U.S. patent application number 16/565424 was filed with the patent office on 2020-07-16 for humanized anti-tau(ps422) antibodies and methods of use.
The applicant listed for this patent is HOFFMANN-LA ROCHE INC.. Invention is credited to Joerg Benz, Bernd Bohrmann, Guy Georges, Ulrich Goepfert, Fiona Grueninger, Hubert Kettenberger, Olaf Mundigl, Michael Schraeml.
Application Number | 20200223912 16/565424 |
Document ID | / |
Family ID | 52347277 |
Filed Date | 2020-07-16 |
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United States Patent
Application |
20200223912 |
Kind Code |
A1 |
Grueninger; Fiona ; et
al. |
July 16, 2020 |
HUMANIZED ANTI-TAU(PS422) ANTIBODIES AND METHODS OF USE
Abstract
The invention provides humanized anti-human Tau(pS422)
antibodies and methods of using the same.
Inventors: |
Grueninger; Fiona;
(Arlesheim, CH) ; Georges; Guy; (Habach, DE)
; Mundigl; Olaf; (Weilhem, DE) ; Schraeml;
Michael; (Penzberg, DE) ; Bohrmann; Bernd;
(Riehen, CH) ; Goepfert; Ulrich; (Penzberg,
DE) ; Benz; Joerg; (Rheinfelden, DE) ;
Kettenberger; Hubert; (Muenchen, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
HOFFMANN-LA ROCHE INC. |
Little Falls |
NJ |
US |
|
|
Family ID: |
52347277 |
Appl. No.: |
16/565424 |
Filed: |
September 9, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15389286 |
Dec 22, 2016 |
10465000 |
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16565424 |
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14575067 |
Dec 18, 2014 |
9562091 |
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15389286 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/4711 20130101;
C07K 2317/34 20130101; C07K 2317/92 20130101; C07K 2317/56
20130101; C07K 16/18 20130101; A61K 39/3955 20130101; C07K 2317/33
20130101; A61P 25/28 20180101; C07K 2317/24 20130101; A61P 43/00
20180101; A61K 2039/507 20130101; C07K 2317/71 20130101 |
International
Class: |
C07K 16/18 20060101
C07K016/18; C07K 14/47 20060101 C07K014/47; A61K 39/395 20060101
A61K039/395 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 20, 2013 |
EP |
13199123.4 |
Jun 26, 2014 |
EP |
14174047.2 |
Claims
1-20. (canceled)
21. A humanized antibody that specifically binds to human
Tau(pS422), wherein the antibody i) specifically binds to a
polypeptide that has the amino acid sequence of SEQ ID NO: 03,
and/or ii) does not bind to full length human Tau (SEQ ID NO: 01)
at 1 .mu.g/mL, and/or iii) specifically binds to full length human
Tau phosphorylated at the serine at position 422 (SEQ ID NO: 02),
and/or iv) specifically binds to aggregates of human Tau
phosphorylated at the serine at position 422 (SEQ ID NO: 02),
and/or v) specifically binds to human Tau that has the amino acid
sequence of SEQ ID NO: 01 and that has the amino acid mutation
S422A.
22. The humanized antibody according to claim 21, wherein the
antibody has in the heavy chain variable domain at positions 4, 24
and 78 a valine residue.
23. The humanized antibody according to claim 22, wherein the
antibody has in the heavy chain variable domain at position 71 an
arginine residue.
24. The humanized antibody according to claim 23, wherein the
antibody comprises a) in the heavy chain variable domain the HVRs
of SEQ ID NO: 08, 18 and 10, or b) in the heavy chain variable
domain the HVRs of SEQ ID NO: 08, 09 and 10.
25. The humanized antibody according to claim 24, further
comprising a) in the light chain variable domain the HVRs of SEQ ID
NO: 13, 14 and 15, or b) in the light chain variable domain the
HVRs of SEQ ID NO: 12, 05 and 15.
26. The humanized antibody according to claim 23, comprising a) in
the heavy chain variable domain the HVRs of SEQ ID NO: 08, 18 and
10, and in the light chain variable domain the HVRs of SEQ ID NO:
13, 14 and 15, or b) in the heavy chain variable domain the HVRs of
SEQ ID NO: 08, 09 and 10, and in the light chain variable domain
the HVRs of SEQ ID NO: 12, 05 and 15, or c) in the heavy chain
variable domain the HVRs of SEQ ID NO: 08, 09 and 10, and in the
light chain variable domain the HVRs of SEQ ID NO: 13, 14 and
15.
27. The humanized antibody according to claims 23, comprising a) a
heavy chain variable domain of SEQ ID NO: 20 and a light chain
variable domain of SEQ ID NO: 17, or b) a heavy chain variable
domain of SEQ ID NO: 19 and a light chain variable domain of SEQ ID
NO: 16, or c) a heavy chain variable domain of SEQ ID NO: 19 and a
light chain variable domain of SEQ ID NO: 17, or d) a heavy chain
variable domain of SEQ ID NO: 21 and a light chain variable domain
of SEQ ID NO: 17.
28. The humanized antibody according to claim 27, wherein the
antibody is effector function silent.
29. The humanized antibody according to claim 21, wherein the
antibody has an EC.sub.50 value for a) the human Tau(pS422)
fragment that has the amino acid sequence of SEQ ID NO: 03 of 6
ng/mL or less, and/or b) the full length human Tau(pS422) that has
the amino acid sequence of SEQ ID NO: 02 of 4.5 ng/mL or less,
and/or c) aggregates of human Tau (pS422) that has the amino acid
sequence of SEQ ID NO: 02 of 30 ng/mL or less, and/or d) the human
Tau that has the amino acid sequence of SEQ ID NO: 01 and that has
the amino acid mutation S422A of 125 ng/mL or less.
30. The humanized antibody according to claim 29, wherein the
antibody specifically binds to human Tau(pS422) (SEQ ID NO: 02) and
does not bind to human Tau (SEQ ID NO: 01).
31. The humanized antibody according to claim 27, wherein the
antibody is a) a full length antibody of the human subclass IgG1,
or b) a full length antibody of the human subclass IgG4, or c) a
full length antibody of the human subclass IgG1 with the mutations
L234A, L235A and P329G, d) a full length antibody of the human
subclass IgG4 with the mutations S228P, L235E and P329G, e) a full
length antibody of the human subclass IgG1 with the mutations
L234A, L235A and P329G in both heavy chains and the mutations T366W
and S354C in one heavy chain and the mutations T366S, L368A, Y407V
and Y349C in the respective other heavy chain, or f) a full length
antibody of the human subclass IgG4 with the mutations S228P, L235E
and P329G in both heavy chains and the mutations T366W and S354C in
one heavy chain and the mutations T366S, L368A, Y407V and Y349C in
the respective other heavy chain.
32. A pharmaceutical formulation comprising the humanized antibody
according to claim 21 and a pharmaceutically acceptable
carrier.
33. The pharmaceutical formulation according to claim 32, further
comprising an anti-human alpha-synuclein antibody or an anti-Abeta
antibody.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to humanized anti-Tau(pS422)
antibodies which specifically bind to phosphorylated Tau fragment
of SEQ ID NO: 03 and their use for the treatment of brain
diseases.
BACKGROUND
[0002] Human Tau (microtubule-associated protein Tau
(neurofibrillary tangle protein, paired helical filament-Tau,
PHF-Tau)) is a neuronal microtubule-associated protein found
predominantly in axons and functions to promote tubulin
polymerization and to stabilize microtubules. Eight isoforms
(isoform A, B, C, D, E, F, G, fetal-Tau) are found in the human
brain, the longest isoform comprising 441 amino acids (isoform F,
Uniprot P10636-8). Tau and its properties are also described by
Reynolds, C. H., et al., J. Neurochem. 69 (1997) 191-198.
[0003] Tau, in its hyperphosphorylated form, is the major component
of paired helical filaments (PHF), the building block of
neurofibrillary lesions in Alzheimer's disease (AD) brain. Tau can
be phosphorylated at its serine or threonine residues by several
different kinases including GSK3beta, cdk5, MARK and members of the
MAP kinase family.
[0004] Tauopathies are characterized by abnormal
hyperphosphorylation of Tau and are according to Iqbal, K., et al.
(Biochim. Biophys. Acta 1739 (2005) 198-210): [0005] Alzheimer
disease, including tangle-only form of the disease [0006] Down
syndrome, adult cases [0007] Guam Parkinsonism dementia complex
[0008] Dementia pugilistica [0009] Pick disease [0010] Dementia
with argyrophilic grains [0011] Fronto-temporal dementia [0012]
Cortico-basal degeneration [0013] Pallido-ponto-nigral degeneration
[0014] Progressive supranuclear palsy [0015]
Gerstmann-Straussler-Scheinker disease with tangles.
[0016] So far nearly 40 serine (S)/threonine (T) phosphorylation
sites have been found in Tau from Alzheimer's disease brains
(Hanger, D. P., et al., J. Biol. Chem. 282 (2007) 23645-23654). The
development of Tau pathology in Alzheimer's disease is related to
its phosphorylation state. However, most of the 40 phosphorylation
sites are not associated with disease pathology since they are also
found in Tau extracted from healthy, fetal brain tissue. Only a few
phosphorylations are unique to the disease state and are presumably
responsible for the abnormal, aggregation and characteristic
insolubility that define Tau in the PHFs of Alzheimer brain
(Morishima-Kawashima, M., et al., J. Biol. Chem. 270 (1995)
823-829). According to Pei, J. J., et al. (J. Alzheimer's Disease
14 (2008) 385-392) the existing literature provides limited and
unclear information about which of these sites are specific to AD
brains. Pei used a list of phospho-specific antibodies to Tau and
measured their levels in homogenates of the medial temporal cortex
from 22 AD patients and 10 controls.
[0017] Bussiere, T., et al. (Acta Neuropathol. 97 (1999) 221-230)
described that phosphorylated serine 422 on Tau proteins is a
pathological epitope found in several diseases with neurofibrillary
degeneration. Augustinack, J. C., et al., (Acta Neuropathol. 103
(2002) 26-35) described p5422 as correlating with the severity of
neuronal pathology in Alzheimer's disease. Guillozet-Bongaarts, A.,
(J. Neurochem. 97 (2006) 1005-1014) described the phosphorylation
of Tau at serine 422 as being part of the maturation process of
PHFs. Tau pS422 is also found in association with developing
pathology in various transgenic mouse models of Alzheimer's
disease. Thus, Deters, N., et al., mentioned in Biochem. Biophys.
Res. Commun. 379 (2009) 400-405 that double-transgenic Dom5/pR5
mice showed 7-fold increased numbers of hippocampal neurons that
contain Tau specifically phosphorylated the pathological S422
epitope. Goetz, J., et al., (Science 293 (2001) 1491-1495) reported
the appearance of Tau phosphorylated at S422 in the brains of Tau
P301L transgenic mice injected with Abeta42 fibrils.
[0018] EP 2 009 104 relates to epitopes of the Tau protein which
occur in a phosphorylated state in Tau protein from Alzheimer's
disease PHFs and to the use of said epitopes for the generation of
antibodies specifically detecting Alzheimer Tau protein. WO
2002/062851 and U.S. Pat. No. 7,446,180 relate to antibodies with a
specificity to an abnormally truncated form of Tau protein and
diagnostic and therapeutic aspects in relation to Alzheimer's
disease and related Tauopathies. WO 98/22120 relates to a method of
treating a patient with Alzheimer's disease comprising the step of
administering to the patient an antibody against phosphorylated Tau
fragment of amino acids about 207 to about 222, amino acids about
224 to about 240, and amino acids about 390 to about 408. Animal
studies where the phosphorylated Tau fragment 379-408
[P-Ser396,404} is used to vaccinate Tau transgenic mice are
mentioned in Asuni, A. A., et al., J. Neuroscience 27 (2007)
9115-9129. US 2008/0050383 relates to methods of treating and
preventing Alzheimer's disease or other Tauopathies in a subject by
administering a Tau protein fragment.
[0019] Hasegawa, M., et al. (FEBS Lett. 384 (1996) 25-30) report
monoclonal antibody (AP422) specific for phosphoserine 422 in
microtubule-associated protein tau.
[0020] In WO 01/55725 an antibody that specifically recognizes tau
and an antibody that specifically recognizes phospho-tau (181) for
use in a method for the in vivo diagnosis of a tauopathy and/or for
the in vivo differential diagnosis of a tauopathy versus a
non-tauopathy is reported.
[0021] In WO 02/027017 an antibody prepared from a polypeptide
immunogen having a phosphorylated serine is reported. WO 02/062851
relates to antibodies with a specificity to an abnormally truncated
form of Tau protein and diagnostic and therapeutic aspects in
relation to Alzheimer's disease and related Tauopathies.
[0022] In WO 2004/016655 an antibody specific to a central nervous
System (CNS) tau protein, wherein the antibody specifically
recognizes a CNS tau protein but not a peripheral tau protein and
wherein the antibody specifically recognizes an amino acid sequence
of a connective portion between the amino acid sequence encoded by
Exon 4 of a gene encoding a tau protein and the amino acid sequence
encoded by Exon 5 thereof as an epitope is reported.
[0023] Monoclonal antibodies against Tau pS422 are described, for
example, in EP 1 876 185. Polyclonal antibodies against Tau pS422
are commercially available (e.g. ProSci Inc. and Biosource
International).
[0024] In WO 2006/055178 a method for inhibiting the
phosphorylation of tau protein at Ser202/Thr205 comprising
contacting a sample containing a tau protein with the antibody or
antigen binding fragment that binds amyloid beta-derived diffusible
ligands thereby inhibiting the phosphorylation of tau protein at
Ser202/Thr205 is reported.
[0025] An antibody preparation that specifically binds to tau
phosphorylated at tyr394 and/or tyr310 is reported in WO
2007/019273. Animal studies where the phosphorylated Tau fragment
379-408 [P-Ser396,404] is used to vaccinate Tau transgenic mice are
mentioned in Asuni, A. A. et al., J. Neuroscience 27 (2007)
9115-9129.
[0026] EP 2 009 104 relates to epitopes of the Tau protein which
occur in a phosphorylated state in Tau protein from Alzheimer's
disease PHFs and to the use of said epitopes for the generation of
antibodies specifically detecting Alzheimer Tau protein.
[0027] US 2008/0050383 relates to methods of treating and
preventing Alzheimer's disease or other Tauopathies in a subject by
administering a Tau protein fragment.
[0028] In WO 2010/037135 an isolated, synthetic or recombinant
polypeptide or peptide comprising a first domain comprising, or
consisting of, a ligand for a blood brain barrier (BBB) receptor or
equivalent and a second domain comprising, or consisting of an
enzyme or composition that slows the rate of aggregation of a
protein aggregate, inhibits the formation of a protein aggregate,
or reverses, digests or dissolves a protein aggregate is reported.
An antibody, particularly a monoclonal antibody or functional parts
thereof, capable of recognizing and binding to a tau protein in
vitro and/or in vivo is reported in WO 2010/115843.
[0029] In WO 2011/026031 a monoclonal antibody or its fragment that
specifically binds tau oligomers and does not bind soluble tau or
tau fibrils, useful for treating tauopathy e.g. Alzheimer's
disease, progressive supranuclear palsy and corticobasal
degeneration is reported. An isolated antibody that specifically
binds human tau protein phosphorylated at one or more of Ser(238)
and Thr(245) is reported in WO 2011/053565.
[0030] In WO 2012/045882 an antibody which specifically binds to a
phospho-epitope on the mammalian Tau protein, useful for treating
neurodegenerative disorders such as tauopathies, and for treating
or alleviating cognitive deficits is reported. A human monoclonal
anti-tau antibody or a tau binding fragment thereof is reported in
WO 2012/049570. A method of preventing or treating Alzheimer's
disease or other tauopathies in a subject, comprising administering
antibodies to a human in need of therapy for Alzheimer's disease or
other tauopathy, the antibodies having specificity to abnormal
forms of tau protein, said antibody showing no binding and/or
reactivity to a normal tau protein and being administered under
conditions and in an amount(s) effective to prevent or treat
Alzheimer's disease or other tauopathy is reported in WO
2012/106363.
[0031] In WO 2012/149365 an antibody which shows reactivity with
aggregated tau and substantially no reactivity with non-aggregated
Tau, wherein the aggregated tau comprises at least two tau proteins
cross-linked to each other, either directly or through a linker, at
one or more cysteine residues is reported.
[0032] A composition useful in treating Tauopathy e.g. Alzheimer
disease comprises antibody binding to Tau, phosphorylated serine
modified compound at specific position specifically binding to
specific phosphorylated Tau and its fragment and carrier is
reported in WO 2010/142423.
[0033] In EP 1 876 185 A an antibody which recognizes
phosphorylated polypeptides is reported. In WO 2013/151762 a
humanized tau antibody is reported. In WO 2014/016737 novel chicken
monoclonal antibodies against human phosphorylated tau and uses
thereof are reported. In WO 2014/016737are reported novel chicken
monoclonal antibodies against human phosphorylated tau and uses
thereof. Antibodies selective for pathological tau dimers and
prefibrillar pathological tau oligomers and their uses in
treatment, diagnosis and monitoring of tauopathies are reported in
WO 2012/149365.
SUMMARY
[0034] The invention provides anti-human Tau(pS422) antibodies,
especially humanized anti-human Tau(pS422) antibodies, and methods
of using the same.
[0035] The humanized antibodies as reported herein were not
available by standard humanization methods. It was required to
introduce non-standard mutations in the amino acid sequence in
order to obtain a humanized antibody with comparable binding
characteristics as the parent rabbit antibody comprising variable
domains with the amino acid sequence of SEQ ID NO: 07 and SEQ ID
NO: 11. This is especially important as the antibodies as reported
herein are intended to cross the human blood-brain-harrier and to
be effective within the human brain. Thus, the generally applied
criteria for the selection of humanized antibodies are not
sufficiently stringent in order to be applied directly in the
current case.
[0036] One aspect as reported herein is a (humanized) antibody that
specifically binds to human Tau(pS422), wherein the antibody [0037]
i) specifically binds to a polypeptide that has the amino acid
sequence of SEQ ID NO: 03, and/or [0038] ii) does not bind to full
length human Tau (SEQ ID NO: 01) at 1 .mu.g/mL, and/or [0039] iii)
specifically binds to full length human Tau phosphorylated at the
serine at position 422 (SEQ ID NO: 02), and/or [0040] iv)
specifically binds to aggregates of human Tau phosphorylated at the
serine at position 422 (SEQ ID NO: 02), and/or [0041] v)
specifically binds to human Tau that has the amino acid sequence of
SEQ ID NO: 01 and that has the amino acid mutation S422A.
[0042] The antibodies as reported herein show a selectivity with
respect to human Tau phosphorylated at the serine at position 422,
with respect to not-phosphorylated wild-type human Tau and the Tau
mutant S422A. The not-phosphorylated wild-type human Tau and the
Tau mutant S422A are not bound at all or with a lower affinity,
respectively.
[0043] One aspect as reported herein is a (humanized) antibody that
specifically binds to human Tau(pS422), characterized in that the
antibody comprises [0044] a) in the heavy chain variable domain the
HVRs of SEQ ID NO: 08, 18 and 10, or [0045] b) in the heavy chain
variable domain the HVRs of SEQ ID NO: 08, 09 and 10.
[0046] In one embodiment the antibody comprises [0047] a) in the
light chain variable domain the HVRs of SEQ ID NO: 13, 14 and 15,
or [0048] b) in the light chain variable domain the HVRs of SEQ ID
NO: 12, 05 and 15.
[0049] In one embodiment the antibody comprises [0050] a) in the
heavy chain variable domain the HVRs of SEQ ID NO: 08, 18 and 10,
and in the light chain variable domain the HVRs of SEQ ID NO: 13,
14 and 15, or [0051] b) in the heavy chain variable domain the HVRs
of SEQ ID NO: 08, 09 and 10, and in the light chain variable domain
the HVRs of SEQ ID NO: 12, 05 and 15, or [0052] c) in the heavy
chain variable domain the HVRs of SEQ ID NO: 08, 09 and 10, and in
the light chain variable domain the HVRs of SEQ ID NO: 13, 14 and
15.
[0053] In one embodiment the antibody comprises [0054] a) a heavy
chain variable domain of SEQ ID NO: 20 and a light chain variable
domain of SEQ ID NO: 17, or [0055] b) a heavy chain variable domain
of SEQ ID NO: 19 and a light chain variable domain of SEQ ID NO:
16, or [0056] c) a heavy chain variable domain of SEQ ID NO: 19 and
a light chain variable domain of SEQ ID NO: 17, or [0057] d) a
heavy chain variable domain of SEQ ID NO: 21 and a light chain
variable domain of SEQ ID NO: 17.
[0058] In one embodiment the antibody is for use in the treatment
of Alzheimer's disease.
[0059] In one embodiment the antibody is effector function silent.
In one embodiment the antibody has no effector function. In one
embodiment the antibody is of the human IgG1 subclass and has the
mutations L234A, L235A and P329G in both heavy chains (numbering
according to the EU index of Kabat).
[0060] In one embodiment the antibody [0061] i) specifically binds
to a polypeptide that has the amino acid sequence of SEQ ID NO: 03,
and/or [0062] ii) does not bind to full length human Tau (SEQ ID
NO: 01) at 1 .mu.g/mL, and/or [0063] iii) specifically binds to
full length human Tau phosphorylated at the serine at position 422
(SEQ ID NO: 02), and/or [0064] iv) specifically binds to aggregates
of human Tau phosphorylated at the serine at position 422 (SEQ ID
NO: 02).
[0065] In one embodiment the antibody has an EC.sub.50 value for
[0066] a) the human Tau(pS422) fragment that has the amino acid
sequence of SEQ ID NO: 03 of 6 ng/mL or less, and/or [0067] b) the
full length human Tau(pS422) that has the amino acid sequence of
SEQ ID NO: 02 of 4.5 ng/mL or less, and/or [0068] c) aggregates of
human Tau (pS422) that has the amino acid sequence of SEQ ID NO: 02
of 30 ng/mL or less, and/or [0069] d) the human Tau that has the
amino acid sequence of SEQ ID NO: 01 and that has the amino acid
mutation S422A of 125 ng/mL or less.
[0070] In one embodiment the antibody specifically binds to human
Tau(pS422) (SEQ ID NO: 02) and does not bind to human Tau (SEQ ID
NO: 01).
[0071] In one embodiment the antibody is a monoclonal antibody.
[0072] In one embodiment the antibody is an antibody fragment that
binds to human Tau(pS422) and [0073] i) specifically binds to a
polypeptide that has the amino acid sequence of SEQ ID NO: 03,
and/or [0074] ii) does not bind to full length human Tau (SEQ ID
NO: 01) at 1 .mu.g/mL, and/or [0075] iii) specifically binds to
full length human Tau phosphorylated at the serine at position 422
(SEQ ID NO: 02), and/or [0076] iv) specifically binds to aggregates
of human Tau phosphorylated at the serine at position 422 (SEQ ID
NO: 02), and/or [0077] v) specifically binds to full length human
Tau that has the amino acid sequence of SEQ ID NO: 01 and that has
the amino acid mutation S422A, and/or [0078] vi) has an EC.sub.50
value for the human Tau(pS422) fragment that has the amino acid
sequence of SEQ ID NO: 03 of 6 ng/mL or less, and/or [0079] vii)
has an EC.sub.50 value for the full length human Tau(pS422) that
has the amino acid sequence of SEQ ID NO: 02 of 4.5 ng/mL or less,
and/or [0080] viii) has an EC.sub.50 value for aggregates of human
Tau (pS422) that has the amino acid sequence of SEQ ID NO: 02 of 30
ng/mL or less, and/or [0081] ix) has an EC.sub.50 value for the
human Tau that has the amino acid sequence of SEQ ID NO: 01 and
that has the amino acid mutation S422A of 125 ng/mL or less.
[0082] In one embodiment the antibody is [0083] a) a full length
antibody of the human subclass IgG 1, or [0084] b) a full length
antibody of the human subclass IgG4, or [0085] c) a full length
antibody of the human subclass IgG1 with the mutations L234A, L235A
and P329G, [0086] d) a full length antibody of the human subclass
IgG4 with the mutations S228P, L235E and P329G, [0087] e) a full
length antibody of the human subclass IgG1 with the mutations
L234A, L235A and P329G in both heavy chains and the mutations T366W
and S354C in one heavy chain and the mutations T366S, L368A, Y407V
and Y349C in the respective other heavy chain, or [0088] f) a full
length antibody of the human subclass IgG4 with the mutations S228P
and P329G in both heavy chains and the mutations T366W and S354C in
one heavy chain and the mutations T366S, L368A, Y407V and Y349C in
the respective other heavy chain.
[0089] One aspect as reported herein is a (humanized) anti-human
Tau(pS422) antibody, characterized in that [0090] a) the antibody
comprises two antibody heavy chains each comprising a heavy chain
variable domain and a heavy chain constant region, wherein [0091]
i) the variable domain comprises the HVRs of SEQ ID NO: 08, SEQ ID
NO: 18 and SEQ ID NO: 10, [0092] ii) the constant region is a human
IgG1 constant region, wherein the C-terminal lysine residue can be
present or absent, and [0093] iii) the constant region comprises
the amino acid changes L234A, L235A and P329G, [0094] b) the
antibody comprises two antibody light chains each comprising a
light chain variable domain and a light chain constant domain,
wherein [0095] i) the variable domain comprises the HVRs of SEQ ID
NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, [0096] ii) the constant
region is a human kappa light chain constant region or a human
lambda light chain constant region, [0097] and [0098] c) the
antibody [0099] i) specifically binds to a polypeptide that has the
amino acid sequence of SEQ ID NO: 03, and/or [0100] ii) does not
bind to full length human Tau (SEQ ID NO: 01) at 1 .mu.g/mL, and/or
[0101] iii) specifically binds to full length human Tau
phosphorylated at the serine at position 422 (SEQ ID NO: 02),
and/or [0102] iv) specifically binds to aggregates of human Tau
phosphorylated at the serine at position 422 (SEQ ID NO: 02),
and/or [0103] v) specifically binds to full length human Tau that
has the amino acid sequence of SEQ ID NO: 01 and that has the amino
acid mutation S422A, and/or [0104] vi) has an EC.sub.50 value for
the human Tau(pS422) fragment that has the amino acid sequence of
SEQ ID NO: 03 of 6 ng/mL or less, and/or [0105] vii) has an
EC.sub.50 value for the full length human Tau(p S422) that has the
amino acid sequence of SEQ ID NO: 02 of 4.5 ng/mL or less, and/or
[0106] viii) has an EC.sub.50 value for aggregates of human Tau
(pS422) that has the amino acid sequence of SEQ ID NO: 02 of 30
ng/mL or less, and/or [0107] ix) has an EC.sub.50 value for the
human Tau that has the amino acid sequence of SEQ ID NO: 01 and
that has the amino acid mutation S422A of 125 ng/mL or less.
[0108] One aspect as reported herein is a (humanized) anti-human
Tau(pS422) antibody, characterized in that [0109] a) the antibody
comprises two antibody heavy chains each comprising a heavy chain
variable domain and a heavy chain constant region, wherein [0110]
i) the variable domain comprises the HVRs of SEQ ID NO: 08, SEQ ID
NO: 09 and SEQ ID NO: 10, [0111] ii) the constant region is a human
IgG1 constant region, wherein the C-terminal lysine residue can be
present or absent, and [0112] iii) the constant region comprises
the amino acid changes L234A, L235A and P329G, [0113] b) the
antibody comprises two antibody light chains each comprising a
light chain variable domain and a light chain constant domain,
wherein [0114] i) the variable domain comprises the HVRs of SEQ ID
NO: 12, SEQ ID NO: 05 and SEQ ID NO: 15, [0115] ii) the constant
region is a human kappa light chain constant region or a human
lambda light chain constant region, [0116] and [0117] c) the
antibody [0118] i) specifically binds to a polypeptide that has the
amino acid sequence of SEQ ID NO: 03, and/or [0119] ii) does not
bind to full length human Tau (SEQ ID NO: 01) at 1 .mu.g/mL, and/or
[0120] iii) specifically binds to full length human Tau
phosphorylated at the serine at position 422 (SEQ ID NO: 02),
and/or [0121] iv) specifically binds to aggregates of human Tau
phosphorylated at the serine at position 422 (SEQ ID NO: 02),
and/or [0122] v) specifically binds to full length human Tau that
has the amino acid sequence of SEQ ID NO: 01 and that has the amino
acid mutation S422A, and/or [0123] vi) has an EC.sub.50 value for
the human Tau(pS422) fragment that has the amino acid sequence of
SEQ ID NO: 03 of 6 ng/mL or less, and/or [0124] vii) has an
EC.sub.50 value for the full length human Tau(p S422) that has the
amino acid sequence of SEQ ID NO: 02 of 4.5 ng/mL or less, and/or
[0125] viii) has an EC.sub.50 value for aggregates of human Tau
(pS422) that has the amino acid sequence of SEQ ID NO: 02 of 30
ng/mL or less, and/or [0126] ix) has an EC.sub.50 value for the
human Tau that has the amino acid sequence of SEQ ID NO: 01 and
that has the amino acid mutation S422A of 125 ng/mL or less.
[0127] One aspect as reported herein is a (humanized) anti-human
Tau(pS422) antibody, characterized in that [0128] a) the antibody
comprises two antibody heavy chains each comprising a heavy chain
variable domain and a heavy chain constant region, wherein [0129]
i) the variable domain comprises the HVRs of SEQ ID NO: 08, SEQ ID
NO: 09 and SEQ ID NO: 10, [0130] ii) the constant region is a human
IgG1 constant region, wherein the C-terminal lysine residue can be
present or absent, and [0131] iii) the constant region comprises
the amino acid changes L234A, L235A and P329G, [0132] b) the
antibody comprises two antibody light chains each comprising a
light chain variable domain and a light chain constant domain,
wherein [0133] i) the variable domain comprises the HVRs of SEQ ID
NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, [0134] ii) the constant
region is a human kappa light chain constant region or a human
lambda light chain constant region, [0135] and [0136] c) the
antibody [0137] i) specifically binds to a polypeptide that has the
amino acid sequence of SEQ ID NO: 03, and/or [0138] ii) does not
bind to full length human Tau (SEQ ID NO: 01) at 1 .mu.g/mL, and/or
[0139] iii) specifically binds to full length human Tau
phosphorylated at the serine at position 422 (SEQ ID NO: 02),
and/or [0140] iv) specifically binds to aggregates of human Tau
phosphorylated at the serine at position 422 (SEQ ID NO: 02),
and/or [0141] v) specifically binds to full length human Tau that
has the amino acid sequence of SEQ ID NO: 01 and that has the amino
acid mutation S422A, and/or [0142] vi) has an EC.sub.50 value for
the human Tau(pS422) fragment that has the amino acid sequence of
SEQ ID NO: 03 of 6 ng/mL or less, and/or [0143] vii) has an
EC.sub.50 value for the full length human Tau(pS422) that has the
amino acid sequence of SEQ ID NO: 02 of 4.5 ng/mL or less, and/or
[0144] viii) has an EC.sub.50 value for aggregates of human Tau
(pS422) that has the amino acid sequence of SEQ ID NO: 02 of 30
ng/mL or less, and/or [0145] ix) has an EC.sub.50 value for the
human Tau that has the amino acid sequence of SEQ ID NO: 01 and
that has the amino acid mutation S422A of 125 ng/mL or less.
[0146] One aspect as reported herein is a (humanized) anti-human
Tau(pS422) antibody, characterized in that [0147] a) the antibody
comprises two antibody heavy chains each comprising a heavy chain
variable domain and a heavy chain constant region, wherein [0148]
i) the variable domain has the amino acid sequence of SEQ ID NO:
20, [0149] ii) the constant region is a human IgG1 constant region,
wherein the C-terminal lysine residue can be present or absent, and
[0150] iii) the constant region comprises the amino acid changes
L234A, L235A and P329G, [0151] b) the antibody comprises two
antibody light chains each comprising a light chain variable domain
and a light chain constant domain, wherein [0152] i) the variable
domain has the amino acid sequence of SEQ ID NO: 17, [0153] ii) the
constant region is a human kappa light chain constant region or a
human lambda light chain constant region, [0154] and [0155] c) the
antibody [0156] i) specifically binds to a polypeptide that has the
amino acid sequence of SEQ ID NO: 03, and/or [0157] ii) does not
bind to full length human Tau (SEQ ID NO: 01) at 1 .mu.g/mL, and/or
[0158] iii) specifically binds to full length human Tau
phosphorylated at the serine at position 422 (SEQ ID NO: 02),
and/or [0159] iv) specifically binds to aggregates of human Tau
phosphorylated at the serine at position 422 (SEQ ID NO: 02),
and/or [0160] v) specifically binds to full length human Tau that
has the amino acid sequence of SEQ ID NO: 01 and that has the amino
acid mutation S422A, and/or [0161] vi) has an EC.sub.50 value for
the human Tau(pS422) fragment that has the amino acid sequence of
SEQ ID NO: 03 of 6 ng/mL or less, and/or [0162] vii) has an
EC.sub.50 value for the full length human Tau(pS422) that has the
amino acid sequence of SEQ ID NO: 02 of 4.5 ng/mL or less, and/or
[0163] viii) has an EC.sub.50 value for aggregates of human Tau
(pS422) that has the amino acid sequence of SEQ ID NO: 02 of 30
ng/mL or less, and/or [0164] ix) has an EC.sub.50 value for the
human Tau that has the amino acid sequence of SEQ ID NO: 01 and
that has the amino acid mutation S422A of 125 ng/mL or less.
[0165] One aspect as reported herein is a (humanized) anti-human
Tau(pS422) antibody, characterized in that [0166] a) the antibody
comprises two antibody heavy chains each comprising a heavy chain
variable domain and a heavy chain constant region, wherein [0167]
i) the variable domain has the amino acid sequence of SEQ ID NO:
19, [0168] ii) the constant region is a human IgG1 constant region,
wherein the C-terminal lysine residue can be present or absent, and
[0169] iii) the constant region comprises the amino acid changes
L234A, L235A and P329G, [0170] b) the antibody comprises two
antibody light chains each comprising a light chain variable domain
and a light chain constant domain, wherein [0171] i) the variable
domain has the amino acid sequence of SEQ ID NO: 16, [0172] ii) the
constant region is a human kappa light chain constant region or a
human lambda light chain constant region, [0173] and [0174] c) the
antibody [0175] i) specifically binds to a polypeptide that has the
amino acid sequence of SEQ ID NO: 03, and/or [0176] ii) does not
bind to full length human Tau (SEQ ID NO: 01) at 1 .mu.g/mL, and/or
[0177] iii) specifically binds to full length human Tau
phosphorylated at the serine at position 422 (SEQ ID NO: 02),
and/or [0178] iv) specifically binds to aggregates of human Tau
phosphorylated at the serine at position 422 (SEQ ID NO: 02),
and/or [0179] v) specifically binds to full length human Tau that
has the amino acid sequence of SEQ ID NO: 01 and that has the amino
acid mutation S422A, and/or [0180] vi) has an EC.sub.50 value for
the human Tau(pS422) fragment that has the amino acid sequence of
SEQ ID NO: 03 of 6 ng/mL or less, and/or [0181] vii) has an
EC.sub.50 value for the full length human Tau(pS422) that has the
amino acid sequence of SEQ ID NO: 02 of 4.5 ng/mL or less, and/or
[0182] viii) has an EC.sub.50 value for aggregates of human Tau
(pS422) that has the amino acid sequence of SEQ ID NO: 02 of 30
ng/mL or less, and/or [0183] ix) has an EC.sub.50 value for the
human Tau that has the amino acid sequence of SEQ ID NO: 01 and
that has the amino acid mutation S422A of 125 ng/mL or less.
[0184] One aspect as reported herein is a (humanized) anti-human
Tau(pS422) antibody, characterized in that [0185] a) the antibody
comprises two antibody heavy chains each comprising a heavy chain
variable domain and a heavy chain constant region, wherein [0186]
i) the variable domain has the amino acid sequence of SEQ ID NO:
19, [0187] ii) the constant region is a human IgG1 constant region,
wherein the C-terminal lysine residue can be present or absent, and
[0188] iii) the constant region comprises the amino acid changes
L234A, L235A and P329G, [0189] b) the antibody comprises two
antibody light chains each comprising a light chain variable domain
and a light chain constant domain, wherein [0190] i) the variable
domain has the amino acid sequence of SEQ ID NO: 17, [0191] ii) the
constant region is a human kappa light chain constant region or a
human lambda light chain constant region, [0192] and [0193] c) the
antibody [0194] i) specifically binds to a polypeptide that has the
amino acid sequence of SEQ ID NO: 03, and/or [0195] ii) does not
bind to full length human Tau (SEQ ID NO: 01) at 1 .mu.g/mL, and/or
[0196] iii) specifically binds to full length human Tau
phosphorylated at the serine at position 422 (SEQ ID NO: 02),
and/or [0197] iv) specifically binds to aggregates of human Tau
phosphorylated at the serine at position 422 (SEQ ID NO: 02),
and/or [0198] v) specifically binds to full length human Tau that
has the amino acid sequence of SEQ ID NO: 01 and that has the amino
acid mutation S422A, and/or [0199] vi) has an EC.sub.50 value for
the human Tau(pS422) fragment that has the amino acid sequence of
SEQ ID NO: 03 of 6 ng/mL or less, and/or [0200] vii) has an
EC.sub.50 value for the full length human Tau(pS422) that has the
amino acid sequence of SEQ ID NO: 02 of 4.5 ng/mL or less, and/or
[0201] viii) has an EC.sub.50 value for aggregates of human Tau
(pS422) that has the amino acid sequence of SEQ ID NO: 02 of 30
ng/mL or less, and/or [0202] ix) has an EC.sub.50 value for the
human Tau that has the amino acid sequence of SEQ ID NO: 01 and
that has the amino acid mutation S422A of 125 ng/mL or less.
[0203] One aspect as reported herein is a (humanized) anti-human
Tau(pS422) antibody, characterized in that [0204] a) the antibody
comprises two antibody heavy chains each comprising a heavy chain
variable domain and a heavy chain constant region, wherein [0205]
i) the variable domain has the amino acid sequence of SEQ ID NO: 21
[0206] ii) the constant region is a human IgG1 constant region,
wherein the C-terminal lysine residue can be present or absent, and
[0207] iii) the constant region comprises the amino acid changes
L234A, L235A and P329G, [0208] b) the antibody comprises two
antibody light chains each comprising a light chain variable domain
and a light chain constant domain, wherein [0209] i) the variable
domain has the amino acid sequence of SEQ ID NO: 17, [0210] ii) the
constant region is a human kappa light chain constant region or a
human lambda light chain constant region, [0211] and [0212] c) the
antibody [0213] i) specifically binds to a polypeptide that has the
amino acid sequence of SEQ ID NO: 03, and/or [0214] ii) does not
bind to full length human Tau (SEQ ID NO: 01) at 1 g/mL, and/or
[0215] iii) specifically binds to full length human Tau
phosphorylated at the serine at position 422 (SEQ ID NO: 02),
and/or [0216] iv) specifically binds to aggregates of human Tau
phosphorylated at the serine at position 422 (SEQ ID NO: 02),
and/or [0217] v) specifically binds to full length human Tau that
has the amino acid sequence of SEQ ID NO: 01 and that has the amino
acid mutation S422A, and/or [0218] vi) has an EC.sub.50 value for
the human Tau(pS422) fragment that has the amino acid sequence of
SEQ ID NO: 03 of 6 ng/mL or less, and/or [0219] vii) has an
EC.sub.50 value for the full length human Tau(pS422) that has the
amino acid sequence of SEQ ID NO: 02 of 4.5 ng/mL or less, and/or
[0220] viii) has an EC.sub.50 value for aggregates of human Tau
(pS422) that has the amino acid sequence of SEQ ID NO: 02 of 30
ng/mL or less, and/or [0221] ix) has an EC.sub.50 value for the
human Tau that has the amino acid sequence of SEQ ID NO: 01 and
that has the amino acid mutation S422A of 125 ng/mL or less.
[0222] In one preferred embodiment of all aspects the anti-human
Tau(pS422) antibody is characterized in that the antibody has in
the heavy chain variable domain at positions 4, 24 and 78 a valine
residue.
[0223] In one preferred embodiment of all aspects the anti-human
Tau(pS422) antibody is characterized in that the antibody has in
the heavy chain variable domain at position 71 an arginine
residue.
[0224] One aspect as reported herein is an isolated nucleic acid
encoding a (humanized) antibody as reported herein.
[0225] One aspect as reported herein is a host cell comprising the
nucleic acid as reported herein.
[0226] One aspect as reported herein is a method of producing a
(humanized) antibody comprising the steps of culturing the host
cell as reported herein so that the antibody is produced.
[0227] In one embodiment the method further comprises the step of
recovering the antibody from the cell or the cultivation
medium.
[0228] One aspect as reported herein is a pharmaceutical
formulation comprising the (humanized) antibody as reported herein
and a pharmaceutically acceptable carrier.
[0229] In one embodiment the pharmaceutical formulation further
comprises an additional therapeutic agent.
[0230] In one embodiment the additional therapeutic agent is an
anti-amyloid therapeutic agent. In one embodiment the anti-amyloid
therapeutic agent is an anti-human alpha-synuclein antibody or an
anti-Abeta antibody.
[0231] One aspect as reported herein is the (humanized) antibody as
reported herein for use as a medicament.
[0232] One aspect as reported herein is the (humanized) antibody as
reported herein for use in treating Alzheimer's disease.
[0233] One aspect as reported herein is the (humanized) antibody as
reported herein for use in treating prodromal Alzheimer's
disease.
[0234] One aspect as reported herein is the (humanized) antibody as
reported herein for use in treating mild Alzheimer's disease.
[0235] One aspect as reported herein is the (humanized) antibody as
reported herein for use in reducing Tau(pS422)-induced
neurodegeneration.
[0236] One aspect as reported herein is the (humanized) antibody as
reported herein for use in maintaining cognition and function.
[0237] One aspect as reported herein is the (humanized) antibody as
reported herein for use in slowing the rate of cognitive and
functional decline.
[0238] One aspect as reported herein is the (humanized) antibody as
reported herein for use in slowing down the rate of neurofibrillary
tangle accumulation.
[0239] In one embodiment of the previous aspects the use is by
reducing neurofibrillary tangle burden by clearing Tau(pS422).
[0240] In one embodiment of the previous aspects the use is by
preventing neurofibrillary tangle build up.
[0241] In one embodiment of the previous aspects the use is by
removing/clearing neurofibrillary tangles.
[0242] In one embodiment the preventing and/or removing is by
promoting the intracellular clearance of Tau aggregates.
[0243] In one embodiment of the previous aspects the use is by
inhibiting neurofibrillary tangle spreading. In one embodiment the
inhibiting is by preventing interneuronal transfer of pathological
Tau farms/seeds.
[0244] Aspects of the current invention are also methods of
treatment comprising administering the (humanized) antibody as
reported herein for treating Alzheimer's disease, for treating
prodromal Alzheimer's disease, for treating mild Alzheimer's
disease, for reducing Tau(pS422)-induced neurodegeneration, for
maintaining cognition and function, for slowing the rate of
cognitive and functional decline, and/or for slowing down the rate
of neurofibrillary tangle accumulation.
[0245] One aspect as reported herein is the use of the (humanized)
antibody as reported herein in the manufacture of a medicament.
[0246] In one embodiment the medicament is for treatment of
Alzheimer's disease.
[0247] In one embodiment the medicament is for treatment of
prodromal Alzheimer's disease.
[0248] In one embodiment the medicament is for treatment of mild
Alzheimer's disease.
[0249] In one embodiment the medicament is for reducing Tau(pS422)
induced neurodegeneration.
[0250] In one embodiment the medicament is for maintaining
cognition and function.
[0251] In one embodiment the medicament is for slowing the rate of
cognitive and functional decline.
[0252] One aspect as reported herein is a method of treating an
individual having Alzheimer's disease comprising administering to
the individual an effective amount of a (humanized) anti-human
Tau(pS422) antibody as reported herein.
[0253] One aspect as reported herein is a method of reducing
Tau(pS422) induced neurodegeneration in an individual comprising
administering to the individual an effective amount of a
(humanized) anti-human Tau(pS422) antibody as reported herein to
reduce Tau(pS422) induced neurodegeneration.
[0254] One aspect as reported herein is a method of maintaining
cognition and function in an individual comprising administering to
the individual an effective amount of a (humanized) anti-human
Tau(pS422) antibody as reported herein to maintain cognition and
function.
[0255] One aspect as reported herein is a method of slowing the
rate of cognitive and functional decline in an individual
comprising administering to the individual an effective amount of a
(humanized) anti-human Tau(pS422) antibody as reported herein to
slow the rate of cognitive and functional decline.
[0256] One aspect as reported herein is the use of a (humanized)
anti-human Tau(pS422) antibody as reported herein in the reduction
of Tau(pS422) induced neurodegeneration.
[0257] One aspect as reported herein is the use of a (humanized)
anti-human Tau(pS422) antibody as reported herein in maintaining
cognition and function.
[0258] One aspect as reported herein is the use of a (humanized)
anti-human Tau(pS422) antibody as reported herein in slowing the
rate of cognitive and functional decline.
[0259] The antibodies as reported herein can be used in the
treatment of Alzheimer's disease.
[0260] With the (humanized) antibodies as reported herein
inhibition/reduction of progression of Alzheimer's disease and
neuropathology can be effected.
[0261] The (humanized) antibodies as reported herein can be used to
protect from development of Alzheimer's disease or even used to
stop the progression of Alzheimer's disease.
[0262] In one embodiment the (humanized) antibody as reported
herein i) binds to Tau(pS422) on brain sections of Tau(pS422)
transgenic mice and Alzheimer's disease patients; and/or labels
Tau(pS422) in Tau(pS422) transgenic cells.
[0263] The (humanized) antibodies as reported herein can be used
for the treatment of Alzheimer's disease.
[0264] One aspect as reported herein is a (humanized) antibody that
specifically binds to the amino acid sequence of SEQ ID NO: 03 in
human Tau(pS422).
[0265] The (humanized) antibodies as reported herein specifically
bind to/recognize early and late stage disease-relevant forms of
human Tau(pS422).
[0266] One aspect as reported herein is the use of the (humanized)
antibody as reported herein for the prevention of human Tau
(pS422)-related Alzheimer's disease spread.
[0267] One aspect as reported herein is the use of the (humanized)
antibody as reported herein for the reduction of lysosomal membrane
disintegration.
[0268] One aspect as reported herein is the use of the (humanized)
antibody as reported herein for the stabilization of lysosome
membrane against human Tau(pS422) induced destabilization and/or
disintegration.
[0269] One aspect as reported herein is the use of the (humanized)
antibody as reported herein for the prevention of Alzheimer's
disease progression.
[0270] The (humanized) antibodies as reported herein function by
antibody mediated inhibition of human Tau(pS422) seeding and
spreading between cells.
[0271] The (humanized) antibodies as reported herein protect
lysosomes from fibrillar damage by binding to human Tau(pS422).
BRIEF DESCRIPTION OF THE FIGURES
[0272] FIG. 1: Sequence alignment of rabbit and humanized light
chain variable domains; CDRs are enboxed.
[0273] FIG. 2: Sequence alignment of rabbit and humanized heavy
chain variable domains; CDRs are enboxed.
[0274] FIG. 3: Biochemical binding of different combinations of
humanized VH and VL to (A) phosphorylated tau peptide, (B)
phosphorylated full-length human tau, (C) not-phosphorylated tau
peptide, (D) not-phosphorylated full-length human tau;
(1)=VH00/VL00, (2)=VH32/VL21, (3)=VH20/VL22, (4)=VH32/VL22,
(5)=VH33/VL22; coating concentrations: phosphorylated tau peptide:
50 ng/ml, all other targets: 1 .mu.g/ml; (comparable results are
obtained if phosphorylated tau peptide is coated with 1 .mu.g/ml
(data not shown)).
[0275] FIG. 4: Biochemical binding of different combinations of
humanized VH and VL to (A)=full length human tau S422A mutant,
(B)=aggregated human Tau(pS422); (1)=VH00/VL00, (2)=VH32/VL21,
(3)=VH20/VL22, (4)=VH32/VL22, (5)=VH33/VL22; coating
concentrations: phosphorylated tau peptide: 50 ng/ml, all other
targets: 1 .mu.g/ml; (comparable results are obtained if
phosphorylated tau peptide is coated with 1 .mu.g/ml (data not
shown)).
[0276] FIG. 5: Western Blot showing the selectivity of selected
humanized VH/VL combinations; (1)=VH00/VL00, (2)=VH32/VL21,
(3)=VH20/VL22, (4)=VH32/VL22, (5)=VH33/VL22.
[0277] FIG. 6: Binding to hyperphosphorylated tau in brain extracts
of Alzheimer's disease patients; (1)=VH00/VL00, (2)=VH32/VL21,
(3)=VH32/VL22.
BRIEF DESCRIPTION OF THE SEQUENCES
[0278] SEQ ID NO: 01 human Tau protein isoform F (441 residues)
[0279] SEQ ID NO: 02 human Tau protein isoform F (441 residues)
phosphorylated at the serine residue at position 422 [0280] SEQ ID
NO: 03 fragment of human Tau protein (residues 416 to 430 of SEQ ID
NO: 01) with phosphorylated serine at position 7 (corresponding to
position 422 of SEQ ID NO: 01):
Ser-Ile-Asp-Met-Val-Asp-Ser(PO.sub.3H.sub.2)-Pro-Gln-Leu-Ala-Thr-Leu-Ala--
Asp [0281] SEQ ID NO: 04 rabbit antibody 086 CDRL1-QSSQSVRTNKLA
[0282] SEQ ID NO: 05 rabbit antibody 086 CDRL2-SASTLDF [0283] SEQ
ID NO: 06 rabbit antibody 086 CDRL3-LGYFDCSIADCVA [0284] SEQ ID NO:
07 rabbit antibody 086 VL00 [0285] SEQ ID NO: 08 rabbit antibody
086 CDRH1-SNAIN [0286] SEQ ID NO: 09 rabbit antibody 086
CDRH2-YIAVSGNTYYASWAKG [0287] SEQ ID NO: 10 rabbit antibody 086
CDRH3-SNI [0288] SEQ ID NO: 11 rabbit antibody 086 VH00 [0289] SEQ
ID NO: 12 humanized CDRL1 variant 1-RSSQSVRTNKLA [0290] SEQ ID NO:
13 humanized CDRL1 variant 2-RSSQSVRTNRLA [0291] SEQ ID NO: 14
humanized CDRL2 variant 1-SASTLDY [0292] SEQ ID NO: 15 humanized
CDRL3 variant 1-LGYFDSSADIVA [0293] SEQ ID NO: 16 humanized VL
variant 1-VL21 [0294] SEQ ID NO: 17 humanized VL variant 2 VL22
[0295] SEQ ID NO: 18 humanized CDRH2-YIAVSGNTYYADSVKG [0296] SEQ ID
NO: 19 humanized VH variant 1-VH32 [0297] SEQ ID NO: 20 humanized
VH variant 2-VH20 [0298] SEQ ID NO: 21 humanized VH variant 3-VH33
[0299] SEQ ID NO: 22 humanized CDRL2 variant 2-SASTLQS [0300] SEQ
ID NO: 23 humanized CDRL2 variant 3-SASTLES [0301] SEQ ID NO: 24
humanized CDRL3 variant 2-LGYFDSSIADSVA [0302] SEQ ID NO: 25
humanized CDRL3 variant 3-LGYFDSSIADRVA [0303] SEQ ID NO: 26
humanized CDRL3 variant 4-LGYFDPSIADPVA [0304] SEQ ID NO: 27
humanized CDRL3 variant 5-LGYFDSSIADIVA [0305] SEQ ID NO: 28
humanized CDRL3 variant 6-LGYFDPSADPIA [0306] SEQ ID NO: 29
humanized CDRL3 variant 7-LGYFDPSADPVA [0307] SEQ ID NO: 30
humanized CDRL1 variant 3-RASQGVRTNKLA [0308] SEQ ID NO: 31
humanized CDRL1 variant 4-RASQSVRTNKLA [0309] SEQ ID NO: 32
humanized VL variant 4-VL01 [0310] SEQ ID NO: 33 humanized VL
variant 5-VL09 [0311] SEQ ID NO: 34 humanized VL variant 6-VL12
[0312] SEQ ID NO: 35 humanized VL variant 7-VL15 [0313] SEQ ID NO:
36 humanized VL variant 8-VL16 [0314] SEQ ID NO: 37 humanized VL
variant 9-VL17 [0315] SEQ ID NO: 38 humanized VL variant 10-VL19
[0316] SEQ ID NO: 39 humanized VL variant 11-VL28 [0317] SEQ ID NO:
40 humanized VL variant 12-VL33 [0318] SEQ ID NO: 41 humanized VL
variant 13-VL35 [0319] SEQ ID NO: 42 humanized VL variant 14-VL39
[0320] SEQ ID NO: 43 humanized VL variant 15-VL40 [0321] SEQ ID NO:
44 humanized VL variant 16-VL41 [0322] SEQ ID NO: 45 humanized VL
variant 17-VL42 [0323] SEQ ID NO: 46 humanized VH variant 4-VH01
[0324] SEQ ID NO: 47 humanized VH variant 5-VH02 [0325] SEQ ID NO:
48 humanized VH variant 6-VH03 [0326] SEQ ID NO: 49 humanized VH
variant 7-VH04 [0327] SEQ ID NO: 50 humanized VH variant 8-VH14
[0328] SEQ ID NO: 51 humanized VH variant 9-VH15 [0329] SEQ ID NO:
52 humanized VH variant 10-VH18 [0330] SEQ ID NO: 53 humanized VH
variant 11-VH19 [0331] SEQ ID NO: 54 humanized VH variant 12 VH22
[0332] SEQ ID NO: 55 humanized VH variant 13-VH23 [0333] SEQ ID NO:
56 humanized VH variant 14-VH24 [0334] SEQ ID NO: 57 humanized VH
variant 15-VH31
TABLE-US-00001 [0334] Sequence correspondence table: variable
domain CDR1 CDR2 CDR3 complete sequence VL00 04 05 06 07 VL01 04 05
06 32 VL09 31 23 06 33 VL12 30 22 06 34 VL15 30 22 24 35 VL16 30 22
25 36 VL17 12 05 06 37 VL19 12 05 06 38 VL21 12 05 15 16 VL22 13 14
15 17 VL28 13 05 29 39 VL33 13 05 27 40 VL35 13 05 27 41 VL39 13 05
26 42 VL40 13 05 29 43 VL41 13 05 28 44 VL42 13 05 28 45 VH00 08 09
10 11 VH01 08 09 10 46 VH02 08 09 10 47 VH03 08 09 10 48 VH04 08 09
10 49 VH14 08 09 10 50 VH15 08 09 10 51 VH18 08 18 10 52 VH19 08 18
10 53 VH20 08 18 10 20 VH22 08 18 10 54 VH23 08 18 10 55 VH24 08 18
10 56 VH31 08 09 10 57 VH32 08 09 10 19 VH33 08 09 10 21
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
I. DEFINITIONS
[0335] An "acceptor human framework" for the purposes herein is a
framework comprising the amino acid sequence of a light chain
variable domain (VL) framework or a heavy chain variable domain
(VH) framework derived from a human immunoglobulin framework or a
human consensus framework, as defined below. An acceptor human
framework "derived from" a human immunoglobulin framework or a
human consensus framework may comprise the same amino acid sequence
thereof, or it may contain amino acid sequence changes. In some
embodiments, the number of amino acid changes are 10 or less, 9 or
less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or
less, or 2 or less. In some embodiments, the VL acceptor human
framework is identical in sequence to the VL human immunoglobulin
framework sequence or human consensus framework sequence.
[0336] "Affinity" refers to the strength of the sum total of
non-covalent interactions between a single binding site of a
molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (kd).
Affinity can be measured by common methods known in the art,
including those described herein. Specific illustrative and
exemplary embodiments for measuring binding affinity are described
in the following.
[0337] An "affinity matured" antibody refers to an antibody with
one or more alterations in one or more hypervariable regions
(HVRs), compared to a parent antibody which does not possess such
alterations, such alterations resulting in an improvement in the
affinity of the antibody for antigen.
[0338] The terms "anti-human Tau(pS422) antibody" and "an antibody
that binds to human Tau(pS422)" refer to an antibody that is
capable of binding human Tau(pS422) with sufficient affinity such
that the antibody is useful as a diagnostic and/or therapeutic
agent in targeting human Tau(pS422). In one embodiment, the extent
of binding of an anti-human Tau(pS422) antibody to an unrelated,
non-human Tau(pS422) protein is less than about 10% of the binding
of the antibody to human Tau(pS422) as measured, e.g., by a
radioimmunoassay (RIA).
[0339] The term "antibody" herein is used in the broadest sense and
encompasses various antibody structures, including but not limited
to monoclonal antibodies, polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), and antibody fragments so
long as they exhibit the desired antigen-binding activity.
[0340] An "antibody fragment" refers to a molecule other than an
intact antibody that comprises a portion of an intact antibody that
binds the antigen to which the intact antibody binds. Examples of
antibody fragments include but are not limited to Fv, Fab, Fab',
Fab'-SH, F(ab').sub.2; diabodies; linear antibodies; single-chain
antibody molecules (e.g. scFv); and multispecific antibodies formed
from antibody fragments.
[0341] An "antibody that binds to the same epitope" as a reference
antibody refers to an antibody that blocks binding of the reference
antibody to its antigen in a competition assay by 50% or more, and
conversely, the reference antibody blocks binding of the antibody
to its antigen in a competition assay by 50% or more. In one
embodiment an antibody binding to the same epitope as a reference
antibody blocks binding of the reference antibody to its antigen by
50% or more. In one embodiment an antibody binding to the same
epitope as a reference antibody blocks binding of the reference
antibody to its antigen by 80% or more. In one embodiment an
antibody binding to the same epitope as a reference antibody blocks
binding of the reference antibody to its antigen by 90% or more. In
one embodiment an antibody binding to the same epitope as a
reference antibody blocks binding of the reference antibody to its
antigen by 95% or more. In one preferred embodiment an antibody
binding to the same epitope as a reference antibody has binding
interactions with the same residues as the reference antibody on
the antigen.
[0342] The term "chimeric" antibody refers to an antibody in which
a portion of the heavy and/or light chain is derived from a
particular source or species, while the remainder of the heavy
and/or light chain is derived from a different source or
species.
[0343] The "class" of an antibody refers to the type of constant
domain or constant region possessed by its heavy chain. There are
five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and
several of these may be further divided into subclasses (isotypes),
e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, and
IgA.sub.2. The heavy chain constant domains that correspond to the
different classes of immunoglobulins are called .alpha., .delta.,
.epsilon., .gamma., and .mu., respectively.
[0344] "Effector functions" refer to those biological activities
attributable to the Fc-region of an antibody, which vary with the
antibody class. Examples of antibody effector functions include:
C1q binding and complement dependent cytotoxicity (CDC); Fc
receptor binding; antibody-dependent cell-mediated cytotoxicity
(ADCC); phagocytosis; down regulation of cell surface receptors
(e.g. B cell receptor); and B cell activation.
[0345] An "effective amount" of an agent, e.g., a pharmaceutical
formulation, refers to an amount effective, at dosages and for
periods of time necessary, to achieve the desired therapeutic or
prophylactic result.
[0346] The term "Fc-region" herein is used to define a C-terminal
region of an immunoglobulin heavy chain that contains at least a
portion of the constant region. The term includes native sequence
Fc-regions and variant Fc-regions. In one embodiment, a human IgG
heavy chain Fc-region extends from Cys226, or from Pro230, to the
carboxyl-terminus of the heavy chain. However, the C-terminal
lysine (Lys447) of the Fc-region may or may not be present. Unless
otherwise specified herein, numbering of amino acid residues in the
Fc-region or constant region is according to the EU numbering
system, also called the EU index, as described in Kabat, E. A. et
al., Sequences of Proteins of Immunological Interest, 5th ed.,
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991), NIH Publication 91-3242.
[0347] "Framework" or "FR" refers to variable domain residues other
than hypervariable region (HVR) residues. The FR of a variable
domain generally consists of four FR domains: FR1, FR2, FR3, and
FR4. Accordingly, the HVR and FR sequences generally appear in the
following sequence in VH (or VL):
FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
[0348] The terms "full length antibody", "intact antibody", and
"whole antibody" are used herein interchangeably to refer to an
antibody having a structure substantially similar to a native
antibody structure or having heavy chains that contain an Fc-region
as defined herein. The term "full length antibody" denotes a
multimeric polypeptide consisting of two antibody light chain
polypeptides and two antibody heavy chain polypeptides linked by
disulfide bonds wherein in the two antibody heavy chain
polypeptides the C-terminal lysine residue (K) can be present or
not.
[0349] The terms "host cell", "host cell line", and "host cell
culture" are used interchangeably and refer to cells into which
exogenous nucleic acid has been introduced, including the progeny
of such cells. Host cells include "transformants" and "transformed
cells," which include the primary transformed cell and progeny
derived therefrom without regard to the number of passages. Progeny
may not be completely identical in nucleic acid content to a parent
cell, but may contain mutations. Mutant progeny that have the same
function or biological activity as screened or selected for in the
originally transformed cell are included herein.
[0350] A "human consensus framework" is a framework which
represents the most commonly occurring amino acid residues in a
selection of human immunoglobulin VL or VH framework sequences.
Generally, the selection of human immunoglobulin VL or VH sequences
is from a subgroup of variable domain sequences. Generally, the
subgroup of sequences is a subgroup as in Kabat, E. A. et al.,
Sequences of Proteins of Immunological Interest, 5th ed., Bethesda
Md. (1991), NIH Publication 91-3242, Vols. 1-3. In one embodiment,
for the VL, the subgroup is subgroup kappa I as in Kabat et al.,
supra. In one embodiment, for the VH, the subgroup is subgroup III
as in Kabat et al., supra.
[0351] A "humanized" antibody refers to a chimeric antibody
comprising amino acid residues from non-human HVRs and amino acid
residues from human FRs. In certain embodiments, a humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the HVRs (e.g., CDRs) correspond to those of a non-human
antibody, and all or substantially all of the FRs correspond to
those of a human antibody. A humanized antibody optionally may
comprise at least a portion of an antibody constant region derived
from a human antibody. A "humanized form" of an antibody, e.g., a
non-human antibody, refers to an antibody that has undergone
humanization.
[0352] The term "hypervariable region" or "HVR", as used herein,
refers to each of the regions of an antibody variable domain which
are hypervariable in sequence ("complementarity determining
regions" or "CDRs") and form structurally defined loops
("hypervariable loops"), and/or contain the antigen-contacting
residues ("antigen contacts"). Generally, antibodies comprise six
HVRs; three in the VH (H1, H2, H3), and three in the VL (L1, L2,
L3).
[0353] HVRs herein include [0354] (a) hypervariable loops occurring
at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32
(H1), 53-55 (H2), and 96-101 (H3) (Chothia, C. and Lesk, A. M., J.
Mol. Biol. 196 (1987) 901-917); [0355] (b) CDRs occurring at amino
acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (HI),
50-65 (H2), and 95-102 (H3) (Kabat, E. A. et al., Sequences of
Proteins of Immunological Interest, 5th ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991), NIH
Publication 91-3242); [0356] (c) antigen contacts occurring at
amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b
(H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol.
262: 732-745 (1996)); and [0357] (d) combinations of (a), (b),
and/or (c), including HVR amino acid residues 46-56 (L2), 47-56
(L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2),
93-102 (H3), and 94-102 (H3).
[0358] Unless otherwise indicated, HVR residues and other residues
in the variable domain (e.g., FR residues) are numbered herein
according to Kabat et al., supra.
[0359] An "immunoconjugate" is an antibody conjugated to one or
more heterologous molecule(s).
[0360] An "individual" or "subject" is a mammal. Mammals include,
but are not limited to, domesticated animals (e.g. cows, sheep,
cats, dogs, and horses), primates (e.g., humans and non-human
primates such as monkeys), rabbits, and rodents (e.g., mice and
rats). In certain embodiments, the individual or subject is a
human.
[0361] An "isolated" antibody is one which has been separated from
a component of its natural environment. In some embodiments, an
antibody is purified to greater than 95% or 99% purity as
determined by, for example, electrophoretic (e.g., SDS-PAGE,
isoelectric focusing (IEF), capillary electrophoresis) or
chromatographic (e.g., ion exchange or reverse phase HPLC). For
review of methods for assessment of antibody purity, see, e.g.,
Flatman, S. et al., J. Chromatogr. B 848 (2007) 79-87.
[0362] An "isolated" nucleic acid refers to a nucleic acid molecule
that has been separated from a component of its natural
environment. An isolated nucleic acid includes a nucleic acid
molecule contained in cells that ordinarily contain the nucleic
acid molecule, but the nucleic acid molecule is present
extrachromosomally or at a chromosomal location that is different
from its natural chromosomal location.
[0363] "Isolated nucleic acid encoding an anti-human Tau(pS422)
antibody" refers to one or more nucleic acid molecules encoding
antibody heavy and light chains (or fragments thereof), including
such nucleic acid molecule(s) in a single vector or separate
vectors, and such nucleic acid molecule(s) present at one or more
locations in a host cell.
[0364] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical and/or bind the same epitope, except for
possible variant antibodies, e.g., containing naturally occurring
mutations or arising during production of a monoclonal antibody
preparation, such variants generally being present in minor
amounts. In contrast to polyclonal antibody preparations, which
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody of a monoclonal
antibody preparation is directed against a single determinant on an
antigen. Thus, the modifier "monoclonal" indicates the character of
the antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by a variety of techniques, including but not
limited to the hybridoma method, recombinant DNA methods,
phage-display methods, and methods utilizing transgenic animals
containing all or part of the human immunoglobulin loci, such
methods and other exemplary methods for making monoclonal
antibodies being described herein.
[0365] "Native antibodies" refer to naturally occurring
immunoglobulin molecules with varying structures. For example,
native IgG antibodies are heterotetrameric glycoproteins of about
150,000 daltons, composed of two identical light chains and two
identical heavy chains that are disulfide-bonded. From N- to
C-terminus, each heavy chain has a variable region (VH), also
called a variable heavy domain or a heavy chain variable domain,
followed by three constant domains (CH1, CH2, and CH3). Similarly,
from N- to C-terminus, each light chain has a variable region (VL),
also called a variable light domain or a light chain variable
domain, followed by a constant light (CL) domain. The light chain
of an antibody may be assigned to one of two types, called kappa
(.kappa.) and lambda (.lamda.), based on the amino acid sequence of
its constant domain.
[0366] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, combination therapy, contraindications
and/or warnings concerning the use of such therapeutic
products.
[0367] "Percent (%) amino acid sequence identity" with respect to a
reference polypeptide sequence is defined as the percentage of
amino acid residues in a candidate sequence that are identical with
the amino acid residues in the reference polypeptide sequence,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)
software. Those skilled in the art can determine appropriate
parameters for aligning sequences, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared. For purposes herein, however, % amino acid sequence
identity values are generated using the sequence comparison
computer program ALIGN-2. The ALIGN-2 sequence comparison computer
program was authored by Genentech, Inc., and the source code has
been filed with user documentation in the U.S. Copyright Office,
Washington D.C., 20559, where it is registered under U.S. Copyright
Registration No. TXU510087. The ALIGN-2 program is publicly
available from Genentech, Inc., South San Francisco, Calif., or may
be compiled from the source code. The ALIGN-2 program should be
compiled for use on a UNIX operating system, including digital UNIX
V4.0D. All sequence comparison parameters are set by the ALIGN-2
program and do not vary.
[0368] In situations where ALIGN-2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical
matches by the sequence alignment program ALIGN-2 in that program's
alignment of A and B, and where Y is the total number of amino acid
residues in B. It will be appreciated that where the length of
amino acid sequence A is not equal to the length of amino acid
sequence B, the % amino acid sequence identity of A to B will not
equal the % amino acid sequence identity of B to A. Unless
specifically stated otherwise, all % amino acid sequence identity
values used herein are obtained as described in the immediately
preceding paragraph using the ALIGN-2 computer program.
[0369] The term "pharmaceutical formulation" refers to a
preparation which is in such form as to permit the biological
activity of an active ingredient contained therein to be effective,
and which contains no additional components which are unacceptably
toxic to a subject to which the formulation would be
administered.
[0370] A "pharmaceutically acceptable carrier" refers to an
ingredient in a pharmaceutical formulation, other than an active
ingredient, which is nontoxic to a subject. A pharmaceutically
acceptable carrier includes, but is not limited to, a buffer,
excipient, stabilizer, or preservative.
[0371] The term "human Tau(pS422)", as used herein, refers to
native human Tau(pS422) (UniProt P37840). The term encompasses
"full-length", unprocessed human Tau(pS422) as well as any form of
human Tau(pS422) that results from processing in the cell. The term
also encompasses naturally occurring variants of human Tau(pS422),
e.g., mutants, splice variants or allelic variants. The amino acid
sequence of human Tau(pS422) is shown in SEQ ID NO: 02.
[0372] As used herein, "treatment" (and grammatical variations
thereof such as "treat" or "treating") refers to clinical
intervention in an attempt to alter the natural course of the
individual being treated, and can be performed either for
prophylaxis or during the course of clinical pathology. Desirable
effects of treatment include, but are not limited to, preventing
occurrence or recurrence of disease, alleviation of symptoms,
diminishment of any direct or indirect pathological consequences of
the disease, preventing metastasis, decreasing the rate of disease
progression, amelioration or palliation of the disease state, and
remission or improved prognosis. In some embodiments, antibodies of
the invention are used to delay development of a disease or to slow
the progression of a disease.
[0373] The term "variable region" or "variable domain" refers to
the domain of an antibody heavy or light chain that is involved in
binding the antibody to antigen. The variable domains of the heavy
chain and light chain (VH and VL, respectively) of a native
antibody generally have similar structures, with each domain
comprising four conserved framework regions (FRs) and three
hypervariable regions (HVRs). (See, e.g., Kindt, T. J. et al. Kuby
Immunology, 6th ed., W.H. Freeman and Co., N.Y. (2007), page 91) A
single VH or VL domain may be sufficient to confer antigen-binding
specificity. Furthermore, antibodies that bind a particular antigen
may be isolated using a VH or VL domain from an antibody that binds
the antigen to screen a library of complementary VL or VH domains,
respectively. See, e.g., Portolano, S. et al., J. Immunol. 150
(1993) 880-887; Clackson, T. et al., Nature 352 (1991)
624-628).
[0374] The term "vector", as used herein, refers to a nucleic acid
molecule capable of propagating another nucleic acid to which it is
linked. The term includes the vector as a self-replicating nucleic
acid structure as well as the vector incorporated into the genome
of a host cell into which it has been introduced. Certain vectors
are capable of directing the expression of nucleic acids to which
they are operatively linked. Such vectors are referred to herein as
"expression vectors".
II. COMPOSITIONS AND METHODS
[0375] A. Exemplary Humanized Anti-Human Tau(pS422) Antibodies
[0376] The humanized antibodies as reported herein were not
available by standard humanization methods. It was required to
introduce non-standard mutations in the amino acid sequence in
order to obtain a humanized antibody with comparable binding
characteristics as the parent rabbit antibody. This is especially
important as the antibodies as reported herein are intended to
cross the human blood-brain-barrier and to be effective within the
human brain. Thus, the generally applied criteria for the selection
of humanized antibodies are not sufficiently stringent in order to
be applied directly in the current case.
[0377] It has been found that in order to obtain a suitable and
developable humanized antibody two cysteines forming a
disulfide-bridge in the CDRL3 (light chain CDR3) had to be replaced
by serine and isoleucine, respectively. In addition to ensure
proper orientation of the same CDRL3 an isoleucine residue present
in the middle of the rabbit CDRL3 was deleted resulting in a
humanized CDRL3 that is one amino acid residue smaller than the
parent rabbit CDRL3.
[0378] Is has further been found that it is advantageous to
maintain three valine amino acid residues in the heavy chain at
positions 4, 24 and 78. Without being bound by this theory it is
assumed that these residues are required to ensure proper
presentation of the antigen binding loops of the heavy chain
variable region. Additionally the presence of an arginine residue
at position 71 is advantageous.
[0379] A sequence alignment of different humanized light chain
variable domains is shown in FIG. 1. A sequence alignment of
different humanized heavy chain variable domains is shown in FIG.
2. All numbering as used herein is based on the Kabat variable
domain numbering scheme.
[0380] In the following Table characteristics of the different
humanized variants of the rabbit light chain variable domain in
combination with the humanized heavy chain variable domains VH14
and VH20, respectively, are shown. Binding partner was human
Tau(pS422).
TABLE-US-00002 t/2 diss ka [1/Ms] kd [1/s] KD [M] [min] T [.degree.
C.] VH14 with VL00 1.04E-03 11 25 VL01 3.82E-03 3 25 VL09 2.35E-03.
5 25 VL12 2.48E-03 5 25 VL15 3.63E-03 3 25 VL16 n.d. VL17 2.39E-03
5 25 VL17 3.03E-03 4 25 VL19 1.98E-03 6 25 VL21 2.93E-03 4 25 VL22
3.30E-03 4 25 VL28 3.84E-03 3 25 VL33 1.02E-02 1 25 VL35 1.10E-02 1
25 VL39 5.22E-03 2 25 VL40 3.01E-03 4 25 VL41 n.d. VL42 n.d. VH20
with VL00 n.d. VL01 n.d. VL09 2.14E-03 5 25 VL12 n.d. VL15 n.d.
VL16 n.d. VL17 5.35E-04 22 25 VL19 3.66E-04 32 25 VL19 1.94E+04
1.13E-03 5.84E-8 10.2 37 VL21 7.88E-04 15 25 VL21 3.03E+04 2.10E-03
6.95E-08 5.5 37 VL22 8.39E-04 14 25 VL22 3.44E+04 2.37E-03 6.90E-08
4.9 37 VL28 1.27E-03 9 25 VL28 2.50E+04 3.61E-03 1.45E-07 3.2 37
VL33 1.61E-03 7 25 VL35 1.59E-03 7 25 VL39 1.91E-03 6 25 VL40
9.98E-04 12 25 VL41 4.29E-03 3 25 VL42 4.57E-03 3 25 Reference
values VH00 with VL00 (rabbit antibody): 25.degree. C.: kd =
2.6E-04; t/2 = 44 min. 37.degree. C.: ka = 3.7E+04, kd = 5.25E-03,
KD = 1.4E-08, t/2 = 22 min.
[0381] In the following Table characteristics of the different
humanized variants of the rabbit light chain variable domain in
combination with the humanized light chain variable domains VL17
and VL19, respectively, are shown.
TABLE-US-00003 ka [1/Ms] kd[1/s] KD [M] t/2 diss [min] T [.degree.
C.] VL17 with VH00 4.98E-04 23 25 VH01 2.3E-03 5 25 VH02 3.71E-03 3
25 VH03 3.93E-03 3 25 VH04 4.16E-03 3 25 VH14 3.0E-03 4 25 VH15
3.26E-03 4 25 VH18 2.3E-03 5 25 VH19 n.d. VH20 5.4E-04 22 25 VH22
2.0E-03 6 25 VH23 7.0E-04 17 25 VH24 7.9E-04 15 25 VH31 n.d. VH32
n.d. VH33 n.d. VL19 with VH00 n.d. VH01 1.9E-03 6 25 VH02 n.d. VH03
n.d. VH04 n.d. VH14 2.0E-03 6 25 VH15 n.d. VH18 1.9E-03 6 25 VH19
2.0E-03 6 25 VH20 3.7E-04 32 25 VH20 1.94E+04 1.13E-03 5.84E-08
10.2 37 VH22 2.1E-03 6 25 VH23 5.7E-04 20 25 VH24 6.3E-04 18 25
VH31 n.d. VH32 n.d. VH33 n.d. Reference values VH00 with VL00
(rabbit antibody): 25.degree. C.: kd = 2.6E-04; t/2 = 44 min.
37.degree. C.: ka = 3.7E+04, kd = 5.25E-03, KD = 1.4E-08, t/2 = 22
min.
[0382] In the following Table the kinetic constants for different
VH/VL combinations are shown.
TABLE-US-00004 KD t/2diss KD t/2diss VH/VL 25.degree. C. 25.degree.
C. 37.degree. C. 37.degree. C. combination [nM] [min] MR [nM] [min]
MR VH00/VL00 8 54 0.6 12 24 0.8 VH20/VL22 37 16 0.4 68 5 0.5
VH32/VL21 18 26 0.5 32 9 0.6 VH32/VL22 14 29 0.5 31 8 0.6 VH33/VL22
20 25 0.4 39 8 0.5
[0383] The biochemical binding of different combinations of
humanized VH and VL is shown in FIGS. 3 and 4.
[0384] In the following Table the binding specificity for different
VH/VL combinations are shown (EC50 values in [ng/ml]).
TABLE-US-00005 tau peptide S422A Tau(pS422) full length full
residues tau mutant fragment Tau(pS422) length tau 416 to 430
microtubuli S422A VH/VL SEQ ID SEQ ID Tau(pS422) SEQ ID of SEQ ID
associated SEQ ID combination NO: 03 NO: 02 aggregates NO: 01 NO:
01 tau NO: 01 VH00/VL00 6.3 5.2 18.1 no binding >1000 no binding
47.9 VH20/VL22 4.8 4.0 27.2 no binding >1000 no binding 110.6
VH32/VL21 4.4 2.9 9.4 no binding 634 no binding 21.5 VH32/VL22 5.6
3.5 8.3 no binding 48 no binding 17.4 VH33/VL22 5.6 3.8 13.5 no
binding 120 no binding 34.5
[0385] The sensitivity of selected humanized VH/VL combinations to
the human tau mutant S422A can be seen from the Western Blots shown
in FIG. 5. All humanized variants selectively bind to human tau
phosphorylated at S422. There is low level x-reactivity to non-S422
phosphoepitopes of the parent rabbit antibody but the humanized
variants shown are less cross-reactive in this respect than the
parental rabbit antibody.
[0386] In FIG. 6 the binding to PHF-tau in brain extracts of
Alzheimer's disease patients for the parental rabbit antibody and
for selected humanized anti-human Tau(pS422) antibodies is
shown.
[0387] The following table summarizes the biological properties of
selected humanized VH/VL combinations.
TABLE-US-00006 VH/VL ELISA combination BIAcore potency specificity
WB IHC in vivo developability VH20/VL22 4 + ++ 4 3 + ++ VH32/VL21 2
+(+) + 2 3 + ++ VH32/VL22 1 +(+) + 1 1 + ++ VH33/VL22 3 + + 3 2 +
++
[0388] In one aspect, the invention provides a (humanized)
anti-human Tau(pS422) antibody comprising at least one, or two, or
three, or four, or five, or six HVRs selected from (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO: 08; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO: 18; (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO: 10; (d) HVR-L1
comprising the amino acid sequence of SEQ ID NO: 13; (e) HVR-L2
comprising the amino acid sequence of SEQ ID NO: 14; and (f) HVR-L3
comprising the amino acid sequence of SEQ ID NO: 15.
[0389] In one aspect, the invention provides a (humanized)
anti-human Tau(pS422) antibody comprising at least one, or two, or
three, or four, or five, or six HVRs selected from (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO: 08; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO: 09; (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO: 10; (d) HVR-L1
comprising the amino acid sequence of SEQ ID NO: 12; (e) HVR-L2
comprising the amino acid sequence of SEQ ID NO: 05; and (f) HVR-L3
comprising the amino acid sequence of SEQ ID NO: 15.
[0390] In one aspect, the invention provides a (humanized)
anti-human Tau(pS422) antibody comprising at least one, or two, or
three, or four, or five, or six HVRs selected from (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO: 08; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO: 09; (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO: 10; (d) HVR-L1
comprising the amino acid sequence of SEQ ID NO: 13; (e) HVR-L2
comprising the amino acid sequence of SEQ ID NO: 14; and (f) HVR-L3
comprising the amino acid sequence of SEQ ID NO: 15.
[0391] In one aspect, the invention provides a (humanized) antibody
comprising at least one, at least two, or all three VH HVR
sequences selected from [0392] i) (a) HVR-H1 comprising the amino
acid sequence of SEQ ID NO: 08; (b) HVR-H2 comprising the amino
acid sequence of SEQ ID NO: 18; and (c) HVR-H3 comprising the amino
acid sequence of SEQ ID NO: 10; or [0393] ii) (a) HVR-H1 comprising
the amino acid sequence of SEQ ID NO: 08; (b) HVR-H2 comprising the
amino acid sequence of SEQ ID NO: 09; and (c) HVR-H3 comprising the
amino acid sequence of SEQ ID NO: 10.
[0394] In one embodiment, the antibody comprises [0395] i) (a)
HVR-H1 comprising the amino acid sequence of SEQ ID NO: 08; (b)
HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; and (c)
HVR-H3 comprising the amino acid sequence of SEQ ID NO: 10; or
[0396] ii) (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO: 08; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:
09; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:
10.
[0397] In another embodiment the antibody further comprises at
least one, at least two, or all three VL HVR sequences selected
from [0398] i) (a) HVR-L1 comprising the amino acid sequence of SEQ
ID NO: 13; (b) HVR-L2 comprising the amino acid sequence of SEQ ID
NO: 14; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID
NO: 15; or [0399] ii) (a) HVR-L1 comprising the amino acid sequence
of SEQ ID NO: 12; (h) HVR-L2 comprising the amino acid sequence of
SEQ ID NO: 05; and (c) HVR-L3 comprising the amino acid sequence of
SEQ ID NO: 15.
[0400] In a further embodiment, the antibody comprises [0401] i)
(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (b)
HVR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and (c)
HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15; or
[0402] ii) (a) HVR-L1 comprising the amino acid sequence of SEQ ID
NO: 12; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:
05; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:
15.
[0403] In one aspect, the invention provides a (humanized) antibody
comprising [0404] i) (a) HVR-HI comprising the amino acid sequence
of SEQ ID NO: 08; (b) HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 18; (c) HVR-H3 comprising the amino acid sequence of SEQ
ID NO: 10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO: 13; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:
14; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:
15, or [0405] ii) (a) HVR-H1 comprising the amino acid sequence of
SEQ ID NO: 08; (b) HVR-H2 comprising the amino acid sequence of SEQ
ID NO: 09; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO: 10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:
12; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 05;
and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15,
or [0406] iii) (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO: 08; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO: 09; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:
10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 13;
(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and
(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15.
[0407] In another embodiment the VH or VL contains substitutions
(e.g. conservative substitutions), insertions, or deletions
relative to the reference sequence, but an anti-human Tau(pS422)
antibody comprising that sequence retains the ability to bind to
human Tau(pS422).
[0408] In a further aspect of the invention, an anti-human
Tau(pS422) antibody according to any of the above embodiments is a
monoclonal antibody, including a chimeric, humanized or human
antibody. In one embodiment, an anti-human Tau(pS422) antibody is
an antibody fragment, e.g., a Fv, Fab, Fab', scFv, diabody, or
F(ab').sub.2 fragment. In another embodiment, the antibody is a
full length antibody, e.g., an intact IgG1 or IgG 4 antibody or
other antibody class or isotype as defined herein.
[0409] The (humanized) antibody as reported herein reduces
Tau(pS422) levels in the brain of transgenic TauPS2APP mice.
[0410] In a further aspect, a (humanized) anti-human Tau(pS422)
antibody according to any of the above embodiments may incorporate
any of the features, singly or in combination, as described in
Sections 1-7 below:
[0411] 1. Antibody Affinity
[0412] In certain embodiments, an antibody provided herein has a
dissociation constant (KD) of .ltoreq.100 nM, .ltoreq.50 nM, or
between 1 nM and 100 nM (e.g. 10.sup.-7M or less, e.g. from
10.sup.-7M to 10.sup.-9M).
[0413] In one embodiment, Kd is measured by a radiolabeled antigen
binding assay (RIA). In one embodiment, an MA is performed with the
Fab version of an antibody of interest and its antigen. For
example, solution binding affinity of FABs for antigen is measured
by equilibrating Fab with a minimal concentration of
(.sup.125I)-labeled antigen in the presence of a titration series
of unlabeled antigen, then capturing bound antigen with an anti-Fab
antibody-coated plate (see, e.g., Chen, Y. et al., J. Mol. Biol.
293 (1999) 865-881). To establish conditions for the assay,
MICROTITER.RTM. multi-well plates (Thermo Scientific) are coated
overnight with 5 .mu.g/ml of a capturing anti-Fab antibody (Cappel
Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked
with 2% (w/v) bovine serum albumin in PBS for two to five hours at
room temperature (approximately 23.degree. C.). In a non-adsorbent
plate (Nunc #269620), 100 pM or 26 pM [.sup.125I]-antigen are mixed
with serial dilutions of a Fab of interest (e.g., consistent with
assessment of the anti-VEGF antibody, Fab-12, in Presta, L. G. et
al., Cancer Res. 57 (1997) 4593-4599). The Fab of interest is then
incubated overnight; however, the incubation may continue for a
longer period (e.g., about 65 hours) to ensure that equilibrium is
reached. Thereafter, the mixtures are transferred to the capture
plate for incubation at room temperature (e.g., for one hour). The
solution is then removed and the plate washed eight times with 0.1%
polysorbate 20 (TWEEN-20.RTM.) in PBS. When the plates have dried,
150 .mu.l/well of scintillant (MICROSCINT-20.TM.; Packard) is
added, and the plates are counted on a TOPCOUNT.TM. gamma counter
(Packard) for ten minutes. Concentrations of each Fab that give
less than or equal to 20% of maximal binding are chosen for use in
competitive binding assays.
[0414] According to another embodiment, Kd is measured using a
BIACORE.RTM. surface plasmon resonance assay. For example, an assay
using a BIACORE.RTM.-2000 or a BIACORE.RTM.-3000 (BIAcore, Inc.,
Piscataway, N.J.) is performed at 25.degree. C. with immobilized
antigen CMS chips at .about.10 response units (RU). In one
embodiment, carboxymethylated dextran biosensor chips (CM5,
BIACORE, Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8,
to 5 .mu.g/ml (.about.0.2 .mu.M) before injection at a flow rate of
5 .mu.l/minute to achieve approximately 10 response units (RU) of
coupled protein. Following the injection of antigen, 1 M
ethanolamine is injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM)
are injected in PBS with 0.05% polysorbate 20 (TWEEN-20.TM.)
surfactant (PBST) at 25.degree. C. at a flow rate of approximately
25 .mu.l/min. Association rates (k.sub.on) and dissociation rates
(k.sub.off) are calculated using a simple one-to-one Langmuir
binding model (BIACORE Evaluation Software version 3.2) by
simultaneously fitting the association and dissociation
sensorgrams. The equilibrium dissociation constant (Kd) is
calculated as the ratio k.sub.off/k.sub.on (see, e.g., Chen, Y. et
al., J. Mol. Biol. 293 (1999) 865-881). If the on-rate exceeds
10.sup.6 M.sup.-1 s.sup.-1 by the surface plasmon resonance assay
above, then the on-rate can be determined by using a fluorescent
quenching technique that measures the increase or decrease in
fluorescence emission intensity (excitation=295 nm; emission=340
nm, 16 nm band-pass) at 25.degree. C. of a 20 .mu.M anti-antigen
antibody (Fab form) in PBS, pH 7.2, in the presence of increasing
concentrations of antigen as measured in a spectrometer, such as a
stop-flow equipped spectrophotometer (Aviv Instruments) or a
8000-series SLM-AMINCO.TM. spectrophotometer (ThermoSpectronic)
with a stirred cuvette.
[0415] 2. Antibody Fragments
[0416] In certain embodiments, an antibody provided herein is an
antibody fragment. Antibody fragments include, but are not limited
to, Fab, Fab', Fab'-SH, F(ab').sub.2, Fv, and scFv fragments, and
other fragments described below. For a review of certain antibody
fragments, see Hudson, P. J. et al., Nat. Med. 9 (2003) 129-134.
For a review of scFv fragments, see, e.g., Plueckthun, A., In; The
Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and
Moore (eds.), Springer-Verlag, New York (1994), pp. 269-315; see
also WO 93/16185; U.S. Pat. No. 5,571,894 and U.S. Pat. No.
5,587,458. For discussion of Fab and F(ab').sub.2 fragments
comprising salvage receptor binding epitope residues and having
increased in vivo half-life, see U.S. Pat. No. 5,869,046.
[0417] Diabodies are antibody fragments with two antigen-binding
sites that may be bivalent or bispecific. See, for example, EP 0
404 097; WO 1993/01161; Hudson, P. J. et al., Nat. Med. 9 (2003)
129-134; and Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90
(1993) 6444-6448. Triabodies and tetrabodies are also described in
Hudson, P. J. et al., Nat. Med. 9 (20039 129-134).
[0418] Single-domain antibodies are antibody fragments comprising
all or a portion of the heavy chain variable domain or all or a
portion of the light chain variable domain of an antibody. In
certain embodiments, a single-domain antibody is a human
single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g.,
U.S. Pat. No. 6,248,516).
[0419] Antibody fragments can be made by various techniques,
including but not limited to proteolytic digestion of an intact
antibody as well as production by recombinant host cells (e.g. E.
coli or phage), as described herein.
[0420] 3. Humanized Antibodies
[0421] Typically, a non-human antibody is humanized to reduce
immunogenicity to humans, while retaining the specificity and
affinity of the parental non-human antibody. Generally, a humanized
antibody comprises one or more variable domains in which HVRs,
e.g., CDRs, (or portions thereof) are derived from a non-human
antibody, and FRs (or portions thereof) are derived from human
antibody sequences. A humanized antibody optionally will also
comprise at least a portion of or a full length human constant
region. In some embodiments, some FR residues in a humanized
antibody are substituted with corresponding residues from a
non-human antibody (e.g. the antibody from which the HVR residues
are derived), e.g. to restore or improve antibody specificity or
affinity.
[0422] Humanized antibodies and methods of making them are
reviewed, e.g., in Almagro, J. C. and Fransson, J., Front. Biosci.
13 (2008) 1619-1633, and are further described, e.g., in Riechmann,
I. et al., Nature 332 (1988) 323-329; Queen, C. et al., Proc. Natl.
Acad. Sci. USA 86 (1989) 10029-10033; U.S. Pat. No. 5,821,337, U.S.
Pat. No. 7,527,791, U.S. Pat. No. 6,982,321, and U.S. Pat. No.
7,087,409; Kashmiri, S. V. et al., Methods 36 (2005) 25-34
(describing specificity determining region (SDR) grafting); Padlan,
E. A., Mol. Immunol. 28 (1991) 489-498 (describing "resurfacing");
Dall'Acqua, W. F. et al., Methods 36 (2005) 43-60 (describing "FR
shuffling"); and Osbourn, J. et al., Methods 36 (2005) 61-68 and
Klinaka, A. et al., Br. J. Cancer 83 (2000) 252-260 (describing the
"guided selection" approach to FR shuffling).
[0423] Human framework regions that may be used for humanization
include but are not limited to: framework regions selected using
the "best-fit" method (see, e.g., Sims, M. J. et al., J. Immunol
151 (1993) 2296-2308; framework regions derived from the consensus
sequence of human antibodies of a particular subgroup of light or
heavy chain variable regions (see, e.g., Carter, P. et al., Proc.
Natl. Acad. Sci. USA 89 (1992) 4285-4289; and Presta, L. G. et al.,
J. Immunol. 151 (1993) 2623-2632); human mature (somatically
mutated) framework regions or human germline framework regions
(see, e.g., Almagro, J. C. and Fransson, J., Front. Biosci. 13
(2008) 1619-1633); and framework regions derived from screening FR
libraries (see, e.g., Baca, M. et al., J. Biol. Chem. 272 (1997)
10678-10684 and Rosok, M. J. et al., J. Biol. Chem. 271 (19969
22611-22618).
[0424] 4. Multispecific Antibodies
[0425] In certain embodiments, an antibody provided herein is a
multispecific antibody, e.g. a bispecific antibody. Multispecific
antibodies are monoclonal antibodies that have binding
specificities for at least two different sites. In certain
embodiments, one of the binding specificities is for human
Tau(pS422) and the other is for any other antigen. In certain
embodiments, bispecific antibodies may bind to two different
epitopes of human Tau(pS422). Bispecific antibodies can be prepared
as full length antibodies or antibody fragments.
[0426] Techniques for making multispecific antibodies include, but
are not limited to, recombinant co-expression of two immunoglobulin
heavy chain-light chain pairs having different specificities (see
Milstein, C. and Cuello, A. C., Nature 305 (1983) 537-540, WO
93/08829, and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659),
and "knob-in-hole" engineering (see, e.g., U.S. Pat. No.
5,731,168). Multi-specific antibodies may also be made by
engineering electrostatic steering effects for making antibody
Fc-heterodimeric molecules (WO 2009/089004); cross-linking two or
more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980,
and Brennan, M. et al., Science 229 (1985) 81-83); using leucine
zippers to produce bi-specific antibodies (see, e.g., Kostelny, S.
A. et al., J. Immunol. 148 (1992) 1547-1553; using "diabody"
technology for making bispecific antibody fragments (see, e.g.,
Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993)
6444-6448); and using single-chain Fv (sFv) dimers (see, e.g.
Gruber, M et al., J. Immunol. 152 (1994) 5368-5374); and preparing
trispecific antibodies as described, e.g., in Tutt, A. et al., J.
Immunol. 147 (1991) 60-69).
[0427] Engineered antibodies with three or more functional antigen
binding sites, including "Octopus antibodies," are also included
herein (see, e.g. US 2006/0025576).
[0428] The antibody or fragment herein also includes a "Dual Acting
Fab" or "DAF" comprising an antigen binding site that binds to
human Tau(pS422) as well as another, different antigen (see, US
2008/0069820, for example).
[0429] The antibody or fragment herein also includes multispecific
antibodies described in WO 2009/080251, WO 2009/080252, WO
2009/080253, WO 2009/080254, WO 2010/112193, WO 2010/115589, WO
2010/136172, WO 2010/145792, and WO 2010/145793.
[0430] 5. Antibody Variants
[0431] In certain embodiments, amino acid sequence variants of the
antibodies provided herein are contemplated. For example, it may be
desirable to improve the binding affinity and/or other biological
properties of the antibody. Amino acid sequence variants of an
antibody may be prepared by introducing appropriate modifications
into the nucleotide sequence encoding the antibody, or by peptide
synthesis. Such modifications include, for example, deletions from,
and/or insertions into and/or substitutions of residues within the
amino acid sequences of the antibody. Any combination of deletion,
insertion, and substitution can be made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics, e.g., antigen-binding.
[0432] a) Substitution, Insertion and Deletion Variants
[0433] In certain embodiments, antibody variants having one or more
amino acid substitutions are provided. Sites of interest for
substitutional mutagenesis include the HVRs and FRs. Conservative
substitutions are shown in the following Table under the heading of
"preferred substitutions". More substantial changes are provided in
the following Table under the heading of "exemplary substitutions",
and as further described below in reference to amino acid side
chain classes. Amino acid substitutions may be introduced into an
antibody of interest and the products screened for a desired
activity, e.g., retained/improved antigen binding, decreased
immunogenicity, or improved ADCC or CDC.
TABLE-US-00007 Original Exemplary Conservative Residue
Substitutions Substitutions His (H) Asn; Gln; Lys; Arg Arg Ile (I)
Leu; Val; Met; Ala; Phe; Leu Norleucine Leu (L) Norleucine; Ile;
Val; Met; Ile Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;
Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala
Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr (Y)
Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Leu
Norleucine
[0434] Amino acids may be grouped according to common side-chain
properties: [0435] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu,
Ile; [0436] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;
[0437] (3) acidic: Asp, Glu; [0438] (4) basic: His, Lys, Arg;
[0439] (5) residues that influence chain orientation: Gly, Pro;
[0440] (6) aromatic: Trp, Tyr, Phe.
[0441] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0442] One type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody (e.g. a
humanized antibody). Generally, the resulting variant(s) selected
for further study will have modifications (e.g. improvements) in
certain biological properties (e.g. increased affinity, reduced
immunogenicity) relative to the parent antibody and/or will have
substantially retained certain biological properties of the parent
antibody. An exemplary substitutional variant is an affinity
matured antibody, which may be conveniently generated, e.g. using
phage display-based affinity maturation techniques such as those
described herein. Briefly, one or more HVR residues are mutated and
the variant antibodies displayed on phage and screened for a
particular biological activity (e.g. binding affinity).
[0443] Alterations (e.g., substitutions) may be made in HVRs, e.g.,
to improve antibody affinity. Such alterations may be made in HVR
"hotspots," i.e., residues encoded by codons that undergo mutation
at high frequency during the somatic maturation process (see, e.g.,
Chowdhury, P. S., Methods Mol. Biol. 207 (2008) 179-196), and/or
residues that contact antigen, with the resulting variant VH or VL
being tested for binding affinity. Affinity maturation by
constructing and reselecting from secondary libraries has been
described, e.g., in Hoogenboom, H. R. et al. in Methods in
Molecular Biology 178 (2002) 1-37. In some embodiments of affinity
maturation, diversity is introduced into the variable genes chosen
for maturation by any of a variety of methods (e.g., error-prone
PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A
secondary library is then created. The library is then screened to
identify any antibody variants with the desired affinity. Another
method to introduce diversity involves HVR-directed approaches, in
which several HVR residues (e.g., 4-6 residues at a time) are
randomized. HVR residues involved in antigen binding may be
specifically identified, e.g., using alanine scanning mutagenesis
or modeling. CDR-H3 and CDR-L3 in particular are often
targeted.
[0444] In certain embodiments, substitutions, insertions, or
deletions may occur within one or more HVRs so long as such
alterations do not substantially reduce the ability of the antibody
to bind antigen. For example, conservative alterations (e.g.,
conservative substitutions as provided herein) that do not
substantially reduce binding affinity may be made in HVRs. Such
alterations may, for example, be outside of antigen contacting
residues in the HVRs. In certain embodiments of the variant VH and
VL sequences provided above, each HVR either is unaltered, or
contains no more than one, two or three amino acid
substitutions.
[0445] A useful method for identification of residues or regions of
an antibody that may be targeted for mutagenesis is called "alanine
scanning mutagenesis" as described by Cunningham, B. C. and Wells,
J. A., Science 244 (1989) 1081-1085. In this method, a residue or
group of target residues (e.g., charged residues such as arg, asp,
his, lys, and glu) are identified and replaced by a neutral or
negatively charged amino acid (e.g., alanine or polyalanine) to
determine whether the interaction of the antibody with antigen is
affected. Further substitutions may be introduced at the amino acid
locations demonstrating functional sensitivity to the initial
substitutions. Alternatively, or additionally, a crystal structure
of an antigen-antibody complex to identify contact points between
the antibody and antigen. Such contact residues and neighboring
residues may be targeted or eliminated as candidates for
substitution. Variants may be screened to determine whether they
contain the desired properties.
[0446] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hunched or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue. Other insertional variants of the
antibody molecule include the fusion to the N- or C-terminus of the
antibody to an enzyme (e.g. for ADEPT) or a polypeptide which
increases the serum half-life of the antibody.
[0447] b) Glycosylation Variants
[0448] In certain embodiments, an antibody provided herein is
altered to increase or decrease the extent to which the antibody is
glycosylated. Addition or deletion of glycosylation sites to an
antibody may be conveniently accomplished by altering the amino
acid sequence such that one or more glycosylation sites is created
or removed.
[0449] Where the antibody comprises an Fc-region, the carbohydrate
attached thereto may be altered. Native antibodies produced by
mammalian cells typically comprise a branched, biantennary
oligosaccharide that is generally attached by an N-linkage to
Asn297 of the CH2 domain of the Fc-region (see, e.g., Wright, A.
and Morrison, S. L., TIBTECH 15 (1997) 26-32). The oligosaccharide
may include various carbohydrates, e.g., mannose, N-acetyl
glucosamine (GlcNAc), galactose, and sialic acid, as well as a
fucose attached to a GlcNAc in the "stem" of the biantennary
oligosaccharide structure. In some embodiments, modifications of
the oligosaccharide in an antibody of the invention may be made in
order to create antibody variants with certain improved
properties.
[0450] c) Fc-Region Variants
[0451] In certain embodiments, one or more amino acid modifications
may be introduced into the Fc-region of an antibody provided
herein, thereby generating an Fc-region variant. The Fc-region
variant may comprise a human Fc-region sequence (e.g. a human IgG1,
IgG2, IgG3 or IgG4 Fc-region) comprising an amino acid modification
(e.g. a substitution) at one or more amino acid positions.
[0452] In certain embodiments, the invention contemplates an
antibody variant that possesses some but not all effector
functions, which make it a desirable candidate for applications in
which the half-life of the antibody in vivo is important yet
certain effector functions (such as complement and ADCC) are
unnecessary or deleterious. In vitro and/or in vivo cytotoxicity
assays can be conducted to confirm the reduction/depletion of CDC
and/or ADCC activities. For example, Fc receptor (FcR) binding
assays can be conducted to ensure that the antibody lacks
Fc.gamma.R binding (hence likely lacking ADCC activity), but
retains FcRn binding ability.
[0453] The primary cells for mediating ADCC, NK cells, express
Fc.gamma.RIII only, whereas monocytes express Fc.gamma.RI,
Fc.gamma.RII and Fc.gamma.RIII. FcR expression on hematopoietic
cells is summarized in Table 3 on page 464 of Ravetch, J. V. and
Kinet, J. P., Annu. Rev. Immunol 9 (1991) 457-492. Non-limiting
examples of in vitro assays to assess ADCC activity of a molecule
of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.
Hellstrom, I. et at, Proc. Natl. Acad. Sci. USA 83 (1986)
7059-7063; and Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA 82
(1985) 1499-1502); U.S. Pat. No. 5,821,337 (see Bruggemann, M. et
al., J. Exp. Med. 166 (1987) 1351-1361). Alternatively,
non-radioactive assays methods may be employed (see, for example,
ACTI.TM. non-radioactive cytotoxicity assay for flow cytometry
(CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96.RTM.
non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Useful
effector cells for such assays include peripheral blood mononuclear
cells (PBMC) and Natural Killer (NK) cells. Alternatively, or
additionally, ADCC activity of the molecule of interest may be
assessed in vivo, e.g. in an animal model such as that disclosed in
Clynes, R. et al., Proc. Natl. Acad. Sci. USA 95 (1998) 652-656.
C1q binding assays may also be carried out to confirm that the
antibody is unable to bind C1q and hence lacks CDC activity. See,
e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO
2005/100402. To assess complement activation, a CDC assay may be
performed (see, for example, Gazzano-Santoro, H. et al., J.
Immunol. Methods 202 (1996) 163-171; Cragg, M. S. et al., Blood 101
(2003) 1045-1052; and Cragg, M. S. and M. J. Glennie, Blood 103
(2004) 2738-2743). FcRn binding and in vivo clearance/half-life
determinations can also be performed using methods known in the art
(see, e.g., Petkova, S. B. et al., Int. Immunol. 18 (2006:
1759-1769).
[0454] Antibodies with reduced effector function include those with
substitution of one or more of Fc-region residues 238, 265, 269,
270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc-region
mutants include Fc-region mutants with substitutions at two or more
of amino acid positions 265, 269, 270, 297 and 327, including the
so-called "DANA" Fc-region mutant with substitution of residues 265
and 297 to alanine (U.S. Pat. No. 7,332,581).
[0455] Certain antibody variants with improved or diminished
binding to FcRs are described (see, e.g., U.S. Pat. No. 6,737,056;
WO 2004/056312, and Shields, R. L. et al., J. Biol. Chem. 276
(2001) 6591-6604).
[0456] In some embodiments, alterations are made in the Fc-region
that result in altered (i.e. diminished) C1q binding and/or
Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S.
Pat. No. 6,194,551, WO 99/51642, and Idusogie, E. E. et al., J.
Immunol. 164 (2000) 4178-4184.
[0457] Antibodies with increased half-lives and improved binding to
the neonatal Fc receptor (FeRn), which is responsible for the
transfer of maternal IgGs to the fetus (Guyer, R. L. et al., J.
Immunol. 117 (1976) 587-593, and Kim, J. K. et al., J. Immunol. 24
(1994) 2429-2434), are described in US 2005/0014934. Those
antibodies comprise an Fc-region with one or more substitutions
therein which improve binding of the Fc-region to FeRn. Such
Fc-region variants include those with substitutions at one or more
of Fc-region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311,
312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,
e.g., substitution of Fc-region residue 434 (U.S. Pat. No.
7,371,826).
[0458] See also Duncan, A. R. and Winter, G., Nature 322 (1988)
738-740; U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO
94/29351 concerning other examples of Fc-region variants.
[0459] d) Cysteine Engineered Antibody Variants
[0460] In certain embodiments, it may be desirable to create
cysteine engineered antibodies, e.g. "thioMAbs," in which one or
more residues of an antibody are substituted with cysteine
residues. In particular embodiments, the substituted residues occur
at accessible sites of the antibody. By substituting those residues
with cysteine, reactive thiol groups are thereby positioned at
accessible sites of the antibody and may be used to conjugate the
antibody to other moieties, such as drug moieties or linker-drug
moieties, to create an immunoconjugate, as described further
herein. In certain embodiments, any one or more of the following
residues may be substituted with cysteine: V205 (Kabat numbering)
of the light chain; A118 (EU numbering) of the heavy chain; and
5400 (EU numbering) of the heavy chain Fc-region. Cysteine
engineered antibodies may be generated as described, e.g., in U.S.
Pat. No. 7,521,541.
[0461] e) Antibody Derivatives
[0462] In certain embodiments, an antibody provided herein may be
further modified to contain additional non-proteinaceous moieties
that are known in the art and readily available. The moieties
suitable for derivatization of the antibody include but are not
limited to water soluble polymers. Non-limiting examples of water
soluble polymers include, but are not limited to, polyethylene
glycol (PEG), copolymers of ethylene glycol/propylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl
pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids (either
homopolymers or random copolymers), and dextran or poly(n-vinyl
pyrrolidone)polyethylene glycol, propropylene glycol homopolymers,
prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated
polyols (e.g. glycerol), polyvinyl alcohol, and mixtures thereof.
Polyethylene glycol propionaldehyde may have advantages in
manufacturing due to its stability in water. The polymer may be of
any molecular weight, and may be branched or unbranched. The number
of polymers attached to the antibody may vary, and if more than one
polymer is attached, they can be the same or different molecules.
In general, the number and/or type of polymers used for
derivatization can be determined based on considerations including,
but not limited to, the particular properties or functions of the
antibody to be improved, whether the antibody derivative will be
used in a therapy under defined conditions, etc.
[0463] In another embodiment, conjugates of an antibody and
non-proteinaceous moiety that may be selectively heated by exposure
to radiation are provided. In one embodiment, the non-proteinaceous
moiety is a carbon nanotube (Kam, N. W. et al., Proc. Natl. Acad.
Sci. USA 102 (2005) 11600-11605). The radiation may be of any
wavelength, and includes, but is not limited to, wavelengths that
do not harm ordinary cells, but which heat the non-proteinaceous
moiety to a temperature at which cells proximal to the
antibody-non-proteinaceous moiety are killed.
[0464] B. Recombinant Methods and Compositions
[0465] Antibodies may be produced using recombinant methods and
compositions, e.g., as described in U.S. Pat. No. 4,816,567. In one
embodiment, isolated nucleic acid encoding an anti-human Tau(pS422)
antibody described herein is provided. Such nucleic acid may encode
an amino acid sequence comprising the VL and/or an amino acid
sequence comprising the VH of the antibody (e.g., the light and/or
heavy chains of the antibody). In a further embodiment, one or more
vectors (e.g., expression vectors) comprising such nucleic acid are
provided. In a farther embodiment, a host cell comprising such
nucleic acid is provided. In one such embodiment, a host cell
comprises (e.g., has been transformed with): (1) a vector
comprising a nucleic acid that encodes an amino acid sequence
comprising the VL of the antibody and an amino acid sequence
comprising the VH of the antibody, or (2) a first vector comprising
a nucleic acid that encodes an amino acid sequence comprising the
VL of the antibody and a second vector comprising a nucleic acid
that encodes an amino acid sequence comprising the VH of the
antibody. In one embodiment, the host cell is eukaryotic, e.g. a
Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0,
Sp20 cell). In one embodiment, a method of making an anti-human
Tau(pS422) antibody is provided, wherein the method comprises
culturing a host cell comprising a nucleic acid encoding the
antibody, as provided above, under conditions suitable for
expression of the antibody, and optionally recovering the antibody
from the host cell (or host cell culture medium).
[0466] For recombinant production of an anti-human Tau(pS422)
antibody, nucleic acid encoding an antibody, e.g., as described
above, is isolated and inserted into one or more vectors for
further cloning and/or expression in a host cell. Such nucleic acid
may be readily isolated and sequenced using conventional procedures
(e.g., by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of the
antibody).
[0467] Suitable host cells for cloning or expression of
antibody-encoding vectors include prokaryotic or eukaryotic cells
described herein. For example, antibodies may be produced in
bacteria, in particular when glycosylation and Fc effector function
are not needed. For expression of antibody fragments and
polypeptides in bacteria, see, e.g., U.S. Pat. No. 5,648,237, U.S.
Pat. No. 5,789,199, and U.S. Pat. No. 5,840,523. (See also
Charlton, K. A., In: Methods in Molecular Biology, Vol. 248, Lo, B.
K. C. (ed.), Humana Press, Totowa, N.J. (2003), pp. 245-254,
describing expression of antibody fragments in E. coli.) After
expression, the antibody may be isolated from the bacterial cell
paste in a soluble fraction and can be further purified.
[0468] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for antibody-encoding vectors, including fungi and yeast strains
whose glycosylation pathways have been "humanized," resulting in
the production of an antibody with a partially or fully human
glycosylation pattern. See Gerngross, T. U., Nat. Biotech. 22
(2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006)
210-215.
[0469] Suitable host cells for the expression of glycosylated
antibody are also derived from multicellular organisms
(invertebrates and vertebrates). Examples of invertebrate cells
include plant and insect cells. Numerous baculoviral strains have
been identified which may be used in conjunction with insect cells,
particularly for transfection of Spodoptera frugiperda cells.
[0470] Plant cell cultures can also be utilized as hosts. See,
e.g., U.S. Pat. No. 5,959,177, U.S. Pat. No. 6,040,498, U.S. Pat.
No. 6,420,548, U.S. Pat. No. 7,125,978, and U.S. Pat. No. 6,417,429
(describing PLANTIBODIES.TM. technology for producing antibodies in
transgenic plants).
[0471] Vertebrate cells may also be used as hosts. For example,
mammalian cell lines that are adapted to grow in suspension may be
useful. Other examples of useful mammalian host cell lines are
monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic
kidney line (293 or 293 cells as described, e.g., in Graham, F. L.
et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells
(BHK); mouse sertoli cells (TM4 cells as described, e.g., in
Mather, J. P., Biol. Reprod. 23 (1980) 243-252); monkey kidney
cells (CV1); African green monkey kidney cells (VERO-76); human
cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo
rat liver cells (BRL 3A); human lung cells (W138); human liver
cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as
described, e.g., in Mather, J. P. et al., Annals N.Y. Acad. Sci.
383 (1982) 44-68; MRC 5 cells; and FS4 cells. Other useful
mammalian host cell lines include Chinese hamster ovary (CHO)
cells, including DHFR.sup.- CHO cells (Urlaub, G. et al., Proc.
Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines
such as Y0, NS0 and Sp2/0. For a review of certain mammalian host
cell lines suitable for antibody production, see, e.g., Yazaki, P.
and Wu, A. M., Methods in Molecular Biology, Vol. 248, Lo, B. K. C.
(ed.), Humana Press, Totowa, N.J. (2004), pp. 255-268.
[0472] C. Assays
[0473] Anti-human Tau(pS422) antibodies provided herein may be
identified, screened for, or characterized for their
physical/chemical properties and/or biological activities by
various assays known in the art.
[0474] 1. Binding Assays and Other Assays
[0475] In one aspect, an antibody of the invention is tested for
its antigen binding activity, e.g., by known methods such as ELISA,
alphaLISA, Western blot, antibody or reverse phase array, etc.
[0476] In an exemplary ELISA or alphaLISA assay, Tau(pS422) in
solution (cell supernatant, cell or tissue lysates, body fluids
etc.) is bound by a capture antibody, which specifically binds to a
first epitope on Tau(pS422), or Tau(pS422) in a certain
conformation and a detection antibody coupled to a detection
entity, which specifically binds to a second epitope or
conformation of Tau(pS422). The readout is based on the detection
entity (chemiluminescence, fluorescence, energy transfer induced
luminescence etc.). In some instances the same antibody can be used
in the same assay as capture and detection antibody to detect
aggregated fauns of Tau(pS422) (see e.g. Tokuda, T. et al.,
Neurology 75 (2010) 1766-1772).
[0477] In the case of antibody array, antibodies are spotted onto
glass or nitrocellulose chips. The slides are blocked and incubated
with Tau(pS422) containing solution, washed to remove unbound
antibodies and bound antibodies are detected with a fluorescently
labeled corresponding secondary antibody. The fluorescence signal
is measured by a fluorescence slide scanner. Similarly for a
reverse phase array, recombinant Tau(pS422), cell supernatant, cell
or tissue lysates, body fluids etc. are spotted onto glass or
nitrocellulose chips. The slides are blocked and individual arrays
are incubated with an antibody against a specific epitope on
Tau(pS422). Unbound antibodies are washed off and bound antibodies
are detected with a fluorescently labeled corresponding secondary
antibody. The fluorescence signal is measured by a fluorescence
slide scanner (Dernick, G., et al., J. Lipid Res. 52 (2011)
2323-2331).
[0478] In the example of Western blot, aggregated recombinant
Tau(pS422) or Tau(pS422) derived from cell supernatant, cell or
tissue lysates, body fluids etc. is separated by molecular weight
in SDS PAGE or native gel conditions and blotted onto a
nitrocellulose or PVDF membrane. After blocking the membrane is
incubated with antibodies specific to amino acid sequence or
conformations of Tau(pS422). Thereafter the membrane is washed to
remove unbound antibody. Bound antibodies are detected by
corresponding secondary antibodies coupled to detection entities
for chemiluminescence or fluorescence or other means of detection.
Antibodies specific to amino acid sequences of Tau(pS422) will bind
to Tau(pS422) in various aggregated forms and hence molecular
weights as long as the epitope is not masked by the aggregation. On
the other hand, conformation specific antibodies will detect only
certain aggregated forms of Tau(pS422) revealing only bands at
specific molecular weights (see e.g. Towbin, H., et al., Proc.
Natl. Acad. Sci. USA 76 (1979) 4350-4353; Burnette, W. N., Anal.
Biochem. 112 (1981) 195-203).
[0479] In another aspect, competition assays may be used to
identify an antibody that competes with the (humanized) antibody as
reported herein for binding to human Tau(pS422). In certain
embodiments, such a competing antibody binds to the same epitope
(e.g., a linear or a conformational epitope) that is bound by the
(humanized) antibody as reported herein. Detailed exemplary methods
for mapping an epitope to which an antibody binds are provided in
Morris, G. E. (ed.), Epitope Mapping Protocols, In: Methods in
Molecular Biology, Vol. 66, Humana Press, Totowa, N.J. (1996).
[0480] In an exemplary competition assay, immobilized human
Tau(pS422) is incubated in a solution comprising a first labeled
antibody that binds to human Tau(pS422) and a second unlabeled
antibody that is being tested for its ability to compete with the
first antibody for binding to human Tau(pS422). As a control,
immobilized human Tau(pS422) is incubated in a solution comprising
the first labeled antibody but not the second unlabeled antibody.
After incubation under conditions permissive for binding of the
first antibody to human Tau(pS422), excess unbound antibody is
removed, and the amount of label associated with immobilized human
Tau(pS422) is measured. If the amount of label associated with
immobilized human Tau(pS422) is substantially reduced in the test
sample relative to the control sample, then that indicates that the
second antibody is competing with the first antibody for binding to
human Tau(pS422) (see e.g., Harlow, E. and Lane, D., Antibodies: A
Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. (1988)).
[0481] 2. Activity Assays
[0482] In one aspect, assays are provided for identifying
anti-human Tau(pS422) antibodies thereof having biological
activity. Biological activity may include, e.g., protection
from/reduction of/inhibition of Tau(pS422) induced cytotoxicity,
and/or protection from/reduction of/inhibition of cell-to-cell
transmission of oligomeric human Tau(pS422), and/or reduction of
Tau(pS422)-induced caspase activity in LUHMES cells. Antibodies
having such biological activity in vivo and/or in vitro are also
provided.
[0483] In certain embodiments, an antibody of the invention is
tested for such biological activity.
[0484] The protective biological activity can be assessed by adding
conditioned medium containing secreted Tau(pS422), which causes
cell death on recipient neuronal cells. This toxicity can be
reversed by adding protective antibodies as described herein. The
toxic nature of secreted Tau(pS422) has been established previously
(Emmanouilidou, E., et al., J. Neurosci., 30 (2010) 6838-6851).
[0485] D. Methods and Compositions for Diagnostics and
Detection
[0486] In certain embodiments, any of the anti-human Tau(pS422)
antibodies provided herein is useful for detecting the presence of
human Tau(pS422) in a biological sample. The term "detecting" as
used herein encompasses quantitative or qualitative detection. In
certain embodiments, a biological sample comprises a cell or
tissue, such as brain tissue.
[0487] In one embodiment, an anti-human Tau(pS422) antibody for use
in a method of diagnosis or detection is provided. In a further
aspect, a method of detecting the presence of human Tau(pS422) in a
biological sample is provided. In certain embodiments, the method
comprises contacting the biological sample with an anti-human
Tau(pS422) antibody as described herein under conditions permissive
for binding of the anti-human Tau(pS422) antibody to human
Tau(pS422), and detecting whether a complex is formed between the
anti-human Tau(pS422) antibody and human Tau(pS422). Such method
may be an in vitro or in viva method. In one embodiment, an
anti-human Tau(pS422) antibody is used to select subjects eligible
for therapy with an anti-human Tau(pS422) antibody, e.g. where
human Tau(pS422) is a biomarker for selection of patients.
[0488] Exemplary disorders that may be diagnosed using an antibody
of the invention include neurodegeneration with brain iron
accumulation type 1 (NBIA1), pure autonomic failure, Down's
syndrome, complex of Guam, and several Lewy body disorders, such as
diffuse Lewy body disease (DLBD), the Lewy body variant of
Alzheimer's disease (LBVAD), certain forms of Gaucher's disease,
and Parkinson's disease dementia (PDD).
[0489] In certain embodiments, labeled anti-human Tau(pS422)
antibodies are provided. Labels include, but are not limited to,
labels or moieties that are detected directly (such as fluorescent,
chromophoric, electron-dense, chemiluminescent, and radioactive
labels), as well as moieties, such as enzymes or ligands, that are
detected indirectly, e.g., through an enzymatic reaction or
molecular interaction. Exemplary labels include, but are not
limited to, the radioisotopes .sup.32P, .sup.14C, .sup.125I,
.sup.3H, and .sup.131I, fluorophores such as rare earth chelates or
fluorescein and its derivatives, rhodamine and its derivatives,
dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and
bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin,
2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),
alkaline phosphatase, .beta.-galactosidase, glucoamylase, lysozyme,
saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and
glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as
uricase and xanthine oxidase, coupled with an enzyme that employs
hydrogen peroxide to oxidize a dye precursor such as HRP,
lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,
bacteriophage labels, stable free radicals, and the like.
[0490] E. Pharmaceutical Formulations
[0491] Pharmaceutical formulations of an anti-human Tau(pS422)
antibody as described herein are prepared by mixing such antibody
having the desired degree of purity with one or more optional
pharmaceutically acceptable carriers (Remington's Pharmaceutical
Sciences, 16th edition, Osol, A. (ed.) (1980)), in the form of
lyophilized formulations or aqueous solutions. Pharmaceutically
acceptable carriers are generally nontoxic to recipients at the
dosages and concentrations employed, and include, but are not
limited to: buffers such as phosphate, citrate, and other organic
acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyl dimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride; benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
poly(vinylpyrrolidone); amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as polyethylene glycol (PEG). Exemplary
pharmaceutically acceptable carriers herein further include
interstitial drug dispersion agents such as soluble neutral-active
hyaluronidase glycoproteins (sHASEGP), for example, human soluble
PH-20 hyaluronidase glycoproteins, such as rhuPH20 (HYLENEX.RTM.,
Baxter International, Inc.). Certain exemplary sHASEGPs and methods
of use, including rhuPH20, are described in US 2005/0260186 and US
2006/0104968. In one aspect, a sHASEGP is combined with one or more
additional glycosaminoglycanases such as chondroitinases.
[0492] Exemplary lyophilized antibody formulations are described in
U.S. Pat. No. 6,267,958. Aqueous antibody formulations include
those described in U.S. Pat. No. 6,171,586 and WO 2006/044908, the
latter formulations including a histidine-acetate buffer.
[0493] The formulation herein may also contain more than one active
ingredients as necessary for the particular indication being
treated, preferably those with complementary activities that do not
adversely affect each other. For example, it may be desirable to
further provide [[list drugs that might be combined with the
anti-human Tau(pS422) antibody]]. Such active ingredients are
suitably present in combination in amounts that are effective for
the purpose intended.
[0494] Active ingredients may be entrapped in microcapsules
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-(methyl methacrylate) microcapsules,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences, 16th edition, Osol, A.
(ed.) (1980).
[0495] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semi-permeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules.
[0496] The formulations to be used for in vivo administration are
generally sterile. Sterility may be readily accomplished, e.g., by
filtration through sterile filtration membranes.
[0497] F. Therapeutic Methods and Compositions
[0498] Any of the anti-human Tau(pS422) antibodies provided herein
may be used in therapeutic methods.
[0499] In one aspect, an anti-human Tau(pS422) antibody for use as
a medicament is provided. In further aspects, an anti-human
Tau(pS422) antibody for use in treating Alzheimer's disease is
provided. In certain embodiments, an anti-human Tau(pS422) antibody
for use in a method of treatment is provided. In certain
embodiments, the invention provides an anti-human Tau(pS422)
antibody for use in a method of treating an individual having
Alzheimer's disease comprising administering to the individual an
effective amount of the anti-human Tau(pS422) antibody. In one such
embodiment, the method further comprises administering to the
individual an effective amount of at least one additional
therapeutic agent, e.g., as described below. In further
embodiments, the invention provides an anti-human Tau(pS422)
antibody for use in inhibiting Tau(pS422) induced cytotoxicity in
human neurons and glia cells, or inhibiting cell-to-cell
transmission of oligomeric human Tau(pS422) between neurons and
glia cells, or reducing Tau(pS422)-induced caspase activity. In
certain embodiments, the invention provides an anti-human
Tau(pS422) antibody for use in a method of inhibiting Tau(pS422)
induced cytotoxicity in human neurons and glia cells, or inhibiting
cell-to-cell transmission of oligomeric human Tau(pS422) between
neurons and glia cells, or reducing Tau(pS422)-induced caspase
activity in an individual comprising administering to the
individual an effective of the anti-human Tau(pS422) antibody to
inhibit Tau(pS422) induced cytotoxicity in human neurons and glia
cells, or inhibit cell-to-cell transmission of oligomeric human
Tau(pS422) between neurons and glia cells, or reduce
Tau(pS422)-induced caspase activity. An "individual" according to
any of the above embodiments is preferably a human.
[0500] In a further aspect, the invention provides for the use of
an anti-human Tau(pS422) antibody in the manufacture or preparation
of a medicament. In one embodiment, the medicament is for treatment
of Alzheimer's disease. In a further embodiment, the medicament is
for use in a method of treating Alzheimer's disease comprising
administering to an individual having Alzheimer's disease an
effective amount of the medicament. In one such embodiment, the
method further comprises administering to the individual an
effective amount of at least one additional therapeutic agent,
e.g., as described below. In a further embodiment, the medicament
is for inhibiting Tau(pS422) induced cytotoxicity in human neurons
and glia cells, or for inhibiting cell-to-cell transmission of
oligomeric human Tau(pS422) between neurons and glia cells, or for
reducing Tau(pS422)-induced caspase activity. In a further
embodiment, the medicament is for use in a method of inhibiting
Tau(pS422) induced cytotoxicity in human neurons and glia cells, or
inhibiting cell-to-cell transmission of oligomeric human Tau(pS422)
between neurons and glia cells, or reducing Tau(pS422)-induced
caspase activity in an individual comprising administering to the
individual an amount effective of the medicament to inhibit
Tau(pS422) induced cytotoxicity in human neurons and glia cells, or
to inhibit cell-to-cell transmission of oligomeric human Tau(pS422)
between neurons and glia cells, or to reduce Tau(pS422)-induced
caspase activity. An "individual" according to any of the above
embodiments may be a human.
[0501] In a further aspect, the invention provides a method for
treating Alzheimer's disease. In one embodiment, the method
comprises administering to an individual having such Alzheimer's
disease an effective amount of an anti-human Tau(pS422) antibody.
In one such embodiment, the method further comprises administering
to the individual an effective amount of at least one additional
therapeutic agent, as described below. An "individual" according to
any of the above embodiments may be a human.
[0502] In a further aspect, the invention provides a method for
inhibiting Tau(pS422) induced cytotoxicity in human neurons and
glia cells, or inhibiting cell-to-cell transmission of oligomeric
human Tau(pS422) between neurons and glia cells, or reducing
Tau(pS422)-induced caspase in an individual. In one embodiment, the
method comprises administering to the individual an effective
amount of an anti-human Tau(pS422) antibody to inhibit Tau(pS422)
induced cytotoxicity in human neurons and glia cells, or to inhibit
cell-to-cell transmission of oligomeric human Tau(pS422) between
neurons and glia cells, or to reduce Tau(pS422)-induced caspase
activity. In one embodiment, an "individual" is a human.
[0503] In a further aspect, the invention provides pharmaceutical
formulations comprising any of the anti-human Tau(pS422) antibodies
provided herein, e.g., for use in any of the above therapeutic
methods. In one embodiment, a pharmaceutical formulation comprises
any of the anti-human Tau(pS422) antibodies provided herein and a
pharmaceutically acceptable carrier. In another embodiment, a
pharmaceutical formulation comprises any of the anti-human
Tau(pS422) antibodies provided herein and at least one additional
therapeutic agent, e.g., as described below.
[0504] Antibodies of the invention can be used either alone or in
combination with other agents in a therapy. For instance, an
antibody of the invention may be co-administered with at least one
additional therapeutic agent.
[0505] Such combination therapies noted above encompass combined
administration (where two or more therapeutic agents are included
in the same or separate formulations), and separate administration,
in which case, administration of the antibody of the invention can
occur prior to, simultaneously, and/or following, administration of
the additional therapeutic agent or agents. In one embodiment,
administration of the anti-human Tau(pS422) antibody and
administration of an additional therapeutic agent occur within
about one month, or within about one, two or three weeks, or within
about one, two, three, four, five, or six days, of each other.
[0506] An antibody of the invention (and any additional therapeutic
agent) can be administered by any suitable means, including
parenteral, intrapulmonary, and intranasal, and, if desired for
local treatment, intralesional administration. Parenteral infusions
include intramuscular, intravenous, intraarterial, intraperitoneal,
or subcutaneous administration. Dosing can be by any suitable
route, e.g. by injections, such as intravenous or subcutaneous
injections, depending in part on whether the administration is
brief or chronic. Various dosing schedules including but not
limited to single or multiple administrations over various
time-points, bolus administration, and pulse infusion are
contemplated herein.
[0507] Antibodies of the invention would be formulated, dosed, and
administered in a fashion consistent with good medical practice.
Factors for consideration in this context include the particular
disorder being treated, the particular mammal being treated, the
clinical condition of the individual patient, the cause of the
disorder, the site of delivery of the agent, the method of
administration, the scheduling of administration, and other factors
known to medical practitioners. The antibody need not be, but is
optionally formulated with one or more agents currently used to
prevent or treat the disorder in question. The effective amount of
such other agents depends on the amount of antibody present in the
formulation, the type of disorder or treatment, and other factors
discussed above. These are generally used in the same dosages and
with administration routes as described herein, or about from 1 to
99% of the dosages described herein, or in any dosage and by any
route that is empirically/clinically determined to be
appropriate.
[0508] For the prevention or treatment of disease, the appropriate
dosage of an antibody of the invention (when used alone or in
combination with one or more other additional therapeutic agents)
will depend on the type of disease to be treated, the type of
antibody, the severity and course of the disease, whether the
antibody is administered for preventive or therapeutic purposes,
previous therapy, the patient's clinical history and response to
the antibody, and the discretion of the attending physician. The
antibody is suitably administered to the patient at one time or
over a series of treatments. Depending on the type and severity of
the disease, about 1 .mu.g/kg to 15 mg/kg (e.g. 0.5 mg/kg-10 mg/kg)
of antibody can be an initial candidate dosage for administration
to the patient, whether, for example, by one or more separate
administrations, or by continuous infusion. One typical daily
dosage might range from about 1 .mu.g/kg to 100 mg/kg or more,
depending on the factors mentioned above. For repeated
administrations over several days or longer, depending on the
condition, the treatment would generally be sustained until a
desired suppression of disease symptoms occurs. One exemplary
dosage of the antibody would be in the range from about 0.05 mg/kg
to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0
mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be
administered to the patient. Such doses may be administered
intermittently, e.g. every week or every three weeks (e.g. such
that the patient receives from about two to about twenty, or e.g.
about six doses of the antibody). An initial higher loading dose,
followed by one or more lower doses may be administered. However,
other dosage regimens may be useful. The progress of this therapy
is easily monitored by conventional techniques and assays.
[0509] It is understood that any of the above formulations or
therapeutic methods may be carried out using an immunoconjugate of
the invention in place of or in addition to an anti-human
Tau(pS422) antibody.
III. ARTICLES OF MANUFACTURE
[0510] In another aspect of the invention, an article of
manufacture containing materials useful for the treatment,
prevention and/or diagnosis of the disorders described above is
provided. The article of manufacture comprises a container and a
label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, syringes,
IV solution bags, etc. The containers may be formed from a variety
of materials such as glass or plastic. The container holds a
composition which is by itself or combined with another composition
effective for treating, preventing and/or diagnosing the condition
and may have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). At least one active
agent in the composition is an antibody of the invention. The label
or package insert indicates that the composition is used for
treating the condition of choice. Moreover, the article of
manufacture may comprise (a) a first container with a composition
contained therein, wherein the composition comprises an antibody of
the invention; and (b) a second container with a composition
contained therein, wherein the composition comprises a further
cytotoxic or otherwise therapeutic agent. The article of
manufacture in this embodiment of the invention may further
comprise a package insert indicating that the compositions can be
used to treat a particular condition. Alternatively, or
additionally, the article of manufacture may farther comprise a
second (or third) container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
[0511] It is understood that any of the above articles of
manufacture may include an immunoconjugate of the invention in
place of or in addition to an anti-human Tau(pS422) antibody.
IV. SPECIFIC EMBODIMENTS
[0512] 1. A humanized antibody that specifically binds to human
Tau(pS422), wherein the antibody [0513] i) specifically binds to a
polypeptide that has the amino acid sequence of SEQ ID NO: 03,
and/or [0514] ii) does not bind to full length human Tau (SEQ ID
NO: 01) at 1 .mu.g/mL, and/or [0515] iii) specifically binds to
full length human Tau phosphorylated at the serine at position 422
(SEQ ID NO: 02), and/or [0516] iv) specifically binds to aggregates
of human Tau phosphorylated at the serine at position 422 (SEQ ID
NO: 02), and/or [0517] v) specifically binds to human Tau that has
the amino acid sequence of SEQ ID NO: 01 and that has the amino
acid mutation S422A. [0518] 2. A humanized antibody that
specifically binds to human Tau(pS422), wherein the antibody
comprises [0519] a) in the heavy chain variable domain the HVRs of
SEQ ID NO: 08, 18 and 10, or [0520] b) in the heavy chain variable
domain the HVRs of SEQ ID NO: 08, 09 and 10. [0521] 3. The
humanized antibody according to item 2, further comprising [0522]
a) in the light chain variable domain the HVRs of SEQ ID NO: 13, 14
and 15, or [0523] b) in the light chain variable domain the HVRs of
SEQ ID NO: 12, 05 and 15. [0524] 4. The humanized antibody
according to any one of items 2 to 3, comprising [0525] a) in the
heavy chain variable domain the HVRs of SEQ ID NO: 08, 18 and 10,
and in the light chain variable domain the HVRs of SEQ ID NO: 13,
14 and 15, or [0526] b) in the heavy chain variable domain the HVRs
of SEQ ID NO: 08, 09 and 10, and in the light chain variable domain
the HVRs of SEQ ID NO: 12, 05 and 15, or [0527] c) in the heavy
chain variable domain the HVRs of SEQ ID NO: 08, 09 and 10, and in
the light chain variable domain the HVRs of SEQ ID NO: 13, 14 and
15. [0528] 5. The humanized antibody according to any one of items
2 to 4, comprising [0529] a) a heavy chain variable domain of SEQ
ID NO: 20 and a light chain variable domain of SEQ ID NO: 17, or
[0530] b) a heavy chain variable domain of SEQ ID NO: 19 and a
light chain variable domain of SEQ ID NO: 16, or [0531] c) a heavy
chain variable domain of SEQ ID NO: 19 and a light chain variable
domain of SEQ ID NO: 17, or [0532] d) a heavy chain variable domain
of SEQ ID NO: 21 and a light chain variable domain of SEQ ID NO:
17. [0533] 6. The humanized antibody according to any one of items
2 to 5, wherein the antibody is for use in the treatment of
Alzheimer's disease. [0534] 7. The humanized antibody according to
any one of items 2 to 6, wherein the antibody is effector function
silent. [0535] 8. The humanized antibody according to any one of
items 2 to 7, wherein the antibody has no effector function. [0536]
9. The humanized antibody according to any one of items 2 to 8,
wherein the antibody [0537] i) specifically binds to a polypeptide
that has the amino acid sequence of SEQ ID NO: 03, and/or [0538]
ii) does not bind to full length human Tau (SEQ ID NO: 01) at 1
.mu.g/mL, and/or [0539] iii) specifically binds to full length
human Tau phosphorylated at the serine at position 422 (SEQ ID NO:
02), and/or [0540] iv) specifically binds to aggregates of human
Tau phosphorylated at the serine at position 422 (SEQ ID NO: 02).
[0541] 10. The humanized antibody according to any one of items 2
to 9, wherein the antibody has an EC.sub.50 value for [0542] a) the
human Tau(pS422) fragment that has the amino acid sequence of SEQ
ID NO: 03 of 6 ng/mL or less, and/or [0543] b) the full length
human Tau(pS422) that has the amino acid sequence of SEQ ID NO: 02
of 4.5 ng/mL or less, and/or [0544] c) aggregates of human Tau
(pS422) that has the amino acid sequence of SEQ ID NO: 02 of 30
ng/mL or less, and/or [0545] d) the human Tau that has the amino
acid sequence of SEQ ID NO: 01 and that has the amino acid mutation
S422A of 125 ng/mL or less. [0546] 11. The humanized antibody
according to any one of items 2 to 10, wherein the antibody
specifically binds to human Tau(pS422) (SEQ ID NO: 02) and does not
bind to human Tau (SEQ ID NO: 01). [0547] 12. The humanized
antibody according to any one of items 1 to 11, wherein the
antibody has in the heavy chain variable domain at positions 4, 24
and 78 a valine residue. [0548] 13. The humanized antibody
according to any one of items 1 to 12, wherein the antibody has in
the heavy chain variable domain at position 71 an arginine residue.
[0549] 14. The humanized antibody according to any one of items 2
to 13, wherein the antibody is a monoclonal antibody. [0550] 15.
The humanized antibody according to any one of items 2 to 10,
wherein the antibody is an antibody fragment that binds to human
Tau(pS422) and [0551] i) specifically binds to a polypeptide that
has the amino acid sequence of SEQ ID NO: 03, and/or [0552] ii)
does not bind to full length human Tau (SEQ ID NO: 01) at 1
.mu.g/mL, and/or [0553] iii) specifically binds to full length
human Tau phosphorylated at the serine at position 422 (SEQ ID NO:
02), and/or [0554] iv) specifically binds to aggregates of human
Tau phosphorylated at the serine at position 422 (SEQ ID NO: 02),
and/or [0555] v) specifically binds to full length human Tau that
has the amino acid sequence of SEQ ID NO: 01 and that has the amino
acid mutation S422A, and/or [0556] vi) has an EC.sub.50 value for
the human Tau(pS422) fragment that has the amino acid sequence of
SEQ ID NO: 03 of 6 ng/mL or less, and/or [0557] vii) has an
EC.sub.50 value for the full length human Tau(pS422) that has the
amino acid sequence of SEQ ID NO: 02 of 4.5 ng/mL or less, and/or
[0558] viii) has an EC.sub.50 value for aggregates of human Tau
(pS422) that has the amino acid sequence of SEQ ID NO: 02 of 30
ng/mL or less, and/or [0559] ix) has an EC.sub.50 value for the
human Tau that has the amino acid sequence of SEQ ID NO: 01 and
that has the amino acid mutation S422A of 125 ng/mL or less. [0560]
16. The humanized antibody according to any one of items 2 to 14,
wherein the antibody is [0561] a) a full length antibody of the
human subclass IgG 1, or [0562] b) a full length antibody of the
human subclass IgG4, or [0563] c) a full length antibody of the
human subclass IgG1 with the mutations L234A, L235A and P329G,
[0564] d) a full length antibody of the human subclass IgG4 with
the mutations S228P, L235E and P329G, [0565] e) a full length
antibody of the human subclass IgG1 with the mutations L234A, L235A
and P329G in both heavy chains and the mutations T366W and S354C in
one heavy chain and the mutations T366S, L368A, Y407V and Y349C in
the respective other heavy chain, or [0566] f) a full length
antibody of the human subclass IgG4 with the mutations S228P and
P329G in both heavy chains and the mutations T366W and S354C in one
heavy chain and the mutations T366S, L368A, Y407V and Y349C in the
respective other heavy chain. [0567] 17. A humanized anti-human
Tau(pS422) antibody, wherein [0568] a) the antibody comprises two
antibody heavy chains each comprising a heavy chain variable domain
and a heavy chain constant region, wherein [0569] i) the variable
domain comprises the HVRs of SEQ ID NO: 08, SEQ ID NO: 18 and SEQ
ID NO: 10, [0570] ii) the constant region is a human IgG1 constant
region, wherein the C-terminal lysine residue can be present or
absent, and [0571] iii) the constant region comprises the amino
acid changes L234A, L235A and P329G, [0572] b) the antibody
comprises two antibody light chains each comprising a light chain
variable domain and a light chain constant domain, wherein [0573]
i) the variable domain comprises the HVRs of SEQ ID NO: 13, SEQ ID
NO: 14 and SEQ ID NO: 15, [0574] ii) the constant region is a human
kappa light chain constant region or a human lambda light chain
constant region, [0575] and [0576] c) the antibody [0577] i)
specifically binds to a polypeptide that has the amino acid
sequence of SEQ ID NO: 03, and/or [0578] ii) does not bind to full
length human Tau (SEQ ID NO: 01) at 1 .mu.g/mL, and/or [0579] iii)
specifically binds to full length human Tau phosphorylated at the
serine at position 422 (SEQ ID NO: 02), and/or [0580] iv)
specifically binds to aggregates of human Tau phosphorylated at the
serine at position 422 (SEQ ID NO: 02), and/or [0581] v)
specifically binds to full length human Tau that has the amino acid
sequence of SEQ ID NO: 01 and that has the amino acid mutation
S422A, and/or [0582] vi) has an EC.sub.50 value for the human
Tau(pS422) fragment that has the amino acid sequence of SEQ ID NO:
03 of 6 ng/mL or less, and/or [0583] vii) has an EC.sub.50 value
for the full length human Tau(pS422) that has the amino acid
sequence of SEQ ID NO: 02 of 4.5 ng/mL or less, and/or [0584] viii)
has an EC.sub.50 value for aggregates of human Tau (pS422) that has
the amino acid sequence of SEQ ID NO: 02 of 30 ng/mL or less,
and/or [0585] ix) has an EC.sub.50 value for the human Tau that has
the amino acid sequence of SEQ ID NO: 01 and that has the amino
acid mutation S422A of 125 ng/mL or less. [0586] 18. A humanized
anti-human Tau(pS422) antibody, wherein [0587] a) the antibody
comprises two antibody heavy chains each comprising a heavy chain
variable domain and a heavy chain constant region, wherein [0588]
i) the variable domain comprises the HVRs of SEQ ID NO: 08, SEQ ID
NO: 09 and SEQ ID NO: 10, [0589] ii) the constant region is a human
IgG1 constant region, wherein the C-terminal lysine residue can be
present or absent, and [0590] iii) the constant region comprises
the amino acid changes L234A, L235A and P329G, [0591] b) the
antibody comprises two antibody light chains each comprising a
light chain variable domain and a light chain constant domain,
wherein [0592] i) the variable domain comprises the HVRs of SEQ ID
NO: 12, SEQ ID NO: 05 and SEQ ID NO: 15, [0593] ii) the constant
region is a human kappa light chain constant region or a human
lambda light chain constant region, [0594] and [0595] c) the
antibody [0596] i) specifically binds to a polypeptide that has the
amino acid sequence of SEQ ID NO: 03, and/or [0597] ii) does not
bind to full length human Tau (SEQ ID NO: 01) at 1 .mu.g/mL, and/or
[0598] iii) specifically binds to full length human Tau
phosphorylated at the serine at position 422 (SEQ ID NO: 02),
and/or [0599] iv) specifically binds to aggregates of human Tau
phosphorylated at the serine at position 422 (SEQ ID NO: 02),
and/or [0600] v) specifically binds to full length human Tau that
has the amino acid sequence of SEQ ID NO: 01 and that has the amino
acid mutation S422A, and/or [0601] vi) has an EC.sub.50 value for
the human Tau(pS422) fragment that has the amino acid sequence of
SEQ ID NO: 03 of 6 ng/mL or less, and/or [0602] vii) has an
EC.sub.50 value for the full length human Tau(pS422) that has the
amino acid sequence of SEQ ID NO: 02 of 4.5 ng/mL or less, and/or
[0603] viii) has an EC.sub.50 value for aggregates of human Tau
(pS422) that has the amino acid sequence of SEQ ID NO: 02 of 30
ng/mL or less, and/or [0604] ix) has an EC.sub.50 value for the
human Tau that has the amino acid sequence of SEQ ID NO: 01 and
that has the amino acid mutation S422A of 125 ng/mL or less. [0605]
19. A humanized anti-human Tau(pS422) antibody, wherein [0606] a)
the antibody comprises two antibody heavy chains each comprising a
heavy chain variable domain and a heavy chain constant region,
wherein [0607] i) the variable domain comprises the HVRs of SEQ ID
NO: 08, SEQ ID NO: 09 and SEQ ID NO: 10, [0608] ii) the constant
region is a human IgG1 constant region, wherein the C-terminal
lysine residue can be present or absent, and [0609] iii) the
constant region comprises the amino acid changes L234A, L235A and
P329G, [0610] b) the antibody comprises two antibody light chains
each comprising a light chain variable domain and a light chain
constant domain, wherein [0611] i) the variable domain comprises
the HVRs of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, [0612]
ii) the constant region is a human kappa light chain constant
region or a human lambda light chain constant region, [0613] and
[0614] c) the antibody [0615] i) specifically binds to a
polypeptide that has the amino acid sequence of SEQ ID NO: 03,
and/or [0616] ii) does not bind to full length human Tau (SEQ ID
NO: 01) at 1 .mu.g/mL, and/or [0617] iii) specifically binds to
full length human Tau phosphorylated at the serine at position 422
(SEQ ID NO: 02), and/or [0618] iv) specifically binds to aggregates
of human Tau phosphorylated at the serine at position 422 (SEQ ID
NO: 02), and/or [0619] v) specifically binds to full length human
Tau that has the amino acid sequence of SEQ ID NO: 01 and that has
the amino acid mutation S422A, and/or [0620] vi) has an EC.sub.50
value for the human Tau(pS422) fragment that has the amino acid
sequence of SEQ ID NO: 03 of 6 ng/mL or less, and/or [0621] vii)
has an EC.sub.50 value for the full length human Tau(pS422) that
has the amino acid sequence of SEQ ID NO: 02 of 4.5 ng/mL or less,
and/or [0622] viii) has an EC.sub.50 value for aggregates of human
Tau (pS422) that has the amino acid sequence of SEQ ID NO: 02 of 30
ng/mL or less, and/or [0623] ix) has an EC.sub.50 value for the
human Tau that has the amino acid sequence of SEQ ID NO: 01 and
that has the amino acid mutation S422A of 125 ng/mL or less. [0624]
20. A humanized anti-human Tau(pS422) antibody, wherein [0625] a)
the antibody comprises two antibody heavy chains each comprising a
heavy chain variable domain and a heavy chain constant region,
wherein [0626] i) the variable domain has the amino acid sequence
of SEQ ID NO: 20, [0627] ii) the constant region is a human IgG1
constant region, wherein the C-terminal lysine residue can be
present or absent, and [0628] iii) the constant region comprises
the amino acid changes L234A, L235A and P329G, [0629] b) the
antibody comprises two antibody light chains each comprising a
light chain variable domain and a light chain constant domain,
wherein [0630] i) the variable domain has the amino acid sequence
of SEQ ID NO: 17, [0631] ii) the constant region is a human kappa
light chain constant region or a human lambda light chain constant
region, [0632] and [0633] c) the antibody [0634] i) specifically
binds to a polypeptide that has the amino acid sequence of SEQ ID
NO: 03, and/or [0635] ii) does not bind to full length human Tau
(SEQ ID NO: 01) at 1 .mu.g/mL, and/or [0636] iii) specifically
binds to full length human Tau phosphorylated at the serine at
position 422 (SEQ ID NO: 02), and/or [0637] iv) specifically binds
to aggregates of human Tau phosphorylated at the serine at position
422 (SEQ ID NO: 02), and/or [0638] v) specifically binds to full
length human Tau that has the amino acid sequence of SEQ ID NO: 01
and that has the amino acid mutation S422A, and/or [0639] vi) has
an EC.sub.50 value for the human Tau(pS422) fragment that has the
amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or less, and/or
[0640] vii) has an EC.sub.50 value for the full length human
Tau(pS422) that has the amino acid sequence of SEQ ID NO: 02 of 4.5
ng/mL or less, and/or
[0641] viii) has an EC.sub.50 value for aggregates of human Tau
(0422) that has the amino acid sequence of SEQ ID NO: 02 of 30
ng/mL or less, and/or [0642] ix) has an EC.sub.50 value for the
human Tau that has the amino acid sequence of SEQ ID NO: 01 and
that has the amino acid mutation S422A of 125 ng/mL or less. [0643]
21. A humanized anti-human Tau(pS422) antibody, wherein [0644] a)
the antibody comprises two antibody heavy chains each comprising a
heavy chain variable domain and a heavy chain constant region,
wherein [0645] i) the variable domain has the amino acid sequence
of SEQ ID NO: 19, [0646] ii) the constant region is a human IgG1
constant region, wherein the C-terminal lysine residue can be
present or absent, and [0647] iii) the constant region comprises
the amino acid changes L234A, L235A and P329G, [0648] b) the
antibody comprises two antibody light chains each comprising a
light chain variable domain and a light chain constant domain,
wherein [0649] i) the variable domain has the amino acid sequence
of SEQ ID NO: 16, [0650] ii) the constant region is a human kappa
light chain constant region or a human lambda light chain constant
region, [0651] and [0652] c) the antibody [0653] i) specifically
binds to a polypeptide that has the amino acid sequence of SEQ ID
NO: 03, and/or [0654] ii) does not bind to full length human Tau
(SEQ ID NO: 01) at 1 .mu.g/mL, and/or [0655] iii) specifically
binds to full length human Tau phosphorylated at the serine at
position 422 (SEQ ID NO: 02), and/or [0656] iv) specifically binds
to aggregates of human Tau phosphorylated at the serine at position
422 (SEQ ID NO: 02), and/or [0657] v) specifically binds to full
length human Tau that has the amino acid sequence of SEQ ID NO: 01
and that has the amino acid mutation S422A, and/or [0658] vi) has
an EC.sub.50 value for the human Tau(pS422) fragment that has the
amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or less, and/or
[0659] vii) has an EC.sub.50 value for the full length human
Tau(pS422) that has the amino acid sequence of SEQ ID NO: 02 of 4.5
ng/mL or less, and/or [0660] viii) has an EC.sub.50 value for
aggregates of human Tau (pS422) that has the amino acid sequence of
SEQ ID NO: 02 of 30 ng/mL or less, and/or [0661] ix) has an
EC.sub.50 value for the human Tau that has the amino acid sequence
of SEQ ID NO: 01 and that has the amino acid mutation S422A of 125
ng/mL or less. [0662] 22. A humanized anti-human Tau(pS422)
antibody, wherein [0663] a) the antibody comprises two antibody
heavy chains each comprising a heavy chain variable domain and a
heavy chain constant region, wherein [0664] i) the variable domain
has the amino acid sequence of SEQ ID NO: 19, [0665] ii) the
constant region is a human IgG1 constant region, wherein the
C-terminal lysine residue can be present or absent, and [0666] iii)
the constant region comprises the amino acid changes L234A, L235A
and P329G, [0667] b) the antibody comprises two antibody light
chains each comprising a light chain variable domain and a light
chain constant domain, wherein [0668] i) the variable domain has
the amino acid sequence of SEQ ID NO: 17, [0669] ii) the constant
region is a human kappa light chain constant region or a human
lambda light chain constant region, [0670] and [0671] c) the
antibody [0672] i) specifically binds to a polypeptide that has the
amino acid sequence of SEQ ID NO: 03, and/or [0673] ii) does not
bind to full length human Tau (SEQ ID NO: 01) at 1 .mu.g/mL, and/or
[0674] iii) specifically binds to full length human Tau
phosphorylated at the serine at position 422 (SEQ ID NO: 02),
and/or [0675] iv) specifically binds to aggregates of human Tau
phosphorylated at the serine at position 422 (SEQ ID NO: 02),
and/or [0676] v) specifically binds to full length human Tau that
has the amino acid sequence of SEQ ID NO: 01 and that has the amino
acid mutation S422A, and/or [0677] vi) has an EC.sub.50 value for
the human Tau(pS422) fragment that has the amino acid sequence of
SEQ ID NO: 03 of 6 ng/mL or less, and/or [0678] vii) has an
EC.sub.50 value for the full length human Tau(pS422) that has the
amino acid sequence of SEQ ID NO: 02 of 4.5 ng/mL or less, and/or
[0679] viii) has an EC.sub.50 value for aggregates of human Tau
(pS422) that has the amino acid sequence of SEQ ID NO: 02 of 30
ng/mL or less, and/or [0680] ix) has an EC.sub.50 value for the
human Tau that has the amino acid sequence of SEQ ID NO: 01 and
that has the amino acid mutation S422A of 125 ng/mL or less. [0681]
23. A humanized anti-human Tau(pS422) antibody, wherein [0682] a)
the antibody comprises two antibody heavy chains each comprising a
heavy chain variable domain and a heavy chain constant region,
wherein [0683] i) the variable domain has the amino acid sequence
of SEQ ID NO: 21, [0684] ii) the constant region is a human IgG1
constant region, wherein the C-terminal lysine residue can be
present or absent, and [0685] iii) the constant region comprises
the amino acid changes L234A, L235A and P329G, [0686] b) the
antibody comprises two antibody light chains each comprising a
light chain variable domain and a light chain constant domain,
wherein [0687] i) the variable domain has the amino acid sequence
of SEQ ID NO: 17, [0688] ii) the constant region is a human kappa
light chain constant region or a human lambda light chain constant
region, [0689] and [0690] c) the antibody [0691] i) specifically
binds to a polypeptide that has the amino acid sequence of SEQ ID
NO: 03, and/or [0692] ii) does not bind to full length human Tau
(SEQ ID NO: 01) at 1 .mu.g/mL, and/or [0693] iii) specifically
binds to full length human Tau phosphorylated at the serine at
position 422 (SEQ ID NO: 02), and/or [0694] iv) specifically binds
to aggregates of human Tau phosphorylated at the serine at position
422 (SEQ ID NO: 02), and/or [0695] v) specifically binds to full
length human Tau that has the amino acid sequence of SEQ ID NO: 01
and that has the amino acid mutation S422A, and/or [0696] vi) has
an EC.sub.50 value for the human Tau(pS422) fragment that has the
amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or less, and/or
[0697] vii) has an EC.sub.50 value for the full length human
Tau(pS422) that has the amino acid sequence of SEQ ID NO: 02 of 4.5
ng/mL or less, and/or [0698] viii) has an EC.sub.50 value for
aggregates of human Tau (pS422) that has the amino acid sequence of
SEQ ID NO: 02 of 30 ng/mL or less, and/or [0699] ix) has an
EC.sub.50 value for the human Tau that has the amino acid sequence
of SEQ ID NO: 01 and that has the amino acid mutation S422A of 125
ng/mL or less. [0700] 25. An isolated nucleic acid encoding a
humanized antibody according to any one of items 1 to 24. [0701]
26. A host cell comprising the nucleic acid according to item 25.
[0702] 27. A method of producing a humanized antibody according to
any one of items 1 to 23 comprising the steps of culturing the host
cell as reported herein so that the humanized antibody is produced.
[0703] 28. The method according to item 27, further comprising the
step of recovering the humanized antibody from the cell or the
cultivation medium. [0704] 29. A pharmaceutical formulation
comprising the humanized antibody according to any one of items 1
to 23 and a pharmaceutically acceptable carrier. [0705] 30. The
pharmaceutical formulation according to item 29, further comprising
an additional therapeutic agent. [0706] 31. The pharmaceutical
formulation according to item 30, wherein the additional
therapeutic agent is an anti-amyloid therapeutic agent. [0707] 32.
The pharmaceutical formulation according to item 31, wherein the
anti-amyloid therapeutic agent is an anti-human alpha-synuclein
antibody or an anti-Abeta antibody. [0708] 33. The humanized
antibody according to any one of items 1 to 23 for use as a
medicament. [0709] 34. The humanized antibody according to any one
of items 1 to 23 for use in treating Alzheimer's disease. [0710]
35. The humanized antibody according to any one of items 1 to 23
for use in treating prodromal Alzheimer's disease. [0711] 36. The
humanized antibody according to any one of items 1 to 23 for use in
treating mild Alzheimer's disease. [0712] 37. The humanized
antibody according to any one of items 1 to 23 for use in reducing
Tau(pS422)-induced neurodegeneration. [0713] 38. The humanized
antibody according to any one of items 1 to 23 for use in
maintaining cognition and function. [0714] 39. The humanized
antibody according to any one of items 1 to 23 for use in slowing
the rate of cognitive and functional decline. [0715] 40. Use of the
humanized antibody according to any one of items 1 to 23 in the
manufacture of a medicament. [0716] 41. Use according to any one of
items 33 and 40, wherein the medicament is for treatment of
Alzheimer's disease. [0717] 42. Use according to any one of items
33 and 40 to 41, wherein the medicament is for treatment of
prodromal Alzheimer's disease. [0718] 43. Use according to any one
of items 33 and 40 to 42, wherein the medicament is for treatment
of mild Alzheimer's disease. [0719] 44. Use according to any one of
items 33 and 40 to 43, wherein the medicament is for reducing
Tau(pS422) induced neurodegeneration. [0720] 45. Use according to
any one of items 33 and 40 to 44, wherein the medicament is for
maintaining cognition and function. [0721] 46. Use according to any
one of items 33 and 40 to 45, wherein the medicament is for slowing
the rate of cognitive and functional decline. [0722] 47. A method
of treating an individual having Alzheimer's disease comprising
administering to the individual an effective amount of the
humanized anti-human Tau(pS422) antibody according to any one of
items 1 to 23. [0723] 48. A method of reducing Tau(pS422) induced
neurodegeneration in an individual comprising administering to the
individual an effective amount of the humanized anti-human
Tau(pS422) antibody according to any one of items 1 to 23 to reduce
Tau(pS422) induced neurodegeneration. [0724] 49. A method of
maintaining cognition and function in an individual comprising
administering to the individual an effective amount of the
humanized anti-human Tau(pS422) antibody according to any one of
items 1 to 23 to maintain cognition and function. [0725] 50. A
method of slowing the rate of cognitive and functional decline in
an individual comprising administering to the individual an
effective amount of the humanized anti-human Tau(pS422) antibody
according to any one of items 1 to 23 to slow the rate of cognitive
and functional decline. [0726] 51. Use of the humanized anti-human
Tau(pS422) antibody according to any one of items 1 to 23 in the
reduction of Tau(pS422) induced neurodegeneration. [0727] 52. Use
of the humanized anti-human Tau(pS422) antibody according to any
one of items 1 to 23 in maintaining cognition and function. [0728]
53. Use of the humanized anti-human Tau(pS422) antibody according
to any one of items 1 to 23 in slowing the rate of cognitive and
functional decline. [0729] 54. The humanized antibody according to
any one of items 1 to 23, wherein the antibody i) binds to
Tau(pS422) on brain sections of Tau(pS422) transgenic mice and
Alzheimer's disease patients; and/or labels Tau(pS422) in
Tau(pS422) transgenic cells. [0730] 55. The humanized antibody
according to any one of items 1 to 23, wherein the antibody
specifically binds to/recognizes early and late stage
disease-relevant forms of human Tau(pS422). [0731] 56. The use of
the humanized antibody according to any one of items 1 to 23 for
the prevention of human Tau (pS422)-related Alzheimer's disease
spread. [0732] 57. The use of the humanized antibody according to
any one of items 1 to 23 for the reduction of lysosomal membrane
disintegration. [0733] 58. The use of the humanized antibody
according to any one of items 1 to 23 for the stabilization of
lysosome membrane against human Tau(pS422) induced destabilization
and/or disintegration. [0734] 59. The use of the humanized antibody
according to any one of items 1 to 23 for the prevention of
Alzheimer's disease progression.
V. EXAMPLES
[0735] The following are examples of methods and compositions of
the invention. It is understood that various other embodiments may
be practiced, given the general description provided above.
[0736] Materials and Methods
[0737] Recombinant DNA Techniques
[0738] Standard methods were used to manipulate DNA as described in
Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The
molecular biological reagents were used according to the
manufacturer's instructions.
[0739] Gene and Oligonucleotide Synthesis
[0740] Desired gene segments were prepared by chemical synthesis at
Geneart GmbH (Regensburg, Germany) The synthesized gene fragments
were cloned into an E. coli plasmid for propagation/amplification.
The DNA sequences of subcloned gene fragments were verified by DNA
sequencing. Alternatively, short synthetic DNA fragments were
assembled by annealing chemically synthesized oligonucleotides or
via PCR. The respective oligonucleotides were prepared by metabion
GmbH (Planegg-Martinsried, Germany)
[0741] Reagents
[0742] All commercial chemicals, antibodies and kits were used as
provided according to the manufacturer's protocol if not stated
otherwise.
Example 1
[0743] Preparation and Purification of Rabbit Antibodies
[0744] Immunization
[0745] NZW rabbits from Charles River Laboratories International,
Inc. were used for immunization. Phosphopeptide Tau
(416-430)[pS422] coupled on KLH was solved in K.sub.3PO.sub.4
puffer pH 7.0 at a concentration of 1 mg/ml and mixed (1:1) with
complete Freund's adjuvant (CFA) till generation of stabile
emulsion. Three rabbits received an intra-dermal (i.d.) injection
of 2 ml of emulsion followed by a second intra muscular (i.m.) and
third subcutaneous (s.c.) injection each with 1 ml in one week
interval. The fourth i.m. injection of 1 ml was performed two weeks
later followed by two further s.c. injections of 1 ml in four weeks
interval. 10 ml peripheral whole blood samples of each animal was
collected 4-6 days after third, fourth, fifth and sixth injection
and used for single cell sorting in FACS. Additional 0.5 ml serum
of each animal was collected at the same time and used for the
determination of Tau (416-463)[pS422] specific antibody
response.
[0746] Antibody Response
[0747] The antibody response to the immunization was determined by
serial dilution of sera using an ELISA, in which 30 ng per well of
biotinylated Tau (416-430)[pS422] was incubated in 1.times. PBS at
4.degree. C. overnight on streptavidin pre-coated 96wells
microtiter plates (MC1347, Micro Coat Biotechnologie GmbH,
Bernried, Germany). For detection, goat anti-rabbit IgG linked to a
horseradish peroxidase (The Jackson laboratory) was used at 1:16000
dilution. BM Blue POD Substrate, precipitating Tetramethyl
benzidine (TMB), ready-to-use solution from Roche Diagnostics GmbH
was used for visualization. Reaction was stopped via 1N HCl and
measured in Tecan Infinite by 450/690 nm.
[0748] B-Cell Cloning
[0749] Coating of Plates
[0750] Sterile streptavidin-coated 6-well plates (cell culture
grade) were incubated with either a mixture of 3 biotinylated
control peptides (non-phosphorylated Tau (416-430), MCAK_Hunaan
(88-102)[95-pSer] and MAP2_Human (1802-1816)[pSer-1802]) or with
the biotinylated phospho-peptide Tau (416-430)[pS422] each in a
concentration at 0.5-1 .mu.g/ml in PBS at room temperature for 1 h.
Plates were washed in sterile PBS three times before use. Cell
culture 6-well plates were coated with 2 .mu.g/ml KLH (keyhole
limpet haemocyanine) in carbonate buffer (0.1 M sodium bicarbonate,
34 mM Disodiumhydrogencarbonate, pH 9.55) over night at 4
C..degree.. Plates were washed in sterile PBS three times before
use.
[0751] Isolation of Rabbit Peripheral Blood Mononuclear Cells
(PBMC)
[0752] EDTA containing whole blood was diluted twofold with
1.times. PBS before density centrifugation on lympholyte mammal
(Cedarlane Laboratories) which was performed to isolate rabbit
PBMC. PBMCs were washed twice before staining with antibodies.
[0753] EL-4 B5 Medium
[0754] RPMI 1640 (Pan Biotech, Aidenbach, Germany) supplemented
with 10% FCS (Hyclone, Logan, Utah, USA), 2 mM Glutamine, 1%
penicillin/streptomycin solution (PAA, Pasching, Austria), 2 mM
sodium pyruvate, 10 mM HEPES (PAN Biotech, Aidenbach, Germany) and
0.05 mM beta-mercaptoethanol (Gibco, Paisley, Scotland)
[0755] Depletion of Macrophages/Monocytes
[0756] Sterile 6-well plates (cell culture grade) were used to
deplete macrophages and monocytes through unspecific adhesion.
Wells were either coated with KLH (keyhole limpet haemocyanine) or
with streptavidin and the control peptides. Each well was filled
with at maximum 4 ml medium and up to 6.times.10.sup.6 peripheral
blood mononuclear cells from the immunized rabbit and allowed to
bind for 1 h at 37.degree. C. in the incubator. 50% of the cells in
the supernatant were used for the panning step; the remaining 50%
of cells were directly subjected to immune fluorescence staining
and single cell sorting.
[0757] Panning B-Cells on Peptides
[0758] 6-well tissue culture plates coated with streptavidin and
the biotinylated peptide Tau (416-430)[pS422] were seeded with up
to 6.times.10.sup.6 cells per 4 ml medium and allowed to bind for 1
h at 37.degree. C. in the incubator. Non-adherent cells were
removed by carefully washing the wells 1-2 times with 1.times. PBS.
The remaining sticky cells were detached by trypsin for 10 min at
37.degree. C. in the incubator and then washed twice in media. The
cells were kept on ice until the immune fluorescence staining.
[0759] Immune Fluorescent Staining and Single Cell Sorting
[0760] Anti-rabbit IgG FITC used for single cell sorting was from
AbD Serotec (STAR121F, Dusseldorf, Germany). For surface staining,
cells from the depletion and panning step were incubated with
anti-rabbit IgG FITC antibody in PBS for 30 min rolling in the cold
room at 4.degree. C. in the dark. Following centrifugation, the
supernatants were removed by aspiration. The PBMCs were subjected
to 2 cycles of centrifugation and washing with ice cold PBS.
Finally the PBMCs were resuspended in ice cold PBS and immediately
subjected to the FACS analyses. Propidium iodide in a concentration
of 5 .mu.g/ml (BD Pharmingen, San Diego, Calif., USA) was added
prior to the FACS analyses to discriminate between dead and live
cells. FACS was performed using a Becton Dickinson FACSAria
equipped the FACSDiva software (BD Biosciences, USA) and single,
FITC-labeled, live cells were deposited in 96-well plates.
[0761] B-Cell Culture
[0762] B-cell cultures were prepared by a method similar to that
described by Zubler, R. H. et al., J. Immunol. 134 (1985)
3662-3668. Briefly, single sorted B cells were cultured in 96-well
plates with 210 .mu.l/well EL-4 B5 medium with Pansorbin Cells
(1:20000) (Calbiochem (Merck), Darmstadt, Deutschland), 5% rabbit
thymocyte supernatant and gamma-irradiated EL-4-B5 murine thymoma
cells (2.times.10.sup.4/well) for 7 days at 37.degree. C. in an
atmosphere of 5% CO.sub.2 in the incubator. B cell culture
supernatants were removed for screening and the cells harvested
immediately for variable region gene cloning or frozen at
-80.degree. C. in 100 .mu.l RLT buffer (Qiagen, Hilden,
Germany).
[0763] B-Cell Clone Screening
[0764] B-cell culture supernatants were screened for binding to
biotinylated Tau (416-430)[pS422] by ELISA. Non-phosphorylated Tau
(416-430), KLH (keyhole limpet haemocyanine) and the unrelated
phospho-peptide MCAK_Human (88-102)[95-pSer] were used as control
antigens. For the preparation of ELISA plates, streptavidin
pre-coated microtiter plates were incubated with biotinylated Tau
(415-430)[pS422] at 50 ng/ml for 1 hour at room temperature.
Coating with KLH or control peptides was performed at 1 B cell
supernatants were diluted 1:5 to 1:10 and were incubated in the
antigen coated microtiter plates for 60 min. After intensive
washing, the binding of the rabbit antibodies was detected using a
sheep anti-rabbit IgG digoxigenin conjugated detection antibody
(Chemicon AQ301D). After incubation with TMB at room temperature,
absorbance at 370 nm-492 nm was measured. B-cell clones yielding
signals above background with biotinylated Tau (416-430)[pS422] but
not with KLH and MCAK_Human (88-102)[95-pSer] were further
considered and subjected to variable region gene cloning.
[0765] PCR Amplification of V-Domains and Sequencing
[0766] Total RNA was prepared using the NucleoSpin.RTM. 8/96 RNA
kit (Macherey&Nagel; 740709.4, 740698) according to
manufacturer's protocol. All steps were done on an epMotion 5075
liquid handling system (Eppendorf). RNA was eluted with 60 .mu.l
RNAse free water. 6 .mu.l of RNA was used to generate cDNA by
reverse transcriptase reaction using the Superscript III
First-Strand Synthesis SuperMix (Invitrogen 18080-400) and an oligo
dT-primer according to the manufacturer's instructions. 4 .mu.l of
cDNA were used to amplify the immunoglobulin heavy and light chain
variable regions (VH and VL) with the AccuPrime SuperMix
(Invitrogen 12344-040) in a final volume of 50 .mu.l using the
primers rbHCfinal.up and rbHCfinal.do for the heavy chain and
rbLCfmal.up and rbLCfinal.do for the light chain (see Table below).
The PCR conditions were as follows: Hot start at 94.degree. C. for
5 min; 35 cycles of 20 s at 94.degree. C., 20 s at 70.degree. C.,
45 s at 68.degree. C., and a final extension at 68.degree. C. for 7
min.
TABLE-US-00008 TABLE rbHCfinal.up AAGCTTGCCACCATGGAGACTGGGCTGCGCTGG
(SEQ ID NO: 61) CTTC rbHCfinal.do CCATTGGTGAGGGTGCCCGAG (SEQ ID NO:
62) rbLCfinal.up AAGCTTGCCACCATGGACAYGAGGGCCCCCACTC (SEQ ID NO: 63)
rbLCfinal.do CAGAGTRCTGCTGAGGTTGTAGGTAC (SEQ ID NO: 64)
[0767] 8 .mu.l of 50 .mu.l PCR solution were loaded on a 48 E-Gel
2% (Invitrogen G8008-02). Positive PCR reactions were cleaned using
the NucleoSpin.RTM. Extract H kit (Macherey&Nagel; 740609250)
according to manufacturer's protocol and eluted in 50 .mu.l elution
buffer. 12 .mu.l of purified PCR products were sequenced directly
in both directions using the rbHCfinal.up and rbHCfinal.do for
heavy chains and rbLCfmal.up and rbLCfinal.do for light chains (see
Table above).
[0768] Recombinant Expression of Rabbit Monoclonal Antibodies and
Rabbit/Mouse Chimeric Antibodies
[0769] For recombinant expression of rabbit monoclonal antibodies,
PCR-products coding for VH or VL were cloned as cDNA into
expression vectors by the overhang cloning method (Haun, R. S. et
al., BioTechniques 13 (1992) 515-518; Li, M. Z., et al., Nature
Methods 4 (2007) 251-256). Linearized expression plasmids coding
for the rabbit kappa or gamma constant region and VL of VH inserts
were amplified by PCR using overlapping primers. Purified PCR
products were incubated with T4 DNA-polymerase which generated
single-strand overhangs. The reaction was stopped by dCTP addition.
In the next step, plasmid and insert were combined and incubated
with RecA which induced site specific recombination. The recombined
plasmids were transformed into E.coli. The next day the grown
colonies were picked and tested for correct recombined plasmid by
plasmid preparation, restriction analysis and DNA-sequencing. For
antibody expression, the isolated HC and LC plasmids were
transiently co-transfected into HEK293 cells and the supernatants
were harvested after 1 week. For cloning and expression of rabbit
mouse chimeric antibodies, the VH and VL regions were amplified by
PCR and sub-cloned into expression vectors containing the mouse
constant kappa or mouse constant gamma 1 region. The rabbit/mouse
chimeric HC and LC plasmids were isolated, tested by restriction
analysis and DNA-sequencing for correct insertion and transiently
co-transfected into HEK293 cells. Supernatants were harvested one
week after transfection.
[0770] Antibody Purification
[0771] Recombinantly expressed rabbit antibodies were purified from
cell culture supernatants on MabSelectSuRe.TM. columns (GE
Healthcare). Prior to sample load the column was equilibrated with
25 mmol/L Tris-HCl, 25 mmol/L NaCl, pH 7.4. Elution of the antibody
was achieved with 50 mmol/L acetate pH 3.14. The eluted sample was
immediately loaded onto a desalting column (Sephadex G25, GE
Healthcare) and eluted in 20 mmol/L His-HCl, 140 mmol/L NaCl pH
6.0. This buffer was also used for the storage of purified
antibody. General storage temperature was 4.degree. C., room
temperature during the purification process and -80.degree. C.
after aliquotation. Recombinantly expressed rabbit/mouse chimaeras
antibodies from cell culture supernatants were purified on
MabSelectSuRe.TM. columns (GE Healthcare). Prior to sample load the
column was equilibrated with 1.times. PBS, pH 7.4. Elution of the
antibodies was achieved with 100 mmol/L citrate pH 3.0. The eluted
sample was immediately neutralized with 2 mol/L Tris/HCl pH 9.0.
Afterwards the antibodies are loaded onto a size exclusion column
(Superdex 200, GE Healthcare) and eluted in 20 mmol/L His-HCl, 140
mmol/L NaCl pH 6.0. This buffer was also used for the storage of
purified antibodies. General storage temperature was 4.degree. C.,
room temperature during the purification process and -80.degree. C.
after aliquotation.
Example 2
[0772] Anti-Tau pS422 Monoclonal Rabbit Antibodies are Highly
Selective for Tau Phosphorylated at pS422 and Bind to Fibrillary
Aggregates of Tau pS422
[0773] ELISA
[0774] Rabbit monoclonal antibodies were recombinantly expressed in
HEK 293 cells. Cell culture supernatants or purified rabbit
antibodies were tested for binding to biotinylated Tau
(416-430)[pS422], non-phosphorylated Tau (416-430), KLH (key hole
limpet haemocyanine) and the unrelated phospho-peptide MCAK Human
(88-102)[95-pSer] by ELISA. For the preparation of ELISA plates,
streptavidin pre-coated microtiter plates were incubated with
biotinylated Tau (415-430)[pS422] at 50 ng/ml for 1 hour at room
temperature. Coating with KLH or control peptides was performed at
1 .mu.g/ml. Rabbit Anti Tau pS422 antibody (Abeam AB51071) or
rabbit antibody containing supernatants were incubated in the
antigen labeled microtiter plates for 60 min at various
concentrations. After intensive washing, the binding of the rabbit
antibodies was detected using a sheep anti-rabbit IgG digoxigenin
conjugated detection antibody (Chemicon AQ301D). After incubation
with TMB at room temperature absorbance at 370 nm-492 nm was
measured. The antibody binding was characterized by its EC50
values. The antibody binding to biotinylated Tau (416-430)[pS422]
and non-phosphorylated Tau (416-430) peptides was characterized by
its EC50 values. Cross-reactivity with KLH or MCAK phosphopeptide
was estimated by single-point measurement at high concentrations,
i.e. at 1:5 dilution of the cell culture supernatants. Results are
shown in the Table below. EC50 values of binding to Tau
phosphopeptide were found to be more than 100 times lower than EC50
values of binding to Tau peptide, indicating at least 100 fold
selectivity for phosphorylated Tau fragment compared to
non-phosphorylated Tau peptide. Binding to KLH and MCAK control
phosphopeptide was at background level with all antibodies, which
is about 1<3% of the maximal value measures with Tau
phosphopeptide.
TABLE-US-00009 TABLE: EC.sub.50 EC.sub.50 IgG phos- non-phos- titer
of OD 1:5 dilution of phorylated phorylated superna- supernatant
Tau peptide Tau peptide tant KLH MCAK (.mu.g/ml) (.mu.g/ml)
(.mu.g/ml) (mE) (mE) Mab 005 <0.003 3.727 5.818 0.026 0.067 Mab
019 <0.003 1.076 6.958 0.026 0.023 Mab 020 0.002 >3.369 3.369
0.016 0.010 Mab 085 0.0009 0.146 6.46 0.029 0.062 Mab 086 0.0011
0.266 8.84 0.046 0.104 Mab 097 0.0013 1.281 19.87 0.042 0.029
[0775] Specificity for soluble and aggregated full-length Tau pS422
was also tested. Fibrillary aggregates of Tau pS422 (300 .mu.g/ml)
were coated to a Polystyrene based Maxisorb microtiter plate (Nunc)
overnight at RT. In similar manner, soluble full-length Tau and Tau
pS422 were coated to the Maxisorb microtiter plate. Rabbit Anti Tau
pS422 antibody control (Abeam AB51071), or purified rabbit
antibodies were added and incubated for 60 min in concentrations up
to 1000 ng/ml. After intensive washing, the binding of the rabbit
antibodies was detected using a sheep anti-rabbit IgG digoxigenin
conjugated detection antibody (Chemicon AQ301D). After incubation
with TMB at room temperature absorbance at 370 nm-492 nm was
measured. The antibody binding was characterized by its EC50
values. Results are shown in the following Table.
TABLE-US-00010 TABLE EC.sub.50 fibrillary Rabbit EC.sub.50 Tau
pS422 EC.sub.50 Tau protein Tau pS422 Mab protein (.mu.g/ml)
(.mu.g/ml) (.mu.g/ml) Mab 005 0.00034 no binding 0.00755 Mab 019
0.00038 no binding 0.00059 Mab 020 0.00036 no binding 0.00042 Mab
085 0.00025 no binding 0.00074 Mab 086 0.00023 no binding 0.00048
Mab 097 0.00040 no binding 0.01358
[0776] Rabbit monoclonal antibodies bound to Tau-pS422 protein with
EC50 values below 1 ng/ml. Fibrillary Tau pS422 was detected with
EC50 values ranging from 0.4 ng/ml to 14 ng/ml. Signals for binding
to non-phosphorylated full-lengths Tau protein were
indistinguishable from background levels. Therefore it was
estimated that each of the antibodies binds to Tau pS422 and
fibrillary Tau pS422 with a selectivity of at least 100-fold
compared to Tau.
[0777] BIAcore.TM.
[0778] Binding to fibrillary Tau pS422 aggregates was further
investigated and confirmed by BIAcore.TM. analysis. Measurements
were performed using the BIAcore 3000 instrument at 37.degree. C.
The system and sample buffer was HBS-EP (10 mM HEPES, 150 mM NaCl,
3.4 mM EDTA, 0.005% Polysorbate 20 (v/v)). A BIAcore.TM. CM5 sensor
chip was subjected to a preconditioning procedure. Sequentially
0.1% SDS, 50 mM NaOH, 10 mM HCl and 100 mM H.sub.3PO.sub.4 were
injected for 30 sec over the flow cells FC1, FC2, FC3 and FC4. The
amine coupling procedure was done according to the manufacturer's
instructions using the BIAcore 3000.TM. wizard v. 4.1. After an
EDC/NHS activation of the sensor surface, a non-phosphoselective
anti-Tau antibody mAb <TAU>M-4/53-IgG was immobilized on
sensor flow cells FC2, FC3 and FC4. As a control, an antibody
against CK-MM (creatine kinase isotype), recognizing an irrelevant
antigen, was captured on the FC1. mAb <TAU>M-4/53-IgG and the
antibody against CK-MM were diluted at 30 .mu.g/ml in 10 mM NaAc pH
5.0 and were injected at 10 .mu.l/min for 7 min contact time to
immobilize 10.000 RU of the antibody capturing system. The surface
was deactivated by saturation with 1M Ethanolamine. The sensor was
conditioned by 5 cycles with phosphorylated filamentous Tau protein
(stock 0.3 mg/ml diluted 1:100 in HBS-EP) as analyte in solution at
10 .mu.l/min for 2 min. Regeneration was performed with 10 mM
Glycine pH 2.5 at 30 .mu.l/min for 3 min. It is assumed, that the
analyte binding to mAb 4/53 does not dissociate the pTau filaments,
because no dissociation of pTau filaments from the mAb 4/53 could
be observed. For all further measurement cycles, 0.3 mg/ml pTau
filaments were diluted 1:100 in HBS-EP buffer and were injected at
10 .mu.l/min for 1 min. in order to present pTau to the respective
antibody analytes in a heterogeneous sandwich-mode. The antibody
analytes were diluted in HBS-EP buffer to a concentration of 100 nM
and were injected into the system at 20 .mu.l/min for 3 min. After
3 min of dissociation the sensor surface was regenerated by 2
injections of a 10 mM Glycine pH 2.5 for 1 min at 100 .mu.l/min
followed by a HBS-wash for 15 sec at 100 .mu.l/min. The association
and dissociation phase of the interactions were monitored. Since
the antibody analyte in solution is bivalent, the avidity-burdened
antibody-pTau kinetics were characterized by a biphasic
dissociation model, consisting of a fast affinity-based early
dissociation step followed by an avidity-stabilized, but
rate-limiting kinetic step in the latter complex dissociation. 10
sec(early) and 50 sec(late) after analyte injection end, the kd and
t/2(diss) were quantified, where possible. The kinetic measurements
were evaluated using a double referencing procedure. First the
signal from the FC1 reference was subtracted to correct the buffer
bulk effect and unspecific binding. Second the 0 nM analyte
injection was subtracted to correct the dissociation of the primary
antibodies from the respective capturing system. The kinetic rates
were evaluated using a Langmuir 1.1 dissociation fit model
according to the BIAcore.TM. evaluation software v.4.1. The
antigen/antibody complex stability halftime (min) was calculated
according to the formula ln(2)/60*kd.
[0779] Results are summarized in the following Table.
TABLE-US-00011 TABLE early (10 s) late (50 s) t/2diss t/2diss Clone
kd (1/s) (min) kd (1/s) (min) Mab 005 2.19E-03 5.3 3.12 .times.
10.sup.-3 4 Mab 019 1.43E-02 0.8 6.17 .times. 10.sup.-4 19 Mab 020
3.28E-03 3.5 4.08 .times. 10.sup.-4 28 Mab 085 n.d. n.d. 6.60
.times. 10.sup.-4 18 Mab 086 1.62E-03 7.2 3.68 .times. 10.sup.-4 32
Mab 097 n.d. n.d. n.d. n.d.
Example 3
[0780] Binding of Anti-Tau pS422 Monoclonal Rabbit Antibodies to
Intracellular pTau in Brain Sections of Alzheimer's Disease
Patients
[0781] The specific and sensitive immunohistochemical detection of
pTau pathology in Alzheimer's disease brain tissue by monoclonal
rabbit anti-Tau pS422 antibodies was investigated by
immunofluorescence staining experiments using cryosections of human
brain tissue from AD patients. The procedure was basically the same
as described in example X (murine antibodies). Rabbit IgGs were
detected by goat anti rabbit Alexa Fluor488.RTM. conjugated
secondary antibodies (Invitrogen/Molecular Probes A11034). Specific
and sensitive staining of pTau deposits and filaments is evident
for clones Mab 005, Mab 019, Mab 020, Mab 085, Mab 086 and Mab 097.
Intracellular pTau deposits, like large neurofibrillary tangles and
elongated neutrophil threads, are noticeable. A minimal effective
concentration ranging between 0.08 and 0.016 .mu.g/ml was
determined for all clones investigated, which indicates highly
sensitive binding to genuine human pTau deposits.
Example 4
[0782] Humanization of Rabbit Anti-Human Tau(pS422) Antibodies
[0783] The "variable domain" (variable domain of a light chain
(VL), variable domain of a heavy chain (VH)) as used herein denotes
each of the pair of light and heavy chain domains which are
involved directly in binding the antibody to the Tau(pS422)
antigen. The variable light and heavy chain domains have the same
general structure and each domain comprises four framework (FR)
regions whose sequences are widely conserved, connected by three
"hypervariable regions".
[0784] The structures of the VH and the VL domain of the rabbit
antibody mAb 086 were analyzed in silico and compared to a
structural database of human VH and VL domains (MGT). A panel of
structurally most similar V domains was chosen for grafting the
CDRs of the rabbit antibody onto the chosen human VH and VL
domains. In addition, similarities in the primary sequence were
taken into account to narrow down the choice of the human V domains
by aligning the primary sequence of the VH and VL domain of the
rabbit antibody to the human V domain repertoire. Backmutations
within the human framework regions to rabbit parent residues were
introduced in some humanization variants. Similarly, mutations in
the CDRs were introduced in some variants where appropriate to
potentially increase the affinity to the antigen, to maintain the
CDR tertiary structure, and to remove unwanted features like
cysteine residues or residues that can undergo modification after
antibody purification.
[0785] The heavy and light chain vectors containing each of the
humanized variant were co-transfected into HEK293 suspension cells
in microtiter culture plates in a matrix manner to obtain cell
cultures expressing full size IgG of all possible light/heavy chain
combinations. After 5 days cultivation at 37.degree. C., the
supernatants were harvested and purified by protein A affinity
chromatography in the microtiter scale.
Example 5
[0786] Generation of Recombinant Expression Vectors
[0787] a) Generation of Vectors for the Expression of
Immunoglobulin Heavy Chains Using the Human IgG1 Constant
Region
[0788] The humanized heavy chain encoding fusion gene comprising
the human IgG1 constant region (CH1, hinge, CH2, CH3) and a
humanized anti-human Tau(pS422) antibody VH domain derived from
rabbit antibody Mab 086 was assembled by fusing a DNA fragment
coding for the respective anti-human Tau(pS422)-specific antibody
VH domain to a sequence element coding the human IgG1 constant
region.
[0789] The human IgG1 constant region has the following amino acid
sequence:
TABLE-US-00012 (SEQ ID NO: 58) ASTKGPSVFP LAPSSKSTSG GTAALGCLVK
DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS
NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS
HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA
LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP
ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT
QKSLSLSPGK.
[0790] The expression vector also comprised an origin of
replication from the vector pUC18, which allows replication of this
plasmid in E. coli, and a beta-lactamase gene which confers
ampicillin resistance in E. coli.
[0791] The transcription unit of the antibody heavy chain comprises
the following functional elements in 5' to 3' direction: [0792] the
immediate early enhancer and promoter from the human
cytomegalovirus (P-CMV) including intron A, [0793] a human heavy
chain immunoglobulin 5'-untranslated region (5'UTR), [0794] a
murine immunoglobulin heavy chain signal sequence, [0795] a heavy
chain variable (VH) domain encoding nucleic acid, [0796] a human
IgG1 constant region encoding nucleic acid, and [0797] the bovine
growth hormone polyadenylation sequence (BGH pA).
[0798] b) Generation of Vectors for the Expression of
Immunoglobulin Light Chains Using the Human Ig-Kappa Constant
Region
[0799] The humanized kappa light chain encoding fusion gene
comprising the human Ig-kappa constant region (CL-kappa) and an
anti-human Tau(pS422) antibody VL (kappa) domain derived from
rabbit antibody Mab 086 was assembled by fusing a DNA fragment
coding for the respective anti-human Tau(pS422) antibody VL (kappa)
domain to a sequence element coding for the human Ig-kappa constant
region.
[0800] The human Ig-kappa constant region has the following amino
acid sequence:
TABLE-US-00013 (SEQ ID NO: 59) RTVAAPSVFI FPPSDEQLKS GTASVVCLLN
NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT
HQGLSSPVTK SFNRGEC.
[0801] The expression vector also comprised an origin of
replication from the vector pUC18, which allows replication of this
plasmid in E. coli, and a beta-lactamase gene which confers
ampicillin resistance in E. coli.
[0802] The transcription unit of the antibody kappa light chain
comprises the following functional elements in 5' to 3' direction:
[0803] the immediate early enhancer and promoter from the human
cytomegalovirus (P-CMV) including intron A, [0804] a human heavy
chain immunoglobulin 5'-untranslated region (5'UTR), [0805] a
murine immunoglobulin heavy chain signal sequence, [0806] a light
chain variable (VL) domain encoding nucleic acid, [0807] a human
Ig-kappa constant region encoding nucleic acid, and [0808] the
bovine growth hormone polyadenylation sequence (BGH pA).
[0809] c) Generation of Vectors for the Expression of
Immunoglobulin Light Chains Using the Human Ig-Lambda Constant
Region
[0810] The humanized lambda light chain encoding fusion gene
comprising the human Ig-lambda constant region (CL-lambda) and an
anti-human Tau(pS422) antibody VL (lambda) domain derived from
rabbit antibody Mab 086 was assembled by fusing a DNA fragment
coding for the respective anti-human Tau(pS422) antibody VL
(lambda) domain to a sequence element coding for the human
Ig-lambda constant region.
[0811] The human Ig-lambda constant region has the following amino
acid sequence:
TABLE-US-00014 (SEQ ID NO: 60) GQPKAAPSVT LFPPSSEELQ ANKATLVCLI
SDFYPGAVTV AWKADSSPVK AGVETTTPSK QSNNKYAASS YLSLTPEQWK SHRSYSCQVT
HEGSTVEKTV APTECS.
[0812] The expression vector also comprised an origin of
replication from the vector pUC18, which allows replication of this
plasmid in E. coli, and a beta-lactamase gene which confers
ampicillin resistance in E. coli.
[0813] The transcription unit of the antibody lambda light chain
comprises the following functional elements in 5' to 3' direction:
[0814] the immediate early enhancer and promoter from the human
cytomegalovirus (P-CMV) including intron A, [0815] a human heavy
chain immunoglobulin 5'-untranslated region (5'UTR), [0816] a
murine immunoglobulin heavy chain signal sequence, [0817] a
variable light chain (VL) domain encoding nucleic acid, [0818] a
human Ig-lambda constant region encoding nucleic acid, and [0819]
the bovine growth hormone polyadenylation sequence (BGH pA).
[0820] d) Generation of Vectors for the Expression of
Immunoglobulin Kappa Light Chains Using the Human Ig-Kappa Constant
Region
[0821] The human Ig-kappa light chain encoding fusion gene
comprising the human Ig-kappa constant region (CL-kappa) and an
anti-human Tau(S422) antibody VL (kappa) domain derived from rabbit
antibody Mab 086 was assembled by fusing a DNA fragment coding for
the respective anti-human Tau(pS422)-antibody VL (kappa) domain to
a sequence element coding for the human Ig-kappa constant region.
The construct was in a genomic organization, i.e. introns were
present in the signal peptide and between the VL (kappa) and the
CL-kappa domains
[0822] The expression vector also comprised an origin of
replication from the vector pUC18, which allows replication of this
plasmid in E. coli, and a beta-lactamase gene which confers
ampicillin resistance in E. coli.
[0823] The transcription unit of the antibody kappa light chain
comprises the following functional elements in 5' to 3' direction:
[0824] the immediate early enhancer and promoter from the human
cytomegalovirus (P-CMV) [0825] a human heavy chain immunoglobulin
5'-untranslated region (5'UTR), [0826] a murine immunoglobulin
heavy chain signal sequence, [0827] a light chain variable (VL)
domain encoding nucleic acid, [0828] a human IgG kappa constant
region, and [0829] the bovine growth hormone polyadenylation
sequence (BGH pA).
[0830] e) Generation of Vectors for the Expression of
Immunoglobulin Lambda Light Chains Using the Human Ig-Lambda
Constant Region
[0831] The human Ig-lambda light chain encoding fusion gene
comprising the human Ig-lambda constant region (CL-lambda) and an
anti-human Tau(S422) antibody VL (lambda) domain derived from
rabbit antibody Mab 086 was assembled by fusing a DNA fragment
coding for the respective anti-human Tau(pS422)-antibody VL
(lambda) domain to a sequence element coding for the human
Ig-lambda constant region. The construct was in a genomic
organization, i.e. introns were present in the signal peptide and
between the VL (lambda) and the CL-lambda domains.
[0832] The expression vector also comprised an origin of
replication from the vector pUC18, which allows replication of this
plasmid in E. coli, and a beta-lactamase gene which confers
ampicillin resistance in E. coli.
[0833] The transcription unit of the antibody lambda light chain
comprises the following functional elements in 5' to 3' direction:
[0834] the immediate early enhancer and promoter from the human
cytomegalovirus (P-CMV) [0835] a human heavy chain immunoglobulin
5'-untranslated region (5'UTR), [0836] a murine immunoglobulin
heavy chain signal sequence, [0837] a light chain variable (VL)
domain encoding nucleic acid, [0838] a human IgG lambda constant
region, and [0839] the bovine growth hormone polyadenylation
sequence (BGH pA).
Example 6
[0840] Recombinant Production of Anti-Human Tau(pS422)
Antibodies
[0841] The antibodies were produced in transiently transfected
HEK293 cells (human embryonic kidney cell line 293-derived)
cultivated in F17 Medium (Invitrogen Corp.). For transfection of
the respective vectors as described in Example 5 "293-Free"
Transfection Reagent (Novagen) was used. The antibodies were
expressed from individual expression plasmids. Transfections were
performed as specified in the manufacturer's instructions.
Recombinant antibody-containing cell culture supernatants were
harvested three to seven days after transfection. Supernatants were
stored at reduced temperature (e.g. -80.degree. C.) until
purification.
[0842] General information regarding the recombinant expression of
human immunoglobulins in e.g. HEK293 cells is given in: Meissner,
P. et al., Biotechnol. Bioeng. 75 (2001) 197-203.
Example 7
[0843] Purification of Recombinant Anti-Human Tau(pS422)
Antibodies
[0844] The antibody-containing culture supernatants were filtered
and purified by two chromatographic steps.
[0845] The antibodies were captured by affinity chromatography
using HiTrap MabSelectSuRe (GE Healthcare) equilibrated with PBS (1
mM KH.sub.2PO.sub.4, 10 mM Na.sub.2HPO.sub.4, 137 mM NaCl, 2.7 mM
KCl), pH 7.4. Unbound proteins were removed by washing with
equilibration buffer, and the antibody was recovered with 25 mM
citrate buffer, pH 3.1, which was immediately after elution
adjusted to pH 6.0 with 1 M Tris-base, pH 9.0.
[0846] Size exclusion chromatography on Superdex 200.TM. (GE
Healthcare) was used as second purification step. The size
exclusion chromatography was performed in 20 mM histidine buffer,
0.14 M NaCl, pH 6.0. The antibody containing solutions were
concentrated with an Ultrafree-CL centrifugal filter unit equipped
with a Biomax-SK membrane (Millipore, Billerica, Mass., USA) and
stored at -80.degree. C.
Example 8
[0847] Kinetic Screening
[0848] The kinetic screening was performed according to Schraeml,
et al. (Schraeml, M. and M. Biehl, Methods Mol. Biol. 901 (2012)
171-181) on a BIAcore 4000 instrument, mounted with a BIAcore CMS
sensor. The BIAcore 4000 instrument was under the control of the
software version V1.1. A BIAcore CM5 series S chip was mounted into
the instrument and was hydrodynamically addressed and
preconditioned according to the manufacturers instructions. The
instrument buffer was HBS-EP buffer (10 mM HEPES (pH 7.4), 150 mM
NaCl, 1 mM EDTA, 0.05% (w/v) P20). An antibody capture system was
prepared on the sensor surface. A polyclonal goat anti-human
antibody with human IgG-Fc specificity (Jackson Lab.) was
immobilized at 30 .mu.g/ml in 10 mM sodium acetate buffer (pH 5) to
spots 1, 2, 4 and 5 in the instrument's flow cells 1, 2, 3 and 4 at
10,000 RU using NHS/EDC chemistry. In each flow cell the antibodies
were captured on spot 1 and spot 5. Spot 2 and spot 4 were used as
reference spots. The sensor was deactivated with a 1 M ethanolamine
solution. Humanized antibody derivatives were applied at
concentrations between 44 nM and 70 nM in instrument buffer
supplemented with 1 mg/ml CMD (carboxymethyldextrane). The
antibodies were injected at a flow rate of 30 .mu.l/min for 2 min.
The capture level (CL) of the surface-presented antibodies was
measured in rel. response units (RU). The analytes in solution,
phosphorylated human tau protein, non-phosphorylated human tau
protein and the phosphorylated human tau mutant protein T422S, were
injected at 300 nM for 3 min. at a flow rate of 30 .mu.l/min. The
dissociation was monitored for 5 min. The capture system was
regenerated by a 1 min. injection of 10 mM glycine buffer pH 1.7 at
30 .mu.L/min. over all flow cells. Two report points, the recorded
signal shortly before the end of the analyte injection, denoted as
binding late (BL) and the recorded signal shortly before the end of
the dissociation time, stability late (SL), were used to
characterize the kinetic screening performance. Furthermore, the
dissociation rate constant kd (1/s) was calculated according to a
Langmuir model and the antibody/antigen complex half-life was
calculated in minutes according to the formula ln(2)/(60*kd). The
molar ratio (MR) was calculated according to the formula
MR=(Binding Late (RU))/(Capture level
(RU))*(MW(antibody)/(MW(antigen)). In case the sensor was
configured with a suitable amount of antibody ligand capture level,
each antibody should be able to functionally bind at least to one
analyte in solution, which is represented by a molar ratio of
MR=1.0. Then, the molar ratio is also an indicator for the valence
mode of analyte binding. The maximum valence can be MR=2 for an
antibody binding two analytes, one with each Fab valence.
[0849] In another embodiment, kinetic rates were determined at
25.degree. C. and 37.degree. C. using the same experimental setup,
but using multiple concentration series of each analyte in solution
at 0 nM (buffer), 1.2 nM, 3.7 nM, 11.1 nM, 33.3 nM, 100 nM and 300
nM. From the concentration-dependent binding behavior the kinetic
data was calculated using the BIAcore evaluation software according
to the manufacturer's instructions and a Langmuir 1.1 model with
RMAX global.
Example 9
[0850] ELISA
[0851] To not-coated Maxisorb plates non-biotinylated
peptide/protein/aggregate and to streptavidin coated Maxisorb
plates biotinylated peptide/protein/aggregate in PBS were added and
incubated over-night. The supernatant was discarded and the wells
washed three times with 90 .mu.l wash buffer (1.times. PBS/0.1%
Tween 20). Remaining reactive spots were blocked with blocking
buffer (1.times.PBS/2% BSA (Bovine Serum Albumin Fraction V. fatty
acid free, Roche, Cat. No.: 10735078001)10.05% Tween 20) by
incubating for 1 h. The supernatant was discarded and the wells
washed three times with 90 .mu.l wash buffer. Samples and control
antibody were prepared in 12 dilutions. (1:2) in ELISA buffer
(1.times.PBS/0.5% BSA (Bovine Serum Albumin Fraction V, fatty acid
free, Roche, Cat. No.: 10735078001)/0.05% Tween 20) with a start
concentration of 500 ng/mL. The incubation time was 60 minutes at
RT on a shaker. The supernatant was discarded and the wells washed
three times with 90 .mu.l wash buffer. Solutions of the secondary
antibody were prepared in ELISA buffer. A total of 25 .mu.l
antibody-mix was transferred in all wells of the assay plate and
the plate was thereafter incubated on shaker for 60 minutes at RT.
The supernatant was discarded and the wells were washed three times
with 90 .mu.l wash buffer. To all wells 25 .mu.l of ABTS solution
was added. The absorbance was read at 405 nm-492 nm.
Sequence CWU 1
1
641441PRTHomo sapiens 1Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met
Glu Asp His Ala Gly1 5 10 15Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln
Gly Gly Tyr Thr Met His 20 25 30Gln Asp Gln Glu Gly Asp Thr Asp Ala
Gly Leu Lys Glu Ser Pro Leu 35 40 45Gln Thr Pro Thr Glu Asp Gly Ser
Glu Glu Pro Gly Ser Glu Thr Ser 50 55 60Asp Ala Lys Ser Thr Pro Thr
Ala Glu Asp Val Thr Ala Pro Leu Val65 70 75 80Asp Glu Gly Ala Pro
Gly Lys Gln Ala Ala Ala Gln Pro His Thr Glu 85 90 95Ile Pro Glu Gly
Thr Thr Ala Glu Glu Ala Gly Ile Gly Asp Thr Pro 100 105 110Ser Leu
Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala Arg Met Val 115 120
125Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys Lys Ala Lys Gly
130 135 140Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala Ala
Pro Pro145 150 155 160Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg Ile
Pro Ala Lys Thr Pro 165 170 175Pro Ala Pro Lys Thr Pro Pro Ser Ser
Gly Glu Pro Pro Lys Ser Gly 180 185 190Asp Arg Ser Gly Tyr Ser Ser
Pro Gly Ser Pro Gly Thr Pro Gly Ser 195 200 205Arg Ser Arg Thr Pro
Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys 210 215 220Lys Val Ala
Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys225 230 235
240Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp Leu Lys Asn Val
245 250 255Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln Pro
Gly Gly 260 265 270Gly Lys Val Gln Ile Ile Asn Lys Lys Leu Asp Leu
Ser Asn Val Gln 275 280 285Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys
His Val Pro Gly Gly Gly 290 295 300Ser Val Gln Ile Val Tyr Lys Pro
Val Asp Leu Ser Lys Val Thr Ser305 310 315 320Lys Cys Gly Ser Leu
Gly Asn Ile His His Lys Pro Gly Gly Gly Gln 325 330 335Val Glu Val
Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser 340 345 350Lys
Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn 355 360
365Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala
370 375 380Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val
Val Ser385 390 395 400Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val
Ser Ser Thr Gly Ser 405 410 415Ile Asp Met Val Asp Ser Pro Gln Leu
Ala Thr Leu Ala Asp Glu Val 420 425 430Ser Ala Ser Leu Ala Lys Gln
Gly Leu 435 4402441PRTHomo
sapiensMISC_FEATURE(422)..(422)X=phosphoserine 2Met Ala Glu Pro Arg
Gln Glu Phe Glu Val Met Glu Asp His Ala Gly1 5 10 15Thr Tyr Gly Leu
Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His 20 25 30Gln Asp Gln
Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu 35 40 45Gln Thr
Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser 50 55 60Asp
Ala Lys Ser Thr Pro Thr Ala Glu Asp Val Thr Ala Pro Leu Val65 70 75
80Asp Glu Gly Ala Pro Gly Lys Gln Ala Ala Ala Gln Pro His Thr Glu
85 90 95Ile Pro Glu Gly Thr Thr Ala Glu Glu Ala Gly Ile Gly Asp Thr
Pro 100 105 110Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala
Arg Met Val 115 120 125Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp
Lys Lys Ala Lys Gly 130 135 140Ala Asp Gly Lys Thr Lys Ile Ala Thr
Pro Arg Gly Ala Ala Pro Pro145 150 155 160Gly Gln Lys Gly Gln Ala
Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro 165 170 175Pro Ala Pro Lys
Thr Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly 180 185 190Asp Arg
Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser 195 200
205Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys
210 215 220Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser
Ala Lys225 230 235 240Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro
Asp Leu Lys Asn Val 245 250 255Lys Ser Lys Ile Gly Ser Thr Glu Asn
Leu Lys His Gln Pro Gly Gly 260 265 270Gly Lys Val Gln Ile Ile Asn
Lys Lys Leu Asp Leu Ser Asn Val Gln 275 280 285Ser Lys Cys Gly Ser
Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly 290 295 300Ser Val Gln
Ile Val Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser305 310 315
320Lys Cys Gly Ser Leu Gly Asn Ile His His Lys Pro Gly Gly Gly Gln
325 330 335Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val
Gln Ser 340 345 350Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro
Gly Gly Gly Asn 355 360 365Lys Lys Ile Glu Thr His Lys Leu Thr Phe
Arg Glu Asn Ala Lys Ala 370 375 380Lys Thr Asp His Gly Ala Glu Ile
Val Tyr Lys Ser Pro Val Val Ser385 390 395 400Gly Asp Thr Ser Pro
Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser 405 410 415Ile Asp Met
Val Asp Xaa Pro Gln Leu Ala Thr Leu Ala Asp Glu Val 420 425 430Ser
Ala Ser Leu Ala Lys Gln Gly Leu 435 440315PRTHomo
sapiensMISC_FEATURE(7)..(7)X=phosphoserine 3Ser Ile Asp Met Val Asp
Xaa Pro Gln Leu Ala Thr Leu Ala Asp1 5 10 15412PRTOryctolagus
cuniculus 4Gln Ser Ser Gln Ser Val Arg Thr Asn Lys Leu Ala1 5
1057PRTOryctolagus cuniculus 5Ser Ala Ser Thr Leu Asp Phe1
5613PRTOryctolagus cuniculus 6Leu Gly Tyr Phe Asp Cys Ser Ile Ala
Asp Cys Val Ala1 5 107112PRTOryctolagus cuniculus 7Ala Gln Val Leu
Thr Gln Thr Thr Ser Pro Val Ser Ala Ala Val Gly1 5 10 15Ser Thr Val
Thr Ile Ser Cys Gln Ser Ser Gln Ser Val Arg Thr Asn 20 25 30Lys Leu
Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45Ile
Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val Pro Ser Arg Phe Ser 50 55
60Ala Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Val Gln65
70 75 80Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Cys
Ser 85 90 95Ile Ala Asp Cys Val Ala Phe Gly Gly Gly Thr Glu Val Val
Val Lys 100 105 11085PRTOryctolagus cuniculus 8Ser Asn Ala Ile Asn1
5916PRTOryctolagus cuniculus 9Tyr Ile Ala Val Ser Gly Asn Thr Tyr
Tyr Ala Ser Trp Ala Lys Gly1 5 10 15103PRTOryctolagus cuniculus
10Ser Asn Ile111108PRTOryctolagus cuniculus 11Gln Ser Val Glu Glu
Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro1 5 10 15Leu Thr Leu Thr
Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Asn Ala 20 25 30Ile Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45Tyr Ile
Ala Val Ser Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60Arg
Phe Thr Ile Ser Lys Ala Ser Thr Thr Val Asp Leu Lys Met Thr65 70 75
80Ser Pro Thr Ala Glu Asp Thr Gly Thr Tyr Phe Cys Gly Lys Ser Asn
85 90 95Ile Trp Gly Pro Gly Thr Leu Val Thr Val Ser Leu 100
1051212PRTArtificial Sequencehumanized sequence 12Arg Ser Ser Gln
Ser Val Arg Thr Asn Lys Leu Ala1 5 101312PRTArtificial
Sequencehumanized sequence 13Arg Ser Ser Gln Ser Val Arg Thr Asn
Arg Leu Ala1 5 10147PRTArtificial Sequencehumanized sequence 14Ser
Ala Ser Thr Leu Asp Tyr1 51512PRTArtificial Sequencehumanized
sequence 15Leu Gly Tyr Phe Asp Ser Ser Ala Asp Ile Val Ala1 5
1016111PRTArtificial Sequencehumanized sequence 16Ala Gln Val Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ser Ser Gln Ser Val Arg Thr Asn 20 25 30Lys Leu
Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45Ile
Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val Pro Ser Arg Phe Ser 50 55
60Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln65
70 75 80Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Ser
Ser 85 90 95Ala Asp Ile Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys 100 105 11017111PRTArtificial Sequencehumanized sequence 17Ala
Gln Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Val Arg Thr Asn
20 25 30Arg Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg
Leu 35 40 45Ile Tyr Ser Ala Ser Thr Leu Asp Tyr Gly Val Pro Ser Arg
Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
Ser Leu Gln65 70 75 80Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly
Tyr Phe Asp Ser Ser 85 90 95Ala Asp Ile Val Ala Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys 100 105 1101816PRTArtificial Sequencehumanized
sequence 18Tyr Ile Ala Val Ser Gly Asn Thr Tyr Tyr Ala Asp Ser Val
Lys Gly1 5 10 1519111PRTArtificial Sequencehumanized sequence 19Glu
Val Gln Val Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Ser Ser Asn
20 25 30Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Gly Tyr Ile Ala Val Ser Gly Asn Thr Tyr Tyr Ala Ser Trp
Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys Gly 85 90 95Lys Ser Asn Ile Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 100 105 11020112PRTArtificial Sequencehumanized
sequence 20Ala Gln Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Val
Arg Thr Asn 20 25 30Lys Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro
Pro Lys Arg Leu 35 40 45Ile Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val
Pro Ser Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu
Thr Ile Ser Ser Leu Gln65 70 75 80Pro Glu Asp Phe Ala Thr Tyr Tyr
Cys Leu Gly Tyr Phe Asp Ser Ser 85 90 95Ile Ala Asp Ile Val Ala Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 11021112PRTArtificial
Sequencehumanizes sequence 21Ala Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ser Ser Gln Ser Val Arg Thr Asn 20 25 30Arg Leu Ala Trp Phe Gln Gln
Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45Ile Tyr Ser Ala Ser Thr
Leu Asp Phe Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly
Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Pro Glu Asp
Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Ser Ser 85 90 95Ile Ala
Asp Ile Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
110227PRTArtificial Sequencehumanized sequence 22Ser Ala Ser Thr
Leu Gln Ser1 5237PRTArtificial Sequencehumanized sequence 23Ser Ala
Ser Thr Leu Glu Ser1 52413PRTArtificial Sequencehumanized sequence
24Leu Gly Tyr Phe Asp Ser Ser Ile Ala Asp Ser Val Ala1 5
102513PRTArtificial Sequencehumanized sequence 25Leu Gly Tyr Phe
Asp Ser Ser Ile Ala Asp Arg Val Ala1 5 102613PRTArtificial
Sequencehumanized sequence 26Leu Gly Tyr Phe Asp Pro Ser Ile Ala
Asp Pro Val Ala1 5 102713PRTArtificial Sequencehumanized sequence
27Leu Gly Tyr Phe Asp Ser Ser Ile Ala Asp Ile Val Ala1 5
102812PRTArtificial Sequencehumanized sequence 28Leu Gly Tyr Phe
Asp Pro Ser Ala Asp Pro Ile Ala1 5 102912PRTArtificial
Sequenceumanized sequence 29Leu Gly Tyr Phe Asp Pro Ser Ala Asp Pro
Val Ala1 5 103012PRTArtificial Sequencehumanized sequence 30Arg Ala
Ser Gln Gly Val Arg Thr Asn Lys Leu Ala1 5 103112PRTArtificial
Sequencehumanized sequence 31Arg Ala Ser Gln Ser Val Arg Thr Asn
Lys Leu Ala1 5 1032112PRTArtificial Sequencehumanized sequence
32Asp Ile Gln Met Thr Gln Ser Thr Ser Thr Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Gln Ser Val Arg Thr
Asn 20 25 30Lys Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys
Arg Leu 35 40 45Ile Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val Pro Ser
Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile
Ser Ser Leu Gln65 70 75 80Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Leu
Gly Tyr Phe Asp Cys Ser 85 90 95Ile Ala Asp Cys Val Ala Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys 100 105 11033112PRTArtificial
Sequencehumanized sequence 33Asp Ile Gln Met Thr Gln Ser Pro Ser
Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Val Arg Thr Asn 20 25 30Lys Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Arg Leu 35 40 45Ile Tyr Ser Ala Ser Thr
Leu Glu Ser Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly
Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Pro Asp Asp
Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Cys Ser 85 90 95Ile Ala
Asp Cys Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
11034112PRTArtificial Sequencehumanized sequence 34Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Gly Val Arg Thr Asn 20 25 30Lys Leu
Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45Ile
Tyr Ser Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55
60Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln65
70 75 80Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Cys
Ser 85 90 95Ile Ala Asp Cys Val Ala Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys 100 105 11035112PRTArtificial Sequencehumanized sequence
35Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Val Arg Thr
Asn
20 25 30Lys Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg
Leu 35 40 45Ile Tyr Ser Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
Ser Leu Gln65 70 75 80Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly
Tyr Phe Asp Ser Ser 85 90 95Ile Ala Asp Ser Val Ala Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys 100 105 11036112PRTArtificial
Sequencehumanized sequence 36Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Gly Val Arg Thr Asn 20 25 30Lys Leu Ala Trp Phe Gln Gln
Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45Ile Tyr Ser Ala Ser Thr
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly
Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Pro Glu Asp
Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Ser Ser 85 90 95Ile Ala
Asp Arg Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
11037112PRTArtificial Sequencehumanized sequence 37Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ser Ser Gln Ser Val Arg Thr Asn 20 25 30Lys Leu
Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45Ile
Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val Pro Ser Arg Phe Ser 50 55
60Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln65
70 75 80Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Cys
Ser 85 90 95Ile Ala Asp Cys Val Ala Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys 100 105 11038112PRTArtificial Sequencehumanized sequence
38Ala Gln Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Val Arg Thr
Asn 20 25 30Lys Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys
Arg Leu 35 40 45Ile Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val Pro Ser
Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile
Ser Ser Leu Gln65 70 75 80Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu
Gly Tyr Phe Asp Cys Ser 85 90 95Ile Ala Asp Cys Val Ala Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys 100 105 11039111PRTArtificial
Sequencehumanized sequence 39Ala Ile Gln Met Thr Gln Ser Pro Ser
Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ser Ser Gln Ser Val Arg Thr Asn 20 25 30Arg Leu Ala Trp Phe Gln Gln
Lys Pro Gly Lys Ala Pro Lys Arg Leu 35 40 45Ile Tyr Ser Ala Ser Thr
Leu Asp Phe Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly
Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Pro Asp Asp
Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Pro Ser 85 90 95Ala Asp
Pro Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
11040112PRTArtificial Sequencehumanized sequence 40Ala Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ser Ser Gln Ser Val Arg Thr Asn 20 25 30Arg Leu
Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45Ile
Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val Pro Ser Arg Phe Ser 50 55
60Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln65
70 75 80Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Ser
Ser 85 90 95Ile Ala Asp Ile Val Ala Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys 100 105 11041112PRTArtificial Sequencehumanized sequence
41Ala Ile Gln Met Thr Gln Ser Thr Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Val Arg Thr
Asn 20 25 30Arg Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys
Arg Leu 35 40 45Ile Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val Pro Ser
Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile
Ser Ser Leu Gln65 70 75 80Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu
Gly Tyr Phe Asp Ser Ser 85 90 95Ile Ala Asp Ile Val Ala Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys 100 105 11042112PRTArtificial
Sequencehumanized sequence 42Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ser Ser Gln Ser Val Arg Thr Asn 20 25 30Arg Leu Ala Trp Phe Gln Gln
Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45Ile Tyr Ser Ala Ser Thr
Leu Asp Phe Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly
Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Pro Glu Asp
Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Pro Ser 85 90 95Ile Ala
Asp Pro Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
11043111PRTArtificial Sequencehumanized sequence 43Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ser Ser Gln Ser Val Arg Thr Asn 20 25 30Arg Leu
Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu 35 40 45Ile
Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val Pro Ser Arg Phe Ser 50 55
60Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln65
70 75 80Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly Tyr Phe Asp Pro
Ser 85 90 95Ala Asp Pro Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys 100 105 11044111PRTArtificial Sequencehumanized sequence 44Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Val Arg Thr Asn
20 25 30Arg Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg
Leu 35 40 45Ile Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val Pro Ser Arg
Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
Ser Leu Gln65 70 75 80Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gly
Tyr Phe Asp Pro Ser 85 90 95Ala Asp Pro Ile Ala Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys 100 105 11045111PRTArtificial Sequencehumanized
sequence 45Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Val
Arg Thr Asn 20 25 30Arg Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala
Pro Lys Arg Leu 35 40 45Ile Tyr Ser Ala Ser Thr Leu Asp Phe Gly Val
Pro Ser Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu
Thr Ile Ser Ser Leu Gln65 70 75 80Pro Glu Asp Phe Ala Thr Tyr Tyr
Cys Leu Gly Tyr Phe Asp Pro Ser 85 90 95Ala Asp Pro Ile Ala Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys 100 105 11046110PRTArtificial
Sequencehumanized sequence 46Gln Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly Ser1 5 10 15Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Ser Leu Ser Ser Asn Ala 20 25 30Ile Asn Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val Gly 35 40 45Tyr Ile Ala Val Ser Gly
Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln65 70 75 80Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gly Lys 85 90 95Ser Asn
Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105
11047110PRTArtificial Sequencehumanized sequence 47Gln Ser Val Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser1 5 10 15Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Ser Asn Ala 20 25 30Ile Asn
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly 35 40 45Tyr
Ile Ala Val Ser Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55
60Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln65
70 75 80Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gly
Lys 85 90 95Ser Asn Ile Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser
100 105 11048110PRTArtificial Sequencehumanized sequence 48Gln Ser
Val Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser1 5 10 15Leu
Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Ser Ser Asn Ala 20 25
30Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly
35 40 45Tyr Ile Ala Val Ser Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys
Gly 50 55 60Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
Leu Gln65 70 75 80Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Gly Lys 85 90 95Ser Asn Ile Trp Gly Pro Gly Thr Leu Val Thr
Val Ser Ser 100 105 11049108PRTArtificial Sequencehumanized
sequence 49Gln Ser Val Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly Ser1 5 10 15Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Ser
Ser Asn Ala 20 25 30Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val Gly 35 40 45Tyr Ile Ala Val Ser Gly Asn Thr Tyr Tyr Ala
Ser Trp Ala Lys Gly 50 55 60Arg Phe Thr Ile Ser Arg Asp Ser Thr Thr
Leu Tyr Leu Gln Met Asn65 70 75 80Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys Gly Lys Ser Asn 85 90 95Ile Trp Gly Pro Gly Thr Leu
Val Thr Val Ser Ser 100 10550110PRTArtificial Sequencehumanized
sequence 50Gln Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly Ser1 5 10 15Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser
Ser Asn Ala 20 25 30Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val Gly 35 40 45Tyr Ile Ala Val Ser Gly Asn Thr Tyr Tyr Ala
Ser Trp Ala Lys Gly 50 55 60Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr Leu Gln65 70 75 80Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys Gly Lys 85 90 95Ser Asn Ile Trp Gly Pro Gly
Thr Leu Val Thr Val Ser Ser 100 105 11051110PRTArtificial
Sequencehumanized sequence 51Gln Ser Val Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly Ser1 5 10 15Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Ser Leu Ser Ser Asn Ala 20 25 30Ile Asn Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val Gly 35 40 45Tyr Ile Ala Val Ser Gly
Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln65 70 75 80Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gly Lys 85 90 95Ser Asn
Ile Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser 100 105
11052110PRTArtificial Sequencehumanized sequence 52Gln Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser1 5 10 15Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Ser Asn Ala 20 25 30Ile Asn
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly 35 40 45Tyr
Ile Ala Val Ser Gly Asn Thr Tyr Tyr Ala Asp Ser Val Lys Gly 50 55
60Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln65
70 75 80Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gly
Lys 85 90 95Ser Asn Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
100 105 11053110PRTArtificial Sequencehumanized sequence 53Gln Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser1 5 10 15Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Ser Asn Ala 20 25
30Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly
35 40 45Tyr Ile Ala Val Ser Gly Asn Thr Tyr Tyr Ala Asp Ser Val Lys
Gly 50 55 60Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr
Leu Gln65 70 75 80Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Gly Lys 85 90 95Ser Asn Ile Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 100 105 11054111PRTArtificial Sequencehumanized
sequence 54Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu
Ser Ser Asn 20 25 30Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Gly Tyr Ile Ala Val Ser Gly Asn Thr Tyr Tyr
Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Gly 85 90 95Lys Ser Asn Ile Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 100 105 11055111PRTArtificial
Sequencehumanized sequence 55Glu Val Gln Val Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Val
Ser Gly Phe Ser Leu Ser Ser Asn 20 25 30Ala Ile Asn Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Tyr Ile Ala Val Ser
Gly Asn Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile
Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr Leu65 70 75 80Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gly 85 90 95Lys Ser
Asn Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105
11056111PRTArtificial Sequencehumanized sequence 56Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe
Ser Leu Ser Ser Asn 20 25 30Ala Ile Asn Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Gly Tyr Ile Ala Val Ser Gly Asn Thr
Tyr Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Lys Asp
Asn Ser Lys Asn Thr Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gly 85 90 95Lys Ser Asn Ile Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105
11057111PRTArtificial Sequencehumanized sequence 57Glu Val Gln Val
Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Ser Ser Asn 20 25 30Ala Ile
Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly
Tyr Ile Ala Val Ser Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
Gly 85 90 95Lys Ser Asn Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 100 105 11058330PRTHomo sapiens 58Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90
95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215
220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 33059107PRTHomo sapiens
59Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1
5 10 15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys 100 10560106PRTHomo sapiens 60Gly Gln Pro Lys Ala Ala Pro
Ser Val Thr Leu Phe Pro Pro Ser Ser1 5 10 15Glu Glu Leu Gln Ala Asn
Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30Phe Tyr Pro Gly Ala
Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 35 40 45Val Lys Ala Gly
Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 50 55 60Lys Tyr Ala
Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys65 70 75 80Ser
His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90
95Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 1056137DNAArtificial
SequencerbHCfinal.up 61aagcttgcca ccatggagac tgggctgcgc tggcttc
376221DNAArtificial SequencerbHCfinal.do 62ccattggtga gggtgcccga g
216334DNAArtificial SequencerbLCfinal.up 63aagcttgcca ccatggacay
gagggccccc actc 346426DNAArtificial SequencerbLCfinal.do
64cagagtrctg ctgaggttgt aggtac 26
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