U.S. patent application number 15/934413 was filed with the patent office on 2018-12-27 for humanized anti-tau antibodies.
The applicant listed for this patent is C2N Diagnostics, LLC. Invention is credited to Diljeet S. Athwal, Francis J. CARR, Robert George Edward HOLGATE, Timothy D. JONES, Tim WEST.
Application Number | 20180371066 15/934413 |
Document ID | / |
Family ID | 54938957 |
Filed Date | 2018-12-27 |
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United States Patent
Application |
20180371066 |
Kind Code |
A1 |
WEST; Tim ; et al. |
December 27, 2018 |
HUMANIZED ANTI-TAU ANTIBODIES
Abstract
Provided herein is an isolated antibody or antigen-binding
fragment that specifically binds tau, the antibody or fragment
comprising a heavy chain variable (VH) region and a light chain
variable (VL) region having amino acid sequences set forth herein.
Also provided are methods of preventing or treating a tauopathy in
a subject, comprising administering to a human in need of therapy
for a tauopathy with one or more antibodies or fragments as
described herein, wherein the antibodies or antigen-binding
fragments are administered under conditions and in an amount
effective to prevent or treat the tauopathy.
Inventors: |
WEST; Tim; (Saint Louis,
MO) ; Athwal; Diljeet S.; (Slough, GB) ;
JONES; Timothy D.; (Cambridgeshire, GB) ; CARR;
Francis J.; (Aberdeen, GB) ; HOLGATE; Robert George
Edward; (Royston, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
C2N Diagnostics, LLC |
Saint Louis |
MO |
US |
|
|
Family ID: |
54938957 |
Appl. No.: |
15/934413 |
Filed: |
March 23, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15257086 |
Sep 6, 2016 |
9957317 |
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15934413 |
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PCT/US2015/038002 |
Jun 26, 2015 |
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15257086 |
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62170036 |
Jun 2, 2015 |
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62080903 |
Nov 17, 2014 |
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62018436 |
Jun 27, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/34 20130101;
C07K 2317/92 20130101; C07K 2317/52 20130101; A61P 25/14 20180101;
A61P 25/28 20180101; C07K 2317/24 20130101; A61P 25/16 20180101;
C07K 16/18 20130101; A61P 25/00 20180101; C07K 2317/33 20130101;
C07K 2317/56 20130101; A61P 21/00 20180101; A61P 43/00 20180101;
A61K 2039/505 20130101; C07K 2317/31 20130101 |
International
Class: |
C07K 16/18 20060101
C07K016/18 |
Claims
1.-59. (canceled)
60. A vector comprising a nucleic acid molecule encoding an
anti-tau antibody comprising: a light chain comprising the amino
acid sequence of SEQ ID NO:2, and a heavy chain comprising the
amino acid sequence of SEQ ID NO:5.
61. The vector of claim 60, wherein the vector is an expression
vector.
62. An isolated host cell comprising the vector of claim 61.
63. The host cell of claim 62, wherein the cell is a prokaryotic or
eukaryotic cell.
64. The host cell of claim 63, wherein the cell is a mammalian
cell.
65. A method of producing an anti-tau antibody, comprising
culturing a cell of claim 64, thereby producing an anti-tau
antibody.
66. The method of claim 65, further comprising isolating the
anti-tau antibody from culture media.
67. The method of claim 65, wherein the expression is at least 11.3
micrograms per mL of culture media.
68. The method of claim 65, wherein the expression is 13.1
micrograms per mL of culture media.
69. The method of claim 65, wherein the expression is 13.4
micrograms per mL of culture media.
70. The vector of claim 60, wherein the vector comprises a nucleic
acid molecule encoding an anti-tau antibody comprising: the light
chain comprises the amino acid sequence of SEQ ID NO: 18, and the
heavy chain comprises the amino acid sequence of SEQ ID NO: 13.
71. The vector of claim 70, wherein the vector is an expression
vector.
72. An isolated host cell comprising the vector of claim 71.
73. The host cell of claim 72, wherein the cell is a prokaryotic or
eukaryotic cell.
74. The host cell of claim 73, wherein the cell is a mammalian
cell.
75. A method of producing an anti-tau antibody, comprising
culturing a cell of claim 74, thereby producing an anti-tau
antibody.
76. The method of claim 75, further comprising isolating the
anti-tau antibody from culture media.
77. The method of claim 75, wherein the expression is at least 11.3
micrograms per mL of culture media.
78. The method of claim 75, wherein the expression is 13.1
micrograms per mL of culture media.
79. The method of claim 75, wherein the expression is 13.4
micrograms per mL of culture media.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/257,086, filed Sep. 6, 2016, which is a continuation of
International Application No. PCT/US2015/038002, filed on Jun. 26,
2015, which claims benefit of priority under 35 U.S.C. .sctn.
119(e) of U.S. Ser. No. 62/170,036, filed Jun. 2, 2015, U.S. Ser.
No. 62/080,903, filed Nov. 17, 2014, and U.S. Ser. No. 62/018,436,
filed Jun. 27, 2014, the entire contents of which are incorporated
herein by reference in their entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Aug. 23, 2018, is named 397835-215C2(159114)_SL.txt and is
43,082 bytes in size.
FIELD OF THE INVENTION
[0003] The present invention relates to the field of humanized
antibodies and antigen-binding fragments thereof that bind to tau
and methods of using such antibodies to treat tauopathies. In
particular, the present invention relates to a humanized antibody
and antigen-binding fragments that bind to specific epitopes of tau
and prevent tau seeding.
BACKGROUND OF THE INVENTION
[0004] Tauopathies have in common the accumulation of insoluble,
hyperphosphorylated tau protein in the brain. More than 20
different neurodegenerative disorders are characterized by some
degree of neurofibrillary degeneration and can be classified as
tauopathies (Williams 2006). Prototypical tauopathies, such as
progressive supranuclear palsy (PSP) and corticobasal degeneration
(CBD) are characterized by tau inclusions being the sole or
predominant central nervous system lesions. Prototypical
tauopathies differ from other tauopathies where tau aggregates are
found in the presence of other neuropathological features, like the
amyloid beta (AP3) plaques found in Alzheimer's disease (AD) or the
Lewy bodies found in Parkinson's disease (PD). In these
non-prototypical tauopathies, it is more uncertain if the tau
pathology represents the primary disease driver or if it is
secondary to other protein misfolding and neurodegeneration.
[0005] Progressive supranuclear palsy (PSP, also known as
Steele-Richardson-Olszewski syndrome) is a progressive
neurodegenerative disorder, with an estimated annual incidence of
5-7 per 100,000 (Golbe 2014). Within the US, the disease affects
approximately 20,000 individuals. There is no apparent
geographical, ethnic, gender, or racial disparity in PSP frequency.
PSP can initially present with clinical symptoms similar to other
brain disorders, including idiopathic Parkinson's disease. For this
reason, correct diagnosis of PSP is sometimes delayed, usually
taking place 1 to 3 years after the initial onset of clinical
symptoms. Symptom onset is most often between the ages of 50 to 70
years and although the clinical course is variable, the typical
survival from time of symptom onset is 5 to 9 years (Houghton,
2007). Though heterogeneity in clinical presentation exists, the
most common and initially described PSP syndrome, now referred to
as Richardson's Syndrome, are the presence of prominent postural
instability and axial rigidity leading to falls, supranuclear gaze
palsy causing range of vision impairment, frontal-subcortical
dementia, and dysphagia leading to aspiration. The course of
disease is progressive and uniformly fatal (Williams and Lees
2009).
[0006] Pathologically, PSP is characterized by the abnormal
accumulation of hyper phosphorylated, insoluble aggregates of tau
protein in neurons and glia in the brainstem, cerebellum, basal
ganglia, and cerebral cortex (Williams and Lees 2009). The degree
and distribution of tau aggregation in PSP is strongly correlated
with PSP symptomatology during life (Schofield et al. 2012). The
National Institute of Neurological Disorders and the Society for
Progressive Supranuclear Palsy (NINDS-SPSP) research criteria which
describe Richardson's Syndrome are highly predictive of underlying
PSP pathology (Litvan et al. 1996). Neuronal loss in various
regions of the brain accompanies neurofibrillary tangles (NFTs)
that are composed of tau aggregates. Multiple neurotransmitter
abnormalities arise as well, including those affecting specific
dopaminergic, cholinergic, GABAergic, and noradrenergic
systems.
[0007] There are no currently approved treatments for PSP (Stamelou
et al. 2010). The negative outcomes of therapeutic efficacy studies
in PSP preclude recommending an evidence-based standard therapy
(Boxer et al. 2014). In the absence of any effective disease
modifying or neuroprotective therapies, PSP represents an urgent
unmet medical need.
[0008] Alzheimer's disease (AD) is a common chronic progressive
neurodegenerative disease in which there is an irreversible loss of
cognitive and behavioral functions. The disease can persevere for
over 10 years, advancing from mild symptoms to extremely severe
manifestations. AD is said to afflict approximately 10% of the
population over the age of 65 and more than 30% of the population
over the age of 80. Alzheimer's disease presents itself
pathologically as extracellular amyloid plaques and intracellular
neurofibrillary tangles. The neurofibrillary tangles are composed,
e.g., of the microtubule-binding protein tau, which is assembled
into paired helical and straight filaments. It has been suggested
that these entities may be functionally linked, although the
mechanisms by which amyloid deposition promotes pathological tau
filament assembly, or vice versa, is not clear.
[0009] The intracellular neurofibrillary structures of tauopathies
(neurofibrillary tangles, dystrophic neurites, and neurophil
threads) have paired helical filaments (PHFs). The major protein
subunit of the PHFs is microtubule associated protein tau in
abnormally hyperphosphorylated form. Neurons with neurofibrillary
changes degenerate, and the degree of this degeneration directly
correlates with the degree of dementia in the affected
individuals.
[0010] Other tauopathies known to have filamentous cellular
inclusions containing microtubule associated protein tau include
Pick's disease (PiD), a group of related disorders collectively
termed frontotemporal dementia with Parkinsonism linked to
chromosome 17 (FTDP-17), amyotropic lateral sclerosis (ALS),
Creutzfeldt-Jakob disease (CJD), dementia pugilistica (DP),
Gerstmann-Straussler-Scheinker disease (GSSD), Lewy body disease,
chronic traumatic encephalopathy (CTE), and Huntington disease.
Although the etiology, clinical symptoms, pathologic findings and
the biochemical composition of filamentous cellular inclusions in
these diseases are different, there is emerging evidence suggesting
that the mechanisms involved in aggregation of normal cellular
proteins to form various filamentous inclusions being comparable.
It is believed, that an initial alteration in conformation of
microtubule associated protein tau, acts to initiate the generation
of nuclei or seeds for filament assembly, is one of the key
features. This process can be influenced by the posttranslational
modification of normal proteins, by mutation or deletion of certain
genes and by factors that bind normal proteins and thus alter their
conformation.
SUMMARY OF THE INVENTION
[0011] As one aspect of the present invention, an isolated antibody
or antigen-binding fragment that specifically binds tau is
provided. The antibody or fragment comprises a heavy chain variable
(VH) region and a light chain variable (VL) region, and each of the
VH and VL regions have a sequence selected from amino acid
sequences set forth in FIGS. 1 and 2. More particularly, the VL
region can have an amino acid sequence selected from the group
consisting of SEQ ID NOs: 1, 2, 3 and 4 [VK1, VK2, VK3, and VK4],
and the VH region can have an amino acid sequence selected from the
group consisting of SEQ ID NOs: 5, 6, 7 and 8 [VH1, VH2, VH3, and
VH4]. In some embodiments, the VL region has an amino acid sequence
of SEQ ID NO: 2 [VK2] and the VH region has an amino acid sequence
of SEQ ID NO: 5 [VH1]. In some embodiments, the antibody comprises
an Fc region, which may be of human IgG1, IgG2, IgG3, IgG4 or
variants thereof, such as a human IgG4 containing a S241P hinge
stabilizing mutation. The antibody can comprise a light chain
constant region of human isotype kappa or variants thereof. In some
embodiments, the antibody or fragment is scFv or Fab. In some
embodiments, the antibody or fragment is a humanized antibody or
fragment or a chimeric antibody or fragment. The antibody or
fragment may be a monoclonal antibody. In some embodiments, the
antibody or fragment competes with HJ8.5 for specific binding to
human tau protein. In some embodiments, the antibody or fragment
binds human tau protein with an equilibrium dissociation constant
(Kd) of at least 10.sup.-4M.
[0012] As another aspect of the present invention, a multi-specific
antibody or antigen-binding fragment having a plurality of
antigen-binding regions is provided. At least one antigen-binding
region of the multi-specific antibody or fragment binds to human
tau protein. Alternatively, a bispecific antibody or
antigen-binding fragment having two antigen-binding regions is
provided. One of the antigen-binding regions of the bispecific
antibody or fragment binds to human tau protein. Alternatively, a
bispecific antibody or antigen-binding fragment is provided where
one arm of the antibody or antigen-binding fragment competes with
HJ8.5 for specific binding to human tau protein. Alternatively, a
bispecific antibody or antigen-binding fragment is provided where
one arm of the antibody or antigen-binding fragment is comprised of
a heavy chain variable (VH) region and a light chain variable (VL)
region, wherein each of the VH and VL regions have a sequence
selected from amino acid sequences set forth in FIGS. 1 and 2.
[0013] Any of the foregoing antibodies or antigen-binding fragments
may further comprise a toxic payload, optionally a drug conjugate,
or a radionuclide.
[0014] As yet another aspect of the present invention, an isolated
nucleic acid molecule is provided which encodes any of the
foregoing antibodies or antigen-binding fragment, or a VH region or
VL region set forth in FIG. 1 or 2. A vector (such as an expression
vector) comprising such a nucleic acid molecule may be provided. An
isolated host cell comprising such a vector may be provided. The
host cell may be a prokaryotic or eukaryotic cell, such as a
mammalian cell.
[0015] As another aspect of the present invention, a pharmaceutical
composition is provided. The pharmaceutical composition comprises
any of the foregoing antibodies or antigen-binding fragments, or a
nucleic acid molecule as described herein, and a pharmaceutically
acceptable carrier.
[0016] As yet another aspect of the present invention, an isolated
amino acid sequence is provided containing the sequence of one of
the light chains as set forth in FIGS. 1 and 2. Alternatively or
additionally, an isolated amino acid sequence is provided
containing the sequence of one of heavy chains as set forth in
FIGS. 1 and 2.
[0017] As a further aspect of the present invention, an isolated
humanized antibody or antigen-binding fragment is provided that
specifically binds an epitope comprising the amino acid sequence
DQGGYT (SEQ ID NO: 9). The antibody or antigen-binding fragment may
contain CDRs of the VH and VL regions are from a donor antibody. In
some embodiments, the antibody comprises an Fc region, such as the
Fc region is of IgG1, IgG2, IgG3, IgG4 or variant thereof. The Fc
region may be a human IgG4 or variant thereof, such a human IgG4
containing the S241P hinge stabilizing mutation. The antibody can
comprise a light chain constant region of human isotype kappa or
variants thereof. In some embodiments, the antibody or fragment is
scFv or Fab. In some embodiments, the antibody or fragment is a
humanized antibody or fragment or a chimeric antibody or fragment.
The antibody or fragment may be a monoclonal antibody. The antibody
or fragment may be a bispecific antibody or antigen-binding
fragment where one arm of the antibody or fragment specifically
binds an epitope comprising the amino acid sequence DQGGYT (SEQ ID
NO: 9). In some embodiments, an immunoconjugate is provided
comprising one of the foregoing antibodies or fragments linked to a
detectable or therapeutic moiety.
[0018] As another aspect, an isolated humanized antibody or
antigen-binding fragment is provided that specifically binds an
epitope comprising the amino acid sequence GYTMHQDQ (SEQ ID NO:
10). The antibody or fragment can have CDRs of the VH and VL
regions from a donor antibody. In some embodiments, the antibody or
fragment comprises an Fc region, such as an Fc region of IgG1,
IgG2, IgG3, IgG4 or a variant thereof. The Fc region may be a human
IgG4 and variants thereof containing the S241P hinge stabilizing
mutation. The antibody may comprise a light chain constant region.
In some embodiments, the antibody or fragment is an scFv or Fab. A
bispecific antibody or antigen-binding fragment is also provided
where one arm of the antibody specifically binds an epitope
comprising the amino acid sequence GYTMHQDQ (SEQ ID NO: 10). In
some embodiments, an immunoconjugate comprising any of the
foregoing antibodies or fragments is linked to a detectable or
therapeutic moiety.
[0019] As a further aspect of the present invention, a method of
preventing or treating a tauopathy in a subject, comprising
administering to a human in need of therapy for a tauopathy with
one or more of the antibodies or fragments described herein. The
antibodies or antigen-binding fragment are administered under
conditions and in an amount effective to prevent or treat the
tauopathy. The tauopathy may be one or more of Alzheimer's disease
(AD), progressive supranuclear palsy (PSP), corticobasal
degeneration (CBD), Pick's disease (PiD), a group of related
disorders collectively termed frontotemporal dementia with
Parkinsonism linked to chromosome 17 (FTDP-17), amyotropic lateral
sclerosis (ALS), Creutzfeldt-Jakob disease (CJD), dementia
pugilistica (DP), Gerstmann-Straussler-Scheinker disease (GSSD),
Lewy body disease, chronic traumatic encephalopathy (CTE), or
Huntington disease.
[0020] A method is provided for treating a tauopathy comprising
administering an anti-tau antibody or fragment to a subject in need
of treatment, wherein the antibody or antigen-binding fragment
specifically binds tau and comprises a heavy chain variable (VH)
region and a light chain variable (VL) region, wherein each of the
VH and VL regions have a sequence selected from amino acid
sequences set forth in FIGS. 1 and 2, and the antibody or fragment
is administered in a dose of from about 0.1 mg/kg to about 250
mg/kg to the subject, alternatively from about 1 mg/kg to about 25
mg/kg. In some embodiments, the antibody or fragment has a VL
region comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 1, 2, 3 and 4 [VK1, VK2, VK3, and VK4];
alternatively or additionally, the antibody or fragment has a VH
region comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 5, 6, 7 and 8 [VH1, VH2, VH3, and
VH4].
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 shows the variable region sequences of the murine
HJ8.5 antibody as well as 4 humanized variant sequences for each of
the heavy and light chains (4 VH and 4 VL/VK sequences). FIG. 1
shows sequences for MuVL (SEQ ID NO: 11); VK1 (SEQ ID NO: 1); VK2
(SEQ ID NO:2); VK3 (SEQ ID NO:3); VK4 (SEQ ID NO:4); MuVH (SEQ ID
NO:12); VH1 (SEQ ID NO:5); VH2 (SEQ ID NO:6); VH3 (SEQ ID NO:7);
VH4 (SEQ ID NO:8).
[0022] FIG. 2A shows the sequence of the humanized variable and
constant region sequences for the heavy chains VH1 (SEQ ID NO:13).
FIG. 2B shows the sequence of the humanized variable and constant
region sequences for the heavy chain VH2 (SEQ ID NO: 14). FIG. 2C
shows the sequence of the humanized variable and constant region
sequences for the heavy chain VH3 (SEQ ID NO:15). FIG. 2D shows the
sequence of the humanized variable and constant region sequences
for the heavy chain VH4 (SEQ ID NO: 16). The variable heavy chain
is grafted to the constant heavy chain of human IgG4 containing a
S241P hinge stabilizing mutation. FIG. 2E shows the sequence of the
humanized variable and constant region sequences for the heavy
chain VL1 (SEQ ID NO:17). FIG. 2F shows the sequence of the
humanized variable and constant region sequences for the heavy
chain VL2 (SEQ ID NO: 18). FIG. 2G shows the sequence of the
humanized variable and constant region sequences for the heavy
chain VL3 (SEQ ID NO:19). FIG. 2H shows the sequence of the
humanized variable and constant region sequences for the heavy
chain VL4 (SEQ ID NO:20).
[0023] FIG. 3 shows expression data from two rounds of transient
expression of cells transfected with polynucleotides encoding VH
and VK regions. Results are summarized for 13 humanized anti tau
antibodies based on different combinations of humanized heavy and
light variable regions, with different levels of expression being
observed.
[0024] FIG. 4 shows data from a potency assay that evaluates the
ability of the present anti-tau antibodies to compete with the
original murine HJ8.5 (parent antibody) for binding to human tau in
an ELISA type format.
[0025] FIG. 5 summarizes the results from surface plasmon resonance
(SPR) analysis, determining the binding kinetics of the six best
expressing humanized constructs against human tau.
[0026] FIG. 6 shows the binding of four humanized antibody variants
to soluble human tau in a sandwich style ELISA.
[0027] FIGS. 7A to 7MM show binding of humanized and control
antibodies to tissue from wild type mice (negative control tissue),
P301S mice (which express human tau having a P301S mutation and
develop age associated tau pathology), and humans with either
Alzheimer's disease or Progressive Supranuclear Palsy (PSP). FIGS.
7A, 7B, 7C, 7D and 7E show binding of chimera (positive control) to
mouse and human tissue; FIGS. 7F, 7G, 7H and 7I show binding of
non-specific human IgG4 (negative control) to mouse and human
tissue. FIGS. 7J, 7K, 7L, 7M, and 7N show binding of VH1/VK2 to
mouse and human tissue. FIGS. 7O, 7P, 7Q, 7R and 7S show binding of
VH1/VK3 to mouse and human tissue. FIGS. 7T, 7U, 7V, 7W and 7X show
binding of VH2/VK2 to mouse and human tissue. FIGS. 7Y, 7Z, 7AA,
7BB and 7CC show binding of VH2/VK3 to mouse and human tissue.
FIGS. 7DD, 7EE, 7FF, 7GG and 7HH show binding of VH3/VK2 to mouse
and human tissue. FIGS. 7II, 7JJ, 7KK, 7LL and 7MM show binding of
VH3/VK3 to mouse and human tissue.
[0028] FIG. 8 shows the epitope mapping for the murine antibody
HJ8.5 against the amino acid sequence of human tau. FIG. 8 shows
human, rhesus monkey and mouse tau sequences (SEQ ID NOs:21, 22,
23, respectively).
[0029] FIG. 9 shows the detailed peptide based epitope mapping of
HJ8.5 and C.sub.2N-8E12. The mapping indicates that the binding
epitope of C.sub.2N-8E12 is .sub.25DQGGYT.sub.30 (SEQ ID NO: 9) and
matches the epitope of the murine parent, HJ8.5. FIG. 9 shows
sequences of peptides PEP_2875800 to PEP_2875830 (SEQ ID NOs:24 to
54, respectively).
[0030] FIG. 10 illustrates the binding results for different anti
human tau antibodies to either human or rhesus monkey tau. The
results demonstrate that C.sub.2N-8E12 and HJ8.5 do not bind to
rhesus tau while they do show positive binding to human tau. HJ8.7
binds to both human and rhesus tau.
[0031] FIG. 11 shows binding of humanized anti-tau antibody to tau
in CSF from human subjects with various tauopathies. The binding of
C.sub.2N-8E12 to tau in CSF samples from subjects diagnosed with a
variety of tauopathies was evaluated.
DESCRIPTION OF THE INVENTION
[0032] Strong experimental evidence and biological rationale exists
to support the tau immunotherapy strategy as a way to counter tau
pathology in neurodegeneration. First, tau is normally a highly
soluble, natively unfolded, and intracellular protein, so an
extracellular antibody is unlikely to affect the normal functions
of tau. Second, the burden of tau pathology correlates with
progressive neuronal dysfunction, synaptic loss, and functional
decline in humans and transgenic mouse models of tauopathy. Third,
under pathological conditions, tau becomes misfolded and aggregates
into intraneuronal neurofibrillary tangles (NFTs) composed of
pathological tau fibrils. In human tauopathies, this pathology
progresses from one brain region to another in disease-specific
patterns. Experimental data suggests that tau aggregates can spread
from cell to cell to induce further tau aggregation and spreading
of tau pathology in brain. This data suggests that aggregates
produced in one cell are released into the extracellular space and
can promote aggregation in neighboring or connected cells. Finally,
prior art exists demonstrating that anti-tau antibodies can prevent
or slow the progression of tau pathology in the brain of mice that
carry a mutated human form of tau.
[0033] A "humanized antibody" is an antibody or a variant,
derivative, analog or fragment thereof which has been modified to
reduce the risk of the non-human antibody eliciting an immune
response in humans following administration. A humanized antibody,
as used herein, immunospecifically binds to the same or similar
epitope as a non-human antibody (donor antibody). In some
embodiments a humanized antibody comprises a framework (FR) region
having substantially the amino acid sequence of a human antibody
and a complementary determining region (CDR) having substantially
the amino acid sequence of a non-human antibody. The term
"substantially" in the context of a CDR refers to a CDR having an
amino acid sequence at least 80%, preferably at least 85%, at least
90%, at least 95%, at least 98% or at least 99% identical to the
amino acid sequence of a non-human antibody CDR. A humanized
antibody comprises substantially all of at least one, and typically
two, variable domains (Fab, Fab', F(ab') 2, FabC, Fv) in which all
or substantially all of the CDR regions correspond to those of a
non-human immunoglobulin (i.e., donor antibody) and all or
substantially all of the framework regions are those of a human
immunoglobulin consensus sequence. Preferably, a humanized antibody
also comprises at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin. A humanized
antibody that comprises a novel framework region is provided in the
invention.
[0034] In some embodiments, a humanized antibody contains both the
light chain as well as at least the variable domain of a heavy
chain. The antibody also may include the CHI, hinge, CH2, CH3, and
CH4 regions of the heavy chain. In some embodiments, a humanized
antibody only contains a humanized light chain. In some
embodiments, a humanized antibody only contains a humanized heavy
chain. In specific embodiments, a humanized antibody only contains
a humanized variable domain of a light chain and/or humanized heavy
chain.
[0035] The antibody can be selected from any class of
immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any
isotype, including without limitation IgG 1, IgG2, IgG3 and IgG4.
The humanized antibody may comprise sequences from more than one
class or isotype, and particular constant domains may be selected
to optimize desired effector functions using techniques well-known
in the art.
[0036] The antibody or antigen-binding fragment thereof is selected
from the group consisting of: a disulfide linked Fv, a monoclonal
antibody, a single-chain variable fragment (scFv), a chimeric
antibody, a CDR-grafted antibody, a diabody, a humanized antibody,
a multispecific antibody, a Fab (fragment antigen-binding), a
bispecific antibody, a F(ab')2 (a dual arm, antigen-binding
fragment typically prepared by cleavage of an antibody with
pepsin), a Fab' (the result of splitting a F(ab')2 into two
antigen-binding fragments, typically by mild reduction), or a Fv
(an antigen-binding variable fragment).
[0037] The term "chimeric antibody" refers to antibodies which
comprise heavy and light chain variable region sequences from one
species and constant region sequences from another species, such as
antibodies having murine heavy and light chain variable regions
linked to human constant regions.
[0038] A "VH region", "VL region" or "VK region" refers to the
variable region of the heavy chain (VH), the variable region of the
light lambda chain (VL) or the variable region of the light kappa
chain (VK), respectively. The VH and VL regions can be further
subdivided into regions of hypervariability, termed complementarity
determining regions (CDR), interspersed with regions that are more
conserved, termed framework regions (FR). Each VH and VL is
composed of three CDRs and four FRs, arranged from amino-terminus
to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2,
FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (e.g.,
IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG2, IgG 3,
IgG4, IgA1 and IgA2) or subclass.
[0039] The term "framework" or "framework sequence" refers to the
remaining sequences of a variable region minus the CDRs. Because
the exact definition of a CDR sequence can be determined by
different systems, the meaning of a framework sequence is subject
to correspondingly different interpretations. The six CDRs (CDR-L1,
-L2, and -L3 of light chain and CDR-H1, -H2, and -H3 of heavy
chain) also divide the framework regions on the light chain and the
heavy chain into four sub-regions (FR1, FR2, FR3 and FR4) on each
chain, in which CDR1 is positioned between FR1 and FR2, CDR2
between FR2 and FR3, and CDR3 between FR3 and FR4. Without
specifying the particular sub-regions as FR1, FR2, FR3 or FR4, a
framework region, as referred by others, represents the combined
FR's within the variable region of a single, naturally occurring
immunoglobulin chain. A FR represents one of the four sub-regions,
and FRs represents two or more of the four sub-regions constituting
a framework region.
[0040] Many humanized immunoglobulins that have been previously
described (Jones et al., Verhoeyen et al., Riechmann et al.) have
comprised a framework that is identical to the framework of a
particular human immunoglobulin chain, the acceptor, and three
CDR's from a non-human donor immunoglobulin chain. A "humanized
anti-tau" antibody refers to an antibody that has been generated
from a non-human (donor) antibody capable of binding tau and said
binding is transferred to a human antibody (acceptor).
[0041] The term "CDR" refers to the complementarity determining
region within antibody variable sequences. There are three CDRs in
each of the variable regions of the heavy chain and the light
chain, which are designated CDR1, CDR2 and CDR3, for each of the
variable regions. The amino acid sequences of the CDRs of the VH
and VL/K regions of the claimed invention are set forth in FIG.
1.
[0042] As used herein, the term single-chain Fv, also termed
single-chain antibody, refers to engineered antibody constructs
prepared by isolating the binding domains (both heavy and light
chain) of a binding antibody, and supplying a linking moiety which
permits preservation of the binding function. A linker peptide
inserted between the two chains allows for the stabilization of the
variable domains without interfering with the proper folding and
creation of an active binding site. This linker can be between 5
and 30 amino acids long and typically consist of repeats of "GGGGS"
((Gly)4Ser) amino acid sequence (SEQ ID NO:55). This forms, in
essence, a radically abbreviated antibody, having only the variable
domain necessary for binding the antigen.
[0043] Diabodies, triabodies, and tetrabodies and higher order
variants are typically created by varying the length of the linker
peptide referred to above, from zero to several amino acids. The
variants are multivalent, multispecific antibodies in which VH and
VL domains are expressed on a polypeptide chain, but using a linker
that is too short to allow for pairing between the two domains on
the same chain, thereby forcing the domains to pair with
complementary domains of another chain and creating two antigen
binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl.
Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure
2: 1121-1123). Such antibody binding portions are known in the art
(Kontermann and Dubel eds., Antibody Engineering (2001)
Springer-Verlag. New York. p. 790 (ISBN 3-540-41354-5).
Alternatively, it is also well known in the art that multivalent
binding antibody variants can be generated using self-assembling
units linked to the variable domain.
[0044] Bispecific, trispecific, or antibodies of multiple
specificities are created by combining the heavy and light chains
of one antibody with the heavy and light chains of one or more
other antibodies. These chains can be covalently linked. For
example, the term "bispecific antibody" refers to full-length
antibodies that are generated by quadroma technology (see Milstein
and Cuello (1983) Nature 305(5934): 537-40), by chemical
conjugation of two different monoclonal antibodies (see Staerz et
al. (1985) Nature 314(6012): 628-31), or by knob-into-hole or
similar approaches which introduces mutations in the Fc region (see
Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90(14):
6444-6448), resulting in multiple different immunoglobulin species
of which only one is the functional bispecific antibody. By
molecular function, a bispecific antibody binds one antigen (or
epitope) on one of its two binding arms (one pair of HC/LC), and
binds a different antigen (or epitope) on its second arm (a
different pair of HC/LC). A bispecific antibody has two distinct
antigen binding arms (in both specificity and CDR sequences), and
is monovalent for each antigen to which it binds.
[0045] A series of murine antibodies capable of bind tau have been
raised using methods known in the art. See Holtzman et al.,
WO2014/08404. Further, these antibodies have been screened to
identify antibodies with specific biological activity that may them
suitable candidates for therapeutic uses.
[0046] In one aspect, the present disclosure provides composite
humanized antibodies. Composite Human Antibody.TM. technology
generates humanized antibodies by identifying potential T cell
epitopes in the variable region (V region) sequences of the donor
antibody and engineering antibodies or antigen-binding fragments in
such a way that binding to the potential T cell epitopes are
eliminated (See EP2,388,871). Unlike other humanization
technologies that use a single human light and heavy chain V region
framework or human consensus framework as light and heavy chain
`acceptors` for the respective complementarity determining regions
(CDRs) from the donor antibody (typically murine); Composite Human
Antibodies.TM. comprise multiple sequence segments ("composites")
derived from V regions of unrelated multiple human antibodies.
[0047] Sequence segments derived from databases of unrelated human
V regions are selected after determining amino acids that are
considered critical for antigen binding of the starting antibody.
All selected sequence segments derived from human V region
databases are filtered for the presence of potential CD4+ T cell
epitopes using in silica tools known in the art. Composite Human
Antibodies.TM. retain affinity and specificity better than standard
humanized antibodies due to the close fit of human sequence
segments with all sections of the starting antibody V regions.
Composite Human Antibodies.TM. are depleted of T cell epitopes and
therefore considered both humanized and de-immunized.
[0048] In one embodiment the murine variable regions from a donor
antibody replace human variable regions in a human acceptor IgG
resulting in a chimeric antibody.
[0049] In a further embodiment the murine CDR sequences from a
donor antibody replace the CDR sequences in a human acceptor IgG,
to create a humanized antibody. Further changes are incorporated
into the humanized antibody to remove potential T cell epitopes and
framework residues considered critical to maintaining the binding
characteristics of the donor antibody. One with skill in the art
will know that other methods such as CDR grafting can be used to
humanize an antibody.
[0050] In a further embodiment non-human antibodies capable of
binding to human tau are humanized.
[0051] The present antibodies may exhibit altered binding affinity
and/or altered immunogenicity as compared to donor antibodies. In
some embodiments, chimeric or humanized antibodies have
substantially the same binding affinity as the donor antibody with
respect to an epitope of tau.
[0052] In a further embodiment, a single-chain variable fragment
based on a humanized antibody as described herein, e.g., humanized
anti-tau antibody, may bind as a monomer.
[0053] In a further embodiment multivalent binding, using antibody
fragments can be achieved by using diabodies, triabodies,
tetrabodies, and other higher order variants, which may be
prepared.
[0054] In a further embodiment the heavy and light chain of the
humanized anti-tau antibody may be combined with the heavy and
light chains of other antibodies to form bispecific or other
additional multi specific antibodies.
[0055] Further the humanized antibodies of the invention, e.g.,
humanized anti-tau antibody may also be in the form of a antibody
fragment, e.g., a Fab, a Fab' monomer, a F(ab)'2 dimer, or a whole
immunoglobulin molecule.
[0056] In one embodiment, the invention provides an isolated
peptide consisting of the amino acid sequence, DQGGYT (SEQ ID NO:
9). This peptide is a core epitope for the antibodies described
herein as C.sub.2N-8E12 or HJ8.5. In one aspect of the invention,
the peptide includes X.sub.(0-8)DQGGYTX.sub.(0-8) (SEQ ID NO: 9)
wherein X is any amino acid. While the illustrative example shows
15 mers (see FIG. 11), one of skill in the art would recognize that
a peptide of different lengths are included in the invention.
Accordingly, the present antibodies or fragments may specifically
bind an epitope containing the amino acid sequence DQGGYT (SEQ ID
NO: 9). The epitope can be a linear or conformational epitopes and
can be from about 6 to 22 amino acids in length.
[0057] In other embodiments, the present methods relate to treating
a tauopathy with the antibody or antigen-binding fragment, wherein
the antibody or fragment is administered in a dose to a subject
having a tauopathy.
[0058] Suitable doses of the antibody or antigen-binding fragment
may be express in terms of mg of drug per kg of subject's body
weight. Suitable doses of the antibody or antigen-binding fragment
include at least about 0.1 mg/kg, alternatively about 0.2 mg/kg,
alternatively about 0.25 mg/kg, alternatively about 0.3 mg/kg,
alternatively about 0.5 mg/kg, alternatively about 0.75 mg/kg,
alternatively about 1 mg/kg, alternatively about 1.25 mg/kg,
alternatively about 1.5 mg/kg, alternatively about 2 mg/kg,
alternatively about 5 mg/kg, alternatively about 7.5 mg/kg,
alternatively about 10 mg/kg, alternatively about 12.5 mg/kg,
alternatively about 15 mg/kg, alternatively about 20 mg/kg,
alternatively about 25 mg/kg, alternatively about 30 mg/kg,
alternatively about 50 mg/kg, alternatively about 100 mg/kg.
Suitable doses of the antibody or antigen-binding fragment may be
at most about 250 mg/kg, alternatively at most about 200 mg/kg,
alternatively at most about 175 mg/kg, alternatively at most about
150 mg/kg, alternatively at most about 125 mg/kg, alternatively at
most about 100 mg/kg, alternatively at most about 75 mg/kg,
alternatively at most about 50 mg/kg, alternatively at most about
25 mg/kg, alternatively at most about 20 mg/kg, alternatively at
most about 15 mg/kg. Any of the foregoing minima and maxima may be
put together to define a range (for example, from about 0.1 mg/kg
to about 250 mg/kg), so long as the minimum value of the range is
lower than the maximum value of the range.
[0059] Suitable doses of the antibody or antigen-binding fragment
may be express in terms of mg of drug administered to a subject.
Suitable doses of the humanized antibody or antigen-binding
fragment include at least about 2.5 mg, alternatively at least
about 5 mg, alternatively at least about 10 mg, alternatively at
least about 15 mg, alternatively at least about 20 mg,
alternatively at least about 25 mg, alternatively at least about 30
mg, alternatively at least about 40 mg, alternatively at least
about 50 mg, alternatively at least about 60 mg, alternatively at
least about 70 mg, alternatively at least about 80 mg,
alternatively at least about 90 mg, alternatively at least about
100 mg, alternatively at least about 125 mg, alternatively at least
about 150 mg, alternatively at least about 175 mg, alternatively at
least about 200 mg, alternatively at least about 250 mg,
alternatively at least about 100 mg, alternatively at least about
125 mg, alternatively at least about 300 mg. Suitable doses of the
antibody or antigen-binding fragment may be at most about 2500 mg,
alternatively at most about 2000 mg, alternatively at most about
1500 mg, alternatively at most about 1000 mg, alternatively at most
about 750 mg, alternatively at most about 500 mg, alternatively at
most about 400 mg, alternatively at most about 300 mg,
alternatively at most about 275 mg, alternatively at most about 250
mg, alternatively at most about 200 mg, alternatively at most about
150 mg. Any of the foregoing minima and maxima may be put together
to define a range (for example, from about 5 mg to about 2500 mg,
so long as the minimum value of the range is lower than the maximum
value of the range.
[0060] C.sub.2N-8E12 is a humanized recombinant IgG4 anti-human tau
antibody. The IgG4 backbone of C.sub.2N-8E12 contains a S241P hinge
stabilizing mutation that minimizes the formation of
half-antibodies. C.sub.2N-8E12 binds to amino acids 25-30 in human
tau (DQGGYT) (SEQ ID NO: 9), a sequence that is present in all
human tau splice variants as well as in amino-terminal fragments of
tau. The antibody binds to both monomeric tau and aggregated tau in
human brain tissue from tauopathies. C.sub.2N-8E12 is highly stable
with very little aggregation or degradation. General physical
properties of C.sub.2N-8E12 are listed in Table 1.
TABLE-US-00001 TABLE 1 Molecular weight 145.72 kDa Stereochemistry
L-amino acids Appearance Clear, colorless to light yellow liquid
Solubility ~130 mg/mL
[0061] Although the invention has been described with reference to
the attached examples, it will be understood that modifications and
variations are encompassed within the spirit and scope of the
invention. The attachments here are illustrative examples of the
invention and herein incorporated by reference in their
entirety.
Example 1
[0062] This example describes efforts and results for humanization
of the murine anti-tau antibody HJ8.5. The efforts yielded four
humanized light chain variable regions (VL or VK) and four
humanized heavy chain variable regions (VH).
[0063] Humanization generally refers to techniques of reducing the
potential immunogenicity associated with using a non-human
monoclonal antibody for chronic treatment. Two methods typically
used to reduce immunogenicity are CDR grafting and deimmunization.
Murine antibody HJ8.5 was de-immunized using a method developed by
Antitope.
[0064] CDR grafting is a protein engineering approach. Briefly, it
relies on both an understanding of the basic architecture of an
antibody and its conservation across species. Murine and human
antibodies share a common/conserved architecture. Antibody
structure is divided into constant and variable regions. The
variable region can be further divided into so called framework
regions and CDR regions. It can be seen that the variable region is
composed of four frameworks (Fwk) and three CDR. The arrangement of
frameworks and CDRs are the same in light and heavy variable
domains.
[0065] In CDR grafting, the non-human constant regions are replaced
with human constant regions, giving rise to a so called chimeric
antibody. In addition, the murine CDR regions are transferred into
human framework regions; the resulting variable domain is a mix of
human frameworks and murine CDR's. As a final step, a number of the
murine framework residues, thought to play a critical role in
maintaining the affinity are transferred (not shown).
[0066] De-immunisation: Composite Human Antibody.TM. technology
from Antitope is said to be a deimmunization technology that is
used in conjunction with identifying both CDRs and key amino acids
in the framework thought to play a role in binding. The resulting
fully-humanized antibodies retain the binding affinity and
specificity of the starting monoclonal antibody and are also devoid
of CD4+ T cell epitopes, which avoids undesirable immunogenicity in
humans.
[0067] Composite Human Antibodies.TM. are generated by combining
multiple segments of human antibody sequences from Antitope's
database comprising 100,000's of unrelated fully-human antibody
variable region sequences. Initial modeling of variable region
sequences of HJ8.5 antibody is used to identify amino acids
critical to antibody binding, which are then used to constrain the
selection of human sequence segments. Individual sequence segments
and the junctions between adjacent segments are then analyzed using
two proprietary in silico technologies (iTope.TM. and TCED.TM.) for
selection of fully-human variable region sequences that are devoid
of CD4+ T cell epitopes. DNA encoding variable regions for
Composite Human Antibodies are synthesized, cloned onto an
expression vector with human constant regions and transfected into
mammalian cells for production of the humanized antibodies.
[0068] Humanization of HJ8.5: Structural models of the HJ8.5 murine
anti-Tau412 antibody V regions were produced using Swiss PDB and
analyzed in order to identify important "constraining" amino acids
in the V regions that were likely to be essential for the binding
properties of the antibody. From the analysis, a number of
constraining framework residues were identified as candidates for
inclusion in the fully humanized V regions. Segments of human
variable region sequences were selected to include one or more of
these residues.
[0069] A preliminary set of human sequence segments that could be
used to create the fully humanized HJ8.5 antibodies were selected
and analyzed using iTope.TM. technology for in silico analysis of
peptide binding to human MHC class II alleles (Perry et al 2008),
and using the TCED.TM. (T Cell Epitope Database) of known antibody
sequence-related T cell epitopes (Bryson et al 2010). Sequence
segments that were identified as significant non-human germline
binders to human MHC class II or that scored significant hits
against the TCED.TM. were discarded. Combinations of sequence
segments were also analyzed to ensure that the junctions between
segments did not contain potential T cell epitopes. Selected
segments were then combined to produce heavy and light chain V
region sequences for synthesis. For HJ8.5, four VH chains and four
VK chains were designed and constructed.
[0070] FIG. 1 shows the variable region sequences of the murine
HJ8.5 antibody as well as 4 humanized variant sequences for each of
the heavy and light chains (4 VH and 4 VL/VK sequences). The amino
acid sequences of those four VH chains and four VK chains are set
forth in FIG. 1. The CDR sequences, as defined by Kabat et al are
highlighted in red (underlined). Framework changes from the
original mouse sequence are highlighted in blue and in bold.
[0071] Table B-1 summarizes the number of framework changes
introduced in each variant of the heavy and light chain variable
domains.
TABLE-US-00002 TABLE B-1 Variable Number of Domain Framework
changes VH1 4 VH2 5 VH3 10 VH4 11 VK1 6 VK2 7 VK3 11 VK4 12
[0072] FIG. 2 shows the sequences of the humanized variable and
constant region sequences for each of the heavy and light chains (4
VH and 4 VL/VK sequences). The variable heavy chain is grafted to
the constant heavy chain of human IgG4 containing the S241P hinge
stabilizing mutation. The variable light chain is grafted to the
constant light chain of human Kappa light chain. This table also
lists the theoretical isoelectric point (PI) and molecular weight
(Mw).
[0073] FIG. 3 shows expression data from 2 rounds of transient
expression of cells transfected with polynucleotides encoding VH
and VK regions. Results for 13 humanized anti tau antibodies are
summarized. Different combinations of heavy and light chains
resulted in markedly different levels of expression being observed.
In Round 1, all variants of VH and VL regions were combined with
each other (only results for 13 are shown of the 16 that were
tested. In Round 2, the 6 best expressing combinations observed in
Round 1 were tested. Expression is shown as .mu.g of antibody
measured per mL of culture media. Higher levels of expression is
advantageous since it suggests that the antibody is correctly
folded, secreted as expected, non toxic and generally stable.
[0074] FIG. 4 shows data from a potency assay. To further
characterize the humanized anti-tau antibody variants, the potency
assay evaluates the ability of antibodies to compete with the
original murine HJ8.5 (parent antibody) for binding to human tau in
an ELISA type format. The assay format involves coating the ELISA
plate with human tau and then allowing the test antibodies as well
as biotinylated HJ8.5 to compete for binding to tau. The assay
enables the relative IC50 value for each humanized antibody variant
to be measured. IC50 values are normalized to that of chimeric
HJ8.5 to enable comparisons to be made between plates. This data
demonstrates that the humanization process has not significantly
changed the binding of the humanized antibodies to human tau.
Example 2
[0075] This study describes the use of the Biacore T200 to measure
and compare the binding characteristics of the interaction between
six fully humanized (VH1/VK2, VH1/VK3, VH2/VK2, VH2/VK3, VH3/VK2
and VH3/VK3, described above in Example 1) monoclonal antibodies
and one chimeric monoclonal antibody based on HJ8.5 with
recombinant human Tau-412 protein. The aim of this study was to use
the Biacore T200 surface plasmon resonance instrument for the high
resolution kinetic characterization of the interactions between
Tau-412 and these seven mAbs.
[0076] The antibodies were stored at 4.degree. C. Tau-412 was
stored at -20.degree. C. as per the manufacturer's instructions.
Once reconstituted the Tau-412 solution was stored on ice and used
within 24 hours. Aliquots of reconstituted Tau-412 were frozen
within 30 minutes of reconstitution and stored at -20.degree.
C.
[0077] The Biacore instrument was run on Biacore T200 Evaluation
Software V1.1 (Uppsala, Sweden). All materials were from Biacore
unless stated:
TABLE-US-00003 Biacore Preventative Maintenance Kit 2 BR-1006-51
Series S CM5 Sensor Chips BR-1006-68 Amine Coupling Kit BR-1000-50
10 mM Acetate pH 4.5 BR-1003-51 HBS-EP Running buffer\ BR-1006-69
10 mM Glycine-HCI pH 1.5 BR-1003-54 10 mM Glycine-HCI pH 2.0
BR-1003-55 Protein A (Sigma) P6031 4M MgCl.sub.2 hexahydrate
(Sigma) M9272-500G
[0078] All experiments were developed with Biacore `wizard`
software. The following Biacore methods were used: Immobilization;
Kinetics/Affinity; and Desorb and Sanitize.
[0079] Before running any samples, and during the study, a system
check (Biacore Preventative Maintenance Kit 2) was performed. All
the systems tested passed (Reagent pump, Refractometer, Injections,
Noise, Mixing and Buffer Selector) indicating that the instrument
was performing to criteria set by the manufacturer.
[0080] Upon insertion of a CM5/Protein A chip the system was primed
and then normalized with BIA normalizing solution (Biacore
Preventative Maintenance Kit 2). All samples were run at 25.degree.
C. with a sample rack incubated at 5.degree. C. The chip was added
to the system with HBS-EP used as the running buffer.
[0081] The mAbs were stored as supplied and diluted to 100 nM for
all immobilization (capture) runs. The antigen Tau-412 was
reconstituted from the dry powder using Milli-Q water to a final
concentration of 1 mg/mL; further dilutions were performed for the
kinetics runs. The mass and molecular weight of Tau-412 used in the
concentration calculation was provided by the reagent manufacture
(100 .mu.g/vial and 42.9 kDa). No carrier protein was added to this
solution. Vials of the antigen were only reconstituted when
required and were stored in their powder form at -20.degree. C.
until use. Once reconstituted, the antigen solution was kept on ice
and used within 24 hours.
[0082] A capture assay with protein A was selected for this study.
The performance of the Protein A surface was superior to the
anti-human, protein A/G, protein G and protein L surfaces that were
also tested. The Protein A chip was prepared through immobilization
using standard amine coupling chemistry. Immobilization was carried
out at a protein concentration of 5 .mu.g/mL in 10 mM Acetate
buffer pH 4.5 to a target response level of 500 RUs on a CM5 Series
S sensor chip (Biacore).
[0083] The final response levels for the Protein A chip `All` and
designated F.sub.cs are shown in Table G-1.
TABLE-US-00004 TABLE G-1 Final Response Ligand Level (RU) F.sub.c1
Protein A 697.1 F.sub.c2 Protein A 691.4 F.sub.c3 Protein A 708.3
F.sub.c4 Protein A 704.6
[0084] For kinetic experiments, the amount of immobilized/captured
ligand needs to be limited to avoid mass transfer effects at the
surface of the chip. For kinetic experiments, a surface should
ideally have a maximum analyte binding level (R.sub.max) of 50-100
RUs. The amount of ligand to immobilize is therefore calculated
using Equation 1:
analyte binding capacity ( RU ) = analyte MW ligand MW immobilize d
ligand ( RU ) Sm ##EQU00001##
[0085] Using an average MW of 42.9 kDa (provided by the reagent
manufacture) for the analyte Tau-412, 150 kDa for the ligand
(estimated value for antibodies) (mAb), 100 RU for R.sub.max, and
the stoichiometry (S.sub.m) as 1, a target of 300 RUs was set for
capture of all the trial antibodies. The capture levels obtained
within the study varied from .sup..about.280-400 RU's. For the
second and third runs the amount of injected antibody was adjusted
to get closer to the desired 300 RU capture level.
[0086] Non-specific binding can be due to either the analyte or
analyte contaminants interacting with either the ligand
(non-specific and difficult to detect), capture protein, or the
sensor chip surface. By analyzing the response of the blank
F.sub.cl, surface after a relatively high concentration (40 nM) 300
second injection of Tau-412, no NSB was observed to the
carboxy-dextran surface, or Protein A capture surface. At Tau-412
concentrations >100 nM, significant NSB was observed to the
carboxy-dextran chip surface; however concentrations within this
range were not required for subsequent kinetic analysis.
[0087] Regeneration scouting was performed and the optimum
conditions for the regeneration of chimeric and VH1/VK2 antibodies
on the Protein A surface were as follows. Three 240 second
injections of 10 mM Glycine-HCl pH 1.7 followed by one 300 second
injection of 4M MgCl.sub.2 all at 40 .mu.L/min. A 600 second wait
step was introduced after the last regeneration injection to allow
the surface to stabilize before starting the next binding
cycle.
[0088] No buffer scouting was performed as initial tests indicated
the selected buffer `HBS-EP` generated a reproducible system
suitable for kinetic analysis.
[0089] The performance of the surface was analyzed by repeated
control injections of 2.5 nM Tau-412 at the start, interspaced and
at the end of a kinetic run. Stable binding was observed throughout
the kinetic run highlighting the suitability of the system for
kinetic analysis.
[0090] Mass transport limitation occurs when the rate of
association contains a significant component associated with the
rate of transport of the analyte to and from the chip surface.
Where mass transfer is found to be significant the resulting
kinetic analysis could be inaccurate. Lowering the density of
immobilized ligand, or increasing the flow rate, can reduce mass
transport limitations. From previous experience of using low
density surfaces and similar Mw antigens, a flow rate of 40
.mu.L/min was selected for this study.
[0091] The linked reaction control experiment is used to assess the
ligand-analyte interaction to check for deviations from a 1-to-1
binding model. The analyte is injected over the surface for
different periods of time (contact times) and the dissociation rate
is analyzed to determine if it varies with the contact time. If
such a relationship is observed, it indicates that a second
interaction event is taking place after the initial binding event
that results in a stabilized complex at the surface.
[0092] From previous experience using capture assay formats, the
apparent binding stoichiometry of 1.5 and that a 1-to-1 model could
be fitted with confidence to the resulting kinetic data, linked
reaction controls were not performed as there was no additional
evidence to support more complex kinetic interactions.
[0093] A 1-to-1 binding model was used to fit the resulting kinetic
data (Equation 2). Due to variations in the amount of antibody
captured the parameter R.sub.max was set to local as opposed to
global analysis for each antibody kinetic analysis.
A + B .fwdarw. ka .rarw. kd AB ##EQU00002##
[0094] Antibody Characterization: The characterization and the
control experiments performed for the Protein A capture surface
suggested this was a suitable system to determine kinetic values
for the Tau-412 interactions. The binding stoichiometry was
assessed by injecting a saturating concentration of Tau-412 (1000
nM) over 277 RU's of captured VH1/VK2 on a trial Protein A surface.
Two sequential injections of 1000 nM Tau-412 appeared to result in
saturated binding at 122 RU's. This resulted in a binding
stoichiometry of 150%, which is higher than expected for one
antibody molecule binding to one Tau-412 molecule. Reasons for this
could include binding of the antibody to two molecules of Tau-412
or Tau-412 oligomerization on the surface of the chip.
[0095] Kinetic data was obtained at a flow rate of 40 .mu.L/min to
minimize any potential mass transfer effects. Two repeats of the
blank (no antigen) and the 2.5 nM concentration of the analyte were
programmed into the kinetic run in order to check the stability of
both the surface and analyte over the kinetic cycles. For the
initial kinetic runs, 2-fold dilutions of Tau-412 from 40 nM to
0.156 nM were run. For kinetic analysis and on subsequent runs, an
analyte range of 20 nM to 0.625 nM was selected. This range covered
multiple analyte concentrations both above and below the reported
K.sub.D.
[0096] The association phase was monitored for 500 seconds to allow
some of the higher concentrations of analyte to reach steady state.
In order to observe a sufficient signal decrease (>10%) during
the dissociation phase of the kinetic cycle, dissociation was
measured for 1200 seconds. As discussed in Section 5, the F.sub.cs
were allowed to stabilize for 600 seconds after each regeneration
step. The signal from the reference channel F.sub.c1 was subtracted
from that of F.sub.c2, F.sub.c3 and F.sub.c4.
[0097] The kinetic parameters for the interaction of Tau-412 with
the 7 mAbs as measured using the Protein A capture system on the
Biacore T200 are shown in Table F-2. To correct for differences in
the capture level of the antibody between each binding cycle, a
local R.sub.max parameter was used in the 1-to-1 binding model.
Kinetic analysis was performed in three independent runs using
fresh preparations of Tau-412 and antibody. Run 1 and runs 2+3 used
different vials of the antigen Tau-412; therefore the reported
errors associated with the mean response probably represent
variation in preparation of the analyte and differences in assay
set-up and run. From run 1 to runs 2+3, the amount of antibody
injected was adjusted to try to get closer to the target 300 RU
capture levels. For run 1, the chimeric antibody was run in
triplicate and an analysis of all three data sets is shown in Table
F-2. The % CV for the K.sub.D derived from these three data sets
was 4.3% indicating that the results were within assay
variability.
TABLE-US-00005 TABLE F-2 Ligand Chip k.sub.a (1/Ms) SE (k.sub.a)
k.sub.d (1/s) SE (k.sub.d) K.sub.D (nM) SD (K.sub.D) Chi.sup.2
Appendix II VH1/VK2 A11/1 2.80 .times. 10.sup.5 4.40 .times.
10.sup.2 6.11 .times. 10.sup.-4 2.80 .times. 10.sup.-7 2.18 1.28
A3-4 VH1/VK2 A11/3 3.25 .times. 10.sup.5 1.00 .times. 10.sup.3 6.32
.times. 10.sup.-4 8.20 .times. 10.sup.-7 1.95 0.44 A5-6 k.sub.a
(1/Ms) SD (k.sub.a) k.sub.d (1/s) SD (k.sub.d) K.sub.D (nM) SD
(K.sub.D) (nM) Mean 3.02 .times. 10.sup.5 3.19 .times. 10.sup.4
6.21 .times. 10.sup.-4 1.48 .times. 10.sup.-5 2.06 0.17 Ligand Chip
k.sub.a (1/Ms) SE (k.sub.a) k.sub.d (1/s) SE (k.sub.d) K.sub.D (nM)
SD (K.sub.D) Chi.sup.2 Appendix II VH1/VK3 A11/1 2.80 .times.
10.sup.5 9.10 .times. 10.sup.2 6.26 .times. 10.sup.-4 8.50 .times.
10.sup.-7 2.24 0.99 A7-8 VH1/VK3 A11/2 2.95 .times. 10.sup.5 4.40
.times. 10.sup.2 5.72 .times. 10.sup.-4 2.70 .times. 10.sup.-7 1.94
0.52 A9-10 k.sub.a (1/Ms) SD (k.sub.a) k.sub.d (1/s) SD (k.sub.d)
K.sub.D (nM) SD (K.sub.D) (nM) Mean 2.87 .times. 10.sup.5 1.07
.times. 10.sup.4 5.99 .times. 10.sup.-4 3.76 .times. 10.sup.-5 2.09
0.21 Ligand Chip k.sub.a (1/Ms) SE (k.sub.a) k.sub.d (1/s) SE
(k.sub.d) K.sub.D (nM) SD (K.sub.D) Chi.sup.2 Appendix II VH2/VK2
A11/1 2.62 .times. 10.sup.5 4.30 .times. 10.sup.2 6.28 .times.
10.sup.-4 2.80 .times. 10.sup.-7 2.40 0.83 A11-12 VH2/VK2 A11/2
2.84 .times. 10.sup.5 4.10 .times. 10.sup.2 5.87 .times. 10.sup.-4
2.60 .times. 10.sup.-7 2.06 0.70 A13-14 k.sub.a (1/Ms) SD (k.sub.a)
k.sub.d (1/s) SD (k.sub.d) K.sub.D (nM) SD (K.sub.D) (nM) Mean 2.73
.times. 10.sup.5 1.59 .times. 10.sup.4 6.08 .times. 10.sup.-4 2.94
.times. 10.sup.-5 2.23 0.24 Ligand Chip k.sub.a (1/Ms) SE (k.sub.a)
k.sub.d (1/s) SE (k.sub.d) K.sub.D (nM) SD (K.sub.D) Chi.sup.2
Appendix II VH2/VK3 A11/1 2.68 .times. 10.sup.5 8.80 .times.
10.sup.2 6.53 .times. 10.sup.-4 9.00 .times. 10.sup.-7 2.44 1.11
A15-16 VH2/VK3 A11/2 2.92 .times. 10.sup.5 4.40 .times. 10.sup.2
5.33 .times. 10.sup.-4 2.60 .times. 10.sup.-7 1.83 0.50 A17-18
k.sub.a (1/Ms) SD (k.sub.a) k.sub.d (1/s) SD (k.sub.d) K.sub.D (nM)
SD (K.sub.D) (nM) Mean 2.80 .times. 10.sup.5 1.71 .times. 10.sup.4
5.93 .times. 10.sup.-4 8.44 .times. 10.sup.-5 2.13 0.43 Ligand Chip
k.sub.a (1/Ms) SE (k.sub.a) k.sub.d (1/s) SE (k.sub.d) K.sub.D (nM)
SD (K.sub.D) Chi.sup.2 Appendix II VH3/VK2 A11/1 2.62 .times.
10.sup.5 3.20 .times. 10.sup.2 5.46 .times. 10.sup.-4 2.20 .times.
10.sup.-7 2.08 0.45 A19-20 VH3/VK2 A11/2 2.90 .times. 10.sup.5 3.70
.times. 10.sup.2 4.85 .times. 10.sup.-4 2.20 .times. 10.sup.-7 1.67
0.71 A21-22 k.sub.a (1/Ms) SD (k.sub.a) k.sub.d (1/s) SD (k.sub.d)
K.sub.D (nM) SD (K.sub.D) (nM) Mean 2.76 .times. 10.sup.5 1.99
.times. 10.sup.4 5.15 .times. 10.sup.-4 4.28 .times. 10.sup.-5 1.88
0.29 Ligand Chip k.sub.a (1/Ms) SE (k.sub.a) k.sub.d (1/s) SE
(k.sub.d) K.sub.D (nM) SD (K.sub.D) Chi.sup.2 Appendix II VH3/VK3
A11/1 2.54 .times. 10.sup.5 3.10 .times. 10.sup.2 5.59 .times.
10.sup.-4 2.00 .times. 10.sup.-7 2.20 0.80 A23-24 VH3/VK3 A11/2
2.76 .times. 10.sup.5 3.80 .times. 10.sup.2 4.54 .times. 10.sup.-4
2.20 .times. 10.sup.-7 1.65 0.80 A25-26 k.sub.a (1/Ms) SD (k.sub.a)
k.sub.d (1/s) SD (k.sub.d) K.sub.D (nM) SD (K.sub.D) (nM) Mean 2.65
.times. 10.sup.5 1.56 .times. 10.sup.4 5.07 .times. 10.sup.-4 7.40
.times. 10.sup.-5 1.92 0.39 Ligand Chip k.sub.a (1/Ms) SE (k.sub.a)
k.sub.d (1/s) SE (k.sub.d) K.sub.D (nM) SD (K.sub.D) Chi.sup.2
Appendix II Chimeric A11/1 7.18 .times. 10.sup.5 1.60 .times.
10.sup.3 1.60 .times. 10.sup.-3 2.40 .times. 10.sup.-4 2.23 1.17
A27-29 Chimeric A11/2 7.22 .times. 10.sup.5 3.00 .times. 10.sup.3
1.30 .times. 10.sup.-3 3.50 .times. 10.sup.-5 1.79 0.97 A30-31
Chimeric A11/3 7.23 .times. 10.sup.5 2.60 .times. 10.sup.3 1.40
.times. 10.sup.-3 3.30 .times. 10.sup.-6 1.94 0.49 A32-33 k.sub.a
(1/Ms) SD (k.sub.a) k.sub.d (1/s) SD (k.sub.d) K.sub.D (nM) SD
(K.sub.D) (nM) Mean 7.21 .times. 10.sup.5 2.87 .times. 10.sup.3
1.43 .times. 10.sup.-4 1.54 .times. 10.sup.-4 1.99 0.22
[0098] The Chi.sup.2 values show how well the association and
dissociation data fits the proposed 1-to-1 binding model--the lower
the value the better the fit. The associated SE values for the rate
constants represent the uncertainty associated with fitting the
data to the model described, and do not represent the total
uncertainty for the true kinetic values. The mean response data
represents the average kinetic values and the associated SD from 2,
or 3 independent analyses.
[0099] Using the mean K.sub.D values from Table F-2, the antibodies
can be ranked based on affinity as follows:
VH3/VK2>VH3/VK3>Chimeric>VH2/VK3>VH1/VK3>VH1/VK2>VH2/VK-
2. The % CV associated with the mean kinetic parameters ranged from
10-20%, and thus it is likely that all antibodies have very similar
affinities and that differences are purely a result of assay
variation. In general, the differences in binding between the
antibodies may be attributable to assay variation, and it is
believed there are no significant differences in K.sub.D values of
the humanized antibodies compared to the chimeric antibody.
[0100] A comparison of the kinetic values determined using the
protein A capture assay on the Biacore T200 for the interaction
between the antibodies and Tau-412 are shown The chimeric antibody
appears to display a significantly different binding profile when
compared to the humanized antibodies, although the affinities are
similar. A k.sub.d versus k.sub.a plot shows the relative kinetic
values of the tested antibodies and Tau-412 interactions as
determined using the Protein A capture assay on the Biacore T200.
The dashed diagonals represent isoaffinity lines. Please note the
axes display different data ranges, with the aim of improving the
clarity of the humanized antibodies on the plot.
[0101] FIG. 5 summarizes the results from surface plasmon resonance
(SPR) analysis, determining the binding kinetics of the 6 best
expressing humanized constructs against human tau. The test
antibody is immobilized on the SPR chip with different
concentrations of human tau then allowed to flow over the chip.
Association and dissociation rates as well as the affinity based on
the measured binding events is calculated for each of the variants.
The chimeric variant was also tested.
Example 3
[0102] FIG. 6 shows the binding of four humanized antibody variants
to soluble human tau in a sandwich style ELISA. Assay methods that
rely on passive adsorption have the potential to create artifactual
binding results. To overcome this possibility, a solution based
method to measure the binding activity of the humanized antibody
variants was employed. In this assay format, antigen (human tau) is
captured by a monoclonal anti-human tau antibody that recognizes a
different epitope than HJ8.5. Subsequent binding of the humanized
anti-tau antibodies to the captured human tau depends on antigen
concentration, while IgG4 isotype controls shows no binding at all.
This assay demonstrates that binding of the humanized anti-tau
antibodies to human tau is specific and that the antibodies bind to
soluble human tau.
Example 4
[0103] FIGS. 7A-7MM show binding of humanized and control
antibodies to tissue from wild type mice (negative control tissue),
P301S mice (which express human tau having a P301S mutation and
develop age associated tau pathology), and humans with either
Alzheimer's disease or Progressive Supranuclear Palsy (PSP). The
aim of this study was to confirm that humanized antibodies retain
the ability to bind to aggregated tau in tissue as compared to the
chimeric form of HJ8.5. The figures show representative images of
staining human and mouse brains with different variants of
humanized HJ8.5 antibody. P301S mice at 4 and 9 month old were
tested, and both time points show pathologic aggregates of tau,
with the 9 month old mice having more tau pathology than the 4
month old mice. For human staining, a sample of brain tissue from
one subject with PSP, and a sample of brain tissue from one subject
with AD was examined. FIGS. 7A-7E illustrate staining with chimeric
HJ8.5 for the mouse and human AD tissue. FIGS. 7F-7I illustrate the
staining with a negative control antibody (non-specific human
IgG4). FIGS. 7J-7MM illustrate the staining with the six humanized
antibodies. All humanized variants of the murine HJ8.5 antibody
bind to tau aggregates found in P301S mouse brain as well as tau
aggregates found in the brain tissue of the subjects diagnosed with
either AD or PSP.
Example 5
[0104] FIG. 8 shows the epitopes of HJ8.5 in human tau. The epitope
was mapped using yeast display. For this method, various peptides
covering the sequence of human tau were expressed using by yeast.
Binding of the HJ8.5 antibody to yeast in culture was measured by
immunofluorescence. Binding to yeast, expressing variants of tau
that included the first 34 amino acids was observed, but no
binding, if the yeast only expressed the first 32 amino acids of
tau. This suggests that the epitope is within the first 34 amino
acids. Additionally, HJ8.5 binds if the peptide includes amino
acids 27-135 but not if the peptide spans amino acids 30-135. This
suggests that the epitope includes amino acids greater than amino
acid 27. Based on this data the epitope is contained within the
27-34 sequence of human tau (GYTMHQDQ (SEQ ID NO: 10). FIG. 8 also
shows the rhesus monkey and mouse tau sequences and highlights in
red the amino acid changes from human tau.
[0105] FIG. 9 shows more detailed, peptide-based epitope mapping of
HJ8.5 and C.sub.2N-8EI2. A peptide library of linear 15 mers
spanning the full sequence of human tau (IN4R, 412 amino acids) was
created. Additionally double alanine versions of these peptides
where amino acids 10 and 11 were changed to alanine were also
produced. For the double alanine library, any naturally occurring
alanines at position 10 or 11 were mutated to glycine. All peptides
were spotted onto a peptide array and then probed with HJ8.5 or
C.sub.2N-8E12 and binding measured. The tau binding epitope(s) of
both antibodies were reliably mapped using these peptide arrays.
The binding epitope of C.sub.2N-8E12 is .sub.25DQGGYT.sub.30 (SEQ
ID NO: 9) and matches the epitope of the murine parent, HJ8.5. The
binding of HJ8.5 and C.sub.2N-8E12 to tau peptides is severely
compromised when amino acids D, Q, Y, or T in the epitope were
replaced with alanine, suggesting that they play a crucial rule in
the antibody binding. However, when the central two glycines in the
epitope were replaced with alanine, the binding of antibodies to
the tau peptides was not as severely compromised (PEP 2875811).
This is likely not an indication that these amino acids are not
important for binding but rather due to the conservative nature of
substitutions between Alanine and Glycine amino acids. The epitope
mapped using these more detailed methods is slightly different from
what was mapped using yeast display (FIG. 8). This difference is
attributed to the difference in the binding assays, with larger
peptides being used on the yeast display system. The 15 mer peptide
array methodology is considered to be superior to the yeast display
methodology.
Example 6
[0106] FIG. 10 illustrates the binding results for different anti
human tau antibodies to either human or rhesus monkey tau. Murine
anti human tau antibodies HJ8.5 and HJ8.7 alongside humanized
variant VH1/VK2 (also referred to as C.sub.2N-8E12) were tested.
FIG. 8 shows that there is a single amino acid difference at
position 32 between human and rhesus tau, in the claimed binding
epitope sequence GYTM(H/L)QDQ (SEQ ID NO:57). FIG. 8 shows that
there is a single amino acid difference at position 27 between
human and rhesus tau, in the claimed binding epitope sequence
DQ(G/E)GYT (SEQ ID NO:58). In order to determine whether these
amino acid difference between the two species of tau, impacts the
ability of antibodies HJ8.5/C.sub.2N-8E12 to bind the following
experiment was performed. Binding of C.sub.2N-8E12, HJ8.5 (murine
precursor of C.sub.2N-8E12), and HJ8.7 (murine anti-human tau
antibody that binds to an epitope of tau where the human and rhesus
amino acid sequence is 100% conserved) to human and rhesus tau by
coating 96 well ELISA plates with either human or rhesus tau at
various concentrations was measured. Our results demonstrated that
C.sub.2N-8E12 and HJ8.5 do not bind to rhesus tau while they do
show positive binding to human tau. As expected, HJ8.7 binds to
both human and rhesus tau.
Example 7
[0107] FIG. 11 shows binding of humanized anti-tau antibody to tau
in CSF from human subjects with various tauopathies. The binding of
C.sub.2N-8E12 to tau in CSF samples from subjects diagnosed with a
variety of tauopathies as well as age matched and young normal
control subjects was evaluated. A sandwich ELISA was used to
demonstrate binding of C.sub.2N-8E12 to tau in human CSF from
subjects with AD, CBD, FTD, or PSP as well as age matched and
young/adult controls. C.sub.2N-8E12 was used as the coating
antibody to capture tau in CSF samples. Biotinylated murine
monoclonal tau antibody HJ8.7 was used as the detection antibody.
Wells coated with control human IgG4 acted as the negative control
for the experiments. A big difference in signal from C.sub.2N-8E12
coated wells vs. control IgG4 coated wells was observed,
demonstrating specific binding of C.sub.2N-8E12 to tau in human CSF
samples. By including a standard curve (recombinant tau), it is
possible to get quantitative information on tau concentration in
these CSF samples.
Example 8
[0108] This study is a randomized, double blind, placebo
controlled, single ascending dose (SAD) phase 1 study to be
conducted in up to ten (10) centers. It is designed to evaluate the
safety, tolerability, immunogenicity, and PK of single-dose
administration of C.sub.2N-8E12 and to establish the MTD to be used
in future repeat dosing studies.
[0109] The primary objective of this study is to determine the
safety, tolerability, immunogenicity, and maximally tolerated dose
(MTD) of a single dose of C.sub.2N-8E12 in subjects with PSP.
Safety assessments will include physical and neurologic
examination, clinical safety laboratory studies, immunogenicity,
adverse events, vital signs and concomitant medication review.
[0110] The secondary objectives are to determine: Single-dose
systemic pharmacokinetics including; Maximum plasma concentration
after single infusion; Area under the curve (AUC) after single
infusion; Time at which the maximum concentration after infusion is
achieved; Terminal half-life of C.sub.2N-8E12; Partition of
C2N-8E12 into cerebrospinal fluid (CSF); and Biologic target
engagement through the measurement of soluble tau levels in blood
and CSF as well as assessing the presence of C.sub.2N-8E12-tau
complexes.
[0111] This study intends to enroll 32 subjects with PSP (24 in the
treatment arm and 8 in the placebo arm). Subjects will be enrolled
in 8 blocks of 4 patients, with one patient in each block
randomized to placebo and 3 to the current estimate of the MTD.
Additional subjects may be enrolled if DLTs occur. No dose may be
skipped, however, during the dose escalation process.
[0112] A continual reassessment method (CRM) for dose escalation
will be used as described in the statistical design section. A
logistic model will be used to identify the probability of DLT by
dose.
[0113] C.sub.2N-8E12 will be shipped to the clinical site as a
frozen liquid in single use bottles at a nominal concentration of
20 mg/mL. Each bottle contains 300 mg C.sub.2N-8E12 and must be
stored frozen at -70.degree. C. to -80.degree. C.
[0114] Patients will undergo screening to assess whether inclusion
and exclusion criteria are met. Screening will also include
assessments of blood and CSF, and MRI. On the day of dosing (Day
0), a single dose of C.sub.2N-8E12 will be administered through an
IV line and subjects will be closely monitored at a clinical
facility for 24 hours after dose administration. This includes
blood samples for safety and PK assessments. During the following 3
days, as well as at one and two weeks after the infusion,
additional clinical examination and blood sampling will occur. An
additional safety MRI and CSF sampling will be performed 4 days
post-infusion. Subjects will be followed every 28 days, for no less
than two months from the date of dosing (e.g., day 56). Monthly
measurements, thereafter, will continue until the earlier
occurrence of any of the following events: (i) C.sub.2N-8E12 is no
longer detectable in blood; (ii) the Sponsor determines completion
of the study; (iii) the subject decides to early discontinue
participation in the study.
[0115] The goal of phase 1 study includes establishment of an MTD
as assessed by safety evaluations including clinical laboratory
tests, physical and neurologic examinations, and occurrences of
adverse events to determine a recommended range of doses for
evaluation in the subsequent phase 2/MAD study. Random assignment
of subjects and inclusion of a placebo arm avoids bias and
increases the likelihood that both known and unknown risk factors
are distributed evenly between treatment groups.
[0116] A data safety monitoring committee (DSMC) will review safety
data on an ongoing basis. The safety monitoring committee will be
minimally comprised of two independent physicians, one
biostatistician, one physician with expertise in PSP, and one
non-voting member from the Sponsor. If any individual study subject
experiences SAEs, all available safety data for the subject will be
reviewed to determine if the event meets the definition of DLT, and
whether the MTD has been established. If MTD has not been
established, and patient enrollment continues, the DSMC will
provide recommendations to the Sponsor whether any further actions
or protocol amendments are necessary to ensure the safety of
subsequently enrolled patients. The Sponsor will make final
determinations on any amendments to or preliminary termination of
the study.
[0117] A dose limiting toxicity is defined as: (i) any Grade 3 or
higher AE per Rheumatology Common Toxicity Criteria v2 for which
there is reasonable possibility that C.sub.2N-8E12 caused the
event; (ii) any Grade 2 AE in the NCI's Common Terminology Criteria
for Adverse Events v4.0. (CTCAE) system organ class of nervous
system and psychiatric disorders that is considered clinically
significant and for which there is reasonable possibility that
C.sub.2N-8E12 caused the event; or (iii) any infusion-related
toxicities (e.g., allergic reaction/hypersensitivity) occurring
during the infusion of C.sub.2N-8E12 or within 24 hours after
completing the infusion that do not resolve promptly with a reduced
infusion rate and/or supportive care.
[0118] Dose Escalation: The assignment of subjects to dose cohorts
is governed by the following rules: (1) Within each block of 4
patients, 1 patient will be allocated to the placebo arm; (2)
Complete toxicity information is required for at least 3 patients
at a dose level before escalation to a higher dose level; (3) The
maximum increment of escalation from one cohort to the next is 1
dose level; and (4) At least 12 subjects (3 blocks) should be dosed
at the MTD dose level.
[0119] Within each 4-patient cohort, patients will be dosed
sequentially, with a minimum interval of at least two days between
dosing of consecutive subjects in order to provide an additional
measure of safety assurance.
[0120] The first cohort will be allocated to d.sub.1. The
statistical model will be updated after complete toxicity
information is available for each cohort. By rule, one additional
cohort may be enrolled before complete information is available for
all subjects on the most recent cohort. Incomplete toxicity data is
allowed for no more than 3 patients before the next cohort is
enrolled and randomized.
[0121] Each subsequent cohort will be assigned to the dose that is
estimated to be the MTD according to the definition above. In the
event of slow accrual, the model may be updated as each patient
enrolls and each subsequent patient dosed to the current estimated
MTD.
[0122] Study Population: This study will enroll male and female
subjects with progressive supranuclear palsy (PSP) aged 50 to 85
years.
[0123] Inclusion Criteria: For inclusion into the study, each
subject must be willing and able to provide informed consent. Prior
to initiation of the treatment protocol, it will be confirmed that
each subject is able to provide consent for the treatment protocol.
Subjects will be invited to participate in the study, and after
signing the informed consent form, screening procedures will take
place. If subjects fail to fulfill the inclusion criteria or meet
any of the exclusion criteria, the subjects will not be enrolled
into the screening assessment or treatment schedule.
[0124] Each subject must meet the following criteria to be enrolled
in this study: Male or female; Between 50 and 85 years of age;
Meets NINDS-SPSP possible or probable criteria as modified for
NNIPPS and AL-108-231 clinical trials, including: (d) supranuclear
gaze palsy or decreased saccade velocity, (ii) gait instability or
falls within the first 3 years of symptoms; Brain MRI at Screening
is consistent with PSP (<4 microhemorrhages, and no large
strokes or severe white matter disease); Score on the PSP rating
scale between 20 and 50; Able to provide informed consent to
participation at baseline or if unable to provide informed consent
can provide assent to participation and has an authorized medical
representative who can provide consent; Has study partner who sees
the patient at least 5 hours per week, who can accompany the
patient to visits and consents to study participation; Other
concurrent non-biologic therapies are allowed but the dose must
have been stable for at least 30 days prior to enrollment; Able to
walk 5 steps with minimal assistance (stabilization of one arm or
use of cane/walker); Stable medications for Parkinsonism for at
least 2 months prior to Screening; including, levodopa, dopamine
agonists, rasagaline, COMT inhibitors, amantadine, memantine or
cholinesterase inhibitors; Agrees to up to 3 lumbar punctures over
4-18 months, up to 6 lumbar punctures if the subject will
participate in both the phase 1/SAD study and the phase 2/MAD
study; Signed and dated written informed consent obtained from the
subject; Agree to use protocol specified methods of contraception
(see below).
[0125] Subjects who meet any of the following criteria will be
excluded from the study: Signs of a progressive neurological
disorder that better meets the criteria for types of neurological
disorders other than PSP, including: (a) meets criteria for
probable Alzheimer's disease or (b) meets research criteria for
Parkinson's Dementia with Lewy Bodies, multiple system atrophy
(MSA), or amyotrophic lateral sclerosis (ALS); Any malignancy
(other than non-metastatic basal cell carcinoma of the skin) within
5 years of screening; Clinically significant renal or hepatic
dysfunction at screening based on professional judgment of
Investigator; Clinically significant cardiovascular event within
three months prior to study entry, based on professional judgment
of Investigator; Clinically significant abnormal hematology or
chemistry laboratory test results during screening, based on
professional judgment of Investigator; Have received any prior
monoclonal antibody therapy for any reason within the last 90 days
or received any other investigational agent within the previous 30
days or 5 half-lives (whichever is longer). Prior administration of
C.sub.2N-8E12 does not apply to this exclusion criteria and,
therefore, does not disqualify a subject from participating in the
phase 2/MAD study; Currently on any other biologic or
immunomodulatory therapy; Disease duration of greater than 5 years
since onset of symptoms; Midbrain volume >8,600 mm.sup.3 on
screening MRI scan; Subjects that reside at a skilled nursing or
dementia care facility; Has clear evidence of motor neuron disease
on examination, consistent with ALS pathology (this has been
described in C90RF72 carriers with CBS presentation); Diagnosis of
any other significant unrelated neurological or psychiatric
disorders that could account for cognitive deficits (e.g., active
seizure disorder, stroke, vascular dementia), based on professional
judgment of Investigator; Untreated major depression at baseline
evaluation, based on clinical judgment and results in GDS; History
of other major psychiatric illness; Any prior history of suicidal
attempts; Severe cognitive impairment as assessed by MMSE (<17)
that in the Investigator's opinion would preclude collection of
outcome measures; Not being able to participate in evaluation
protocol; Significant, abnormal values in general from blood
samples taken at screening that would pose a safety risk or
interfere with appropriate interpretation of study data; Current or
recent history (within four weeks prior to Screening) of a
clinically significant bacterial, fungal, or mycobacterial
infection; Unable to tolerate MRI scan at Screening or any other
contraindication to MRI; Any contraindication to or unable to
tolerate lumbar puncture at Screening, including use of
anti-coagulant medications such as warfarin. Daily administration
of 81 mg aspirin or similar anticoagulants will be allowed as long
as the dose is stable for 30 days prior to Screening; Subjects who,
in the opinion of the Investigator, are unable or unlikely to
comply with the dosing schedule or study evaluations; Participation
in another interventional clinical trial within 3 months of
Screening; Treatment with another investigational drug within 30
days of Screening; Any preexisting QTcF duration exceeding 450 ms;
Subject is employee or family member of the Sponsor or
investigational site staff member or their family members.
[0126] Subjects must agree to use (and/or have their partner use)
acceptable methods of contraception beginning at the baseline visit
throughout the study and until 56 days after the last dose of study
drug in the last treatment period. Acceptable methods of
contraception are listed below.
[0127] Study drug: The phase 1/SAD study will use C.sub.2N-8E12
from DP Lot #1018775--the Research Cell Bank (RCB) material
currently being used in the Expanded Access IND 119404. It is
formulated in 25 mM acetate buffer at pH 5.5, and it is provided at
a concentration of 20 mg/mL. Placebo: Placebo is formulated
identically to C.sub.2N-8E12 without the active study drug.
[0128] Dose Rationale: The maximum recommended starting dose (MRSD)
was calculated using the Food and Drug Administration (FDA)
Guidance for Industry "Estimating the Safe Starting Dose in the
Clinical Trials for Therapeutics in Adult Healthy Volunteers". Per
the guidance, for investigational therapeutic proteins with
molecular weight >100,000 Daltons that are administered IV, the
MRSD should be estimated by normalizing across species instead of
via body surface area scaling. Based upon the No Observed Effect
Level (NOEL) observed of 250 mg/kg in the mouse toxicology study, a
standard safety factor of 10 limits the dose to 25 mg/kg.
[0129] The starting dose for phase 1/SAD study will be 2.5 mg/kg
dosed IV. This starting dose is 10 times lower than the maximally
allowed starting dose based on a 4-week mouse toxicology study and
6 times lower than the current maximal dose (15 mg/kg) administered
in the Expanded Access and compassionate use human treatment
protocols involving C.sub.2N-8E12 (see Investigator's
Brochure).
[0130] Based on preliminary plasma PK from a single patient trial,
it is possible to estimate the percent of tau in CSF that is bound
by C.sub.2N-8E12 at various times after a 2.5 mg/kg dose of
C.sub.2N-8EI2. For this calculation, it is assumed that the CSF
concentration for a humanized antibodies is 0.1% of the plasma
concentration and that the concentration of tau in CSF is 500
pg/mL. Based on these assumptions, a dose of 2.5 mg/kg will lead to
a CSF concentration of C.sub.2N-8E12 over the first month that is
between 3 to 40 times higher than the molar concentration of tau in
CSF. Based on the K.sub.D of C.sub.2N8E12, the 2.5 mg/kg dose will
lead to 3-26% of tau in CSF being bound by the antibody. Similar
modeling has been performed on the PK data from the highest dose
administered to humans to date (15 mg/kg) and estimated that the
average tau binding over the 28 day period is around 50% (max 72%
bound, min 40%). There will likely also be an abundance of
extracellular tau present in the brain that is not accessible
through the CSF compartment, but to which the antibody will be able
to bind. Therefore, dose escalation will proceed to 25 mg/kg to
assess safety of a dose that will likely lead to significant target
engagement in the brain.
[0131] Unless approved by the Investigator, during the treatment
period, no study subject should receive: Any other biologic or
immunomodulatory therapy; Any other investigational agent;
Warfarin; Any anticoagulant (other than 81 mg daily aspirin) for a
condition for which temporary cessation of the treatment prior to
CSF sampling would provide a medical hazard.
[0132] Informed Consent: After providing informed consent, each
subject will undergo screening assessments to reconfirm that the
inclusion criteria can be fulfilled, and that no contraindications
exist to receiving treatment under this protocol. Specifically,
each subject will be assessed at screening with a clinical and
neurological examination to confirm the diagnosis. A brief
cognitive screening with PSP ratings scale and a depression scale
with interview will be made and a complete medical and
drug/medication history will be obtained. If the subject fulfills
all inclusion criteria and lacks exclusion criteria, further
investigations will be performed. The Screening visit occurs 28
days to 7 days before the Baseline/Day 0 Visit.
[0133] Pharmacokinetic Assessments: Samples will be collected for
PK analysis at various time points described in Table 3 below.
TABLE-US-00006 TABLE 3 Post Pre- 15 min 28 28 dose (#) 3 hrs 6 hrs
12 hrs 24 hrs 48 hrs 168 hrs 336 hrs days day X X X X X X X X X X
(@)
(*) If an early termination occurs, a final blood sample will be
obtained at the Early Termination Visit for final PK assessment;
(#) Within 15 minutes of infusion completion; (@) Post day 28, PK
samples will be taken every 28 days until the earlier occurrence of
either of the following events: (i) study termination; (ii) the
absence of any detectable blood levels of C.sub.2N-8EI2.
[0134] Adverse Events Assessments: The following safety assessment
will be conducted in order to monitor for AEs: Vital signs (blood
pressure, pulse/heart rate, temperature, respiratory rate, SPO2);
Complete neurologic exam, including a cognitive assessment (mental
status tests); Laboratory tests: (1) Hematology panel: complete
blood count (CBC) with differential, hematocrit, and hemoglobin
(Hb), platelet count; (2) Chemistry panel: serum electrolytes,
glucose, uric acid, blood urea nitrogen (BUN), creatinine, total
protein, albumin, bilirubin (total, direct and indirect), alkaline
phosphatase, lactate dehydrogenase (LDH), liver enzymes (AST, ALT
and GGT), iron, cholesterol panel, CPK, amylase, and lipase; (3)
Coagulation panel: Prothrombin Time (PT), INR, and Partial
Thromboplastin Time (PTT); Urinalysis, including measurement of Hb,
WBC, and protein content; ECG--continuous monitoring or 12 lead
ECG; MRI brain imaging, including fluid attenuated inversion
recovery (FLAIR); A CSF sampling with measurement of cell counts
(WBC and RBC), total protein and glucose.
[0135] Determination of C2N-8E12 in human plasma and CSF: Sandwich
ELISA assays have been developed for measuring the concentration of
C2N-8E12 in plasma and CSF. Charles River Laboratories has
validated these assays for use in a variety of different matrices
(See Table 4).
TABLE-US-00007 TABLE 4 CRL Study # Study Title 20056682 Validation
of an Enzyme-Linked Immunosorbent Completed Assay (ELISA) Method
for the Determination of C.sub.2N-8E12 in Human Plasma (K2EDTA)
20057088 Validation of an Enzyme-Linked Immunosorbent Assay Ongoing
(ELISA) Method for the Determination of C.sub.2N-8E12 in Human
Cerebrospinal Fluid
[0136] Determination of anti-C2N-8E12 antibodies in human plasma:
Blood samples will be collected for assessment of anti-drug
antibody (ADA) development prior to initial dosing and on day 14
and 28 post-administration of drug. A final measurement will occur
at termination. An ECL based sandwich ELISA assay has been
developed for measuring the presence of antibodies against
C.sub.2N-8E12 in plasma (ADA). Charles River Laboratories has
validated this assay for detection of ADA response in human plasma
(See Table 5).
TABLE-US-00008 TABLE 5 CRL Study # Study Title 20056686 Validation
of a Qualitative Electrochemiluminescent (ECL) Complete Assay for
the Detection of anti-C.sub.2N-8E12 Antibodies in Human Plasma
(K2EDTA)
[0137] Clinical and functional assessments include Colombia-Suicide
Severity Rating Scale: As a safety parameter, the Columbia-Suicide
Severity Rating Scale (C-SSRS) for suicidal ideation will be used
(Posner et al. 2011). Geriatric Depression Scale: Similar to the
C-SSRS, the Geriatric Depression Scale (GDS) will be used to assess
overall mood during the study. The Geriatric Depression Scale (GDS)
is a 30-item self-report assessment used to identify depression in
the elderly (Yesavage et al. 1983). PSP Rating Scale: The PSP
Rating Scale (PSPRS) will be used at screening for inclusion as
well as baseline and end of the study to assess changes in the
scale over time (Golbe and Ohman-Strickland 2007). Clinical Global
Impressions: The clinical Global Impressions rate of change (CGIc)
and severity (CGIs) scales will be used to assess severity of
symptoms. Schwab and England Activities of Daily Living: The Schwab
and England Activities of Daily Living (SEADL) scale will be used
as a means of assessing the subjects ability to perform daily
activities (Schwab and England 1969). Clinical Dementia Rating Sum
of Boxes Frontotemporal Lobe Dementia: The Clinical Dementia Rating
Sum of Boxes Frontotemporal Lobe Dementia (CDR-SB-FTLD) is a
version of the CDR-SB cognitive assessment test that includes
assessment of behavior, comportment, personality, and language
(Knopman et al. 2008). Mini Mental State Examination: The mini
mental state examination (MMSE) is a reliable 30-point
questionnaire that measures cognitive impairment (Folstein,
Folstein, and McHugh 1975).
[0138] Cerebrospinal fluid: CSF will be drawn from the L3-4
interspace by lumbar puncture. If CSF sampling is not successful
CT/fluoro guided lumbar puncture can be used at the discretion of
the local clinical site staff as per local protocol. Safety labs on
CSF will be analyzed locally at the applicable clinical site after
each lumbar puncture/CSF collection. These measures include: cell
counts (WBC and RBC), total protein and glucose. Other CSF
measurements (e.g., C2N-8E12 concentration, target engagement and
other exploratory biomarkers) will be analyzed by the applicable
designated laboratory.
[0139] Imaging: The subject will also receive a baseline MRI scan
with structural, FLAIR, diffusion-weighted and susceptibility
weighted imaging. Post-dosing imaging analyses will be performed
according to the Schedule of Events.
[0140] Exploratory pharmacogenomic analysis: A blood sample will be
collected for DNA extraction at baseline. All individuals will
undergo an extended MAPT haplotype sequence analysis to determine
H1(A-D) and H2 carrier status. DNA will be extracted from the
samples and the DNA shipped to the designated pharmacogenomic core
for this study.
[0141] Subjects will be enrolled in this study until the earlier
occurrence of any of the following events: (i) they complete their
participation and the entire Schedule of Events; (ii) they or the
Investigator decide(s) to terminate their participation; or (iii)
the applicable subject experiences any DLT or any SAE that an
Investigator deems to preclude further participation in this study
and precludes eligibility for the subsequent phase 2/MAD study.
Additionally, at the discretion of the Investigator, subjects who
cease to meet any inclusion criterion, or meet one or more
exclusion criterion during the study, may be determined ineligible
to continue participating in the study or for subsequent
participation in the phase 2/MAD study.
Sequence CWU 1
1
581111PRTHomo sapiens 1Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu
Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys Arg Ala
Ser Gln Ser Val Ser Thr Ser 20 25 30 Arg Tyr Ser Tyr Ile His Trp
Tyr Gln Gln Lys Pro Gly Gln Phe Pro 35 40 45 Lys Leu Leu Ile Lys
Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro
Leu Glu Glu Glu Asp Phe Ala Thr Tyr Tyr Cys His His Ser Trp 85 90
95 Glu Ile Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
105 110 2111PRTHomo sapiens 2Asp Ile Val Leu Thr Gln Ser Pro Asp
Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys
Arg Ala Ser Gln Ser Val Ser Thr Ser 20 25 30 Arg Tyr Ser Tyr Ile
His Trp Tyr Gln Gln Lys Pro Gly Gln Phe Pro 35 40 45 Lys Leu Leu
Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ser 50 55 60 Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70
75 80 Pro Leu Glu Pro Glu Asp Phe Ala Thr Tyr Tyr Cys His His Ser
Trp 85 90 95 Glu Ile Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys 100 105 110 3111PRTHomo sapiens 3Asp Ile Val Leu Thr Gln
Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr
Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ser 20 25 30 Arg Tyr
Ser Tyr Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Phe Pro 35 40 45
Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ser 50
55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys His
His Ser Trp 85 90 95 Glu Ile Pro Leu Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys 100 105 110 4111PRTHomo sapiens 4Asp Ile Val Leu
Met Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg
Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ser 20 25 30
Arg Tyr Ser Tyr Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35
40 45 Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro
Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
Cys His His Ser Trp 85 90 95 Glu Ile Pro Leu Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105 110 5124PRTHomo sapiens 5Glu Val
Lys Val Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Met Lys Leu Ser Cys Val Val Ser Gly Phe Thr Phe Ser Asn Tyr 20
25 30 Trp Val Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala Gln Ile Arg Leu Lys Ser Asp Asn Tyr Ala Thr His
Tyr Glu Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asp Ser Lys Ser Ser 65 70 75 80 Val Tyr Leu Gln Met Asn Asn Leu Arg
Ala Glu Asp Ser Gly Ile Tyr 85 90 95 Tyr Cys Thr Asn Trp Glu Asp
Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe 115 120 6124PRTHomo sapiens 6Glu Val
Lys Val Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Lys Leu Ser Cys Val Val Ser Gly Phe Thr Phe Ser Asn Tyr 20
25 30 Trp Val Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala Gln Ile Arg Leu Lys Ser Asp Asn Tyr Ala Thr His
Tyr Glu Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asp Ser Lys Ser Ser 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Gly Ile Tyr 85 90 95 Tyr Cys Thr Asn Trp Glu Asp
Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe 115 120 7124PRTHomo sapiens 7Glu Val
Gln Val Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Lys Leu Ser Cys Val Val Ser Gly Phe Thr Phe Ser Asn Tyr 20
25 30 Trp Val Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala Gln Ile Arg Leu Lys Ser Asp Asn Tyr Ala Thr His
Tyr Glu Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asp Ser Lys Asn Ser 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Thr Asn Trp Glu Asp
Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe 115 120 8124PRTHomo sapiens 8Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Lys Leu Ser Cys Val Val Ser Gly Phe Thr Phe Ser Asn Tyr 20
25 30 Trp Val Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala Gln Ile Arg Leu Lys Ser Asp Asn Tyr Ala Thr His
Tyr Glu Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Thr Asn Trp Glu Asp
Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe 115 120 96PRTHomo sapiens 9Asp Gln Gly
Gly Tyr Thr 1 5 108PRTHomo sapiens 10Gly Tyr Thr Met His Gln Asp
Gln 1 5 11111PRTHomo sapiens 11Asp Ile Val Leu Thr Gln Ser Pro Ala
Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys
Arg Ala Ser Gln Ser Val Ser Thr Ser 20 25 30 Arg Tyr Ser Tyr Ile
His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu
Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70
75 80 Pro Leu Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys His His Ser
Trp 85 90 95 Glu Ile Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu
Leu Lys 100 105 110 12124PRTHomo sapiens 12Glu Val Lys Val Glu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Met Lys Leu
Ser Cys Val Val Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Val
Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val 35 40 45
Ala Gln Ile Arg Leu Lys Ser Asp Asn Tyr Ala Thr His Tyr Glu Glu 50
55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser
Ser 65 70 75 80 Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Ser
Gly Ile Tyr 85 90 95 Tyr Cys Thr Asn Trp Glu Asp Tyr Trp Gly Gln
Gly Thr Thr Leu Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe 115 120 13442PRTHomo sapiens 13Glu Val Lys Val Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Met Lys Leu
Ser Cys Val Val Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Val
Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Gln Ile Arg Leu Lys Ser Asp Asn Tyr Ala Thr His Tyr Glu Glu 50
55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser
Ser 65 70 75 80 Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Ser
Gly Ile Tyr 85 90 95 Tyr Cys Thr Asn Trp Glu Asp Tyr Trp Gly Gln
Gly Thr Thr Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro 115 120 125 Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180
185 190 Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr
Lys 195 200 205 Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys
Pro Pro Cys 210 215 220 Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val Ser
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp 260 265 270 Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln
Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305
310 315 320 Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Gln Glu Glu 340 345 350 Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu Tyr Ser
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn 405 410 415 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425
430 Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 14442PRTHomo
sapiens 14Glu Val Lys Val Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Val Val Ser Gly Phe Thr
Phe Ser Asn Tyr 20 25 30 Trp Val Asn Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ala Gln Ile Arg Leu Lys Ser Asp
Asn Tyr Ala Thr His Tyr Glu Glu 50 55 60 Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Val Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Ile Tyr 85 90 95 Tyr Cys
Thr Asn Trp Glu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115
120 125 Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu
Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Lys Thr Tyr Thr Cys
Asn Val Asp His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Arg
Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys 210 215 220 Pro Ala
Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 225 230 235
240 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
245 250 255 Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
Asn Trp 260 265 270 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu 275 280 285 Glu Gln Phe Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu 290 295 300 His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn 305 310 315 320 Lys Gly Leu Pro Ser
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 325 330 335 Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu 340 345 350 Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360
365 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
370 375 380 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe 385 390 395 400 Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
Trp Gln Glu Gly Asn 405 410 415 Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr 420 425 430 Gln Lys Ser Leu Ser Leu Ser
Leu Gly Lys 435 440 15442PRTHomo sapiens 15Glu Val Gln Val Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu
Ser Cys Val Val Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Val
Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Gln Ile Arg Leu Lys Ser Asp Asn Tyr Ala Thr His Tyr Glu Glu 50
55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn
Ser 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Ile Tyr 85 90 95 Tyr Cys Thr Asn Trp Glu Asp Tyr Trp Gly Gln
Gly Thr Thr Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro 115 120 125 Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180
185
190 Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys
195 200 205 Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro
Pro Cys 210 215 220 Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val Ser Gln
Glu Asp Pro Glu Val Gln Phe Asn Trp 260 265 270 Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln Phe
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310
315 320 Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Gln Glu Glu 340 345 350 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu Tyr Ser Arg
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn 405 410 415 Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430
Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 16442PRTHomo
sapiens 16Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Val Val Ser Gly Phe Thr
Phe Ser Asn Tyr 20 25 30 Trp Val Asn Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ala Gln Ile Arg Leu Lys Ser Asp
Asn Tyr Ala Thr His Tyr Glu Glu 50 55 60 Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys
Thr Asn Trp Glu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115
120 125 Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu
Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Lys Thr Tyr Thr Cys
Asn Val Asp His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Arg
Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys 210 215 220 Pro Ala
Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 225 230 235
240 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
245 250 255 Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
Asn Trp 260 265 270 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu 275 280 285 Glu Gln Phe Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu 290 295 300 His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn 305 310 315 320 Lys Gly Leu Pro Ser
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 325 330 335 Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu 340 345 350 Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360
365 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
370 375 380 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe 385 390 395 400 Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
Trp Gln Glu Gly Asn 405 410 415 Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr 420 425 430 Gln Lys Ser Leu Ser Leu Ser
Leu Gly Lys 435 440 17218PRTHomo sapiens 17Asp Ile Val Leu Thr Gln
Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr
Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ser 20 25 30 Arg Tyr
Ser Tyr Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45
Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ser 50
55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile
His 65 70 75 80 Pro Leu Glu Glu Glu Asp Phe Ala Thr Tyr Tyr Cys His
His Ser Trp 85 90 95 Glu Ile Pro Leu Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180
185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
18218PRTHomo sapiens 18Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu
Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys Arg Ala
Ser Gln Ser Val Ser Thr Ser 20 25 30 Arg Tyr Ser Tyr Ile His Trp
Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Lys
Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro
Leu Glu Pro Glu Asp Phe Ala Thr Tyr Tyr Cys His His Ser Trp 85 90
95 Glu Ile Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val
Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 19218PRTHomo sapiens
19Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1
5 10 15 Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr
Ser 20 25 30 Arg Tyr Ser Tyr Ile His Trp Tyr Gln Gln Lys Pro Gly
Gln Pro Pro 35 40 45 Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu
Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys His His Ser Trp 85 90 95 Glu Ile Pro Leu Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135
140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg
Gly Glu Cys 210 215 20218PRTHomo sapiens 20Asp Ile Val Met Thr Gln
Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr
Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ser 20 25 30 Arg Tyr
Ser Tyr Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45
Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ser 50
55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys His
His Ser Trp 85 90 95 Glu Ile Pro Leu Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180
185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
2160PRTHomo sapiens 21Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met
Glu Asp His Ala Gly 1 5 10 15 Thr Tyr Gly Leu Gly Asp Arg Lys Asp
Gln Gly Gly Tyr Thr Met His 20 25 30 Gln Asp Gln Glu Gly Asp Thr
Asp Ala Gly Leu Lys Glu Ser Pro Leu 35 40 45 Gln Thr Pro Thr Glu
Asp Gly Ser Glu Glu Pro Gly 50 55 60 2260PRTHomo sapiens 22Met Ala
Glu Pro Arg Gln Glu Phe Asp Val Met Glu Asp His Ala Gly 1 5 10 15
Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Glu Gly Tyr Thr Met Leu 20
25 30 Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro
Leu 35 40 45 Gln Thr Pro Ala Glu Asp Gly Ser Glu Glu Leu Gly 50 55
60 2349PRTMus musculus 23Met Ala Asp Pro Arg Gln Glu Phe Asp Thr
Met Glu Asp His Ala Gly 1 5 10 15 Asp Tyr Thr Leu Leu Gln Asp Gln
Glu Gly Asp Met Asp His Gly Leu 20 25 30 Lys Glu Ser Pro Pro Gln
Pro Pro Ala Asp Asp Gly Ala Glu Glu Pro 35 40 45 Gly 2415PRTHomo
sapiens 24Asp His Ala Gly Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln
Gly 1 5 10 15 2515PRTHomo sapiens 25Asp His Ala Gly Thr Tyr Gly Leu
Gly Ala Ala Lys Asp Gln Gly 1 5 10 15 2615PRTHomo sapiens 26His Ala
Gly Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly 1 5 10 15
2715PRTHomo sapiens 27His Ala Gly Thr Tyr Gly Leu Gly Asp Ala Ala
Asp Gln Gly Gly 1 5 10 15 2815PRTHomo sapiens 28Ala Gly Thr Tyr Gly
Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr 1 5 10 15 2915PRTHomo
sapiens 29Ala Gly Thr Tyr Gly Leu Gly Asp Arg Ala Ala Gln Gly Gly
Tyr 1 5 10 15 3015PRTHomo sapiens 30Gly Thr Tyr Gly Leu Gly Asp Arg
Lys Asp Gln Gly Gly Tyr Thr 1 5 10 15 3115PRTHomo sapiens 31Gly Thr
Tyr Gly Leu Gly Asp Arg Lys Ala Ala Gly Gly Tyr Thr 1 5 10 15
3215PRTHomo sapiens 32Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly
Gly Tyr Thr Met 1 5 10 15 3315PRTHomo sapiens 33Thr Tyr Gly Leu Gly
Asp Arg Lys Asp Ala Ala Gly Tyr Thr Met 1 5 10 15 3415PRTHomo
sapiens 34Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met
His 1 5 10 15 3515PRTHomo sapiens 35Tyr Gly Leu Gly Asp Arg Lys Asp
Gln Ala Ala Tyr Thr Met His 1 5 10 15 3615PRTHomo sapiens 36Gly Leu
Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His Gln 1 5 10 15
3715PRTHomo sapiens 37Gly Leu Gly Asp Arg Lys Asp Gln Gly Ala Ala
Thr Met His Gln 1 5 10 15 3815PRTHomo sapiens 38Leu Gly Asp Arg Lys
Asp Gln Gly Gly Tyr Thr Met His Gln Asp 1 5 10 15 3915PRTHomo
sapiens 39Leu Gly Asp Arg Lys Asp Gln Gly Gly Ala Ala Met His Gln
Asp 1 5 10 15 4015PRTHomo sapiens 40Gly Asp Arg Lys Asp Gln Gly Gly
Tyr Thr Met His Gln Asp Gln 1 5 10 15 4115PRTHomo sapiens 41Gly Asp
Arg Lys Asp Gln Gly Gly Tyr Ala Ala His Gln Asp Gln 1 5 10 15
4215PRTHomo sapiens 42Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His
Gln Asp Gln Glu 1 5 10 15 4315PRTHomo sapiens 43Asp Arg Lys Asp Gln
Gly Gly Tyr Thr Ala Ala Gln Asp Gln Glu 1 5 10 15 4415PRTHomo
sapiens 44Arg Lys Asp Gln Gly Gly Tyr Thr Met His Gln Asp Gln Glu
Gly 1 5 10 15 4515PRTHomo sapiens 45Arg Lys Asp Gln Gly Gly Tyr Thr
Met Ala Ala Asp Gln Glu Gly 1 5 10 15 4615PRTHomo sapiens 46Lys Asp
Gln Gly Gly Tyr Thr Met His Gln Asp Gln Glu Gly Asp 1 5 10 15
4715PRTHomo sapiens 47Lys Asp Gln Gly Gly Tyr Thr Met His Ala Ala
Gln Glu Gly Asp 1 5 10 15 4815PRTHomo sapiens 48Asp Gln Gly Gly Tyr
Thr Met His Gln Asp Gln Glu Gly Asp Thr 1 5 10 15 4915PRTHomo
sapiens 49Asp Gln Gly Gly Tyr Thr Met His Gln Ala Ala Glu Gly Asp
Thr 1 5 10 15 5015PRTHomo sapiens 50Gln Gly Gly Tyr Thr Met His Gln
Asp Gln Glu Gly Asp Thr Asp 1 5 10 15 5115PRTHomo sapiens 51Gln Gly
Gly Tyr Thr Met His Gln Asp Ala Ala Gly Asp Thr Asp 1 5 10 15
5215PRTHomo sapiens 52Gly Gly Tyr Thr Met His Gln Asp Gln Glu Gly
Asp Thr Asp Ala 1 5 10 15 5315PRTHomo sapiens 53Gly Gly Tyr Thr Met
His Gln Asp Gln Ala Ala Asp Thr Asp Ala 1 5 10 15
5415PRTHomo sapiens 54Gly Tyr Thr Met His Gln Asp Gln Glu Gly Asp
Thr Asp Ala Gly 1 5 10 15 555PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 55Gly Gly Gly Gly Ser 1 5
5622PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(8)Any amino acid or not
presentMOD_RES(15)..(22)Any amino acid or not present 56Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Asp Gln Gly Gly Tyr Thr Xaa Xaa 1 5 10 15 Xaa
Xaa Xaa Xaa Xaa Xaa 20 578PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(5)..(5)His or Leu
57Gly Tyr Thr Met Xaa Gln Asp Gln 1 5 586PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(3)..(3)Gly or Glu 58Asp Gln Xaa Gly Tyr Thr 1 5
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