
SGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCWQGTHEPYTEGQGTKLEIKR 20 VL
4D10hum Residues 24-39 of KSSQSLLDIDGKTYLN CDR-L1 SEQ ID NO: 12 21
VL 4D10hum Residues 55-61 of LVSKLDS CDR-L2 SEQ ID NO: 12 22 VL
4D10hum Residues 94-102 of WQGTHFPYT CDR-L3 SEQ ID NO: 12 *CDRs are
underlined in humanized light and heavy chains.
[0211] 4. Anti-A.beta.(20-42) Globulomer Humanized Antibodies
[0212] Humanized antibodies are antibody molecules from non-human
species antibody that binds the desired antigen having one or more
complementarity determining regions (CDRs) from the non-human
species and framework regions from a human immunoglobulin
molecule.
[0213] Known human Ig sequences are disclosed, e.g., Kabat et al.,
Sequences of Proteins of Immunological Interest, U.S. Dept. Health
(1983), each entirely incorporated herein by reference. Such
imported sequences can be used to reduce immunogenicity or reduce,
enhance or modify binding, affinity, on-rate, off-rate, avidity,
specificity, half-life, or any other suitable characteristic, as
known in the art.
[0214] Framework residues in the human framework regions may be
substituted with the corresponding residue from the CDR donor
antibody to alter, preferably improve, antigen binding. These
framework substitutions are identified by methods well known in the
art, e.g., by modeling of the interactions of the CDR and framework
residues to identify framework residues important for antigen
binding and sequence comparison to identify unusual framework
residues at particular positions. (See, e.g., Queen et al., U.S.
Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which
are incorporated herein by reference in their entireties.)
Three-dimensional immunoglobulin models are commonly available and
are familiar to those skilled in the art. Computer programs are
available which illustrate and display probable three-dimensional
conformational structures of selected candidate immunoglobulin
sequences. Inspection of these displays permits analysis of the
likely role of the residues in the functioning of the candidate
immunoglobulin sequence, i.e., the analysis of residues that
influence the ability of the candidate immunoglobulin to bind its
antigen. In this way, FR residues can be selected and combined from
the consensus and import sequences so that the desired antibody
characteristic, such as increased affinity for the target
antigen(s), is achieved. In general, the CDR residues are directly
and most substantially involved in influencing antigen binding.
Antibodies can be humanized using a variety of techniques known in
the art, such as but not limited to those described in Jones et
al., Nature 321:522 (1986); Verhoeyen et al., Science 239:1534
(1988), Sims et al., J. Immunol. 151: 2296 (1993); Chothia and
Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl.
Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol.
151:2623 (1993), Padlan, Molecular Immunology 28(4/5):489-498
(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);
Roguska. et al., PNAS 91:969-973 (1994); PCT publication WO
91/09967, PCT/: US98/16280, US96/18978, US91/09630, US91/05939,
US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443,
WO90/14424, WO90/14430, EP 229246, EP 592,106; EP 519,596, EP
239,400, U.S. Pat. Nos. 5,565,332, 5,723,323, 5,976,862, 5,824,514,
5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766886, 5,714,352,
6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539;
4,816,567, each entirely incorporated herein by reference, included
references cited therein.
[0215] Table 6 below illustrates the sequences of humanized
antibodies of the present invention (4D10hum antibodies) and the
CDRs contained therein.
TABLE-US-00009 TABLE 6 LIST OF AMINO ACID SEQUENCES OF VH AND VL
REGIONS OF HUMANIZED ANTIBODIES SEQ SEQUENCE ID NO PROTEIN REGION
123456789012345678901234567890 5 4D10hum_VH.1
EVQLVESGGGLVQPGGSLRLSCAASGFTVS SYGVHWVRQAPGKGLEWVSVIWRGGRIDYN
AAFMSRFTISRDNSKNTLYLQMNSLRAEDT AVYYCARNSDVWGQGTTVTVSS 6
4D10hum_VH.1a EVQLVESGGGLVQPGGSLRLSCAVSGFTLS
SYGVHWVRQAPGKGLEWLGVIWRGGRIDYN AAFMSRLTISKDNSKSTVYLQMNSLRAEDT
AVYYCARNSDVWGQGTTVTVSS 7 4D10hum_VH.1b
EVQLVESGGGLIQPGGSLRLSCAASGFTLS SYGVHWVRQAPGKGLEWVSVIWRGGRIDYN
AAFMSRFTISKDNSKNTLYLQMNSLRAEDT AVYYCARNSDVWGQGTTVTVSS 9
4D10hum_VH.2 EVQLQESGPGLVKPSETLSLTCTVSGGSIS
SYGVHWIRQPPGKGLEWIGVIWRGGRIDYN AAFMSRVTISVDTSKNQFSLKLSSVTAADT
AVYYCARNSDVWGQGTTVTVSS 10 4D10hum_VH.2a
EVQLQESGPGLVKPSETLSLTCTVSGFSLS SYGVHWVRQPPGKGLEWLGVIWRGGRIDYN
AAFMSRLTISKDTSKSQVSLKLSSVTAADT AVYYCARNSDVWGQGTTVTVSS 11
4D10hum_VH.2b EVQLQESGPGLVKPSETLSLTCTVSGFSLS
SYGVHWIRQPPGKGLEWIGVIWRGGRIDYN AAFMSRVTISKDTSKNQFSLKLSSVTAADT
AVYYCARNSDVWGQGTTVTVSS 17 VH 4D10hum Residues 31-35 of SYGVH CDR-H1
SEQ ID NOs: 5, 6, 7, 9, 10, 11 18 VH 4D10hum Residues 50-65 of
VIWRGGRIDYNAAFMS CDR-H2 SEQ ID NOs: 5, 6, 7, 9, 10, 11 19 VH
4D10hum Residues 98-101 of NSDV CDR-H3 SEQ ID NOs: 5, 6, 7, 9, 10,
11 13 4D10hum_V.kappa..1 DVVMTQTPLSLPVTPGQPASISCKSSQSLL
DIDGKTYLNWELQKPGQSPQRLIYLVSKLD SGVPDRFSGSGSGTDFTLKISRVEAEDVGV
YYCWQGTHEPYTEGQGTKLEIKR 14 4D10hum_V.kappa..1a
DVVMTQTPLSLPVTPGQPASISCKSSQSLL DIDGKTYLNWLLQKPGQSPQRLIYLVSKLD
SGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCWQGTHEPYTEGQGTKLEIKR 15
4D10hum_V.kappa..1b DVVMTQTPLSLPVTLGQPASISCKSSQSLL
DIDGKTYLNWLLQRPGQSPRRLIYLVSKLD SGVPDRFSGSGSGTDFTLKISRVEAEDVGV
YYCWQGTHEPYTEGQGTKLEIKR 16 4D10hum_V.kappa..1c
DVVMTQTPLSLPVTLGQPASISCKSSQSLL DIDGKTYLNWELQKPGQSPRRLIYLVSKLD
SGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCWQGTHEPYTEGQGTKLEIKR 20 VL
4D10hum Residues 24-39 of KSSQSLLDIDGKTYLN CDR-L1 SEQ ID NOs: 13,
14, 15, 16 21 VL 4D10hum Residues 55-61 of LVSKLDS CDR-L2 SEQ ID
NOs: 13, 14, 15, 16 22 VL 4D10hum Residues 94-102 of WQGTHFPYT
CDR-L3 SEQ ID NOs: 13, 14, 15, 16 *CDRs are underlined in humanized
light and heavy chains.
[0216] C. Antibodies and Antibody-Producing Cell Lines
[0217] According to one aspect, anti-A.beta.(20-42) globulomer
antibodies of the present invention or antibodies against any other
targeted A.beta. form exhibit a high capacity to reduce or to
neutralize activity of A.beta.(20-42) globulomer (and/or any other
targeted A.beta. form).
[0218] In certain embodiments, the antibody comprises a heavy chain
constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM
or IgD constant region. According to one aspect, the heavy chain
constant region is an IgG1 heavy chain constant region or an IgG4
heavy chain constant region. According to a further aspect, the
antibody comprises a light chain constant region, either a kappa
light chain constant region or a lambda light chain constant
region. According to one aspect, the antibody comprises a kappa
light chain constant region. An antibody portion can be, for
example, a Fab fragment or a single chain Fv fragment.
[0219] Replacements of amino acid residues in the Fc portion to
alter antibody effector function are known in the art (Winter, et
al. U.S. Pat. Nos. 5,648,260 and 5,624,821). The Fc portion of an
antibody mediates several important effector functions e.g.
cytokine induction, ADCC, phagocytosis, complement dependent
cytotoxicity (CDC) and half-life/clearance rate of antibody and
antigen-antibody complexes. In some cases these effector functions
are desirable for therapeutic antibody but in other cases might be
unnecessary or even deleterious, depending on the therapeutic
objectives. Certain human IgG isotypes, particularly IgG1 and IgG3,
mediate ADCC and CDC via binding to Fc.gamma.Rs and complement C1q,
respectively. Neonatal Fc receptors (FcRn) are the critical
components determining the circulating half-life of antibodies. In
still another embodiment at least one amino acid residue is
replaced in the constant region of the antibody, for example the Fc
region of the antibody, such that effector functions of the
antibody are altered.
[0220] One embodiment provides a labeled antibody wherein an
antibody of the invention is derivatized or linked to another
functional molecule (e.g., another peptide or protein). For
example, a labeled antibody of the invention can be derived by
functionally linking an antibody of the invention (by chemical
coupling, genetic fusion, noncovalent association or otherwise) to
one or more other molecular entities, such as another antibody
(e.g., a bispecific antibody or a diabody), a detectable agent, a
pharmaceutical agent, and/or a protein or peptide that can mediate
association of the antibody with another molecule (such as a
streptavidin core region or a polyhistidine tag).
[0221] Useful detectable agents with which an antibody of the
invention may be derivatized include fluorescent compounds.
Exemplary fluorescent detectable agents include fluorescein,
fluorescein isothiocyanate, rhodamine,
5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin and
the like. An antibody may also be derivatized with detectable
enzymes, such as alkaline phosphatase, horseradish peroxidase,
glucose oxidase and the like. When an antibody is derivatized with
a detectable enzyme, it is detected by adding additional reagents
that the enzyme uses to produce a detectable reaction product. For
example, when the detectable agent horseradish peroxidase is
present, the addition of hydrogen peroxide and diaminobenzidine
leads to a colored reaction product, which is detectable. An
antibody may also be derivatized with biotin, and detected through
indirect measurement of avidin or streptavidin binding.
[0222] Another embodiment of the invention provides a crystallized
antibody. According to one aspect, the invention relates to
crystals of whole anti-A.beta.(20-42) globulomer antibodies and
fragments thereof as disclosed herein, and formulations and
compositions comprising such crystals. According to a further
aspect, the crystallized antibody has a greater half-life in vivo
than the soluble counterpart of the antibody. According to a
further aspect, the antibody retains biological activity after
crystallization.
[0223] Crystallized antibody of the invention may be produced
according methods known in the art and as disclosed in WO02/072636,
incorporated herein by reference.
[0224] Another embodiment of the invention provides a glycosylated
antibody wherein the antibody comprises one or more carbohydrate
residues. Nascent in vivo protein production may undergo further
processing, known as post-translational modification. In
particular, sugar (glycosyl) residues may be added enzymatically, a
process known as glycosylation. The resulting proteins bearing
covalently linked oligosaccharide side chains are known as
glycosylated proteins or glycoproteins.
[0225] Antibodies are glycoproteins with one or more carbohydrate
residues in the Fc domain, as well as the variable domain.
Carbohydrate residues in the Fc domain have important effect on the
effector function of the Fc domain, with minimal effect on antigen
binding or half-life of the antibody (R. Jefferis, Biotechnol.
Prog. 21 (2005), pp. 11-16). In contrast, glycosylation of the
variable domain may have an effect on the antigen binding activity
of the antibody. Glycosylation in the variable domain may have a
negative effect on antibody binding affinity, likely due to steric
hindrance (Co, M. S., et al., Mol. Immunol. (1993) 30:1361-1367),
or result in increased affinity for the antigen (Wallick, S. C., et
al., Exp. Med. (1988) 168:1099-1109; Wright, A., et al., EMBO J.
(1991) 10:2717 2723).
[0226] One aspect of the present invention is directed to
generating glycosylation site mutants in which the O- or N-linked
glycosylation site of the antibody has been mutated. One skilled in
the art can generate such mutants using standard well-known
technologies. The creation of glycosylation site mutants that
retain the biological activity but have increased or decreased
binding activity is another object of the present invention.
[0227] In still another embodiment, the glycosylation of the
antibody of the invention is modified. For example, an aglycoslated
antibody can be made (i.e., the antibody lacks glycosylation).
Glycosylation can be altered to, for example, increase the affinity
of the antibody for antigen. Such carbohydrate modifications can be
accomplished by, for example, altering one or more sites of
glycosylation within the antibody sequence. For example, one or
more amino acid substitutions can be made that result in
elimination of one or more variable region glycosylation sites to
thereby eliminate glycosylation at that site. Such aglycosylation
may increase the affinity of the antibody for antigen. Such an
approach is described in further detail in International Appln.
Publication No. WO03/016466A2, and U.S. Pat. Nos. 5,714,350 and
6,350,861, each of which is incorporated herein by reference in its
entirety.
[0228] Additionally or alternatively, a modified antibody of the
invention can be made that has an altered type of glycosylation,
such as a hypofucosylated antibody having reduced amounts of
fucosyl residues or an antibody having increased bisecting GlcNAc
structures. Such altered glycosylation patterns have been
demonstrated to increase the ADCC ability of antibodies. Such
carbohydrate modifications can be accomplished by, for example,
expressing the antibody in a host cell with altered glycosylation
machinery. Cells with altered glycosylation machinery have been
described in the art and can be used as host cells in which to
express recombinant antibodies of the invention to thereby produce
an antibody with altered glycosylation. See, for example, Shields,
R. L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al.
(1999) Nat. Biotech. 17:176-1, as well as, European Patent NO.:
EP1,176,195; International Appln. Publication Nos. WO03/035835 and
WO99/54342 80, each of which is incorporated herein by reference in
its entirety.
[0229] Protein glycosylation depends on the amino acid sequence of
the protein of interest, as well as the host cell in which the
protein is expressed. Different organisms may produce different
glycosylation enzymes (e.g., glycosyltransferases and
glycosidases), and have different substrates (nucleotide sugars)
available. Due to such factors, protein glycosylation pattern, and
composition of glycosyl residues, may differ depending on the host
system in which the particular protein is expressed. Glycosyl
residues useful in the invention may include, but are not limited
to, glucose, galactose, mannose, fucose, n-acetylglucosamine and
sialic acid. According to one aspect, the glycosylated antibody
comprises glycosyl residues such that the glycosylation pattern is
human.
[0230] It is known to those skilled in the art that differing
protein glycosylation may result in differing protein
characteristics. For instance, the efficacy of a therapeutic
protein produced in a microorganism host, such as yeast, and
glycosylated utilizing the yeast endogenous pathway may be reduced
compared to that of the same protein expressed in a mammalian cell,
such as a CHO cell line. Such glycoproteins may also be immunogenic
in humans and show reduced half-life in vivo after administration.
Specific receptors in humans and other animals may recognize
specific glycosyl residues and promote the rapid clearance of the
protein from the bloodstream. Other adverse effects may include
changes in protein folding, solubility, susceptibility to
proteases, trafficking, transport, compartmentalization, secretion,
recognition by other proteins or factors, antigenicity, or
allergenicity. Accordingly, a practitioner may prefer a therapeutic
protein with a specific composition and pattern of glycosylation,
for example glycosylation composition and pattern identical, or at
least similar, to that produced in human cells or in the
species-specific cells of the intended subject animal.
[0231] Expressing glycosylated proteins different from that of a
host cell may be achieved by genetically modifying the host cell to
express heterologous glycosylation enzymes. Using techniques known
in the art a practitioner may generate antibodies exhibiting human
protein glycosylation. For example, yeast strains have been
genetically modified to express non-naturally occurring
glycosylation enzymes such that glycosylated proteins
(glycoproteins) produced in these yeast strains exhibit protein
glycosylation identical to that of animal cells, especially human
cells (U.S Patent Application Publication Nos. 20040018590 and
20020137134; and WO05/100584).
[0232] Another embodiment is directed to an anti-idiotypic
(anti-Id) antibody specific for such antibodies of the invention.
An anti-Id antibody is an antibody, which recognizes unique
determinants generally associated with the antigen-binding region
of another antibody. The anti-Id can be prepared by immunizing an
animal with the antibody or a CDR containing region thereof. The
immunized animal will recognize, and respond to the idiotypic
determinants of the immunizing antibody and produce an anti-Id
antibody. The anti-Id antibody may also be used as an "immunogen"
to induce an immune response in yet another animal, producing a
so-called anti-anti-Id antibody.
[0233] Further, it will be appreciated by one skilled in the art
that a protein of interest may be expressed using a library of host
cells genetically engineered to express various glycosylation
enzymes, such that member host cells of the library produce the
protein of interest with variant glycosylation patterns. A
practitioner may then select and isolate the protein of interest
with particular novel glycosylation patterns. According to a
further aspect, the protein having a particularly selected novel
glycosylation pattern exhibits improved or altered biological
properties.
[0234] D. Uses of Anti-A.beta.(20-42) Globulomer Antibodies
[0235] Given their ability to bind to A.beta.(20-42) globulomer,
the anti-A.beta.(20-42) globulomer antibodies, or antibodies
against any other targeted A.beta. form, of the invention can be
used to detect A.beta.(20-42) globulomer and/or any other targeted
A.beta. form (e.g., in a biological sample such as serum, CSF,
brain tissue or plasma), using a conventional immunoassay, such as
an enzyme linked immunosorbent assays (ELISA), an radioimmunoassay
(RIA) or tissue immunohistochemistry. The invention provides a
method for detecting A.beta.(20-42) globulomer and/or any other
targeted A.beta. form in a biological sample comprising contacting
a biological sample with an antibody of the invention and detecting
either the antibody bound to A.beta.(20-42) globulomer (and/or
and/or any other targeted A.beta. form) or unbound antibody, to
thereby detect A.beta.(20-42) globulomer, and/or any other targeted
A.beta. form in the biological sample. The antibody is directly or
indirectly labeled with a detectable substance to facilitate
detection of the bound or unbound antibody. Suitable detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials and radioactive materials.
Examples of suitable enzymes include horseradish peroxidase,
alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; and examples of suitable radioactive material include
.sup.3H, .sup.14C, .sup.35S, .sup.90Y, .sup.99Tc, .sup.111In,
.sup.125I, .sup.131I, .sup.177Lu, .sup.166Ho, and .sup.153Sm.
[0236] Alternative to labeling the antibody, A.beta.(20-42)
globulomer and/or any other targeted A.beta. form can be assayed in
biological fluids by a competition immunoassay utilizing
A.beta.(20-42) globulomer standards labeled with a detectable
substance and an unlabeled anti-A.beta.(20-42) globulomer antibody.
In this assay, the biological sample, the labeled A.beta.(20-42)
globulomer standards and the anti-A.beta.(20-42) globulomer
antibody are combined and the amount of labeled A.beta.(20-42)
globulomer standard bound to the unlabeled antibody is determined.
The amount of A.beta.(20-42) globulomer, and/or any other targeted
A.beta. form in the biological sample is inversely proportional to
the amount of labeled A.beta.(20-42) globulomer standard bound to
the anti-A.beta.(20-42) globulomer antibody.
[0237] According to one aspect of the invention, the antibodies of
the invention are capable of neutralizing A.beta.(20-42) globulomer
activity, and/or activity of any other targeted A.beta. form both
in vitro and in vivo. Accordingly, such antibodies of the invention
can be used to inhibit (i.e. reduce) A.beta.(20-42) globulomer
activity, and/or activity of any other targeted A.beta. form, e.g.,
in a cell culture containing A.beta.(20-42) globulomer, and/or any
other targeted A.beta. form in human subjects or in other mammalian
subjects having A.beta.(20-42) globulomer, and/or any other
targeted A.beta. form with which an antibody of the invention
cross-reacts. In one embodiment, the invention provides a method
for inhibiting (i.e. reducing) A.beta.(20-42) globulomer activity,
and/or activity of any other targeted A.beta. form comprising
contacting A.beta.(20-42) globulomer, and/or any other targeted
A.beta. form with an antibody of the invention such that
A.beta.(20-42) globulomer activity, and/or activity of any other
targeted A.beta. form is inhibited (i.e. reduced). For example, in
a cell culture containing, or suspected of containing
A.beta.(20-42) globulomer, and/or any other targeted A.beta. form
an antibody of the invention can be added to the culture medium to
inhibit (i.e. reduce) A.beta.(20-42) globulomer activity, and/or
activity of any other targeted A.beta. form in the culture.
[0238] In another embodiment, the invention provides a method for
inhibiting (i.e. reducing) activity of a targeted A.beta. form in a
subject, advantageously in a subject suffering from a disease or
disorder in which activity of said A.beta. form is detrimental, or
a disease or disorder or disorder which is selected from the group
consisting of Alpha1-antitrypsin-deficiency, C1-inhibitor
deficiency angioedema, Antithrombin deficiency thromboembolic
disease, Kuru, Creutzfeld-Jacob disease/scrapie, Bovine spongiform
encephalopathy, Gerstmann-Straussler-Scheinker disease, Fatal
familial insomnia, Huntington's disease, Spinocerebellar ataxia,
Machado-Joseph atrophy, Dentato-rubro-pallidoluysian atrophy,
Frontotemporal dementia, Sickle cell anemia, Unstable hemoglobin
inclusion-body hemolysis, Drug-induced inclusion body hemolysis,
Parkinson's disease, Systemic AL amyloidosis, Nodular AL
amyloidosis, Systemic AA amyloidosis, Prostatic amyloidosis,
Hemodialysis amyloidosis, Hereditary (Icelandic) cerebral
angiopathy, Huntington's disease, Familial visceral amyloidosis,
Familial visceral polyneuropathy, Familial visceral amyloidosis,
Senile systemic amyloidosis, Familial amyloid neurophathy, Familial
cardiac amyloidosis, Alzheimer's disease, Down syndrome, Medullary
carcinoma thyroid and Type 2 diabetes mellitus (T2DM).
[0239] The invention provides methods for inhibiting (i.e.
reducing) the activity of a targeted A.beta. form in a subject
suffering from such a disease or disorder, which method comprises
administering to the subject an antibody of the invention such that
the activity of said A.beta. form in the subject is inhibited (i.e.
reduced). In one aspect of the invention, said targeted A.beta.
form is a human A.beta. form, and the subject is a human subject.
Alternatively, the subject can be a non-human mammal expressing APP
or any A.beta.-form resulting in the generation of a targeted
A.beta. form to which an antibody of the invention is capable of
binding. Still further the subject can be a non-human mammal into
which a targeted A.beta. form has been introduced (e.g., by
administration of the targeted A.beta. form or by expression of APP
or any other A.beta.-form resulting in the generation of the
targeted A.beta. form. An antibody of the invention can be
administered to a human subject for therapeutic purposes. Moreover,
an antibody of the invention can be administered to a non-human
mammal wherein expression of APP or any A.beta.-form resulting in
the generation of a targeted A.beta. form with which the antibody
is capable of binding for veterinary purposes or as an animal model
of human disease. Regarding the latter, such animal models may be
useful for evaluating the therapeutic efficacy of antibodies of the
invention (e.g., testing of dosages and time courses of
administration).
[0240] Another embodiment is a method for inhibiting (i.e.
reducing) activity of a targeted A.beta. form in a subject
suffering from an amyloidosis, such as Alzheimer's disease or Down
syndrome.
[0241] A disorder in which activity of a targeted A.beta. form is
detrimental includes diseases and other disorders in which the
presence of a targeted A.beta. form in a subject suffering from the
disorder has been shown to be or is suspected of being either
responsible for the pathophysiology of the disorder or a factor
that contributes to a worsening of the disorder. Accordingly, a
disorder in which activity of a targeted A.beta. form is
detrimental is a disorder in which inhibition (i.e. reduction) of
the activity said A.beta. form is expected to alleviate some or all
of the symptoms and/or progression of the disorder. Such disorders
may be evidenced, for example, by an increase in the concentration
of a targeted A.beta. form in a biological fluid of a subject
suffering from the disorder (e.g., an increase in the concentration
of the targeted A.beta. form in serum, brain tissue, plasma,
cerebrospinal fluid, etc. of the subject), which can be detected,
for example, using an anti-A.beta.(20-42) globulomer antibody
and/or antibody against any other targeted A.beta. form as
described above or any antibody to any A.beta. form that comprises
the globulomer epitope with which the antibodies of the present
invention are reactive. Non-limiting examples of disorders that can
be treated with the antibodies of the invention include those
disorders disclosed herein and those discussed in the section below
pertaining to pharmaceutical compositions of the antibodies of the
invention.
[0242] In still yet another embodiment, the present invention
relates to a method for preventing the progression (e.g.,
worsening) of a disease condition described herein. The method
comprises administering to the subject in need of treatment thereof
(e.g., a mammal, such as a human) a therapeutically effective
amount of any of the binding proteins or antibodies as described
herein. Alternatively, the method comprises administering to the
subject a therapeutically effective amount of any of the proteins
as described herein, in combination with a therapeutically
effective amount of at least one therapeutic agent.
[0243] In the above described methods for preventing the
development or progression of a disorder described herein one or
more biomarkers, diagnostic tests or combination of biomarkers and
diagnostic tests known to those skilled the art can be used to
determine whether or not (1) a subject is at risk of developing one
or more of the disorders described herein; or (2) the disorders
described herein in the subject previously diagnosed with one or
more of the aforementioned disorders is progressing (e.g.,
worsening).
[0244] One or more biomarkers, diagnostic tests or combinations of
biomarkers and diagnostic tests known in the art can be used to
identify subjects who are at risk of developing a disorder
described herein. Likewise, one or more biomarkers, diagnostic
tests or combinations of biomarkers and diagnostic tests known in
the art can be used to determine the progression of the disease or
condition of subjects who have been identified as suffering from a
disorder described herein. For example, one or more biological
markers, neuroimaging markers or combination of biological or
neuroimaging markers (e.g., MRI, etc.) can be used to identify
subjects at risk of developing Alzheimer's disease or, for those
subjects identified as suffering from Alzheimer's disease, the
progression of the disease. Biological markers that can be examined
include, but are not limited to, beta-amyloid.sub.1-42, tau,
phosphorylated tau (ptau), plasma A.beta. antibodies,
.alpha.-antichymotrypsin, amyloid precursor protein, APP isoform
ratio in platelets, .beta.-secretase (also known as BACE), CD59,
8-hydroxy-deoxyguanine, glutamine synthetase, glial fibrillary
acidic protein (GFAP), antibodies to GFAP, interleukin-6-receptor
complex, kallikrein, melanotransferrin, neurofilament proteins,
nitrotyrosine, oxysterols, sulphatides, synaptic markers, 510013,
NPS, plasma signaling proteins, etc., or any combinations thereof
(See, Shaw, L., et al., Nature Reviews 2007, 6, 295-303. Borroni,
B., et al., Current Med Chem. 2007, 14, 1171-1178. Phillips, K., et
al., Nature Reviews 2006, 5 463-469. Bouwman, F. H., et al.,
Neurology 2007, 69, 1006-1011; Ray, S., et al., Nature Medicine
2007, 13(11), 1359-1362. Cummings, J., et al., Neurology 2007, 69,
1622-1634.).
[0245] E. Pharmaceutical Compositions
[0246] The invention also provides pharmaceutical compositions
comprising an antibody of the invention and a pharmaceutically
acceptable carrier. The pharmaceutical compositions comprising
antibodies of the invention are for use in, but not limited to,
diagnosing, detecting, or monitoring a disorder, in preventing,
treating, managing, or ameliorating of a disorder or one or more
symptoms thereof, and/or in research. In a specific embodiment, a
composition comprises one or more antibodies of the invention. In
another embodiment, the pharmaceutical composition comprises one or
more antibodies of the invention and one or more prophylactic or
therapeutic agents other than antibodies of the invention for
treating a disorder in which activity of a targeted A.beta. form is
detrimental. In a further embodiment, the prophylactic or
therapeutic agents are known to be useful for, or have been, or are
currently being used in the prevention, treatment, management, or
amelioration of a disorder, or one or more symptoms thereof. In
accordance with these embodiments, the composition may further
comprise of a carrier, diluent or excipient.
[0247] The antibodies of the invention can be incorporated into
pharmaceutical compositions suitable for administration to a
subject. Typically, the pharmaceutical composition comprises an
antibody of the invention and a pharmaceutically acceptable
carrier. As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like that are physiologically compatible.
Examples of pharmaceutically acceptable carriers include one or
more of water, saline, phosphate buffered saline, dextrose,
glycerol, ethanol and the like, as well as combinations thereof. In
many cases, it will be preferable to include isotonic agents, for
example, sugars, polyalcohols such as mannitol, sorbitol, or sodium
chloride in the composition. Pharmaceutically acceptable carriers
may further comprise minor amounts of auxiliary substances such as
wetting or emulsifying agents, preservatives or buffers, which
enhance the shelf life or effectiveness of the antibody.
[0248] In a further embodiment, the pharmaceutical composition
comprises at least one additional therapeutic agent for treating a
disorder as disclosed herein.
[0249] Various delivery systems are known and can be used to
administer one or more antibodies of the invention or the
combination of one or more antibodies of the invention and a
prophylactic agent or therapeutic agent useful for preventing,
managing, treating, or ameliorating a disorder or one or more
symptoms thereof, e.g., encapsulation in liposomes, microparticles,
microcapsules, recombinant cells capable of expressing the antibody
or antibody fragment, receptor-mediated endocytosis (see, e.g., Wu
and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a
nucleic acid as part of a retroviral or other vector, etc. Methods
of administering a prophylactic or therapeutic agent of the
invention include, but are not limited to, parenteral
administration (e.g., intradermal, intramuscular, intraperitoneal,
intravenous and subcutaneous), epidurala administration,
intratumoral administration, and mucosal administration (e.g.,
intranasal and oral routes). In addition, pulmonary administration
can be employed, e.g., by use of an inhaler or nebulizer, and
formulation with an aerosolizing agent. See, e.g., U.S. Pat. Nos.
6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913,
5,290,540, and 4,880,078; and PCT Publication Nos. WO 92/19244,
WO97/32572, WO97/44013, WO98/31346, and WO99/66903, each of which
is incorporated herein by reference their entireties. In one
embodiment, an antibody of the invention, combination therapy, or a
composition of the invention is administered using Alkermes
AIR.RTM. pulmonary drug delivery technology (Alkermes, Inc.,
Cambridge, Mass.). In a specific embodiment, prophylactic or
therapeutic agents of the invention are administered
intramuscularly, intravenously, intratumorally, orally,
intranasally, pulmonary, or subcutaneously. The prophylactic or
therapeutic agents may be administered by any convenient route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically active agents. Administration can be systemic or
local.
[0250] In a specific embodiment, it may be desirable to administer
the antibodies of the invention locally to the area in need of
treatment; this may be achieved by, for example, and not by way of
limitation, local infusion, by injection, or by means of an
implant, said implant being of a porous or non-porous material,
including membranes and matrices, such as sialastic membranes,
polymers, fibrous matrices (e.g., Tissuel.RTM.), or collagen
matrices. In one embodiment, an effective amount of one or more
antibodies of the invention is administered locally to the affected
area to a subject to prevent, treat, manage, and/or ameliorate a
disorder or a symptom thereof. In another embodiment, an effective
amount of one or more antibodies of the invention is administered
locally to the affected area in combination with an effective
amount of one or more therapies (e.g., one or more prophylactic or
therapeutic agents) other than an antibody of the invention of a
subject to prevent, treat, manage, and/or ameliorate a disorder or
one or more symptoms thereof.
[0251] In another embodiment, the antibody can be delivered in a
controlled release or sustained release system. In one embodiment,
a pump may be used to achieve controlled or sustained release (see
Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20;
Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N.
Engl. J. Med. 321:574). In another embodiment, polymeric materials
can be used to achieve controlled or sustained release of the
therapies of the invention (see e.g., Medical Applications of
Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton,
Fla. (1974); Controlled Drug Bioavailability, Drug Product Design
and Performance, Smolen and Ball (eds.), Wiley, New York (1984);
Ranger and Peppas, 1983, J., Macromol. Sci. Rev. Macromol. Chem.
23:61; see also Levy et al., 1985, Science 228:190; During et al.,
1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 7
1:105); U.S. Pat. No. 5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat.
No. 5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat. No. 5,128,326;
PCT Publication No. WO99/15154; and PCT Publication No. WO99/20253.
Examples of polymers used in sustained release formulations
include, but are not limited to, poly(-hydroxy ethyl methacrylate),
poly(methyl methacrylate), poly(acrylic acid),
poly(ethylene-co-vinyl acetate), poly(methacrylic acid),
polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone),
poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol),
polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and
polyorthoesters. In a particular embodiment, the polymer used in a
sustained release formulation is inert, free of leachable
impurities, stable on storage, sterile, and biodegradable. In yet
another embodiment, a controlled or sustained release system can be
placed in proximity of the prophylactic or therapeutic target, thus
requiring only a fraction of the systemic dose (see, e.g., Goodson,
in Medical Applications of Controlled Release, supra, vol. 2, pp.
115-138 (1984)).
[0252] Controlled release systems are discussed in the review by
Langer (1990, Science 249:1527-1533). Any technique known to one of
skill in the art can be used to produce sustained release
formulations comprising one or more antibodies of the invention.
See, e.g., U.S. Pat. No. 4,526,938, PCT publication WO91/05548, PCT
publication WO96/20698, Ning et al., 1996, "Intratumoral
Radioimmunotheraphy of a Human Colon Cancer Xenograft Using a
Sustained-Release Gel," Radiotherapy & Oncology 39:179-189,
Song et al., 1995, "Antibody Mediated Lung Targeting of
Long-Circulating Emulsions," PDA Journal of Pharmaceutical Science
& Technology 50:372-397, Cleek et al., 1997, "Biodegradable
Polymeric Carriers for a bFGF Antibody for Cardiovascular
Application," Pro. Int'l. Symp. Control. Rel. Bioact. Mater.
24:853-854, and Lam et al., 1997, "Microencapsulation of
Recombinant Humanized Monoclonal Antibody for Local Delivery,"
Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of
which is incorporated herein by reference in their entireties.
[0253] In a specific embodiment, where the composition of the
invention is a nucleic acid encoding an antibody, the nucleic acid
can be administered in vivo to promote expression of its encoded
antibody, by constructing it as part of an appropriate nucleic acid
expression vector and administering it so that it becomes
intracellular, e.g., by use of a retroviral vector (see U.S. Pat.
No. 4,980,286), or by direct injection, or by use of microparticle
bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with
lipids or cell-surface receptors or transfecting agents, or by
administering it in linkage to a homeobox-like peptide which is
known to enter the nucleus (see, e.g., Joliot et al., 1991, Proc.
Natl. Acad. Sci. USA 88:1864-1868). Alternatively, a nucleic acid
can be introduced intracellularly and incorporated within host cell
DNA for expression by homologous recombination.
[0254] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include, but are not limited
to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral,
intranasal (e.g., inhalation), transdermal (e.g., topical),
transmucosal, and rectal administration. In a specific embodiment,
the composition is formulated in accordance with routine procedures
as a pharmaceutical composition adapted for intravenous,
subcutaneous, intramuscular, oral, intranasal, or topical
administration to human beings. Typically, compositions for
intravenous administration are solutions in sterile isotonic
aqueous buffer. Where necessary, the composition may also include a
solubilizing agent and a local anesthetic such as lignocamne to
ease pain at the site of the injection.
[0255] If the compositions of the invention are to be administered
topically, the compositions can be formulated in the form of an
ointment, cream, transdermal patch, lotion, gel, shampoo, spray,
aerosol, solution, emulsion, or other form well-known to one of
skill in the art. See, e.g., Remington's Pharmaceutical Sciences
and Introduction to Pharmaceutical Dosage Forms, 19.sup.th ed.,
Mack Pub. Co., Easton, Pa. (1995). For non-sprayable topical dosage
forms, viscous to semi-solid or solid forms comprising a carrier or
one or more excipients compatible with topical application and
having a dynamic viscosity greater than water are typically
employed.
[0256] Suitable formulations include, without limitation,
solutions, suspensions, emulsions, creams, ointments, powders,
liniments, salves, and the like, which are, if desired, sterilized
or mixed with auxiliary agents (e.g., preservatives, stabilizers,
wetting agents, buffers, or salts) for influencing various
properties, such as, for example, osmotic pressure. Other suitable
topical dosage forms include sprayable aerosol preparations wherein
the active ingredient, for example in combination with a solid or
liquid inert carrier, is packaged in a mixture with a pressurized
volatile (e.g., a gaseous propellant, such as freon) or in a
squeeze bottle. Moisturizers or humectants can also be added to
pharmaceutical compositions and dosage forms if desired. Examples
of such additional ingredients are well-known in the art.
[0257] If the method of the invention comprises intranasal
administration of a composition, the composition can be formulated
in an aerosol form, spray, mist or in the form of drops. In
particular, prophylactic or therapeutic agents for use according to
the present invention can be conveniently delivered in the form of
an aerosol spray presentation from pressurized packs or a
nebuliser, with the use of a suitable propellant (e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas).
In the case of a pressurized aerosol the dosage unit may be
determined by providing a valve to deliver a metered amount.
Capsules and cartridges (composed of, e.g., gelatin) for use in an
inhaler or insufflator may be formulated containing a powder mix of
the compound and a suitable powder base such as lactose or
starch.
[0258] If the method of the invention comprises oral
administration, compositions can be formulated orally in the form
of tablets, capsules, cachets, gelcaps, solutions, suspensions, and
the like. Tablets or capsules can be prepared by conventional means
with pharmaceutically acceptable excipients such as binding agents
(e.g., pregelatinised maize starch, polyvinylpyrrolidone, or
hydroxypropyl methylcellulose); fillers (e.g., lactose,
microcrystalline cellulose, or calcium hydrogen phosphate);
lubricants (e.g., magnesium stearate, talc, or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well-known in the art. Liquid preparations for
oral administration may take the form of, but not limited to,
solutions, syrups or suspensions, or they may be presented as a dry
product for constitution with water or other suitable vehicle
before use. Such liquid preparations may be prepared by
conventional means with pharmaceutically acceptable additives such
as suspending agents (e.g., sorbitol syrup, cellulose derivatives,
or hydrogenated edible fats); emulsifying agents (e.g., lecithin or
acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl
alcohol, or fractionated vegetable oils); and preservatives (e.g.,
methyl or propyl-p-hydroxybenzoates or sorbic acid). The
preparations may also contain buffer salts, flavoring, coloring,
and sweetening agents as appropriate. Preparations for oral
administration may be suitably formulated for slow release,
controlled release, or sustained release of a prophylactic or
therapeutic agent(s).
[0259] The method of the invention may comprise pulmonary
administration, e.g., by use of an inhaler or nebulizer, of a
composition formulated with an aerosolizing agent. See, e.g., U.S.
Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064,
5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO
92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903,
each of which is incorporated herein by reference their entireties.
In a specific embodiment, an antibody of the invention, combination
therapy, and/or composition of the invention is administered using
Alkermes AIR.RTM. pulmonary drug delivery technology (Alkermes,
Inc., Cambridge, Mass.).
[0260] The method of the invention may comprise administration of a
composition formulated for parenteral administration by injection
(e.g., by bolus injection or continuous infusion). Formulations for
injection may be presented in unit dosage form (e.g., in ampoules
or in multi-dose containers) with an added preservative. The
compositions may take such forms as suspensions, solutions or
emulsions in oily or aqueous vehicles, and may contain formulatory
agents such as suspending, stabilizing and/or dispersing agents.
Alternatively, the active ingredient may be in powder form for
constitution with a suitable vehicle (e.g., sterile pyrogen-free
water) before use. The methods of the invention may additionally
comprise of administration of compositions formulated as depot
preparations. Such long acting formulations may be administered by
implantation (e.g., subcutaneously or intramuscularly) or by
intramuscular injection. Thus, for example, the compositions may be
formulated with suitable polymeric or hydrophobic materials (e.g.,
as an emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives (e.g., as a sparingly soluble
salt).
[0261] The methods of the invention encompass administration of
compositions formulated as neutral or salt forms. Pharmaceutically
acceptable salts include those formed with anions such as those
derived from hydrochloric, phosphoric, acetic, oxalic, tartaric
acids, etc., and those formed with cations such as those derived
from sodium, potassium, ammonium, calcium, ferric hydroxides,
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
[0262] Generally, the ingredients of compositions are supplied
either separately or mixed together in unit dosage form, for
example, as a dry lyophilized powder or water free concentrate in a
hermetically sealed container such as an ampoule or sachette
indicating the quantity of active agent. Where the mode of
administration is infusion, composition can be dispensed with an
infusion bottle containing sterile pharmaceutical grade water or
saline. Where the mode of administration is by injection, an
ampoule of sterile water for injection or saline can be provided so
that the ingredients may be mixed prior to administration.
[0263] In particular, the invention also provides that one or more
of the antibodies, or pharmaceutical compositions, of the invention
is packaged in a hermetically sealed container such as an ampoule
or sachette indicating the quantity of the antibody. In one
embodiment, one or more of the antibodies, or pharmaceutical
compositions of the invention is supplied as a dry sterilized
lyophilized powder or water free concentrate in a hermetically
sealed container and can be reconstituted (e.g., with water or
saline) to the appropriate concentration for administration to a
subject. In one embodiment, one or more of the antibodies or
pharmaceutical compositions of the invention is supplied as a dry
sterile lyophilized powder in a hermetically sealed container at a
unit dosage of at least 5 mg, for example at least 10 mg, at least
15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50
mg, at least 75 mg, or at least 100 mg. The lyophilized antibodies
or pharmaceutical compositions of the invention should be stored at
between 2.degree. C. and 8.degree. C. in its original container and
the antibodies, or pharmaceutical compositions of the invention
should be administered within 1 week, for example within 5 days,
within 72 hours, within 48 hours, within 24 hours, within 12 hours,
within 6 hours, within 5 hours, within 3 hours, or within 1 hour
after being reconstituted. In an alternative embodiment, one or
more of the antibodies or pharmaceutical compositions of the
invention is supplied in liquid form in a hermetically sealed
container indicating the quantity and concentration of the
antibody. In a further embodiment, the liquid form of the
administered composition is supplied in a hermetically sealed
container at least 0.25 mg/ml, for example at least 0.5 mg/ml, at
least 1 mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8
mg/ml, at least 10 mg/ml, at least 15 mg/kg, at least 25 mg/ml, at
least 50 mg/ml, at least 75 mg/ml or at least 100 mg/ml. The liquid
form should be stored at between 2.degree. C. and 8.degree. C. in
its original container.
[0264] The antibodies of the invention can be incorporated into a
pharmaceutical composition suitable for parenteral administration.
In one aspect, antibodies will be prepared as an injectable
solution containing 0.1-250 mg/ml antibody. The injectable solution
can be composed of either a liquid or lyophilized dosage form in a
flint or amber vial, ampule or pre-filled syringe. The buffer can
be L-histidine (1-50 mM), optimally 5-10 mM, at pH 5.0 to 7.0
(optimally pH 6.0). Other suitable buffers include but are not
limited to, sodium succinate, sodium citrate, sodium phosphate or
potassium phosphate. Sodium chloride can be used to modify the
toxicity of the solution at a concentration of 0-300 mM (optimally
150 mM for a liquid dosage form). Cryoprotectants can be included
for a lyophilized dosage form, principally 0-10% sucrose (optimally
0.5-1.0%). Other suitable cryoprotectants include trehalose and
lactose. Bulking agents can be included for a lyophilized dosage
form, principally 1-10% mannitol (optimally 2-4%). Stabilizers can
be used in both liquid and lyophilized dosage forms, principally
1-50 mM L-Methionine (optimally 5-10 mM). Other suitable bulking
agents include glycine, arginine, can be included as 0-0.05%
polysorbate-80 (optimally 0.005-0.01%). Additional surfactants
include but are not limited to polysorbate 20 and BRIJ surfactants.
The pharmaceutical composition comprising the antibodies of the
invention prepared as an injectable solution for parenteral
administration, can further comprise an agent useful as an
adjuvant, such as those used to increase the absorption, or
dispersion of the antibody. A particularly useful adjuvant is
hyaluronidase, such as Hylenex.RTM. (recombinant human
hyaluronidase). Addition of hyaluronidase in the injectable
solution improves human bioavailability following parenteral
administration, particularly subcutaneous administration. It also
allows for greater injection site volumes (i.e. greater than 1 ml)
with less pain and discomfort, and minimum incidence of injection
site reactions. (See International Appln. Publication No. WO
04/078140 and U.S. Patent Appln. Publication No. US2006104968,
incorporated herein by reference.)
[0265] The compositions of this invention may be in a variety of
forms. These include, for example, liquid, semi-solid and solid
dosage forms, such as liquid solutions (e.g., injectable and
infusible solutions), dispersions or suspensions, tablets, pills,
powders, liposomes and suppositories. The preferred form depends on
the intended mode of administration and therapeutic application.
Compositions can be in the form of injectable or infusible
solutions, such as compositions similar to those used for passive
immunization of humans with other antibodies. In one embodiment,
the antibody is administered by intravenous infusion or injection.
In another embodiment, the antibody is administered by
intramuscular or subcutaneous injection.
[0266] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
dispersion, liposome, or other ordered structure suitable to high
drug concentration. Sterile injectable solutions can be prepared by
incorporating the active compound (i.e., a binding protein, e.g. an
antibody, of the present invention) in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle that contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile, lyophilized powders for the
preparation of sterile injectable solutions, methods of preparation
comprise vacuum drying and spray-drying that yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof. The proper fluidity
of a solution can be maintained, for example, by the use of a
coating such as lecithin, by the maintenance of the required
particle size in the case of dispersion and by the use of
surfactants. Prolonged absorption of injectable compositions can be
brought about by including, in the composition, an agent that
delays absorption, for example, monostearate salts and gelatin.
[0267] The antibodies of the present invention can be administered
by a variety of methods known in the art. For many therapeutic
applications, the route/mode of administration may be subcutaneous
injection, intravenous injection or infusion. As will be
appreciated by the skilled artisan, the route and/or mode of
administration will vary depending upon the desired results. In
certain embodiments, the active compound may be prepared with a
carrier that will protect the compound against rapid release, such
as a controlled release formulation, including implants,
transdermal patches, and microencapsulated delivery systems.
Biodegradable, biocompatible polymers can be used, such as ethylene
vinyl acetate, polyanhydrides, polyglycolic acid, collagen,
polyorthoesters, and polylactic acid. Many methods for the
preparation of such formulations are patented or generally known to
those skilled in the art. See, e.g., Sustained and Controlled
Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker,
Inc., New York, 1978.
[0268] In certain embodiments, an antibody of the invention may be
orally administered, for example, with an inert diluent or an
assimilable edible carrier. The antibody (and other ingredients, if
desired) may also be enclosed in a hard or soft shell gelatin
capsule, compressed into tablets, or incorporated directly into the
subject's diet. For oral therapeutic administration, the antibody
may be incorporated with excipients and used in the form of
ingestible tablets, buccal tablets, troches, capsules, elixirs,
suspensions, syrups, wafers, and the like. To administer an
antibody of the invention by other than parenteral administration,
it may be necessary to coat the antibody with, or co-administer the
antibody with, a material to prevent its inactivation.
[0269] Supplementary active compounds can also be incorporated into
the compositions. In certain embodiments, an antibody of the
invention is coformulated with and/or coadministered with one or
more additional therapeutic agents that are useful for treating
disorders or diseases described herein. For example, an
anti-A.beta.(20-42) globulomer antibody of the invention may be
coformulated and/or coadministered with one or more additional
antibodies that bind other targets (e.g., antibodies that bind
other soluble antigens or that bind cell surface molecules).
Furthermore, one or more antibodies of the invention may be used in
combination with two or more of the foregoing therapeutic agents.
Such combination therapies may advantageously utilize lower dosages
of the administered therapeutic agents, thus avoiding possible
toxicities or complications associated with the various
monotherapies.
[0270] In certain embodiments, an antibody of the invention is
linked to a half-life extending vehicle known in the art. Such
vehicles include, but are not limited to, the Fc domain,
polyethylene glycol, and dextran. Such vehicles are described,
e.g., in U.S. application Ser. No. 09/428,082 and published PCT
Application No. WO 99/25044, which are hereby incorporated by
reference for any purpose.
[0271] In a specific embodiment, nucleic acid sequences comprising
nucleotide sequences encoding an antibody of the invention are
administered to treat, prevent, manage, or ameliorate a disorder or
one or more symptoms thereof by way of gene therapy. Gene therapy
refers to therapy performed by the administration to a subject of
an expressed or expressible nucleic acid. In this embodiment of the
invention, the nucleic acids produce their encoded antibody of the
invention that mediates a prophylactic or therapeutic effect.
[0272] Any of the methods for gene therapy available in the art can
be used according to the present invention. For general reviews of
the methods of gene therapy, see Goldspiel et al., 1993, Clinical
Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95;
Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;
Mulligan, Science 260:926-932 (1993); and Morgan and Anderson,
1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIBTECH
11(5):155-215. Methods commonly known in the art of recombinant DNA
technology which can be used are described in Ausubel et al.
(eds.), Current Protocols in Molecular Biology, John Wiley &
Sons, N Y (1993); and Kriegler, Gene Transfer and Expression, A
Laboratory Manual, Stockton Press, NY (1990). Detailed description
of various methods of gene therapy are disclosed in US20050042664
A1 which is incorporated herein by reference.
[0273] Antibodies of the invention can be used alone or in
combination to treat diseases such as Alzheimer's disease, Down
syndrome, dementia, Parkinson's disease, or any other disease or
condition associated with a build up of amyloid beta protein within
the brain. The antibodies of the present invention may be used to
treat "conformational diseases". Such diseases arise from secondary
to tertiary structural changes within constituent proteins with
subsequent aggregation of the altered proteins (Hayden et al., JOP.
J Pancreas 2005; 6(4):287-302). In particular, the antibodies of
the present invention may be used to treat one or more of the
following conformational diseases: Alpha1-antitrypsin-deficiency,
C1-inhibitor deficiency angioedema, Antithrombin deficiency
thromboembolic disease, Kuru, Creutzfeld-Jacob disease/scrapie,
Bovine spongiform encephalopathy, Gerstmann-Straussler-Scheinker
disease, Fatal familial insomnia, Huntington's disease,
Spinocerebellar ataxia, Machado-Joseph atrophy,
Dentato-rubro-pallidoluysian atrophy, Frontotemporal dementia,
Sickle cell anemia, Unstable hemoglobin inclusion-body hemolysis,
Drug-induced inclusion body hemolysis, Parkinson's disease,
Systemic AL amyloidosis, Nodular AL amyloidosis, Systemic AA
amyloidosis, Prostatic amyloidosis, Hemodialysis amyloidosis,
Hereditary (Icelandic) cerebral angiopathy, Huntington's disease,
Familial visceral amyloidosis, Familial visceral polyneuropathy,
Familial visceral amyloidosis, Senile systemic amyloidosis,
Familial amyloid neurophathy, Familial cardiac amyloidosis,
Alzheimer's disease, Down syndrome, Medullary carcinoma thyroid and
Type 2 diabetes mellitus (T2DM) Preferably, the antibodies of the
present invention may be utilized to treat an amyloidosis, for
example, Alzheimer's disease and Down syndrome.
[0274] It should be understood that the antibodies of the invention
can be used alone or in combination with one or more additional
agents, e.g., a therapeutic agent (for example, a small molecule or
biologic), said additional agent being selected by the skilled
artisan for its intended purpose. For example, the additional
therapeutic agent can be a "cognitive enhancing drug," which is a
drug that improves impaired human cognitive abilities of the brain
(namely, thinking, learning, and memory). Cognitive enhancing drugs
work by altering the availability of neurochemicals (e.g.,
neurotransmitters, enzymes, and hormones), by improving oxygen
supply, by stimulating nerve growth, or by inhibiting nerve damage.
Examples of cognitive enhancing drugs include a compound that
increases the activity of acetylcholine such as, but not limited
to, an acetylcholine receptor agonist (e.g., a nicotinic .alpha.-7
receptor agonist or allosteric modulator, an .alpha.4.beta.2
nicotinic receptor agonist or allosteric modulators), an
acetylcholinesterase inhibitor (e.g., donepezil, rivastigmine, and
galantamine), a butyrylcholinesterase inhibitor, an
N-methyl-D-aspartate (NMDA) receptor antagonist (e.g., memantine),
an activity-dependent neuroprotective protein (ADNP) agonist, a
serotonin 5-HT1A receptor agonist (e.g., xaliproden), a 5-HT.sub.4
receptor agonist, a 5-HT.sub.6 receptor antagonist, a serotonin 1A
receptor antagonist, a histamine H.sub.3 receptor antagonist, a
calpain inhibitor, a vascular endothelial growth factor (VEGF)
protein or agonist, a trophic growth factor, an anti-apoptotic
compound, an AMPA-type glutamate receptor activator, a L-type or
N-type calcium channel blocker or modulator, a potassium channel
blocker, a hypoxia inducible factor (HIF) activator, a HIF prolyl
4-hydroxylase inhibitor, an anti-inflammatory agent, an inhibitor
of amyloid A.beta. peptide or amyloid plaque, an inhibitor of tau
hyperphosphorylation, a phosphodiesterase 5 inhibitor (e.g.,
tadalafil, sildenafil), a phosphodiesterase 4 inhibitor, a
monoamine oxidase inhibitor, or pharmaceutically acceptable salt
thereof. Specific examples of such cognitive enhancing drugs
include, but are not limited to, cholinesterase inhibitors such as
donepezil (Aricept.RTM.), rivastigmine (Exelon.RTM.), galanthamine
(Reminyl.RTM.), N-methyl-D-aspartate antagonists such as memantine
(Namenda.RTM.). At least one cognitive enhancing drug can be
administered simultaneously with the antibodies of the present
invention or sequentially with the antibodies of the present
invention (and in any order) including those agents currently
recognized, or in the future being recognized, as useful to treat
the disease or condition being treated by an antibody of the
present invention). Additionally, it is believed that the
combinations described herein may have additive or synergistic
effects when used in the above-described treatment. The additional
agent also can be an agent that imparts a beneficial attribute to
the therapeutic composition, e.g., an agent that affects the
viscosity of the composition.
[0275] It should further be understood that the combinations which
are to be included within this invention are those combinations
useful for their intended purpose. The agents set forth above are
illustrative for purposes and not intended to be limited. The
combinations, which are part of this invention, can comprise an
antibody of the present invention and at least one additional agent
selected from the lists below. The combination can also include
more than one additional agent, e.g., two or three additional
agents if the combination is such that the formed composition can
perform its intended function.
[0276] The pharmaceutical compositions of the invention may include
a "therapeutically effective amount" or a "prophylactically
effective amount" of an antibody of the invention. A
"therapeutically effective amount" refers to an amount effective,
at dosages and for periods of time necessary, to achieve the
desired therapeutic result. A therapeutically effective amount of
the antibody may be determined by a person skilled in the art and
may vary according to factors such as the disease state, age, sex,
and weight of the individual, and the ability of the antibody to
elicit a desired response in the individual. A therapeutically
effective amount is also one in which any toxic or detrimental
effects of the antibody are outweighed by the therapeutically
beneficial effects. A "prophylactically effective amount" refers to
an amount effective, at dosages and for periods of time necessary,
to achieve the desired prophylactic result. Typically, since a
prophylactic dose is used in subjects prior to or at an earlier
stage of disease, the prophylactically effective amount will be
less than the therapeutically effective amount.
[0277] Dosage regimens may be adjusted to provide the optimum
desired response (e.g., a therapeutic or prophylactic response).
For example, a single bolus may be administered, several divided
doses may be administered over time or the dose may be
proportionally reduced or increased as indicated by the exigencies
of the therapeutic situation. It is especially advantageous to
formulate parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the mammalian subjects to be treated; each unit
containing a predetermined quantity of active compound calculated
to produce the desired therapeutic effect in association with the
required pharmaceutical carrier. The specification for the dosage
unit forms of the invention are dictated by and directly dependent
on (a) the unique characteristics of the active compound and the
particular therapeutic or prophylactic effect to be achieved, and
(b) the limitations inherent in the art of compounding such an
active compound for the treatment of sensitivity in
individuals.
[0278] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of an antibody of the invention
is 0.1-20 mg/kg, for example 1-10 mg/kg. It is to be noted that
dosage values may vary with the type and severity of the condition
to be alleviated. It is to be further understood that for any
particular subject, specific dosage regimens should be adjusted
over time according to the individual need and the professional
judgment of the person administering or supervising the
administration of the compositions, and that dosage ranges set
forth herein are exemplary only and are not intended to limit the
scope or practice of the claimed composition.
[0279] It will be readily apparent to those skilled in the art that
other suitable modifications and adaptations of the methods of the
invention described herein are obvious and may be made using
suitable equivalents without departing from the scope of the
invention or the embodiments disclosed herein. Having now described
the present invention in detail, the same will be more clearly
understood by reference to the following examples, which are
included for purposes of illustration only and are not intended to
be limiting of the invention.
EXAMPLES
Example 1: Preparation of Globulomers
[0280] a) A.beta.(1-42) Globulomer:
[0281] The A.beta.(1-42) synthetic peptide (H-1368, Bachem,
Bubendorf, Switzerland) was suspended in 100%
1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at 6 mg/ml and incubated
for complete solubilization under shaking at 37.degree. C. for 1.5
h. The HFIP acts as a hydrogen-bond breaker and is used to
eliminate pre-existing structural inhomogeneities in the A.beta.
peptide. HFIP was removed by evaporation in a SpeedVac and
A.beta.(1-42) resuspended at a concentration of 5 mM in
dimethylsulfoxide and sonicated for 20 s. The HFIP-pre-treated
A.beta.(1-42) was diluted in phosphate-buffered saline (PBS) (20 mM
NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4) to 400 .mu.M and 1/10
volume 2% sodium dodecyl sulfate (SDS) (in H.sub.2O) added (final
concentration of 0.2% SDS). An incubation for 6 h at 37.degree. C.
resulted in the 16/20-kDa A.beta.(1-42) globulomer (short form for
globular oligomer) intermediate. The 38/48-kDa A.beta.(1-42)
globulomer was generated by a further dilution with three volumes
of H.sub.2O and incubation for 18 h at 37.degree. C. After
centrifugation at 3000 g for 20 min the sample was concentrated by
ultrafiltration (30-kDa cut-off), dialysed against 5 mM
NaH.sub.2PO.sub.4, 35 mM NaCl, pH 7.4, centrifuged at 10,000 g for
10 min and the supernatant comprising the 38/48-kDa A.beta.(1-42)
globulomer withdrawn. As an alternative to dialysis the 38/48-kDa
A.beta.(1-42) globulomer could also be precipitated by a ninefold
excess (v/v) of ice-cold methanol/acetic acid solution (33%
methanol, 4% acetic acid) for 1 h at 4.degree. C. The 38/48-kDa
A.beta.(1-42) globulomer is then pelleted (10 min at 16200 g),
resuspended in 5 mM NaH.sub.2PO.sub.4, 35 mM NaCl, pH 7.4, and the
pH adjusted to 7.4.
[0282] b) A.beta.(20-42) Globulomer:
[0283] 1.59 ml of A.beta.(1-42) globulomer preparation prepared
according to Example 1a were admixed with 38 ml of buffer (50 mM
MES/NaOH, pH 7.4) and 200 .mu.l of a 1 mg/ml thermolysin solution
(Roche) in water. The reaction mixture was stirred at RT for 20 h.
Then, 80 .mu.l of a 100 mM EDTA solution, pH 7.4, in water were
added and the mixture was furthermore adjusted to an SDS content of
0.01% with 400 .mu.l of a 1% strength SDS solution. The reaction
mixture was concentrated to approximately 1 ml via a 15 ml 30 kDa
Centriprep tube. The concentrate was admixed with 9 ml of buffer
(50 mM MES/NaOH, 0.02% SDS, pH 7.4) and again concentrated to 1 ml.
The concentrate was dialyzed at 6.degree. C. against 1 l of buffer
(5 mM sodium phosphate, 35 mM NaCl) in a dialysis tube for 16 h.
The dialysate was adjusted to an SDS content of 0.1% with a 2%
strength SDS solution in water. The sample was centrifuged at
10,000 g for 10 min and the A.beta.(20-42) globulomer supernatant
was withdrawn.
[0284] c) A.beta.(12-42) Globulomer:
[0285] 2 ml of an A.beta.(1-42) globulomer preparation prepared
according to Example 1a were admixed with 38 ml buffer (5 mM sodium
phosphate, 35 mM sodium chloride, pH 7.4) and 150 .mu.l of a 1
mg/ml GluC endoproteinase (Roche) in water. The reaction mixture
was stirred for 6 h at RT, and a further 150 .mu.l of a 1 mg/ml
GluC endoproteinase (Roche) in water were subsequently added. The
reaction mixture was stirred at RT for another 16 h, followed by
addition of 8 .mu.l of a 5 M DIFP solution. The reaction mixture
was concentrated to approximately 1 ml via a 15 ml 30 kDa
Centriprep tube. The concentrate was admixed with 9 ml of buffer (5
mM sodium phosphate, 35 mM sodium chloride, pH 7.4) and again
concentrated to 1 ml. The concentrate was dialyzed at 6.degree. C.
against 1 l of buffer (5 mM sodium phosphate, 35 mM NaCl) in a
dialysis tube for 16 h. The dialysate was adjusted to an SDS
content of 0.1% with a 1% strength SDS solution in water. The
sample was centrifuged at 10,000 g for 10 min and the
A.beta.(12-42) globulomer supernatant was withdrawn.
[0286] d) Cross-Linked A.beta.(1-42) Globulomer:
[0287] The A.beta.(1-42) synthetic peptide (H-1368, Bachem,
Bubendorf, Switzerland) was suspended in 100%
1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at 6 mg/ml and incubated
for complete solubilization under shaking at 37.degree. C. for 1.5
h. The HFIP acts as a hydrogen-bond breaker and was used to
eliminate pre-existing structural inhomogeneities in the A.beta.
peptide. HFIP was removed by evaporation by a SpeedVac and
A.beta.(12-42) globulomer A.beta.(1-42) resuspended at a
concentration of 5 mM in dimethylsulfoxide and sonicated for 20 s.
The HFIP-pre-treated A.beta.(1-42) was diluted in PBS (20 mM
NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4) to 400 .mu.M and 1/10 vol.
2% SDS (in water) added (final conc. Of 0.2% SDS). An incubation
for 6 h at 37.degree. C. resulted in the 16/20-kDa A.beta.(1-42)
globulomer (short form for globulomer oligomer) intermediate. The
38/48-kDa A.beta.(1-42) globulomer was generated by a further
dilution with 3 volumes of water and incubation for 18 h at
37.degree. C. Cross-linking of the 38/48-kDa A.beta.(1-42)
globulomer was now performed by incubation with 1 mM glutaraldehyde
for 2 h at 21.degree. C. room temperature followed by ethanolamine
(5 mM) treatment for 30 min at room temperature.
Example 2: Generation, Isolation and Characterization of Humanized
Anti-A.beta.(20-42) Globulomer Antibodies
Example 2.1: Selection of Human Antibody Frameworks
[0288] Selection of human antibody frameworks was based on
similarity of canonical structures and amino acid sequence homology
of human antibodies. Further, the retention of amino acid residues
which support loop structures and VH/VL interface as well as the
retention of amino acid residues of the Vernier zone was taken into
account when identifying suitable acceptor VL and VH framework
sequences based on amino acid sequence homology of human VH and
V.kappa. germline sequences. Moreover, immunogenicity of VH and VL
sequences resulting from grafting 4D10 CDRs into potentially
suitable acceptor VL and VH framework sequences was evaluated in
silico based on the predicted affinity of overlapping peptides to a
variety of MHC class I and/or MHC class II alleles. VH and VL were
adapted to the consensus of the respective VH or VL family to
further minimize potential immunogenicity. Selected backmutations
to murine amino acid residues were performed to retain amino acids
which support loop structures and VH/VL interface. The frequencies
of these backmutations in corresponding pools of naturally
occurring human VH or VL sequences having the respective VH or VL
germline gene were determined by amino acid sequence alignments.
The VH and VL sequences resulting from the considerations described
above were checked for potential N-linked glycosylation sites (NXS
or NXT, wherein X is any amino acid except P).
Example 2.2: Humanization of Murine Anti-A.beta.(20-42) Globulomer
Antibody
[0289] 4D10hum_VH.1z (SEQ ID NO:4): The heavy chain CDR sequences
from the murine anti-A.beta.(20-42) globulomer antibody 4D10
described in Table 4 were grafted into an acceptor framework of
human VH3-53 and JH6 sequences.
[0290] 4D10hum_VH.1 (SEQ ID NO:5): The heavy chain CDR sequences
from the murine anti-A.beta.(20-42) globulomer antibody 4D10
described in Table 4 were grafted into an acceptor framework of
human VH3-53 and JH6 sequences comprising VH3 consensus change
I12V.
[0291] 4D10hum_VH.1a (SEQ ID NO:6): The heavy chain CDR sequences
from the murine anti-A.beta.(20-42) globulomer antibody 4D10
described in Table 4 were grafted into an acceptor framework of
human VH3-53 and JH6 sequences comprising VH3 consensus change I12V
and framework backmutations A24V, V29L, V48L, S49G, F67L, R71K,
N76S and L78V.
[0292] 4D10hum_VH.1b (SEQ ID NO:7): The heavy chain CDR sequences
from the murine anti-A.beta.(20-42) globulomer antibody 4D10
described in Table 4 were grafted into an acceptor framework of
human VH3-53 and JH6 sequences comprising backmutations V29L and
R71K.
[0293] 4D10hum_VH.2z (SEQ ID NO:8): The heavy chain CDR sequences
from the murine anti-A.beta.(20-42) globulomer antibody 4D10
described in Table 4 were grafted into an acceptor framework of
human VH4-59 and JH6 sequences.
[0294] 4D10hum_VH.2 (SEQ ID NO:9): The heavy chain CDR sequences
from the murine anti-A.beta.(20-42) globulomer antibody 4D10
described in Table 4 were grafted into an acceptor framework of
human VH4-59 and JH6 sequences comprising a Q1E change to prevent
N-terminal pyroglutamate formation.
[0295] 4D10hum_VH.2a (SEQ ID NO:10): The heavy chain CDR sequences
from the murine anti-A.beta.(20-42) globulomer antibody 4D10
described in Table 4 were grafted into an acceptor framework of
human VH4-59 and JH6 sequences comprising a Q1E change to prevent
N-terminal pyroglutamate formation, and framework backmutations
G27F, I29L, I37V, I48L, V67L, V71K, N76S and F78V.
[0296] 4D10hum_VH.2b (SEQ ID NO:11): The heavy chain CDR sequences
from the murine anti-A.beta.(20-42) globulomer antibody 4D10
described in Table 4 were grafted into an acceptor framework of
human VH4-59 and JH6 sequences comprising a Q1E change to prevent
N-terminal pyroglutamate formation, and framework backmutations
G27F, I29L and V71K.
[0297] 4D10hum_V.kappa..1z (SEQ ID NO:12): The light chain CDR
sequences from the murine anti-A.beta.(20-42) globulomer antibody
4D10 described in Table 4 were grafted into an acceptor framework
of human V.kappa. A17/2-30 and J.kappa.2 sequences.
[0298] 4D10hum_V.kappa..1 (SEQ ID NO:13): The light chain CDR
sequences from the murine anti-A.beta.(20-42) globulomer antibody
4D10 described in Table 4 were grafted into an acceptor framework
of human V.kappa. A17/2-30 and J.kappa.2 sequences comprising
V.kappa.2 consensus changes S7T, L15P, Q37L, R39K and R45Q.
[0299] 4D10hum_V.kappa..1a (SEQ ID NO:14): The light chain CDR
sequences from the murine anti-A.beta.(20-42) globulomer antibody
4D10 described in Table 4 were grafted into an acceptor framework
of human V.kappa. A17/2-30 and J.kappa.2 sequences comprising
V.kappa.2 consensus changes S7T, LISP, Q37L, R39K and R45Q, and
framework backmutation F36L which affects the VL/VH interface.
[0300] 4D10hum_V.kappa..1b (SEQ ID NO:15): The light chain CDR
sequences from the murine anti-A.beta.(20-42) globulomer antibody
4D10 described in Table 4 were grafted into an acceptor framework
of human V.kappa. A17/2-30 and J.kappa.2 sequences comprising
V.kappa.2 consensus changes S7T and Q37L.
[0301] 4D10hum_V.kappa..1c (SEQ ID NO:16): The light chain CDR
sequences from the murine anti-A.beta.(20-42) globulomer antibody
4D10 described in Table 4 were grafted into an acceptor framework
of human V.kappa. A17/2-30 and J.kappa.2 sequences comprising
V.kappa.2 consensus changes S7T, Q37L and R39K.
[0302] Some of said VH and V.kappa. back-mutations, consensus
changes or the Q1E mutation in 4D10hum_VH.2, 4D10hum_VH.2a or
4D10hum_VH.2b may be removed during a subsequent affinity
maturation.
Example 2.3: Construction of Humanized Antibodies
[0303] In silico constructed humanized antibodies described above
will be constructed de novo using oligonucleotides. For each
variable region cDNA, 6 oligonucleotides of 60-80 nucleotides each
will be designed to overlap each other by 20 nucleotides at the 5'
and/or 3' end of each oligonucleotide. In an annealing reaction,
all 6 oligos will be combined, boiled, and annealed in the presence
of dNTPs. Then DNA polymerase I, Large (Klenow) fragment (New
England Biolabs #M0210, Beverley, Mass.) will be added to fill-in
the approximately 40 bp gaps between the overlapping
oligonucleotides. PCR will then be performed to amplify the entire
variable region gene using two outermost primers containing
overhanging sequences complementary to the multiple cloning site in
a modified pBOS vector (Mizushima, S. and Nagata, S., (1990)
Nucleic acids Research Vol 18, No. 17)). The PCR products derived
from each cDNA assembly will be separated on an agarose gel and the
band corresponding to the predicted variable region cDNA size will
be excised and purified. The variable heavy region will be inserted
in-frame onto a cDNA fragment encoding the human IgG1 constant
region containing 2 hinge-region amino acid mutations by homologous
recombination in bacteria. These mutations are a leucine to alanine
change at position 234 (EU numbering) and a leucine to alanine
change at position 235 (Lund et al., 1991, J. Immunol., 147:2657).
The variable light chain region will be inserted in-frame with the
human kappa constant region by homologous recombination. Bacterial
colonies will be isolated and plasmid DNA extracted; cDNA inserts
will be sequenced in their entirety. Correct humanized heavy and
light chains corresponding to each antibody will be co-transfected
into COS cells to transiently produce full-length humanized
anti-A.beta. globulomer antibodies. Cell supernatants containing
recombinant chimeric antibody will be purified by Protein A
Sepharose chromatography and bound antibody will be eluted by
addition of acid buffer. Antibodies will be neutralized and
dialyzed into PBS. (Dieder Moechars et al J Biol Chem 274:6483-6492
(1999); Ausubel, F. M. et al. eds., Short Protocols In Molecular
Biology (4th Ed. 1999) John Wiley & Sons, NY. (ISBN
0-471-32938-X); Lu and Weiner eds., Cloning and Expression Vectors
for Gene Function Analysis (2001) BioTechniques Press. Westborough,
Mass. 298 pp. (ISBN 1-881299-21-X); Kontermann and Dubel eds.,
Antibody Engineering (2001) Springer-Verlag. New York. 790 pp.
(ISBN 3-540-41354-5); Old, R. W. & S. B. Primrose, Principles
of Gene Manipulation: An Introduction To Genetic Engineering (3d
Ed. 1985) Blackwell Scientific Publications, Boston. Studies in
Microbiology; V.2:409 pp. (ISBN 0-632-01318-4); Sambrook, J. et al.
eds., Molecular Cloning: A Laboratory Manual (2d Ed. 1989) Cold
Spring Harbor Laboratory Press, NY. Vols. 1-3. (ISBN
0-87969-309-6); Winnacker, E. L. From Genes To Clones: Introduction
To Gene Technology (1987) VCH Publishers, NY (translated by Horst
Ibelgaufts). 634 pp. (ISBN 0-89573-614-4); all of which are
incorporated by reference in their entirety).
[0304] Although a number of embodiments and features have been
described above, it will be understood by those skilled in the art
that modifications and variations of the described embodiments and
features may be made without departing from the present disclosure
or the invention as defined in the appended claims.
Example 2:4: Expression and Purification of Humanized Antibodies in
HEK293 Cells
[0305] DNA constructs encoding an antibody heavy chain as set forth
in SEQ ID NO:46; an antibody heavy chain as set forth in SET ID
NO:47, and an antibody light chain construct encoding a polypeptide
as set forth in SEQ ID NO:48 were prepared as described in example
2.3. After DNA confirmation by sequencing, all heavy chain and
light chain DNA constructs were expanded in E. coli and DNA was
purified using Qiagen Endo Free Plasmid Maxi Prep (cat#12362,
QIAGEN) according to the manufacturer's protocol.
[0306] For expression of a monoclonal antibody 4D10hum#1, HEK293
(EBNA) cells were transiently cotransfected with plasmids encoding
the heavy chain set forth in SEQ ID NO:46 and the light chain set
forth in SEQ ID NO:48. For expression of a monoclonal antibody
4D10hum#2, HEK293 (EBNA) cells were transiently cotransfected with
plasmids encoding the heavy chain set forth in SEQ ID NO:47 and the
light chain set forth in SEQ ID NO:48. Before transfection, HEK293
(EBNA) cells were propagated in Freestyle 293 media (Invitrogen,
Carlsbad Calif.) at a 0.5 l scale in culture flasks (2 L Corning
Cat#431198) shaking in a CO.sub.2 incubator (8% CO.sub.2, 125 rpm,
37.degree. C.). When the cell cultures reached a density of
1.times.10.sup.6 cells/ml, cells were transfected by adding
transfection complex. The transfection complex was prepared by
first mixing 150 .mu.g of the plasmid encoding the light chain, 100
.mu.g of the plasmid encoding the heavy chain and 25 ml Freestyle
medium, followed by the addition of 500 .mu.l PEI solution (1 mg/ml
(pH 7.0) linear 25 kDa polyethylenimine, Polysciences Cat#23966).
The transfection complex was mixed by inversion and incubated at
room temperature for 15 min prior to being added to the cell
culture. After transfection, cultures continued to be grown in the
CO.sub.2 incubator (8% CO.sub.2, 125 rpm, 37.degree. C.).
Twenty-four hours after transfection, the culture media were
supplemented with 50 ml of a 5% Tryptone N1 solution (Organo
Technie, La Courneuve France Cat#19553). Six days after
transfection, the cells were pelleted by centrifugation (16,000 g,
30 min), the supernatant containing the expressed antibodies was
sterile filtered (0.2 .mu.m PES filter) and placed at 4.degree. C.
until initiation of the purification step. The expressed antibodies
were purified from the supernatants by Protein A sepharose affinity
chromatography using Pierce Thermo Scientific reagents and protocol
according to manufacturer's instructions. The protein eluates were
dialyzed against PBS (pH 7). The purified 4D10hum antibodies were
spectrophotometrically quantified at 280 nm, and analyzed by mass
spectrometry and size exclusion chromatography (SEC).
Example 2:5: Affinity Analysis of Humanized Antibodies
[0307] Interaction of the purified humanized antibodies 4D10hum#1
and 4D10hum#2 to A.beta.(20-42) globulomer was evaluated by surface
plasmon resonance (SPR) analysis using a BIAcore device. Goat
anti-human IgG Fc (10,000 RU) was directly immobilized on a CMS
sensor chip by an amine coupling procedure according to the
manufacturer's instructions (BIAcore). The respective 4D10hum
antibody was captured on the goat anti-human IgG Fc coated surface
of the chip by injecting 5.0 .mu.l of a 1 .mu.g/ml 4D10hum antibody
solution at a flow rate of 10-15 .mu.l/min. Interaction of soluble
A.beta.(20-42) globulomer with the 4D10hum antibody on the sensor
chip was examined by injecting globulomer solutions (concentration
range: 20-0.3125 nM) at a flow rate of 50 .mu.l/min. The
association rate was monitored for 5.0 min and the dissociation
rate was monitored for 10 min. From the resulting sensorgrams the
association rate constant (k.sub.on), dissociation rate constant
(k.sub.off) and equilibrium dissociation constant (K.sub.D) were
determined using to the manufacturer's software and instructions.
The kinetic and equilibrium constants determined for three
different preparations of 4D10hum#1 and two different preparations
of 4D10hum#2 are summarized in table 7. Table 7 also shows affinity
data of antibodies #3, #4 and #5 having chimeric and humanized
chains. The heavy chains of antibodies #4 and #5 are as in
4D10hum#1 or #2, and the light chains are chimeras of m4D10 VL (SEQ
ID NO:24) and the human Ig kappa constant region (SEQ ID NO:27).
The light chains of antibody #3 are as in 4D10hum#1 and #2, and the
heavy chains are chimeras of m4D10 VH (SEQ ID NO23) and the human
Ig gamma-1 constant region (SEQ ID NO:25).
TABLE-US-00010 TABLE 7 AFFINITY OF 4D10HUM ANTIBODIES FOR
A.beta.(20-42) GLOBULOMER k.sub.on k.sub.off K.sub.D Antibody
Antibody Lot Experiment [M.sup.-1s.sup.-1] [s.sup.-1] [M] 4D10hum#1
#1759115 1 5.22 .times. 10.sup.5 3.02 .times. 10.sup.-4 5.78
.times. 10.sup.-10 2 5.39 .times. 10.sup.5 3.61 .times. 10.sup.-4
6.71 .times. 10.sup.-10 average 5.31 .times. 10.sup.5 3.32 .times.
10.sup.-4 6.25 .times. 10.sup.-10 #1763976 1 4.86 .times. 10.sup.5
2.81 .times. 10.sup.-4 5.78 .times. 10.sup.-10 2 5.13 .times.
10.sup.5 3.04 .times. 10.sup.-4 5.93 .times. 10.sup.-10 average
5.00 .times. 10.sup.5 2.93 .times. 10.sup.-4 5.86 .times.
10.sup.-10 #1773662 1 4.66 .times. 10.sup.5 2.49 .times. 10.sup.-4
5.35 .times. 10.sup.-10 2 5.18 .times. 10.sup.5 2.87 .times.
10.sup.-4 5.53 .times. 10.sup.-10 average 4.92 .times. 10.sup.5
2.68 .times. 10.sup.-4 5.44 .times. 10.sup.-10 4D10hum#2 #1759119 1
5.93 .times. 10.sup.5 2.70 .times. 10.sup.-4 4.54 .times.
10.sup.-10 2 5.46 .times. 10.sup.5 3.32 .times. 10.sup.-4 6.09
.times. 10.sup.-10 average 5.70 .times. 10.sup.5 3.01 .times.
10.sup.-4 5.32 .times. 10.sup.-10 #1773659 1 5.07 .times. 10.sup.5
2.68 .times. 10.sup.-4 5.29 .times. 10.sup.-10 2 6.86 .times.
10.sup.5 2.98 .times. 10.sup.-4 4.35 .times. 10.sup.-10 average
5.97 .times. 10.sup.5 2.83 .times. 10.sup.-4 4.82 .times.
10.sup.-10 k.sub.on k.sub.off K.sub.D Antibody Experiment
[M.sup.-1s.sup.-1] [s.sup.-1] [M] 4D10#3 1 6.03 .times. 10.sup.5
3.17 .times. 10.sup.-4 5.25 .times. 10.sup.-10 (chimeric heavy
chain; light 2 5.22 .times. 10.sup.5 3.49 .times. 10.sup.-4 6.69
.times. 10.sup.-10 chain as 4D10hum#1 and #2) average 5.63 .times.
10.sup.5 3.33 .times. 10.sup.-4 5.97 .times. 10.sup.-10 4D10#4 1
4.62 .times. 10.sup.5 2.94 .times. 10.sup.-4 6.35 .times.
10.sup.-10 (heavy chain as 4D10hum#1; 2 5.06 .times. 10.sup.5 3.32
.times. 10.sup.-4 6.57 .times. 10.sup.-10 chimeric light chain)
average 4.84 .times. 10.sup.5 3.13 .times. 10.sup.-4 6.46 .times.
10.sup.-10 4D10#5 1 4.94 .times. 10.sup.5 2.62 .times. 10.sup.-4
5.30 .times. 10.sup.-10 (heavy chain as 4D10hum#2; 2 4.72 .times.
10.sup.5 2.92 .times. 10.sup.-4 6.19 .times. 10.sup.-10 chimeric
light chain) average 4.83 .times. 10.sup.5 2.77 .times. 10.sup.-4
5.75 .times. 10.sup.-10
Example 2.6: Analysis of Antibody Selectivity Via Dot Blot
[0308] In order to characterize the selectivity of monoclonal anti
A.beta.(20-42) globulomer antibodies, they were tested for binding
to different A.beta.-forms. To this end, serial dilutions of the
individual A.beta.(1-42) forms ranging from 100 pmol/.mu.l to
0.00001 pmol/.mu.l in PBS supplemented with 0.2 mg/ml BSA were
prepared. 1 .mu.l of each dilution was blotted onto a
nitrocellulose membrane. Detection was performed by incubating with
the corresponding antibody (0.2 .mu.g/ml) followed by
immunostaining using Peroxidase conjugated anti-human-IgG and the
staining reagent BM Blue POD Substrate (Roche).
[0309] A.beta.-Standards for Dot-Blot:
[0310] 1. A.beta.(1-42) globulomer
[0311] A.beta.(1-42) globulomer was prepared as described in
Example 1a (buffer exchange by dialysis).
[0312] 2. A.beta.(20-42) globulomer
[0313] A.beta.(20-42) globulomer was prepared as described in
Example 1b.
[0314] 3. A.beta.(1-40) monomer, 0.1% NaOH
[0315] 2.5 mg A.beta.(1-40) (Bachem Inc., cat. no. H-1368) was
dissolved in 0.5 ml 0.1% NaOH in H.sub.2O (freshly prepared) (=5
mg/ml) and immediately shaken for 30 sec. at room temperature to
obtain a clear solution. The sample was stored at -20.degree. C.
until use.
[0316] 4. A.beta.(1-42) monomer, 0.1% NaOH
[0317] 2.5 mg A.beta.(1-42) (Bachem Inc., cat. no. H-1368) was
dissolved in 0.5 ml 0.1% NaOH in H.sub.2O (freshly prepared) (=5
mg/ml) and immediately shaken for 30 sec. at room temperature to
obtain a clear solution. The sample was stored at -20.degree. C.
until use.
[0318] 5. A.beta.(1-42) fibrils
[0319] 1 mg A.beta.(1-42) (Bachem Inc. cat. no.: H-1368) was
dissolved in 500 .mu.l aqueous 0.1% NH.sub.4OH (Eppendorf tube) and
stirred for 1 min at room temperature. 100 .mu.l of this freshly
prepared A.beta.(1-42) solution were neutralized with 300 .mu.l 20
mM NaH.sub.2PO.sub.4; 140 mM NaCl, pH 7.4. The pH was adjusted to
pH 7.4 with 1% HCl. The sample was incubated for 24 h at 37.degree.
C. and centrifuged (10 min at 10000 g). The supernatant was
discarded and the fibril pellet resuspended with 400 .mu.l 20 mM
NaH.sub.2PO.sub.4; 140 mM NaCl, pH 7.4 by vortexing for 1 min.
[0320] 6. sAPP.alpha.
[0321] Supplied by Sigma (cat. no. 59564; 25 .mu.g in 20 mM
NaH.sub.2PO.sub.4; 140 mM NaCl; pH 7.4). The sAPP.alpha. was
diluted to 0.1 mg/ml (=1pmol/.mu.l) with 20 mM NaH.sub.2PO.sub.4,
140 mM NaCl, pH 7.4, 0.2 mg/ml BSA.
[0322] 7. A.beta.(12-42) globulomer
[0323] A.beta.(12-42) globulomer was prepared as described in
Example 1c.
[0324] Materials for Dot Blot:
[0325] Serial dilution of A.beta.-standards (see above 1. to 7.) in
20 mM NaH.sub.2PO.sub.4, 140 mM NaCl, pH 7.4+0.2 mg/ml BSA to
obtain concentrations of: 100 pmol/.mu.l, 10 pmol/.mu.l, 1
pmol/.mu.l, 0.1 pmol/.mu.l, 0.01 pmol/.mu.l, 0.001 pmol/.mu.l,
0.0001 pmol/.mu.l, and 0.00001 pmol/.mu.l.
[0326] Nitrocellulose: Trans-Blot Transfer medium, Pure
Nitrocellulose Membrane (0.2 .mu.m); BIO-RAD
[0327] Anti-human-POD: cat no: 109-035-003 (Jackson Immuno
Research)
[0328] Detection reagent: BM Blue POD Substrate, precipitating, cat
no: 11442066001 (Roche)
[0329] Bovine serum albumin, (BSA): Cat no: 11926 (Serva)
[0330] Blocking reagent: 5% low fat milk in TBS
[0331] Buffer solutions: [0332] TBS: 25 mM Tris/HCl buffer pH
7.5+150 mM NaCl [0333] TTBS: 25 mM Tris/HCl-buffer pH 7.5+150 mM
NaCl+0.05% Tween 20 [0334] PBS+0.2 mg/ml BSA: 20 mM NaH2PO4 buffer
pH 7.4+140 mM NaCl+0.2 mg/ml BSA
[0335] Antibody solution I: 0.2 .mu.g/ml antibody in 20 ml 1% low
fat milk in TBS
[0336] Antibody: humanized monoclonal anti-A.beta. antibody
4D10hum#1; 4.7 mg/ml OD 280 nm; stored at -80.degree. C.
[0337] Antibody solution II: 1:5000 dilution of anti-human-POD in
1% low fat milk in TBS
[0338] Dot Blot Procedure: [0339] 1) 1 .mu.l of each of the 8
concentrations of the different A.beta.-standards (obtained by
serial dilution) was dotted onto the nitrocellulose membrane in a
distance of approximately 1 cm from each other. [0340] 2) The dots
of A.beta.-standards were allowed to dry on the nitrocellulose
membrane on air for at least 10 min at room temperature (RT). (=dot
blot) [0341] 3) Blocking: [0342] The dot blot was incubated with 30
ml 5% low fat milk in TBS for 1.5 h at RT. [0343] 4) Washing:
[0344] The blocking solution was discarded and the dot blot was
incubated under shaking with 20 ml TTBS for 10 min at RT. [0345] 5)
Antibody solution I: [0346] The washing buffer was discarded and
the dot blot was incubated with antibody solution I for 2 h at RT
[0347] 6) Washing: [0348] The antibody solution I was discarded and
the dot blot was incubated under shaking with 20 ml TTBS for 10 min
at RT. The washing solution was discarded and the dot blot was
incubated under shaking with 20 ml TTBS for 10 min at RT. The
washing solution was discarded and the dot blot was incubated under
shaking with 20 ml TBS for 10 min at RT. [0349] 7) Antibody
solution II: [0350] The washing buffer was discarded and the dot
blot was incubated with antibody solution II for 1 h at RT [0351]
8) Washing: [0352] The antibody solution II was discarded and the
dot blot was incubated under shaking with 20 ml TTBS for 10 min at
RT. The washing solution was discarded and the dot blot was
incubated under shaking with 20 ml TTBS for 10 min at RT. The
washing solution was discarded and the dot blot was incubated under
shaking with 20 ml TBS for 10 min at RT. [0353] 9) Development:
[0354] The washing solution was discarded. The dot blot was
developed with 7.5 ml BM Blue POD Substrate for 10 min. The
development was stopped by intense washing of the dot blot with
H.sub.2O. Quantitative evaluation was done based on a densitometric
analysis (GS800 densitometer (BioRad) and software package Quantity
one, Version 4.5.0 (BioRad)) of the dot intensity. Only dots were
evaluated that had a relative density of greater than 20% of the
relative density of the last optically unambiguously identified dot
of the A.beta.(20-42) globulomer. This threshold value was
determined for every dot blot independently. The calculated value
indicates the relation between recognition of A.beta.(20-42)
globulomer and the respective A.beta. form for the given
antibody.
[0355] Dot blot analysis was performed with humanized monoclonal
anti-A.beta. antibody 4D10hum#1. The individual A.beta. forms were
applied in serial dilutions and incubated with the respective
antibodies for immune reaction (1=A.beta.(1-42) globulomer;
2=A.beta.(20-42) globulomer; 3=A.beta.(1-40) monomer, 0.1% NaOH;
4=A.beta.(1-42) monomer, 0.1% NaOH; 5=A.beta.(1-42) fibril
preparation; 6=sAPP.alpha. (Sigma); (first dot: 1pmol)). Results
are summarized in Table 8.
TABLE-US-00011 TABLE 8 DOT BLOT QUANTIFICATION DATA ANTIBODY:
ANTIGEN 4D10hum#1 A.beta.(1-42) globulomer >10000 A.beta.(20-42)
globulomer 1 A.beta.(1-40) monomer in 0.1% NaOH 72000 A.beta.(1-42)
monomer in 0.1% NaOH 72000 A.beta.(1-42) fibril >10000
sAPP.alpha. >100 A.beta.(12-42) globulomer 11
Example 3: Determination of Platelet Factor 4 Cross-Reaction
Example 3.1: Determination of Cross-Reaction with Platelet Factor 4
in Cynomolgus Monkey Plasma Via Sandwich-ELISA
[0356] Reagent List:
[0357] F96 Cert. Maxisorp NUNC-Immuno Plate cat. no. 439454
[0358] Binding antibodies in experiment E1: [0359] Humanized
monoclonal anti-A.beta. antibody 4D10hum#1; 2.36 mg/ml OD 280 nm;
stored at -80.degree. C. [0360] Humanized monoclonal anti-A.beta.
antibody 4D10hum#2; 1.74 mg/ml OD 280 nm; stored at -80.degree. C.
[0361] Human/mouse chimeric anti-A.beta. monoclonal antibody clone
h1G5 wild type Fc-frame (chim h1G5 wt); 0.99 mg/ml OD 280 nm;
stored at -80.degree. C. (used as a positive control) [0362]
Affinity purified human polyclonal antibody hIgG1 (Chemicon
(Millipore), Cat#AG502); 1.00 mg/ml OD 280 nm; stored at
-80.degree. C. (used as a negative control)
[0363] Binding antibodies in reference experiment R1: [0364]
Anti-HPF4 monoclonal antibody; 4.2 mg/ml OD 280 nm; Abcam cat. no.
ab49735; stored at -30.degree. C. (used as a positive control)
[0365] Anti-A.beta. monoclonal antibody clone m1G5; 1.70 mg/ml OD
280 nm; stored at -80.degree. C. [0366] Anti-A.beta. monoclonal
antibody clone m4D10; 8.60 mg/ml OD 280 nm; stored at -80.degree.
C. [0367] Monoclonal antibody clone mIgG2a; 7.89 mg/ml OD 280 nm;
stored at -80.degree. C. (used as a negative control)
[0368] Coating buffer: 100 mM sodium hydrogen carbonate; pH 9.6
[0369] Blocking reagent for ELISA; Roche Diagnostics GmbH cat. no.:
1112589
[0370] PBST buffer: 20 mM NaH.sub.2PO.sub.4; 140 mM NaCl; 0.05%
Tween 20; pH 7.4
[0371] PBST+0.5% BSA buffer: 20 mM NaH.sub.2PO.sub.4; 140 mM NaCl;
0.05% Tween 20; pH 7.4+0.5% BSA; Serva cat. no. 11926
[0372] Cynomolgus plasma: Cynomolgus EDTA plasma pool from 13
different donors; stored at -30.degree. C.
[0373] Trypsin inhibitor: Sigma cat. no. T7902
[0374] Primary antibody: pRAb-HPF4; 0.5 mg/ml; Abcam cat. no.
ab9561
[0375] Label reagent: anti-rabbit-POD conjugate; Jackson
ImmunoResearch Ltd. cat. no.: 111-036-045
[0376] Staining Solution: 42 mM TMB (Roche Diagnostics GmbH cat.
no.: 92817060) in DMSO; 3% H.sub.2O.sub.2 in water; 100 mM sodium
acetate, pH 4.9
[0377] Stop solution: 2 M sulfonic acid
[0378] Method Used in Preparation of Reagents:
[0379] Binding Antibody:
[0380] The binding antibodies were diluted to 10 .mu.g/ml in
coating buffer.
[0381] Blocking Solution:
[0382] Blocking reagent was dissolved in 100 ml water to prepare
the blocking stock solution and aliquots of 10 ml were stored at
-20.degree. C. 3 ml blocking stock solution was diluted with 27 ml
water for each plate to block.
[0383] Preparation of Cynomolgus (Macaca fascicularis) Plasma Stock
Solution:
[0384] 2 ml Cynomolgus plasma pool were centrifuged for 10 min at
10,000 g. 1.58 ml of the supernatant was removed and diluted with
3.42 ml PBST+0.5% BSA buffer (=1:3.16 dilution). Then 50 .mu.l 10
mg/ml trypsin inhibitor in H.sub.2O were added. After incubation
for 10 min at room temperature the sample was filtrated through a
0.22 .mu.m filter (Millipore cat. no. SLGS0250S).
[0385] Dilution Series of Cynomolgus Plasma Stock Solution:
TABLE-US-00012 Volume of Volume of PBST + Final dilution of No
cynomolgus plasma dilution 0.5% BSA buffer cynomolgus plasma 1 250
.mu.l stock solution 0 ml 1:3.16 2 79 .mu.l (1) 171 .mu.l 1:10 3 79
.mu.l (2) 171 .mu.l 1:31.6 4 79 .mu.l (3) 171 .mu.l 1:100 5 79
.mu.l (4) 171 .mu.l 1:316 6 79 .mu.l (5) 171 .mu.l 1:1000 7 79
.mu.l (6) 171 .mu.l 1:3160 8 0 .mu.l 250 .mu.l buffer only
[0386] Primary Antibody Solution:
[0387] The primary antibody was diluted to 1 .mu.g/ml in PBST+0.5%
BSA buffer. The dilution factor was 1:500. The antibody solution
was used immediately.
[0388] Label Reagent:
[0389] Anti-rabbit-POD conjugate lyophilizate was reconstituted in
0.5 ml water. 500 .mu.l glycerol was added and aliquots of 100
.mu.l were stored at -20.degree. C. for further use. The
concentrated label reagent was diluted in PBST buffer. The dilution
factor was 1:10000. The reagent was used immediately.
[0390] TMB Solution:
[0391] 20 ml 100 mM of sodium acetate, pH 4.9, was mixed with 200
.mu.l of the TMB stock solution and 29.5 .mu.l 3% peroxide
solution. The solution was used immediately.
[0392] Standard Plate Setup for Experiment E1. Dilutions of
cynomolgus plasma. Note that each sample was run in duplicate.
TABLE-US-00013 1 2 7 8 Positive control 3 4 5 6 Negative control
chim h1G5 wt 4D10hum#1 4D10hum#2 hIgG1 9 10 11 12 A 1:3.16 1:3.16
1:3.16 1:3.16 1:3.16 1:3.16 1:3.16 1:3.16 none none none none B
1:10 1:10 1:10 1:10 1:10 1:10 1:10 1:10 none none none none C
1:31.6 1:31.6 1:31.6 1:31.6 1:31.6 1:31.6 1:31.6 1:31.6 none none
none none D 1:100 1:100 1:100 1:100 1:100 1:100 1:100 1:100 none
none none none E 1:316 1:316 1:316 1:316 1:316 1:316 1:316 1:316
none none none none F 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000
1:1000 1:1000 none none none none G 1:3160 1:3160 1:3160 1:3160
1:3160 1:3160 1:3160 1:3160 none none none none H 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 none none none none
[0393] Standard Plate Setup for Reference Experiment R1. Dilutions
of cynomolgus plasma. Note that each sample was run in
duplicate.
TABLE-US-00014 1 2 7 8 Positive control 3 4 5 6 Negative control
anti-HPF4 mAb m1G5 mAb m4D10 mIgG2a 9 10 11 12 A 1:3.16 1:3.16
1:3.16 1:3.16 1:3.16 1:3.16 1:3.16 1:3.16 none none none none B
1:10 1:10 1:10 1:10 1:10 1:10 1:10 1:10 none none none none C
1:31.6 1:31.6 1:31.6 1:31.6 1:31.6 1:31.6 1:31.6 1:31.6 none none
none none D 1:100 1:100 1:100 1:100 1:100 1:100 1:100 1:100 none
none none none E 1:316 1:316 1:316 1:316 1:316 1:316 1:316 1:316
none none none none F 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000
1:1000 1:1000 none none none none G 1:3160 1:3160 1:3160 1:3160
1:3160 1:3160 1:3160 1:3160 none none none none H 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 none none none none
[0394] Procedure used: [0395] 1. 100 .mu.l binding antibody
solution per well were applied and incubated overnight at 4.degree.
C. [0396] 2. The antibody solution was discarded and the wells were
washed three times with 250 .mu.l PBST buffer. [0397] 3. 265 .mu.l
blocking solution per well were added and incubated 1.5 h at room
temperature. [0398] 4. The blocking solution was discarded and the
wells were washed three times with 250 .mu.l PBST buffer. [0399] 5.
After preparation of the cynomolgus plasma dilution series, 100
.mu.l per well of these dilutions were applied to the plate. The
plate was incubated 2 h at room temperature. [0400] 6. The
cynomolgus plasma dilutions were discarded and the wells were
washed three times with 250 .mu.l PBST buffer. [0401] 7. 100 .mu.l
of primary antibody solution per well were added and incubated 1 h
at room temperature. [0402] 8. The primary antibody solution was
discarded and the wells were washed three times with 250 .mu.l PBST
buffer. [0403] 9. 200 .mu.l label solution per well were added and
incubated 1 h at room temperature. [0404] 10. The label solution
was discarded and the wells were washed three times with 250 .mu.l
PBST buffer. [0405] 11. 100 .mu.l of TMB solution were added to
each well. [0406] 12. Plate colour was monitored during development
(5-15 min at ambient temperature) and the reaction was terminated
by adding 50 .mu.l/well of stop solution when an appropriate colour
had developed. [0407] 13. The absorbance was read at 450 nm.
[0408] Data Analysis:
[0409] Plasma dilution factors (X-values) were log-transformed
using the equation: X=log(X). Data were plotted using the
log-transformed X-values on the X-axis expressed as dilution of
plasma (1:X). The OD.sub.450 nm value of the respective PBST blank
in row H was subtracted from the values of the plasma dilution
series of each column in row A-G. The resulting background
corrected OD.sub.450 nm values were plotted on the Y-axis. The
dilution effect curves were calculated from these data points by
curve fitting using a non-linear regression "four parameter
logistic equation" with a "least squares (ordinary) fit" fitting
method (that equals the fitting method "sigmoidal dose-response
(variable slope)") using the Data analysis software package
GraphPadPrism (Version 5.03; GraphPad Software Inc.). Curve fitting
was performed for the sole purpose of data visualization but not as
basis for any further calculations i.e. the area under the curve
calculation. The area under the curve (AUC, or total peak area) was
determined based on non-curve fitted data, the log-transformed
X-values and the OD.sub.450 nm values in the measured range (final
plasma dilutions from 1:3.16 to 1:3160). The following calculation
settings were used within the Data analysis software package
GraphPadPrism (Version 5.03; GraphPad Software Inc.): [0410] The
baseline was set to Y=0.0. [0411] Minimum peak height: Ignore peaks
that are less than 10% of the distance from minimum to maximum Y.
[0412] Peak direction: By definition, all peaks must go above the
baseline
[0413] For each individual antibody a PF4 discrimination factor was
calculated using the commercially available anti-HPF4 antibody
(Abcam cat. no.: ab49735) as a reference antibody for PF4
recognition, wherein
[ PF 4 discrimination factor ] = [ total peak area of anti - HPF 4
antibody ab 49735 ] [ total peak area of antibody to be determined
] ##EQU00001##
[0414] Note: The PF4 discrimination factor was calculated based on
the anti-HPF4 antibody AUCs obtained in the reference experiment as
no human version of an anti-HPF4 exists.
[0415] Results of experiment E1 and reference experiment R1 are
shown in FIGS. 20A and 22A as well as in Tables 9A and 9B.
Example 3.2: Determination of Cross-Reaction with Platelet Factor 4
in Human Plasma Via Sandwich-ELISA
[0416] The same reagents and procedures for reagent preparation
were used as for Example 3.1 except from:
[0417] Human plasma (Human EDTA plasma pool from 4 different
donors; stored at -30.degree. C.) spiked with human PF4 (7.3 mg/ml;
Molecular Innovation cat. no. HPF4; stored at -30.degree. C.) was
used instead of cynomolgus plasma. HPF4-spiked human plasma stock
solution was prepared as follows.
[0418] A) Preparation of Human Plasma Dilution:
[0419] 2 ml human plasma pool were centrifuged for 10 min at 10000
g. 1.58 ml of the supernatant was removed and diluted with 3.42 ml
PBST+0.5% BSA (=1:3.16 dilution). Then 50 .mu.l 10 mg/ml trypsin
inhibitor in H.sub.2O were added. After incubation for 10 min at
room temperature the sample was filtrated through a 0.22 .mu.m
filter (Millipore cat. no. SLGS0250S).
[0420] B) Preparation of HPF4 Stock Solution:
[0421] 1 .mu.l HPF4 was added to 99 .mu.l PBST+0.5% BSA buffer=73
.mu.g/ml.
[0422] C) Preparation of Human Plasma Stock Solution Spiked with 10
ng/ml HPF4:
[0423] 0.69 .mu.l of 73 .mu.g/ml HPF4 stock solution were added to
5 ml 1:3.16 diluted human plasma resulting in 10 ng/ml HPF4 spiking
of the human plasma stock dilution.
[0424] The preparation of a dilution series, the standard plate
setup, the experimental procedure and the data analysis for
sandwich-ELISA with HPF4-spiked human plasma were analogous to
those described for sandwich-ELISA with cynomolgus plasma in
Example 3.1.
[0425] Binding antibodies in experiment E2: same as used in
experiment E1 in Example 3.1
[0426] Binding antibodies in reference experiment R2: same as used
in reference experiment R1 in Example 3.1.
[0427] Results of experiment E2 and reference experiment R2 are
shown in FIGS. 20B and 22B as well as in Tables 9A and 9B.
TABLE-US-00015 TABLE 9A AUC (OR TOTAL PEAK AREA) CALCULATED FROM
LOG- TRANSFORMED DATA OF EXPERIMENTS E1 and E2 DEPICTED IN FIGS.
20A AND 20B Positive Negative control mAb mAb control chim h1G5
wt.sup.1 4D10hum#1 4D10hum#2 hIgG1 Cynomolgus Area Under
Curve.sup.2 1.255 0.042 0.075 0.075 plasma Ratio 2 64 36 27 (data
from HPF4/aA.beta. FIG. 20A) antibody Human plasma Area Under
Curve.sup.2 0.949 0.067 0.116 0.113 (data from Ratio 2 30 17 18
FIG. 20B) HPF4/aA.beta. antibody .sup.1chim h1G5 wt is an antibody
as described in WO 2007/062853, i.e. a monoclonal antibody having a
binding affinity to the A.beta.(20-42) globulomer that is greater
than its binding affinity to the A.beta.(1-42) globulomer.
.sup.2Area under curve was calculated as described in example
3.1.
TABLE-US-00016 TABLE 9B AUC (OR TOTAL PEAK AREA) CALCULATED FROM
LOG- TRANSFORMED DATA OF REFERENCE EXPERIMENTS R1 and R2 DEPICTED
IN FIGS. 22A AND 22B Positive Negative control mAb mAb control
anti-HPF4 m1G5.sup.1 m4D10 mIgG2a Cynomolgus plasma Area Under
Curve.sup.2 2.681 0.861 0.086 0.005 (data from FIG. 22A) Ratio 1 3
31 517 HPF4/aA.beta. antibody Human plasma Area Under Curve.sup.2
1.986 0.311 0.093 0.006 (data from FIG. 22B) Ratio 1 6 21 331
HPF4/aA.beta. antibody .sup.1m1G5 is an antibody as described in WO
2007/062853, i.e. a monoclonal antibody having a binding affinity
to the A.beta.(20-42) globulomer that is greater than its binding
affinity the A.beta.(1-42) globulomer. .sup.2Area under curve was
calculated as described in example 3.1.
Example 3.3: Determination of Cross-Reaction with Platelet Factor 4
in Cynomolgus Monkey Plasma Via Aligned Sandwich-ELISA
[0428] The reagents described in Example 3.1 and aligning
antibodies anti-mouse IgG (Fc specific; produced in goat; Sigma
cat. no.: M3534; 2.3 mg/ml; stored at -20.degree. C. for murine
binding antibodies in reference experiment R3) and anti-human IgG
(Fc specific; produced in goat; Sigma cat. no.: 12136; 2.2 mg/ml;
stored at -20.degree. C., for human, humanized and human/mouse
chimeric binding antibodies in experiment E3) were used.
[0429] Methods Used in Preparation of Reagents:
[0430] Blocking solution, primary antibody and TMB solution were
prepared as described in Example 3.1.
[0431] Each aligning antibody was diluted to 10 .mu.g/ml in coating
buffer.
[0432] Binding antibodies in experiment E3: same as used in
experiment E1 in Example 3.1 Binding antibodies in reference
experiment R3: same as used in reference experiment R1 in Example
3.1.
[0433] Each binding antibody was diluted with PBST+0.5% BSA buffer
to 10 .mu.g/ml (stock solution), and dilution series were prepared
as follows:
TABLE-US-00017 Volume of Volume of Final antibody No antibody
dilution PBST + 0.5% BSA buffer concentration 1 250 .mu.l stock
solution 0 ml 10000 ng/ml 2 79 .mu.l (1) 171 .mu.l 3160 ng/ml 3 79
.mu.l (2) 171 .mu.l 1000 ng/ml 4 79 .mu.l (3) 171 .mu.l 316 ng/ml 5
79 .mu.l (4) 171 .mu.l 100 ng/ml 6 79 .mu.l (5) 171 .mu.l 31.6
ng/ml 7 79 .mu.l (6) 171 .mu.l 10 ng/ml 8 0 .mu.l 250 .mu.l buffer
only
[0434] Cynomolgus Plasma:
[0435] 400 .mu.l Cynomolgus plasma pool were centrifuged for 10 min
at 10000 g. 158 .mu.l of the supernatant was removed and diluted
with 684 .mu.l PBST+0.5% BSA (=1:3.16 dilution). Then 10 .mu.l 10
mg/ml trypsin inhibitor in H.sub.2O were added. After incubation
for 10 min at room temperature the sample was filtrated through a
0.22 .mu.m filter (Millipore cat. no. SLGS0250S). Afterwards 500
.mu.l of this 1:3.16 diluted plasma sample was again diluted 1:31.6
with 15.3 ml PBST+0.5% BSA buffer resulting in a total dilution of
1:100.
[0436] Label Reagent:
[0437] Anti-rabbit-POD conjugate lyophilised was reconstituted in
0.5 ml water. 500 .mu.l glycerol was added and aliquots of 100
.mu.l were stored at -20.degree. C. for further use. The
concentrated label reagent was diluted in PBST buffer. The dilution
factor was 1:5000. The reagent was used immediately.
[0438] Binding Antibody Plate Setup for Experiment E2. Dilutions of
binding antibodies. Note that each concentration of each binding
antibody was run in duplicate.
TABLE-US-00018 1 2 7 8 Positive control 3 4 5 6 Negative control
chim h1G5 wt 4D10hum#1 4D10hum#2 hIgG1 9 10 11 12 A 10000 10000
10000 10000 10000 10000 10000 10000 none none none none B 3160 3160
3160 3160 3160 3160 3160 3160 none none none none C 1000 1000 1000
1000 1000 1000 1000 1000 none none none none D 316 316 316 316 316
316 316 316 none none none none E 100 100 100 100 100 100 100 100
none none none none F 31.6 31.6 31.6 31.6 31.6 31.6 31.6 31.6 none
none none none G 10 10 10 10 10 10 10 10 none none none none H 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 none none none none
[0439] Binding Antibody Plate Setup for Reference Experiment R3.
Dilutions of binding antibodies. Note that each concentration of
each binding antibody was run in duplicate.
TABLE-US-00019 1 2 7 8 Positive control 3 4 5 6 Negative control
anti-HPF4 mAb m1G5 mAb m4D10 mIgG2a 9 10 11 12 A 10000 10000 10000
10000 10000 10000 10000 10000 none none none none B 3160 3160 3160
3160 3160 3160 3160 3160 none none none none C 1000 1000 1000 1000
1000 1000 1000 1000 none none none none D 316 316 316 316 316 316
316 316 none none none none E 100 100 100 100 100 100 100 100 none
none none none F 31.6 31.6 31.6 31.6 31.6 31.6 31.6 31.6 none none
none none G 10 10 10 10 10 10 10 10 none none none none H 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 none none none none
[0440] Procedure Used: [0441] 1. 100 .mu.l of the respective
aligning antibody solution (anti-human IgG for experiment E3;
anti-murine IgG for reference experiment R3) per well were applied
and incubated overnight at 4.degree. C. [0442] 2. The antibody
solution was discarded and the wells were washed three times with
250 .mu.l PB ST-buffer. [0443] 3. 265 .mu.l blocking solution per
well were added and incubated 2 h at room temperature. [0444] 4.
The blocking solution was discarded and the wells were washed three
times with 250 .mu.l PBST buffer. [0445] 5. After preparation of
the dilution series of each binding antibody, 100 .mu.l per well of
these antibody dilutions were applied to the plate. The plate was
incubated 2 h at room temperature. [0446] 6. The antibody solutions
were discarded and the wells were washed three times with 250 .mu.l
PBST buffer. [0447] 7. 100 .mu.l 1:100 dilution of cynomolgus
plasma per well were added and incubated 2 h at room temperature.
[0448] 8. The plasma solution was discarded and the wells were
washed three times with 250 .mu.l PBST buffer. [0449] 9. 100 .mu.l
primary antibody solution per well were added and incubated 1 h at
room temperature. [0450] 10. The primary antibody solution was
discarded and the wells were washed three times with 250 .mu.l PBST
buffer. [0451] 11. 200 .mu.l label reagent per well were added and
incubated 1 h at room temperature. [0452] 12. The label reagent was
discarded and the wells were washed three times with 250 .mu.l PBST
buffer. [0453] 13. 100 .mu.l of TMB solution were added to each
well. [0454] 14. Plate colour was monitored during development
(5-15 min at ambient temperature) and the reaction was terminated
by adding 50 .mu.l/well of stop solution when an appropriate colour
had developed. [0455] 15. The absorbance was read at 450 nm.
[0456] Data analysis was performed as described for sandwich-ELISA
with cynomolgus plasma in Example 3.1, except that not plasma
dilution factors but the amounts of antibody (expressed in ng/ml)
were used as X-values and thus concentration effect curves were
calculated. Accordingly, area under curve was determined based on
non-curve fitted data, the log-transformed X-values and the
OD.sub.450 nm values in the measured range (final antibody
concentrations from 10 ng/ml to 10000 ng/ml).
[0457] Results of experiment E3 and reference experiment R3 are
shown in FIGS. 21A and 23A as well as in Tables 10A and 10B.
Example 3.4: Determination of Cross-Reaction with Platelet Factor 4
in Human Plasma Via Aligned Sandwich-ELISA
[0458] The same reagents and procedures for reagent preparation
were used as for Example 3.3 except from:
[0459] Each aligning antibody used for experiment E4 was diluted to
10 .mu.g/ml in coating buffer, and each aligning antibody used for
experiment R4 was diluted to 50 .mu.g/ml in coating
[0460] Human plasma (Human EDTA plasma pool from 4 different
donors; stored at -30.degree. C.) spiked with human PF4 (7.3 mg/ml;
Molecular Innovation cat. no. HPF4; stored at -30.degree. C.) was
used instead of cynomolgus plasma. HPF4-spiked human plasma stock
solution was prepared as follows.
[0461] A) Preparation of Human Plasma Dilution:
[0462] 4 ml human plasma pool were centrifuged for 10 min at 10000
g. 3.16 ml of the supernatant was removed and diluted with 6.84 ml
PBST+0.5% BSA (=1:3.16 dilution). Then 100 .mu.l 10 mg/ml trypsin
inhibitor in H.sub.2O were added. After incubation for 10 min at
room temperature the sample was filtrated through a 0.22 .mu.m
filter (Millipore cat. no. SLGS0250S). Afterwards 5 ml of this
1:3.16 diluted plasma sample was again diluted 1:3.16 with 10.8 ml
PBST+0.5% BSA buffer resulting in a total dilution of 1:10.
[0463] B) Preparation of HPF4 Stock Solution:
[0464] 1 .mu.l HPF4 was added to 99 .mu.l PBST+0.5% BSA buffer=73
.mu.g/ml.
[0465] C) Preparation of Human Plasma Stock Solution Spiked with 10
ng/ml HPF4:
[0466] 1.64 .mu.l of 73 .mu.g/ml HPF4 stock solution were added to
12 ml 1:10 diluted human plasma resulting in 10 ng/ml HPF4 spiking
of the human plasma stock dilution.
[0467] The preparation of dilution series of the binding
antibodies; the binding antibody plate setup; the preparation of
blocking solution, primary antibody, reagent and TMB solution were
the same as in Example 3.3.
[0468] Aligning antibody and binding antibodies in experiment E4:
same as used in experiment E3 in Example 3.3
[0469] Aligning antibody and binding antibodies in reference
experiment R4: same as used in reference experiment R4 in Example
3.3.
[0470] The experimental procedure (but using 1:10 diluted human
plasma in step 7) and data analysis for aligned sandwich-ELISA with
HPF4-spiked human plasma were analogous to that described for
aligned sandwich-ELISA with cynomolgus plasma in Example 3.3.
[0471] Results of experiment E4 and reference experiment R4 are
shown in FIGS. 21B and 23B as well as in Tables 10A and 10B.
TABLE-US-00020 TABLE 10A AUC (OR TOTAL PEAK AREA) CALCULATED FROM
LOG- TRANSFORMED DATA OF EXPERIMENTS E3 and E4 DEPICTED IN FIGS.
21A AND 21B Positive mAb mAb Negative control 4D10 4D10 control
chim h1G5 wt.sup.1 hum#1 hum#2 hIgG1 Cynomolgus plasma Area Under
Curve.sup.2 0.290 0.030 0 0 (data from FIG. 21A) Ratio 16 158
>158.sup.3 >158.sup.3 HPF4/aA.beta. antibody Human plasma
Area Under Curve.sup.2 0.106 0.168 0.051 0.024 (data from FIG. 21B)
Ratio 36 23 75 157 HPF4/aA.beta. antibody .sup.1)chim h1G5 wt is an
antibody as described in WO 2007/062853, i.e. a monoclonal antibody
having a binding affinity to the A.beta.(20-42) globulomer that is
greater than its binding affinity to the A.beta.(1-42) globulomer.
.sup.2)Area under curve was calculated as described in example 3.3.
.sup.3)For antibodies 4D10hum#2 and hIgG1 the HPF4 binding activity
was so low that the AUC was calculated to be 0. Therefore the ratio
HPF4/aA.beta. antibody could not be calculated and was indicated to
be >158 (the highest ratio achieved by another antibody
(4D10hum#1) in this assay).
[0472] .sup.1) chim h1G5 wt is an antibody as described in WO
2007/062853, i.e. a monoclonal antibody having a binding affinity
to the A.beta.(20-42) globulomer that is greater than its binding
affinity to the A.beta.(1-42) globulomer.
[0473] .sup.2) Area under curve was calculated as described in
example 3.3.
[0474] .sup.3) For antibodies 4D10hum#2 and hIgG1 the HPF4 binding
activity was so low that the AUC was calculated to be 0. Therefore
the ratio HPF4/aA.beta. antibody could not be calculated and was
indicated to be >158 (the highest ratio achieved by another
antibody (4D10hum#1) in this assay).
TABLE-US-00021 TABLE 10B AUC (OR TOTAL PEAK AREA) CALCULATED FROM
LOG- TRANSFORMED DATA OF REFERENCE EXPERIMENTS R3 and R4 DEPICTED
IN FIGS. 21A AND 21B Positive Negative control mAb mAb control
anti-HPF4 m1G5.sup.1 m4D10 mIgG2a Cynomolgus plasma Area Under
Curve.sup.2 4.781 0.2768 0.04066 0.01473 (data from FIG. 23A) Ratio
1 17 118 325 HPF4/aA.beta. antibody Human plasma Area Under
Curve.sup.2 3.844 0.165 0.141 0.033 (data from FIG. 23B) Ratio 1 23
27 118 HPF4/aA.beta. antibody .sup.1m1G5 is an antibody as
described in WO 2007/062853, i.e. a monoclonal antibody having a
binding affinity to the A.beta.(20-42) globulomer that is greater
than its binding affinity to both the A.beta.(1-42) globulomer.
.sup.2Area under curve was calculated as described in example 3.3.
Sequence CWU 1
1
481113PRTArtificial Sequencesyntheticmisc_feature(7)..(7)Xaa can be
Ser or Thrmisc_feature(15)..(15)Xaa can be Leu or
Promisc_feature(41)..(41)Xaa can be Phe or
Leumisc_feature(42)..(42)Xaa can be Gln or
Leumisc_feature(44)..(44)Xaa can be Arg or
Lysmisc_feature(50)..(50)Xaa can be Arg or Gln 1Asp Val Val Met Thr
Gln Xaa Pro Leu Ser Leu Pro Val Thr Xaa Gly 1 5 10 15 Gln Pro Ala
Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ile 20 25 30 Asp
Gly Lys Thr Tyr Leu Asn Trp Xaa Xaa Gln Xaa Pro Gly Gln Ser 35 40
45 Pro Xaa Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro
50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105 110 Arg 2112PRTArtificial
Sequencesyntheticmisc_feature(12)..(12)Xaa can be Ile or
Valmisc_feature(24)..(24)Xaa can be Ala or
Valmisc_feature(29)..(29)Xaa can be Val or
Leumisc_feature(48)..(48)Xaa can be Val or
Leumisc_feature(49)..(49)Xaa can be Ser or
Glymisc_feature(67)..(67)Xaa can be Phe or
Leumisc_feature(71)..(71)Xaa can be Arg or
Lysmisc_feature(76)..(76)Xaa can be Asn or
Sermisc_feature(78)..(78)Xaa can be Leu or Val 2Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Xaa Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Xaa Ser Gly Phe Thr Xaa Ser Ser Tyr 20 25 30 Gly
Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Xaa 35 40
45 Xaa Val Ile Trp Arg Gly Gly Arg Ile Asp Tyr Asn Ala Ala Phe Met
50 55 60 Ser Arg Xaa Thr Ile Ser Xaa Asp Asn Ser Lys Xaa Thr Xaa
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala 85 90 95 Arg Asn Ser Asp Val Trp Gly Gln Gly Thr
Thr Val Thr Val Ser Ser 100 105 110 3112PRTArtificial
Sequencesyntheticmisc_feature(1)..(1)Xaa can be Gln or
Glumisc_feature(27)..(27)Xaa can be Gly or
Phemisc_feature(29)..(29)Xaa can be Ile or
Leumisc_feature(37)..(37)Xaa can be Ile or
Valmisc_feature(48)..(48)Xaa can be Ile or
Leumisc_feature(67)..(67)Xaa can be Val or
Leumisc_feature(71)..(71)Xaa can be Val or
Lysmisc_feature(76)..(76)Xaa can be Asn or
Sermisc_feature(78)..(78)Xaa can be Phe or Val 3Xaa Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Xaa Ser Xaa Ser Ser Tyr 20 25 30 Gly
Val His Trp Xaa Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Xaa 35 40
45 Gly Val Ile Trp Arg Gly Gly Arg Ile Asp Tyr Asn Ala Ala Phe Met
50 55 60 Ser Arg Xaa Thr Ile Ser Xaa Asp Thr Ser Lys Xaa Gln Xaa
Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
Tyr Tyr Cys Ala 85 90 95 Arg Asn Ser Asp Val Trp Gly Gln Gly Thr
Thr Val Thr Val Ser Ser 100 105 110 4112PRTArtificial
Sequencesynthetic 4Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile
Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Val Ser Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Trp Arg Gly
Gly Arg Ile Asp Tyr Asn Ala Ala Phe Met 50 55 60 Ser Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95
Arg Asn Ser Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 100
105 110 5112PRTArtificial Sequencesynthetic 5Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Tyr 20 25 30 Gly
Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Val Ile Trp Arg Gly Gly Arg Ile Asp Tyr Asn Ala Ala Phe Met
50 55 60 Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala 85 90 95 Arg Asn Ser Asp Val Trp Gly Gln Gly Thr
Thr Val Thr Val Ser Ser 100 105 110 6112PRTArtificial
Sequencesynthetic 6Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly
Phe Thr Leu Ser Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Arg Gly
Gly Arg Ile Asp Tyr Asn Ala Ala Phe Met 50 55 60 Ser Arg Leu Thr
Ile Ser Lys Asp Asn Ser Lys Ser Thr Val Tyr Leu 65 70 75 80 Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95
Arg Asn Ser Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 100
105 110 7112PRTArtificial Sequencesynthetic 7Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Ser Tyr 20 25 30 Gly
Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Val Ile Trp Arg Gly Gly Arg Ile Asp Tyr Asn Ala Ala Phe Met
50 55 60 Ser Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala 85 90 95 Arg Asn Ser Asp Val Trp Gly Gln Gly Thr
Thr Val Thr Val Ser Ser 100 105 110 8112PRTArtificial
Sequencesynthetic 8Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val
Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly
Gly Ser Ile Ser Ser Tyr 20 25 30 Gly Val His Trp Ile Arg Gln Pro
Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Trp Arg Gly
Gly Arg Ile Asp Tyr Asn Ala Ala Phe Met 50 55 60 Ser Arg Val Thr
Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu
Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95
Arg Asn Ser Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 100
105 110 9112PRTArtificial Sequencesynthetic 9Glu Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30 Gly
Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45 Gly Val Ile Trp Arg Gly Gly Arg Ile Asp Tyr Asn Ala Ala Phe Met
50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
Tyr Tyr Cys Ala 85 90 95 Arg Asn Ser Asp Val Trp Gly Gln Gly Thr
Thr Val Thr Val Ser Ser 100 105 110 10112PRTArtificial
Sequencesynthetic 10Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val
Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly
Phe Ser Leu Ser Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro
Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Arg Gly
Gly Arg Ile Asp Tyr Asn Ala Ala Phe Met 50 55 60 Ser Arg Leu Thr
Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Lys Leu
Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95
Arg Asn Ser Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 100
105 110 11112PRTArtificial Sequencesynthetic 11Glu Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Gly
Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45 Gly Val Ile Trp Arg Gly Gly Arg Ile Asp Tyr Asn Ala Ala Phe Met
50 55 60 Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Phe
Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
Tyr Tyr Cys Ala 85 90 95 Arg Asn Ser Asp Val Trp Gly Gln Gly Thr
Thr Val Thr Val Ser Ser 100 105 110 12113PRTArtificial
Sequencesynthetic 12Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro
Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser
Gln Ser Leu Leu Asp Ile 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp
Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr
Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg
Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95
Thr His Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
105 110 Arg 13113PRTArtificial Sequencesynthetic 13Asp Val Val Met
Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Gln Pro
Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ile 20 25 30
Asp Gly Lys Thr Tyr Leu Asn Trp Phe Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Gln Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg 14113PRTArtificial
Sequencesynthetic 14Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro
Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser
Gln Ser Leu Leu Asp Ile 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp
Leu Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Arg Leu Ile Tyr
Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg
Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95
Thr His Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
105 110 Arg 15113PRTArtificial Sequencesynthetic 15Asp Val Val Met
Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro
Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ile 20 25 30
Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35
40 45 Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg 16113PRTArtificial
Sequencesynthetic 16Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro
Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser
Gln Ser Leu Leu Asp Ile 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp
Phe Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr
Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg
Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95
Thr His Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
105 110 Arg 175PRTArtificial Sequencesynthetic 17Ser Tyr Gly Val
His 1 5 1816PRTArtificial Sequencesynthetic 18Val Ile Trp Arg Gly
Gly Arg Ile Asp Tyr Asn Ala Ala Phe Met Ser 1 5 10 15
194PRTArtificial Sequencesynthetic 19Asn Ser Asp Val 1
2016PRTArtificial Sequencesynthetic 20Lys Ser Ser Gln Ser Leu Leu
Asp Ile Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15 217PRTArtificial
Sequencesynthetic 21Leu Val Ser Lys Leu Asp Ser 1 5
229PRTArtificial Sequencesynthetic 22Trp Gln Gly Thr His Phe Pro
Tyr Thr 1 5 23112PRTArtificial Sequencesynthetic 23Gln Val Gln Leu
Lys Gln Ser Gly Pro Ser Leu Ile Gln Pro Ser Gln 1 5 10 15 Ser Leu
Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35
40 45 Gly Val Ile Trp Arg Gly Gly Arg Ile Asp Tyr Asn Ala Ala Phe
Met 50 55 60 Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln
Val Phe Phe 65 70 75 80 Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala
Ile Tyr Tyr Cys Ala 85 90 95 Arg Asn Ser Asp Val Trp Gly Thr Gly
Thr Thr Val Thr Val Ser Ser 100 105 110 24113PRTArtificial
Sequencesynthetic 24Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser
Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser
Gln Ser Leu Leu Asp Ile 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp
Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro
Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55
60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp
Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 105 110 Arg 25330PRTArtificial
Sequencesynthetic 25Ala Ser Thr Lys Gly Pro Ser Val Phe Phe Leu Ala
Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100
105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225
230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 325 330 26330PRTArtificial
Sequencesynthetic 26Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100
105 110 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225
230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 325 330 27106PRTArtificial
Sequencesynthetic 27Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln 1 5 10 15 Leu Lys Ser Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr 20 25 30 Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser 35 40 45 Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60 Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 65 70 75 80 His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 28105PRTArtificial
Sequencesynthetic 28Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro
Pro Ser Ser Glu 1 5 10 15 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val
Cys Leu Ile Ser Asp Phe 20 25 30 Tyr Pro Gly Ala Val Thr Val Ala
Trp Lys Ala Asp Ser Ser Pro Val 35 40 45 Lys Ala Gly Val Glu Thr
Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 50 55 60 Tyr Ala Ala Ser
Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 65 70 75 80 His Arg
Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 85 90 95
Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 2943PRTArtificial
Sequencesynthetic 29Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val
His His Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp Val Gly Ser
Asn Lys Gly Ala Ile Ile 20 25 30 Gly Leu Met Val Gly Gly Val Val
Ile Ala Thr 35 40 3042PRTArtificial Sequencesynthetic 30Asp Ala Glu
Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 10 15 Leu
Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25
30 Gly Leu Met Val Gly Gly Val Val Ile Ala 35 40 3142PRTArtificial
Sequencesynthetic 31Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val
His His Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp Val Gly Ser
Asn Lys Gly Ala Ile Ile 20 25 30 Gly Leu Met Val Gly Gly Val Val
Ile Ala 35 40 3231PRTArtificial Sequencesynthetic 32Val His His Gln
Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn 1 5 10 15 Lys Gly
Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala 20 25 30
3323PRTArtificial Sequencesynthetic 33Phe Ala Glu Asp Val Gly Ser
Asn Lys Gly Ala Ile Ile Gly Leu Met 1 5 10 15 Val Gly Gly Val Val
Ile Ala 20 3430PRTArtificial Sequencesynthetic 34Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser 20 25 30
3514PRTArtificial Sequencesynthetic 35Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val Ser 1 5 10 3632PRTArtificial
Sequencesynthetic 36Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys Ala Arg 20 25 30 3711PRTArtificial
Sequencesynthetic 37Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 1 5
10 3830PRTArtificial Sequencesynthetic 38Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu
Thr Cys Thr Val Ser Gly Gly Ser Ile Ser 20 25 30 3914PRTArtificial
Sequencesynthetic 39Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
Ile Gly 1 5 10 4032PRTArtificial Sequencesynthetic 40Arg Val Thr
Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys 1 5 10 15 Leu
Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25
30 4111PRTArtificial Sequencesynthetic 41Trp Gly Gln Gly Thr Thr
Val Thr Val Ser Ser 1 5 10 4223PRTArtificial Sequencesynthetic
42Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1
5 10 15 Gln Pro Ala Ser Ile Ser Cys 20 4315PRTArtificial
Sequencesynthetic 43Trp Phe Gln Gln Arg Pro Gly Gln Ser Pro Arg Arg
Leu Ile Tyr 1 5 10 15 4432PRTArtificial Sequencesynthetic 44Gly Val
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys 20
25 30 4511PRTArtificial Sequencesynthetic 45Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys Arg 1 5 10 46442PRTArtificial Sequencesynthetic
46Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Leu Ser Ser
Tyr 20 25 30 Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Leu 35 40 45 Gly Val Ile Trp Arg Gly Gly Arg Ile Asp Tyr
Asn Ala Ala Phe Met 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Asn
Ser Lys Ser Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asn Ser Asp Val
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 100 105 110 Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 115 120 125 Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 130 135
140 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
145 150 155 160 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 165 170 175 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr 180 185 190 Tyr Ile Cys Asn Val Asn His Lys Pro
Ser Asn Thr Lys Val Asp Lys 195 200 205 Lys Val Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Cys Pro Pro Cys 210 215 220 Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 225 230 235 240 Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250 255
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 260
265 270 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu 275 280 285 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu 290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 305 310 315 320 Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly 325 330 335 Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 340 345 350 Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360 365 Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375 380
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 385
390 395 400 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 405 410 415 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr 420 425 430 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 47442PRTArtificial Sequencesynthetic 47Glu Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Gly
Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40
45 Gly Val Ile Trp Arg Gly Gly Arg Ile Asp Tyr Asn Ala Ala Phe Met
50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val
Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
Tyr Tyr Cys Ala 85 90 95 Arg Asn Ser Asp Val Trp Gly Gln Gly Thr
Thr Val Thr Val Ser Ser 100 105 110 Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys 115 120 125 Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 130 135 140 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 145 150 155 160 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 165 170
175 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
180 185 190 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 195 200 205 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys 210 215 220 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 260 265 270 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295
300 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
305 310 315 320 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu 340 345 350 Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln
Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe 385 390 395 400 Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn 405 410 415 Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430 Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 435 440 48219PRTArtificial
Sequencesynthetic 48Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro
Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser
Gln Ser Leu Leu Asp Ile 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp
Leu Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Arg Leu Ile Tyr
Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg
Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95
Thr His Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val
Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
* * * * *