Note: Descriptions are shown in the official language in which they were submitted.
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DOSING REGIMES FOR TREATMENT OF SYNUCLEINOPATH1ES
SEQUENCE LISTING
[0001] A sequence listing, comprising SEQ ID NOs: 1-151, is attached and
incorporated by
reference in its entirety. The listing, named 5037255EQLI5T.txt, was created
on September
27, 2017, in ASCII format and is 127,151 bytes in size.
BACKGROUND
[0002] Lewy body diseases (LBDs) are characterized by degeneration of the
dopaminergic
system, motor alterations, cognitive impairment, and formation of Lewy bodies
(LBs) and/or
Lewy neurites. (McKeith et al., Neurology (1996) 47:1113-24). Lewy Body
disease include
Parkinson's disease (including idiopathic Parkinson's disease), and Dementia
with Lewy
Bodies (DLB). Lewy body diseases are a common cause for movement disorders and
cognitive deterioration in the aging population (Galasko et al., Arch. Neurol.
(1994) 51:888-
95). Constipation is another common symptom of subjects with Lewy body disease
(Ondo et
al., Neurology 2012;78;1650-1654; Ashraf et al., Movement Disorders 12, 946-
951 (1997)).
[0003] Alpha-synuclein is a protein that is normally associated with synapses
and is
believed to play a role in neural plasticity, learning and memory. Alpha-
synuclein is also
implicated with a central role in pathogenesis of Lewy body disease. The
protein can
aggregate to form insoluble fibrils in pathological conditions. For example,
alpha-synuclein
accumulates in LBs (Spillantini et al., Nature (1997) 388:839-40; Takeda et
al., J. Pathol.
(1998) 152:367-72; Wakabayashi et al., Neurosci. Lett. (1997) 239:45-8).
Mutations in the
alpha-synuclein gene co-segregate with rare familial forms of parkinsonism
(Kruger et al.,
Nature Gen. (1998) 18:106-8; Polymeropoulos, et al., Science (1997) 276:2045-
7). Over
expression of alpha-synuclein in transgenic mice (Masliah et al., Science
(2000) 287:1265-9)
and Drosophila (Feany et al., Nature (2000) 404:394-8) mimics several
pathological aspects
of Lewy body disease. Soluble oligomers of alpha-synuclein may be neurotoxic
(Conway et
al., Proc Natl Acad Sci USA (2000) 97:571-576. The accumulation of alpha-
synuclein with
similar morphological and neurological alterations in species and animal
models as diverse as
humans, mice, and flies suggests that this molecule contributes to the
development of Lewy
body disease.
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[0004] Immunotherapy directed against alpha-synuclein has been reported to
reduce alpha-
synuclein deposits and behavioral symptoms in mouse models of Lewy body
disease
(Masliah et at, PLoS ONE 6(4): e19338. doi:10.1371/journal.pone.0019338).
SUMMARY OF THE CLAIMED INVENTION
[0005] In one aspect, the invention provides a method of treating or effecting
prophylaxis
of a subject having or at risk of a synucleinopathy comprising intravenously
administering to
the subject a dose of 3000-5000 mg of an antibody against alpha-synuclein at
intervals of 3-5
weeks, or administering another regime that delivers the antibody to the
subject with
substantially the same area under the curve. In some such methods the dose is
3500-4500
mg.
[0006] In some such methods, the antibody is administered to a plurality of
subjects having
or at risk of the synucleinopathy wherein subjects receive either one or two
fixed doses. In
some such methods, subjects with a weight less than 65 kg receive a dose of
3500 mg
antibody and subjects with a weight greater or equal to 65 kg receive a dose
of 4500 mg.
[0007] In some such methods, the dose of 45-75 mg/kg. In some such methods,
the dose is
50-70 mg/kg.
[0008] In some such methods, the intervals are 28 days. In some such methods,
the subject
receives at least six doses of the antibody at the intervals of 3-5 weeks. In
some such
methods, the subject receives at least 12 doses of the antibody at the
intervals of 3-5 weeks.
In some such methods, the subject receives at least 18 doses of the antibody
at the intervals of
3-5 weeks. In some such methods, the interval is 4 weeks. In some such
methods, the
subject receives the antibody every 4 weeks for at least 52 weeks.
[0009] Some such methods further comprise monitoring the subject for a change
in
movement, cognitive deficit, autonomic dysfunction, gastrointestinal
dysfunction, visual
hallucination or a psychological symptom.
[0010] In some such methods, the monitoring comprises
(a) providing a subject with a mobile device programmed to receive and
transmit data acquired from sensors internal and/or external to the device
relating to
movement deficits of a subject having or suspected of having a
synucleinopathy, whereafter
the subject undergoes a series of movements to reveal movement deficits, if
present, and the
internal or external sensors of the device acquire data relating to the
movements;
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(b) collecting data transmitted from the mobile device; and
(c) comparing the data acquired from the subjects with control data to assess
presence or extent of movement deficits in the subject.
[0011] In some such methods, the mobile device is programmed to receive and
transmit
data from at least two external sensors attached to upper and lower limbs of
the subject. In
some such methods, the mobile device acquires data from sensors on the upper
and lower
limbs of the subject. In some such methods, the mobile device is carried by
the subject and
acquires data from an internal sensor. In some such methods, the series of
movements
includes tapping the device, sitting and standing. In some such methods, the
monitoring
indicates a reduced movement deficit or a reduced cognitive deficit responsive
to
administering the antibody.
[0012] In some such methods, the antibody binds within residues 115-130 of
alpha
synuclein.
[0013] In some such methods, the antibody binds with residues 118-126 of alpha
synuclein.
[0014] In some such methods, the antibody binds within residues 117-123 of
alpha
synuclein. In some such methods, the antibody is NI-202.21D11. In some such
methods, the
antibody comprises three heavy CDRs designated SEQ ID NOs:139-141 respectively
and
three light chain CDRs designated SEQ ID NOs: 143-145 respectively. In some
such
methods, the antibody comprises a heavy chain variable region designated SEQ
ID NO:138
and a light chain variable region designated SEQ ID NO:142.
[0015] In some such methods, the antibody comprises the three light chain
Kabat CDRs of
SEQ ID NO:5 and the three heavy chain Kabat CDRs of SEQ ID NO:10. In some such
methods, the antibody comprises a heavy chain variable region of any of SEQ ID
NOs:8-11
and a light chain variable region of any of SEQ ID NO:3-5, preferably wherein
the heavy
chain variable region is of SEQ ID NO:10 and a light chain variable region of
SEQ ID NO:5.
[0016] In some such methods, the antibody is of human IgG1 isotype. In some
such
methods, the antibody comprises a heavy chain constant region of SEQ ID NO:35
provided
the C-terminal lysine of SEQ ID NO:35 may be absent and a light chain constant
region of
SEQ ID NO:30. In some such methods, the antibody comprises a heavy chain
constant
region of SEQ ID NO:35 provided the C-terminal lysine of SEQ ID NO:35 may be
absent
and a light chain constant region of SEQ ID NO:13.
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[0017] In some such methods, the antibody is 9E4. In some such methods, the
antibody
comprises three heavy CDRs designated SEQ ID NOs: 146-148 respectively and
three light
chain CDRs designated SEQ ID NOs: 149-151 respectively. In some such methods,
the
antibody comprises a heavy chain designated SEQ ID NO:37 and a light chain
designated
SEQ ID NO:32, wherein the C-terminal lysine of SEQ ID NO:37 may be absent.
[0018] In some such methods, the antibody binds within residues 1-20 of alpha-
synuclein.
In some such methods, the antibody binds with residues 4-15 of alpha-
synuclein.
[0019] In some such methods, the antibody is NI-202.12F4. In some such
methods, the
antibody comprises three heavy CDRs designated SEQ ID NOs:131-133 respectively
and
three light chain CDRs designated SEQ ID NOs: 135-137 respectively. In some
such
methods, the antibody comprises a heavy chain variable region designated SEQ
ID NO:130
and a light chain variable region designated SEQ ID NO:134.
[0020] In some such methods, the subject has a weight less than 65 kg and is
administered
a dose of 3500 mg antibody every 4 weeks.
[0021] In some such methods, the administration of the antibody within the
range of 3000-
5000 mg is preceded by administering a loading dose of 2000 mg of the antibody
and
optionally uptitration at one or more subsequent dose at greater or equal to
2000 mg but less
than 3500 mg until a dose of 3500 mg is reached, all doses being separated by
intervals of 3-5
weeks..
[0022] In some such methods, the 3500 mg dose is administered in the next
interval after
the 2000 mg dose. In some such methods, the dose is gradually increased over
several
intervals prior to administration of the 3500 mg dose. In some such methods,
the subject has
a weight greater than 65 kg and is administered a dose of 4500 mg antibody
every 4 weeks.
[0023] In some such methods, the antibody is administered at 4500 mg preceded
by
administration of a loading dose of 2000 mg of the antibody and optionally
uptitration with
one or more subsequent doses of greater than or equal to 2000 mg but less than
4500 mg until
a dose of 4500 mg is reached with all doses being separated by intervals of 3-
5 weeks..
[0024] In some such methods, the 4500 mg dose is administered in the next
interval after
the 2000 mg dose. In some such methods, the dose is gradually increased over
several
intervals prior to administration of the 4500 mg dose.
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[0025] Some such methods further comprise monitoring the subject for a change
in
movement, cognitive deficit autonomic dysfunction, gastrointestinal
dysfunction, visual
hallucination or a psychological symptom.
[0026] In some such methods, the monitoring comprises
(a) providing a subject with a mobile device programmed to receive and
transmit data acquired from sensors internal and/or external to the device
relating to
movement deficits of a subject having or suspected of having a
synucleinopathy, whereafter
the subject undergoes a series of movements to reveal movement deficits, if
present, and the
internal or external sensors of the device acquire data relating to the
movements;
(b) collecting data transmitted from the mobile device; and
(c) comparing the data acquired from the subjects with control data to assess
presence or extent of movement deficits in the subject.
[0027] In some such methods, the mobile device is programmed to receive and
transmit
data from at least two external sensors attached to upper and lower limbs of
the subject. In
some such methods, the mobile device acquires data from sensors on the upper
and lower
limbs of the subject. In some such methods, the mobile device is carried by
the subject and
acquires data from an internal sensor. In some such methods, the series of
movements
includes tapping the device, sitting and standing. In some such methods, the
monitoring
indicates a reduced movement deficit or a reduced cognitive deficit responsive
to
administering the antibody.
[0028] In some such methods, the synucleinopathy is Parkinson's disease. In
some such
methods, the synucleinopathy is dementia with Lewy bodies. In some such
methods, the
synucleinopathy is multiple system atrophy. In some such methods, the
synucleinopathy is
progressive supra nuclear palsy. In some such methods, the synucleinopathy is
REM sleep
behavior disorder. In some such methods, the synucleinopathy is Alzheimer's
disease with
amygdala Lewy bodies. In some such methods, the subject has the disease.
[0029] In some such methods, the subject is not receiving symptomatic
treatment for
Parkinson's disease concomitant with the antibody. In some such methods, the
antibody is
administered concomitantly with levodopa.
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[0030] In another aspect, the invention provides a method of treating or
effecting
prophylaxis of a subject having a Lewy body disease comprising intravenously
administering
to the subject a dose of 1300-1700 mg of an antibody against alpha-synuclein
at intervals of
3-5 weeks, or administering another regime that delivers the antibody to the
subject with
substantially the same area under the curve.
[0031] In some such methods, the dose is 1500 mg. In some such methods, the
antibody is
administered to a plurality of subjects having Lewy body disease wherein
subjects receive
either one or two fixed doses. In some such methods, the subject receives a
dose of 18-25
mg/kg. In some such methods, the subject receives a dose of 20 mg/kg.
[0032] In some such methods, the intervals are 28 days. In some such methods,
the subject
receives at least six doses of the antibody at the intervals of 3-5 weeks. In
some such
methods, the subject receives at least 12 doses of the antibody at the
intervals of 3-5 weeks.
In some such methods, the subject receives at least 18 doses of the antibody
at the intervals of
3-5 weeks. In some such methods, the interval is 4 weeks. In some such
methods, the
subject receives the antibody every 4 weeks for at least 52 weeks.
[0033] Some such methods further comprise monitoring the subject for a change
in
movement, cognitive deficit autonomic dysfunction, gastrointestinal
dysfunction, visual
hallucination or a psychological symptom.
[0034] In some such methods, the monitoring comprises
(a) providing a subject with a mobile device programmed to receive and
transmit data acquired from sensors internal and/or external to the device
relating to
movement deficits of a subject having or suspected of having a Lewy body
disease,
whereafter the subject undergoes a series of movements to reveal movement
deficits, if
present, and the internal or external sensors of the device acquire data
relating to the
movements;
(b) collecting data transmitted from the mobile device; and
(c) comparing the data acquired from the subjects with control data to assess
presence or extent of movement deficits in the subject.
[0035] In some such methods, the mobile device is programmed to receive and
transmit
data from at least two external sensors attached to upper and lower limbs of
the subject.
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[0036] In some such methods, the mobile device acquires data from sensors on
the upper
and lower limbs of the subject. In some such methods, the mobile device is
carried by the
subject and acquires data from an internal sensor. In some such methods, the
series of
movements includes tapping the device, sitting and standing. In some such
methods, the
monitoring indicates a reduced movement deficit or a reduced cognitive deficit
responsive to
administering the antibody.
[0037] In some such methods, the antibody binds within residues 115-130 of
alpha
synuclein.
[0038] In some such methods, the antibody binds with residues 118-126 of alpha
synuclein.
[0039] In some such methods, the antibody binds within residues 117-123 of
alpha
synuclein.
[0040] In some such methods, the antibody is NI-202.21D11. In some such
methods, the
antibody comprises three heavy CDRs designated SEQ ID NOs:139-141 respectively
and
three light chain CDRs designated SEQ ID NOs: 143-145 respectively. In some
such
methods, the antibody comprises a heavy chain variable region designated SEQ
ID NO:138
and a light chain variable region designated SEQ ID NO:142.
[0041] In some such methods, the antibody comprises the three light chain
Kabat CDRs of
SEQ ID NO:5 and the three heavy chain Kabat CDRs of SEQ ID NO:10. In some such
methods, the antibody comprises a heavy chain variable region of any of SEQ ID
NOs:8-11
and a light chain variable region of any of SEQ ID NO:3-5, preferably wherein
the heavy
chain variable region is of SEQ ID NO:10 and a light chain variable region of
SEQ ID NO:5.
[0042] In some such methods, the antibody is of human IgG1 isotype.
[0043] In some such methods, the antibody comprises a heavy chain constant
region of
SEQ ID NO:35 provided the C-terminal lysine may be absent and a light chain
constant
region of SEQ ID NO:30. In some such methods, the antibody comprises a heavy
chain
constant region of SEQ ID NO:35 provided the C-terminal lysine may be absent
and a light
chain constant region of SEQ ID NO:13.
[0044] In some such methods, the antibody comprises three heavy CDRs
designated SEQ
ID NOs: 146-148 respectively and three light chain CDRs designated SEQ ID NOs:
149-151
respectively. In some such methods, the antibody comprises a heavy chain
designated SEQ
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ID NO:37 and a light chain designated SEQ ID NO:32, wherein the C-terminal
lysine of SEQ
ID NO:37 may be absent.
[0045] In some such methods, the antibody binds within residues 1-20 of alpha-
synuclein.
In some such methods, the antibody binds with residues 4-15 of alpha-
synuclein.
[0046] In some such methods, the antibody is NI-202.12F4. In some such
methods, the
antibody comprises three heavy CDRs designated SEQ ID NOs:131-133 respectively
and
three light chain CDRs designated SEQ ID NOs: 135-137 respectively. In some
such
methods, the antibody comprises a heavy chain variable region designated SEQ
ID NO:130
and a light chain variable region designated SEQ ID NO:134.
[0047] In some such methods, the synucleinopathy is Parkinson's disease. In
some such
methods, the synucleinopathy is dementia with Lewy bodies. In some such
methods, the
synucleinopathy is multiple system atrophy. In some such methods, the
synucleinopathy is
progressive supra nuclear palsy. In some such methods, the synucleinopathy is
REM sleep
behavior disorder. In some such methods, the synucleinopathy is Alzheimer's
disease with
amygdala Lewy bodies. In some such methods, the subject has the disease.
[0048] In some such methods, the subject is not receiving symptomatic
treatment for
Parkinson's disease concomitant with the antibody. In some such methods, the
antibody is
administered concomitantly with levodopa.
DEFINITIONS
[0049] The term "antibody" includes intact antibodies and binding fragments
thereof.
Typically, fragments compete with the intact antibody from which they were
derived for
specific binding to the target. Fragments include separate heavy chains,
separate light chains,
Fab, Fab', F(ab')2, F(ab)c, Fv, single chain antibodies, and single domain
antibodies. The
term "antibody" also includes a bispecific antibody. A bispecific or
bifunctional antibody is
an artificial hybrid antibody having two different heavy/light chain pairs and
two different
binding sites (see, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol.,
79:315-321 (1990);
Kostelny et al., J. Immunol., 148:1547-53 (1992)).
[0050] The basic antibody structural unit is a tetramer of subunits. Each
tetramer includes
two identical pairs of polypeptide chains, each pair having one "light" chain
(about 25 kDa)
and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each
chain
includes a variable region of about 100 to 110 or more amino acids primarily
responsible for
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antigen recognition. When initially expressed, this variable region is
typically linked to a
cleavable signal peptide. The variable region without the signal peptide is
sometimes
referred to as a mature variable region. Thus, for example, a light chain
mature variable
region means a light chain variable region without the light chain signal
peptide. The
carboxy-terminal portion of each chain defines a constant region primarily
responsible for
effector function. A constant region can include any or all of a CH1 region,
hinge region,
CH2 region, and CH3 region.
[0051] Light chains are classified as either kappa or lambda. Heavy chains are
classified as
gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG,
IgM, IgA, IgD
and IgE, respectively. Within light and heavy chains, the variable and
constant regions are
joined by a "J" region of about 12 or more amino acids, with the heavy chain
also including a
"D" region of about 10 or more amino acids. (See generally, Fundamental
Immunology
(Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989), Ch. 7) (incorporated by
reference in its
entirety for all purposes).
[0052] The mature variable regions of each light/heavy chain pair form the
antibody
binding site. Thus, an intact antibody has two binding sites. Except for
bifunctional or
bispecific antibodies, the two binding sites are the same. The chains all
exhibit the same
general structure of relatively conserved framework regions (FR) joined by
three
hypervariable regions, also called complementarity determining regions (CDRs).
The CDRs
from the two chains of each pair are aligned by the framework regions,
enabling binding to a
specific epitope. From N-terminal to C-terminal, both light and heavy chains
comprise the
regions FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids
to
each region is in accordance with the definitions of Kabat, Sequences of
Proteins of
Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and
1991), or
Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature
342:878-883
(1989). Kabat also provides a widely used numbering convention (Kabat
numbering) in
which corresponding residues between different heavy chains or between
different light
chains are assigned the same number.
[0053] Percentage sequence identities are determined with antibody sequences
maximally
aligned by the Kabat numbering convention. After alignment, if a subject
antibody region
(e.g., the entire mature variable region of a heavy or light chain) is being
compared with the
same region of a reference antibody, the percentage sequence identity between
the subject
and reference antibody regions is the number of positions occupied by the same
amino acid in
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both the subject and reference antibody region divided by the total number of
aligned
positions of the two regions (with gaps not counted) multiplied by 100 to
convert to
percentage.
[0054] For purposes of classifying amino acids substitutions as conservative
or non-
conservative, amino acids are grouped as follows: Group I (hydrophobic
sidechains):
Norleucine, Met, Ala, Val, Leu, Ile; Group II (neutral hydrophilic side
chains): Cys, Ser, Thr;
Group III (acidic side chains): Asp, Glu; Group IV (basic side chains): Asn,
Gln, His, Lys,
Arg; Group V (residues influencing chain orientation): Gly, Pro; and Group VI
(aromatic side
chains): Trp, Tyr, Phe. Conservative substitutions involve substitutions
between amino acids
in the same class. Non-conservative substitutions constitute exchanging a
member of one of
these classes for a member of another.
[0055] Antibodies of the invention typically bind to their designated target
with an affinity
constant of at least 106, 107, 108, 109, or 1010 M-1. Such binding is specific
binding in that it
is detectably higher in magnitude and distinguishable from non-specific
binding occurring to
at least one unrelated target. Specific binding can be the result of formation
of bonds
between particular functional groups or particular spatial fit (e.g., lock and
key type) whereas
nonspecific binding is usually the result of van der Wails forces. Specific
binding does not,
however, necessarily imply that a monoclonal antibody binds one and only one
target.
[0056] The term "symptom" refers to subjective evidence of a disease, such as
altered gait,
as perceived by a subject. A "sign" refers to objective evidence of a disease
as observed by a
physician.
[0057] An individual is at increased risk of a disease if the subject has at
least one known
risk-factor (e.g., genetic, biochemical, family history, situational exposure)
placing
individuals with that risk factor at a statistically significant greater risk
of developing the
disease than individuals without the risk factor. Statistical significance
means p<0.05.
[0058] Unless otherwise apparent from the context, the term "about"
encompasses values
within the standard deviation of the mean of a stated value or +/- 5% of a
stated value,
whichever is greater.
[0059] The term "9E4 antibody" refers to any antibody in which each of the
CDRs is
substantially that of 9E4, and thus includes murine, chimeric, veneered, and
humanized 9E4.
References to other antibodies, such as 5C1 and 1H7 have corresponding
meanings.
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[0060] Antibodies of the invention can be administered concomitant with
another treatment
for the same indication as the antibody, meaning that the other treatment is
administered at
least once during the period in which the antibody is administered, such
period beginning one
month before the first dosing and ending one month after the last dosing of
the antibody. The
other treatment can be administered at recurring intervals during this period,
which may or
may not be the same as the intervals at which the antibody is administered.
The other
treatment may be a symptomatic treatment.
[0061] A treatment is symptomatic if it only affects one or more symptoms of a
disease, not
its cause, i.e., its etiology.
[0062] The term "mobile device" as used herein refers to any portable device
which
comprises a sensor and data-recording equipment suitable for obtaining the
dataset of activity
measurements. Typically, the mobile device comprises a sensor for measuring
the activity.
This may also require a data processor and storage unit as well as a display
for electronically
simulating an activity test on the mobile device. Moreover, from the activity
of the subject
data shall be recorded and compiled to a dataset which is to be evaluated by
the method of the
present invention either on the mobile device itself or on a second device.
Depending on the
specific setup envisaged, it may be necessary that the mobile device comprises
data
transmission equipment in order to transfer the acquired dataset from the
mobile device to
one or more further devices. Particular well suited as mobile devices
according to the present
invention are smartphones, smartwatches, wearable sensors, portable multimedia
devices or
tablet computers. Alternatively, portable sensors with data recording and,
optionally,
processing equipment may be used. Further, depending on the kind of activity
test to be
performed, the mobile device shall be adapted to display instructions for the
subject regarding
the activity to be carried out for the test.
[0063] Unless otherwise apparent from the context, reference to a range
includes any
integer within the range.
[0064] A treatment regime refers to a combination of parameters characterizing
administration of an immunotherapeutic agent including any or all of dose,
frequency of
administration, route of administration, and total duration of administration.
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BRIEF DESCRIPTION OF THE SEQUENCES
[0065] SEQ ID NO:1 is an m9E4VL variable region.
[0066] SEQ ID NO:2 is an 63102889Hu9E4VLFr variable region.
[0067] SEQ ID NO:3 is an Hu9E4VLv1 variable region.
[0068] SEQ ID NO:4 is an Hu9E4VLv2 (No backmutation) variable region.
[0069] SEQ ID NO:5 is an Hu9E4VLv3 variable region.
[0070] SEQ ID NO:6 is an m9E4VH variable region.
[0071] SEQ ID NO:7 is an1791009Hu9E4VHFr variable region.
[0072] SEQ ID NO:8 is an Hu9E4VHv1 variable region.
[0073] SEQ ID NO:9 is an Hu9E4VHv2 variable region.
[0074] SEQ ID NO:10 is an Hu9E4VHv3 variable region.
[0075] SEQ ID NO:11 is an Hu9E4VHv4 (no backmutation) variable region.
[0076] SEQ ID NO:12 is the amino acid sequence of natural human wildtype alpha-
synuclein.
[0077] SEQ ID NO:13 is a humanized 9E4 light chain constant region (with R)
(common
for vi, v2, v3).
[0078] SEQ ID NO:14 is a humanized 9E4 light chain constant region (with R)
nucleotide
sequence (common for vi, v2, v3).
[0079] SEQ ID NO:15 is an amino acid sequence of an exemplary human IgG1
constant
region.
[0080] SEQ ID NO:16 is a nucleic acid sequence encoding an exemplary human
IgG1
constant region.
[0081] SEQ ID NO:17 is an Hu9E4VLvl nucleotide sequence variable region.
[0082] SEQ ID NO:18 is an Hu9E4VLv2 nucleotide sequence (no backmutation)
variable
region.
[0083] SEQ ID NO:19 is an Hu9E4VLv3 nucleotide sequence variable region.
[0084] SEQ ID NO:20 is an Hu9E4VHvl nucleotide sequence variable region.
[0085] SEQ ID NO:21 is an Hu9E4VHv2 nucleotide sequence variable region.
[0086] SEQ ID NO:22 is an Hu9E4VHv3 nucleotide sequence variable region.
[0087] SEQ ID NO:23 is an Hu9E4VHv4 nucleotide sequence (no backmutation)
variable
region.
[0088] SEQ ID NO:24 is an Hu9E4VL signal peptide (common for vi, v2, v3).
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[0089] SEQ ID NO:25 is an Hu9E4VL signal peptide nucleotide sequence (common
for vi,
v2, v3).
[0090] SEQ ID NO:26 is an Hu9E4VH signal peptide (common for vi, v2, v3).
[0091] SEQ ID NO:27 is an Hu9E4VH signal peptide nucleotide sequence (common
for
vi, v2, v3, v4).
[0092] SEQ ID NO:28 is an Hu9E4VL alternative.
[0093] SEQ ID NO:29 is an Hu9E4VH alternative.
[0094] SEQ ID NO:30 is an amino acid sequence of an exemplary human kappa
light chain
constant region.
[0095] SEQ ID NO:31 is a humanized 9E4 light chain constant region (without R)
nucleotide.
[0096] sequence (common for vi, v2, v3).
[0097] SEQ ID NO:32 is a humanized 9E4 light chain version 3 (variable region
+ constant
region with Arginine).
[0098] SEQ ID NO:33 is a humanized 9E4 light chain version 3 (variable region
+ constant
region without Arginine).
[0099] SEQ ID NO:34 is a humanized 9E4 heavy chain version 3 (variable region
+
constant region).
[0100] SEQ ID NO:35 is a humanized 9E4 heavy chain constant region (G1m3
allotype;
BIP version).
[0101] SEQ ID NO:36 is a humanized 9E4 heavy chain version 3 (variable region
+
constant region).
[0102] SEQ ID NO:37 is a humanized 9E4 heavy chain version 3 (variable region
+
alternative constant region Glm3 allotype).
[0103] SEQ ID NO:38 is an m5C1 antibody mature heavy chain variable region
nucleotide
sequence.
[0104] SEQ ID NO:39 is an m5C1 antibody heavy chain variable region amino acid
sequence.
[0105] SEQ ID NO:40 is an m5C1 antibody heavy chain signal peptide nucleotide
sequence.
[0106] SEQ ID NO:41 is an m5C1 antibody heavy chain signal peptide amino acid
sequence.
[0107] SEQ ID NO:42 is an m5C1 antibody mature light chain variable region
nucleotide
sequence.
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[0108] SEQ ID NO:43 is an m5C1 antibody light chain variable region amino acid
sequence.
[0109] SEQ ID NO:44 is an m5C1 antibody light chain signal peptide nucleotide
sequence.
[0110] SEQ ID NO:45 is an m5C1 antibody light chain signal peptide amino acid
sequence.
[0111] SEQ ID NO:46 is a 5C1 immunogen.
[0112] SEQ ID NO:47 is an m5C1 heavy chain CDR1.
[0113] SEQ ID NO:48 is an m5C1 heavy chain CDR2.
[0114] SEQ ID NO:49 is an m5C1 heavy chain CDR3.
[0115] SEQ ID NO:50 is an m5C1 light chain CDR1.
[0116] SEQ ID NO:51 is an m5C1 light chain CDR2.
[0117] SEQ ID NO:52 is an m5C1 light chain CDR3.
[0118] SEQ ID NO:53 is a murine 5C1 heavy chain variable region nucleotide
sequence
with sequence encoding signal peptide.
[0119] SEQ ID NO:54 is a murine 5C1 heavy chain variable region sequence with
signal
peptide.
[0120] SEQ ID NO:55 is a murine 5C1 light chain variable region nucleotide
sequence
with sequence encoding signal peptide.
[0121] SEQ ID NO:56 is a murine 5C1 light chain variable region sequence with
signal
peptide.
[0122] SEQ ID NO:57 is a human VH Acceptor FR (Acc#AAY42876.1).
[0123] SEQ ID NO:58 is a humanized 5C1H1.
[0124] SEQ ID NO:59 is a humanized 5C1H2.
[0125] SEQ ID NO:60 is a humanized 5C1H3.
[0126] SEQ ID NO:61 is a humanized 5C1H4.
[0127] SEQ ID NO:62 is a humanized 5C1H5.
[0128] SEQ ID NO:63 is a nucleic acid sequence encoding humanized 5C1 Hl.
[0129] SEQ ID NO:64 is a nucleic acid sequence encoding humanized 5C1 H2.
[0130] SEQ ID NO:65 is a nucleic acid sequence encoding humanized 5C1H3.
[0131] SEQ ID NO:66 is a nucleic acid sequence encoding humanized 5C1 H4.
[0132] SEQ ID NO:67 is a nucleic acid sequence encoding humanized 5C1H5.
[0133] SEQ ID NO:68 is a human VL Acceptor FR (Acc#CAB51293.1).
[0134] SEQ ID NO:69 is a humanized 5C1L1.
[0135] SEQ ID NO:70 is a humanized 5C1L2.
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[0136] SEQ ID NO:71 is a humanized 5C1L3.
[0137] SEQ ID NO:72 is a humanized 5C1L4.
[0138] SEQ ID NO:73 is a nucleic acid sequence encoding humanized 5C1 Ll.
[0139] SEQ ID NO:74 is a nucleic acid sequence encoding humanized 5C1 L2.
[0140] SEQ ID NO:75 is a nucleic acid sequence encoding humanized 5C1L3.
[0141] SEQ ID NO:76 is a nucleic acid sequence encoding humanized 5C1 L4.
[0142] SEQ ID NO:77 is a nucleic acid sequence encoding an exemplary human
kappa
light chain constant region.
[0143] SEQ ID NO:78 is a non-amyloid component (NAC) domain of alpha-synuclein
as
reported by Jensen et al.
[0144] SEQ ID NO:79 is a non-amyloid component (NAC) domain of alpha-synuclein
as
reported by Ueda et al.
[0145] SEQ ID NO:80 is an m1H7 antibody heavy chain variable nucleotide
sequence.
[0146] SEQ ID NO:81 is an m1H7 antibody heavy chain variable amino acid
sequence.
[0147] SEQ ID NO:82 is an m1H7 antibody light chain variable nucleotide
sequence.
[0148] SEQ ID NO:83 is an m1H7 antibody light chain variable amino acid
sequence.
[0149] SEQ ID NO:84 is a mature ml H7 antibody heavy chain variable nucleotide
sequence.
[0150] SEQ ID NO:85 is a mature ml H7 antibody heavy chain variable amino acid
sequence.
[0151] SEQ ID NO:86 is a mature ml H7 antibody light chain variable nucleotide
sequence.
[0152] SEQ ID NO:87 is a mature ml H7 antibody light chain variable amino acid
sequence.
[0153] SEQ ID NO:88 is an m1H7 antibody heavy chain CDR1 (Kabat definition).
[0154] SEQ ID NO:89 is an m1H7 antibody heavy chain CDR2 (Kabat definition).
[0155] SEQ ID NO:90 is an m1H7 antibody heavy chain CDR3 (Kabat definition).
[0156] SEQ ID NO:91 is an m1H7 antibody light chain CDR1 (Kabat definition).
[0157] SEQ ID NO:92 is an m1H7 antibody light chain CDR2 (Kabat definition).
[0158] SEQ ID NO:93 is an m1H7 antibody light chain CDR3 (Kabat definition).
[0159] SEQ ID NO:94 is an HulH7VHvl nucleotide sequence.
[0160] SEQ ID NO:95 is an HulH7VHvl amino acid sequence.
[0161] SEQ ID NO:96 is an HulH7VHv2 nucleotide sequence.
[0162] SEQ ID NO:97 is an HulH7VHv2 amino acid sequence.
[0163] SEQ ID NO:98 is an HulH7VHv3 nucleotide sequence.
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[0164] SEQ ID NO:99 is an Hu1H7VHv3 amino acid sequence.
[0165] SEQ ID NO:100 is an Hu1H7VHv4 nucleotide sequence.
[0166] SEQ ID NO:101 is an Hu1H7VHv4 amino acid sequence.
[0167] SEQ ID NO:102 is an Hu1H7VHv5 nucleotide sequence.
[0168] SEQ ID NO:103 is an Hu1H7VHv5 amino acid sequence.
[0169] SEQ ID NO:104 is an Hu1H7VH signal peptide nucleotide sequence.
[0170] SEQ ID NO:105 is an Hu1H7VH signal peptide amino acid sequence.
[0171] SEQ ID NO:106 is an Hu1H7VH signal peptide nucleotide sequence.
[0172] SEQ ID NO:107 is an Hu1H7VH signal peptide amino acid sequence.
[0173] SEQ ID NO:108 is an Hu1H7VLv1 nucleotide sequence.
[0174] SEQ ID NO:109 is an Hu1H7VLv1 amino acid sequence.
[0175] SEQ ID NO:110 is an Hu1H7VLv2 nucleotide sequence.
[0176] SEQ ID NO:111 is an HulH7VLv2 amino acid sequence.
[0177] SEQ ID NO:112 is an Hu1H7VLv3 nucleotide sequence.
[0178] SEQ ID NO:113 is an Hu1H7VLv3 amino acid sequence.
[0179] SEQ ID NO:114 is an Hu1H7VLv4 nucleotide sequence.
[0180] SEQ ID NO:115 is an Hu1H7VLv4 amino acid sequence.
[0181] SEQ ID NO:116 is an Hu1H7VL signal peptide nucleotide sequence.
[0182] SEQ ID NO:117 is an amino acid sequence of BACO2037 (GI-21670055) human
acceptor used for heavy chain framework.
[0183] SEQ ID NO:118 is an amino acid sequence of AAY33358 GI-63102905) human
acceptor used for light chain framework.
[0184] SEQ ID NO:119 is an Hu1H7VH with no backmutation or CDR mutation.
[0185] SEQ ID NO:120 is an Hu1H7VL with no backmutation or CDR mutation.
[0186] SEQ ID NO:121 is an Hu1H7VH alternative.
[0187] SEQ ID NO:122 is an Hu1H7VL alternative.
[0188] SEQ ID NO:123 is an Hu1H7VH CDR3 alternative.
[0189] SEQ ID NO:124 is a humanized 1H7 light chain version 3 (variable region
+
constant region with Arginine).
[0190] SEQ ID NO:125 is a humanized 1H7 light chain version 3 (variable region
+
constant region without Arginine).
[0191] SEQ ID NO:126 is a humanized 1H7 heavy chain version 3 (variable region
+
constant region).
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[0192] SEQ ID NO:127 is a humanized 1H7 heavy chain version 3 (variable region
+
constant region G1m3 allotype).
[0193] SEQ ID NO:128 is a humanized 1H7 heavy chain constant region (IgG2).
[0194] SEQ ID NO:129 is a humanized 1H7 heavy chain constant region (Glml
allotype).
[0195] SEQ ID NO:130 is a NI-202.12F4-VHA1b variable heavy chain (VH)
sequence.
[0196] SEQ ID NO:131 is a NI-202.12F4-VHA1b CDR1 sequence.
[0197] SEQ ID NO:132 is a NI-202.12F4-VHA1b CDR2 sequence
[0198] SEQ ID NO:133 is a NI-202.12F4-VHA1b CDR3 sequence.
[0199] SEQ ID NO:134 is a NI-202.12F4-VLa1 variable light chain (VL) sequence.
[0200] SEQ ID NO:135 is a NI-202.12F4-VLa1 CDR1 sequence.
[0201] SEQ ID NO:136 is a NI-202.12F4-VLa1 CDR2 sequence.
[0202] SEQ ID NO:137 is a NI-202.12F4-VLa1 CDR3 sequence.
[0203] SEQ ID NO:138 is a NI-202.21D11-VH sequence.
[0204] SEQ ID NO:139 is a NI-202.21D11-VH CDR1 sequence.
[0205] SEQ ID NO:140 is a NI-202.21D11-VH CDR2 sequence.
[0206] SEQ ID NO:141 is a NI-202.21D11-VH CDR3 sequence.
[0207] SEQ ID NO:142 is a NI-202.21D11-VK sequence.
[0208] SEQ ID NO:143 is a NI-202.21D11 VK CDR1 sequence.
[0209] SEQ ID NO:144 is a NI-202.21D11 VK CDR2 sequence.
[0210] SEQ ID NO:145 is a NI-202.21D11 VK CDR3 sequence.
[0211] SEQ ID NO:146 is a 9E4 VH CDR1 sequence.
[0212] SEQ ID NO:147 is a 9E4 VH CDR2 sequence.
[0213] SEQ ID NO:148 is a 9E4 VH CDR3 sequence.
[0214] SEQ ID NO:149 is a 9E4 VL CDR1 sequence.
[0215] SEQ ID NO:150 is a 9E4 VL CDR2 sequence.
[0216] SEQ ID NO:151 is a 9E4 VL CDR3 sequence.
DETAILED DESCRIPTION
I. GENERAL
[0217] The invention provides dosing regimes for immunotherapy of
synucleinopathies.
Subjects receive an antibody against alpha-synuclein at a dose of 3000-5000 mg
or 1300-
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1700 mg intravenously every 3-5 weeks. Such doses can be preceded by one or
more lower
loading doses.
TARGET MOLECULE
[0218] Natural human wild type alpha-synuclein is a peptide of 140 amino acids
having the
following amino acid sequence:
MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH
GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL
GKNEEGAPQE GILEDMPVDP DNEAYEMP SE EGYQDYEPEA (SEQ ID NO:12)
(Ueda et al., Proc. Natl. Acad. Sci. USA (1993) 90:11282-6); GenBank accession
number:
P37840. The protein has three recognized domains: an-N-terminal KTKE repeat
domain
covering amino acids 1-61; a NAC (Non-amyloid component) domain running from
about
amino acids 60-95; and a C-terminal acidic domain running from about amino
acid 98 to 140.
[0219] Unless otherwise apparent from the context, reference to alpha-
synuclein or its
fragments includes the natural human wildtype amino acid sequences indicated
above, and
human allelic variants thereof, particularly those associated with Lewy body
disease (e.g.,
variants E46K, A3OP and A53T, with the first letter indicating the amino acid
in SEQ ID
NO:12, the number indicating the codon position in SEQ ID NO:12, and the
second letter
indicating the amino acid in the allelic variant). Such variants can
optionally be present
individually or in any combination in any of the aspects of the invention
described below.
SYNUCLEINOPATHIES
[0220] Synucleinopathies are characterized by excessive accumulation of alpha-
synuclein,
which may or may not involve the formation of Lewy bodies (LBs). Lewy Body
Diseases
(LBD), for example, are characterized by degeneration of the dopaminergic
system, motor
alterations, cognitive impairment, and formation of Lewy bodies. (McKeith et
al., Neurology
(1996) 47:1113-24). Lewy Bodies are spherical protein deposits found in nerve
cells. Their
presence in the brain disrupts the brain's normal function interrupting the
action of chemical
messengers including acetylcholine and dopamine. Synucleinopathies include
Parkinson's
disease (including idiopathic Parkinson's disease), Dementia with Lewy Bodies
(DLB) (also
known as Diffuse Lewy Body Disease (DLBD)), Lewy Body variant of Alzheimer's
disease
(LBV), Combined Alzheimer's and Parkinson disease, progressive supra nuclear
palsy (PSP),
and multiple system atrophy (MSA; e.g., Olivopontocerebellar Atrophy,
Striatonigral
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Degeneration, and Shy-Drager Syndrome). DLB shares symptoms of both
Alzheimer's and
Parkinson's disease. DLB differs from Parkinson's disease mainly in the
location of Lewy
Bodies. In DLB Lewy Bodies form mainly in the cortex. In Parkinson's disease,
they form
mainly in the substantia nigra. Other synucleinopathies include Pure Autonomic
Failure,
Lewy Body dysphagia, Incidental LBD, and Inherited LBD (e.g., mutations of the
alpha-
synuclein gene, PARK3 and PARK4).
IV. IMMUNOTHERAPY AGENTS
[0221] Passive immunotherapy involves treatment with an antibody specifically
binding to
alpha-synuclein.
1. Antibodies
A. Binding Specificity and Functional Properties
[0222] The present methods can include antibodies specifically binding to an
epitope
within residues 1-15, 4-15, 1-20, 91-99, 115-130, 117-123 or 118-126 of human
alpha-
synuclein. Antibodies can be monoclonal or polyclonal. A polyclonal serum can
be specific
to an epitope or range of amino acids within alpha-synuclein in that it lacks
antibodies
binding outside the epitope or range. Antibodies can be non-human, chimeric,
veneered,
humanized, or human among other possiblities.
[0223] Some antibodies are bispecific, one arm of which is an antibody against
alpha-
synculein and the other arm of which is an antibody that binds to a receptor
expressed on the
blood brain barrier, such as an insulin receptor, an insulin-like growth
factor (IGF) receptor,
a leptin receptor, or a lipoprotein receptor, or preferably a transferrin
receptor (Friden et at,
PNAS 88:4771-4775, 1991; Friden et al., Science 259:373-377, 1993). Such a
bispecific
antibody can be transferred cross the blood brain barrier by receptor-mediated
transcytosis.
Brain uptake of the bispecific antibody can be further enhanced by engineering
the bi-
specific antibody to reduce its affinity to the blood brain barrier receptor.
Reduced affinity
for the receptor resulted in a broader distributioin in the brain (see, e.g.,
Atwal. et al. ScL
Trans. Med. 3, 84ra43, 2011; Yu et al. Sci. Trans. Med. 3, 84ra44, 2011).
[0224] Exemplary bispecific antibodies can also be (1) a dual-variable-domain
antibody
(DVD-Ig), where each light chain and heavy chain contains two variable domains
in tandem
through a short peptide linkage (Wu et al., Generation and Characterization of
a Dual
Variable Domain Immunoglobulin (DVD-IgTM) Molecule, In: Antibody Engineering,
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Springer Berlin Heidelberg (2010)); (2) a Tandab, which is a fusion of two
single chain
diabodies resulting in a tetravalent bispecific antibody that has two binding
sites for each of
the target antigens; (3) a flexibody, which is a combination of scFvs with a
diabody resulting
in a multivalent molecule; (4) a so called "dock and lock" molecule, based on
the
"dimerization and docking domain" in Protein Kinase A, which, when applied to
Fabs, can
yield a trivalent bispecific binding protein consisting of two identical Fab
fragments linked to
a different Fab fragment; (5) a so-called Scorpion molecule, comprising, e.g.,
two scFvs
fused to both termini of a human Fc-region. Examples of platforms useful for
preparing
bispecific antibodies include BiTE (Micromet), DART (MacroGenics), Fcab and
Mab2 (F-
star) , Fc-engineered IgG1(Xencor) or DuoBody (based on Fab arm exchange,
Genmab).
[0225] Another exemplary antibody is mAb 9E4, which binds to an epitope within
residues
118-126 of human alpha-synuclein. The cell line designated
JH17.9E4.3.37.1.14.2 producing
the antibody 9E4 has the ATCC accession number PTA-8221 having been deposited
under
the provisions of the Budapest Treaty with the American Type Culture
Collection (ATCC,
P.O. Box 1549, Manassas, Va. 20108) on Feb. 26, 2007. The murine light and
heavy chain
variable sequences are SEQ ID NO:1 and SEQ ID NO:6, respectively. Murine mAb
9E4
comprises a VH-CDR1 of SEQ ID NO:146, a VH-CDR2 of SEQ ID NO:147, a VH-CDR3 of
SEQ ID NO:148, a VL-CDR1 of SEQ ID NO: 149, a VL-CDR2 of SEQ ID NO:150, and a
VL-CDR3 of SEQ ID NO: 151.
[0226] Another exemplary antibody is mAb 5C1. The murine light and heavy chain
variable sequences are SEQ ID NO:43 and SEQ ID NO:39, respectively.
[0227] Another exemplary antibody is mAb 1H7. The cell line designated
JH17.1H7.4.24.34 producing the antibody 1H7 has the ATCC accession number PTA-
8220
having been deposited under the provisions of the Budapest Treaty with the
American Type
Culture Collection (ATCC, P.O. Box 1549, Manassas, Va. 20108) on Feb. 26,
2007. The
murine light and heavy chain variable sequences are SEQ ID NO:87 and SEQ ID
NO:85,
respectively.
[0228] Such antibodies are described in WO 06/020581 and US Application Nos.
61/591,835, 61/711,207, 13/750,983, 61/711,204, 61/719,281, 61/840,432,
61/872,366,
14/049,169, 61/553,131, 61/711,204, 61/843,011, and 13/662,261, all of which
are
incorporated herein by reference for all purposes.
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[0229] Another exemplary antibody is NI-202.12F4, which is a human antibody
reported to
bind to an epitope within residues 4-15 of alpha-synuclein with residue 10
being a major
contributor to the epitope (see US 8,940,276; W02012177972, US 9,580,493). NI-
202.12F4
comprises a variable heavy chain (VHAlb) sequence of SEQ ID NO:130 and a
variable light
chain (VLal) sequence of SEQ ID NO:134. NI-202.12F4 comprises a VHA1b-CDR1 of
SEQ ID NO:131, a VHA1b-CDR2 of SEQ ID NO:132, a VHA1b-CDR3 sequence of SEQ ID
NO:133 (Ala-His), a VLa1-CDR1 sequence of SEQ ID NO:135, a VLa1-CDR2 sequence
of
SEQ ID NO: 136, and VLa1-CDR3 sequence of SEQ ID NO:137. Another exemplary
antibody is NI-202.21D11, which has been reported to bind to an epitope within
residues
117-123 of alpha-synuclein with residues 121 and 122 contributing most to the
epitope (see
US 9,580,493). NI-202.21D11 comprises a variable heavy chain (VH) sequence of
SEQ ID
NO:138 and a variable light chain (VK) sequence of SEQ ID NO:142. NI-202.21D11
comprises a VH-CDR1 of SEQ ID NO:139, a VH-CDR2 of SEQ ID NO:140, a VH-CDR3
sequence of SEQ ID NO:141, a VK-CDR1 sequence of SEQ ID NO:143, a VK-CDR2
sequence of SEQ ID NO: 144, and VK-CDR3 sequence of SEQ ID NO:145.
B. Humanized Antibodies
[0230] A humanized antibody is a genetically engineered antibody in which the
CDRs from a
non-human "donor" antibody are grafted into human "acceptor" antibody
sequences (see,
e.g., Queen et at, U55,530,101 and 5,585,089; Winter et al., US 5,225,539,
Carter, US
6,407,213, Adair, US 5,859,205 6,881,557, Foote, US 6,881,557). The acceptor
antibody
sequences can be, for example, a mature human antibody variable region
sequence, a
composite of such sequences, a consensus sequence of human antibody sequences
(e.g., light
and heavy chain variable region consensus sequences of Kabat, 1991, supra), or
a germline
variable region sequence. Thus, a humanized antibody of the invention includes
antibodies
having three light chain and three heavy chain CDRs as defined by Kabat
entirely or
substantially from a murine antibody, such as 9E4, 5C1 or 1H7 (donor antibody)
and mature
variable region framework sequences and constant regions, if present, entirely
or
substantially from human antibody sequences. Likewise a humanized heavy chain
includes
heavy chains having three heavy chain CDRs as defined by Kabat from the heavy
chain of
the donor antibody, and a mature heavy chain variable sequence and heavy chain
constant
region sequence, if present, entirely or substantially from human antibody
heavy chain
sequences. Likewise a humanized light chain includes light chains having three
light chain
CDRs as defined by Kabat from the light chain of the donor antibody, and a
mature light
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chain variable sequence and light chain constant region sequence, if present,
entirely or
substantially from human antibody light chain sequences. The mature variable
region
framework sequences of an antibody chain or the constant region sequence of an
antibody
chain are substantially from a human mature variable region framework sequence
or human
constant region sequence, respectively, when at least 85%, 90%, 95%, 96%, 97%,
98%, 99%
or 100% of corresponding residues defined by Kabat are identical. A CDR is
substantially
from a corresponding donor CDR if showing at least 85% identity thereto,
except in the case
of CDRH2 wherein at least 65% identify is required.
[0231] Certain amino acids from the human mature variable region framework
residues can
be selected for substitution based on their possible influence on CDR
conformation and/or
binding to antigen. Investigation of such possible influences is by modeling,
examination of
the characteristics of the amino acids at particular locations, or empirical
observation of the
effects of substitution or mutagenesis of particular amino acids.
[0232] For example, when an amino acid differs between a murine mature
variable region
framework residue and a selected human mature variable region framework
residue, the
human framework amino acid can be substituted by the equivalent framework
amino acid
from the mouse antibody when it is reasonably expected that the amino acid:
(1) noncovalently binds antigen directly,
(2) is adjacent to a CDR region,
(3) otherwise interacts with a CDR region (e.g. is within about 6 A of a
CDR
region)
(4) mediates interaction between the heavy and light chains.
[0233] Exemplary humanized forms of the mouse 9E4 antibody including three
exemplified humanized light chain mature variable regions (Hu9E4VLv1-v3; SEQ
ID NOs:3-
5) and four exemplified humanized heavy chain mature variable regions
(Hu9E4VHv1 -v4;
SEQ ID NOs:8-11).
[0234] The exemplary light and heavy chain mature variable regions can be
paired in any
combination. An exemplary combination is Hu9E4VLv3 (SEQ ID NO:5) and Hu9E4VHv3
(SEQ ID NO:10). An exemplary heavy chain constant region is SEQ ID NO:35, with
or
without the C-terminal lysine. This constant region can be linked to any of
the heavy chain
variable regions. An exemplary light chain constant region is SEQ ID NO:30.
This constant
region can be linked to any of the light chain variable regions. An exemplary
full length
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heavy chain has the amino acid sequence of SEQ ID NO:37, with or without the C-
terminal
lysine. An exemplary full length light chain has the amino acid sequence of
SEQ ID NO:32.
An exemplary combination is an antibody comprising SEQ ID NO:37 (with or
without the C-
terminal lysine) and SEQ ID NO:32. The nucleic acids encoding the various
heavy and light
chains just described are provided in SEQ ID NOs:17-23. An exemplified
humanized
antibody has heavy and light chains characterized by SEQ ID NOs: 37 (with or
without the
C-terminal lysine) and 32.
[0235] Exemplary humanized forms of the mouse 5C1 antibody include four
exemplified
humanized light chain mature variable regions (5C1L1-L4; SEQ ID NOs:69-72, and
five
exemplified humanized heavy chain mature variable regions (5C1H1-H4; SEQ ID
NOs:58-
62). The exemplary light and heavy chain mature variable regions can be paired
in any
combination. An exemplary heavy chain constant region is SEQ ID NO:35, with or
without
the C-terminal lysine. This constant region can be linked to any of the heavy
chain variable
regions. An exemplary light chain constant region is SEQ ID NO:30. This
constant region
can be linked to any of the light chain variable regions. The nucleic acids
encoding the
various heavy and light chains just described are provided in SEQ ID NOs:73-76
and 63-67.
[0236] Exemplary humanized forms of the mouse 1H7 antibody include four
exemplified
humanized light chain mature variable regions (Hu1H7VLv1 -v4; SEQ ID NOs:109,
111,
113õ and 115, respectively and five exemplified humanized heavy chain mature
variable
regions (Hu1H7VHv1-v5; SEQ ID NOs:95, 97, 99, 101, and 103, respectively). The
exemplary light and heavy chain mature variable regions can be paired in any
combination.
An exemplary combination is Hu1H7VHv3 (SEQ ID NO:99) and Hu1H7VLv3 (SEQ ID
NO:113). An exemplary heavy chain constant region is SEQ ID NO:35, with or
without the
C-terminal lysine. This constant region can be linked to any of the heavy
chain variable
regions. An exemplary light chain constant region is SEQ ID NO:30. This
constant region
can be linked to any of the light chain variable regions. An exemplified full
length heavy
chain has the amino acid sequence of SEQ ID NO:127 (with the G1m3). An
exemplary full
length light chain has the amino acid sequence of SEQ ID NO:125 (without
Arginine). An
exemplary combination is an antibody comprising SEQ ID NO:127 and SEQ ID
NO:125.
The nucleic acids encoding the various heavy and light chains just described
are provided in
SEQ ID NOs 108, 110, 112, 114,94, 96, 98, 100, 102.
[0237] Such humanized antibodies are described in WO 06/020581 and
US13/750,983,
14/049,169, 13/662,261, and 61/843,011.
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[0238] In these or other antibodies described herein, one or more terminal
residues can be
cleaved in the course of posttranslational processing, particularly for
terminal lysines of
heavy chain constant regions.
C. Chimeric and Veneered Antibodies
[0239] The invention further provides chimeric and veneered forms of non-human
antibodies, particularly 9E4, 5C1 or1H7.
[0240] A chimeric antibody is an antibody in which the mature variable regions
of light and
heavy chains of a non-human antibody (e.g., a mouse) are combined with light
and heavy
chain constant regions from an antibody of a different species. Typically, the
light and heavy
chain constant regions are of human origin, but the constant regions can
originate from a
different non-human species, such as a rat, as needed (e.g., to facilitate
testing of the non-
human antibody in an appropriate animal model). Such antibodies substantially
or entirely
retain the binding specificity of the non-human (e.g., mouse) antibody
supplying the variable
regions, and are about two-thirds human (or different non-human species)
sequence.
[0241] A veneered antibody is a type of humanized antibody that retains some
and usually
all of the CDRs and some of the non-human variable region framework residues
of a non-
human antibody but replaces other variable region framework residues that may
contribute to
B- or T-cell epitopes, for example exposed residues (Padlan, Mol. Immunol.
28:489, 1991)
with residues from the corresponding positions of a human antibody sequence.
The result is
an antibody in which the CDRs are entirely or substantially from a non-human
antibody and
the variable region frameworks of the non-human antibody are made more human-
like by the
substitutions. Veneered forms of 9E4 are included in the invention.
D. Selection of Constant Region
[0242] The heavy and light chain variable regions of chimeric, veneered or
humanized
antibodies can be linked to at least a portion of a human constant region. The
choice of
constant region depends, in part, whether antibody-dependent cell-mediated
cytotmdcity,
antibody dependent cellular phagocytosis and/or complement dependent
cytotmdcity are
desired. For example, human isotopes IgG1 and IgG3 have complement-dependent
cytotoxicity and human isotypes IgG2 and IgG4 do not. Human IgG1 and IgG3 also
induce
stronger cell mediated effector functions than human IgG2 and IgG4. Light
chain constant
regions can be lambda or kappa. An exemplary human light chain kappa constant
region has
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the amino acid sequence of SEQ ID NO:13. Some such light chain kappa constant
regions
can be encoded by a nucleic acid sequence. The N-terminal arginine of SEQ ID
NO:13 can
be omitted, in which case light chain kappa constant region has the amino acid
sequence of
SEQ ID NO:30. Some such light chain kappa constant regions can be encoded by a
nucleic
acid sequence. An exemplary human IgG1 heavy chain constant region has the
amino acid
sequence of SEQ ID NO:15 (with or without the C-terminal lysine) or the heavy
chain
constant region component of SEQ ID NO:37 (with or without the C-terminal
lysine). Some
such heavy chain constant regions can be encoded by a nucleic acid sequence.
Antibodies
can be expressed as tetramers containing two light and two heavy chains, as
separate heavy
chains, light chains, as Fab, Fab', F(ab')2, and Fv, or as single chain
antibodies in which
heavy and light chain mature variable domains are linked through a spacer.
[0243] Human constant regions show allotypic variation and isoallotypic
variation between
different individuals, that is, the constant regions can differ in different
individuals at one or
more polymorphic positions. Isoallotypes differ from allotypes in that sera
recognizing an
isoallotype bind to a non-polymorphic region of a one or more other isotypes.
Thus, for
example, another heavy chain constant region is of IgG1 Glm3 allotype and has
the amino
acid sequence encoding a constant region of SEQ ID NO:37. Yet another heavy
chain
constant region has the amino acid sequence encoding a constant region of SEQ
ID NO:37
except that it lacks the C-terminal lysine.
[0244] One or several amino acids at the amino or carboxy terminus of the
light and/or
heavy chain, such as the C-terminal lysine of the heavy chain, may be missing
or derivatized
in a proportion or all of the molecules. Substitutions can be made in the
constant regions to
reduce or increase effector function such as complement-mediated cytotoxicity
or ADCC
(see, e.g., Winter et al., US 5,624,821; Tso et al., US 5,834,597; and Lazar
et al., Proc. Natl.
Acad. Sci. USA 103:4005, 2006), or to prolong half-life in humans (see, e.g.,
Hinton et al., J.
Biol. Chem. 279:6213, 2004). Exemplary substitutions include a Gln at position
250 and/or a
Leu at position 428 (EU numbering is used in this paragraph for the constant
region) for
increasing the half-life of an antibody. Substitution at any or all of
positions 234, 235, 236
and/or 237 reduce affinity for Fey receptors, particularly FeyRI receptor
(see, e.g., US
6,624,821). Some antibodies have alanine substitution at positions 234, 235
and 237 of
human IgG1 for reducing effector functions. Optionally, positions 234, 236
and/or 237 in
human IgG2 are substituted with alanine and position 235 with glutamine (see,
e.g., US
5,624,821).
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E. Expression of Recombinant Antibodies
[0245] Antibodies can be produced by recombinant expression. Nucleic acids
encoding the
antibodies can be codon-optimized for expression in the desired cell-type
(e.g., CHO or
Sp2/0). Recombinant nucleic acid constructs typically include an expression
control
sequence operably linked to the coding sequences of antibody chains, including
naturally-
associated or heterologous promoter regions. The expression control sequences
can be
eukaryotic promoter systems in vectors capable of transforming or transfecting
eukaryotic
host cells. Once the vector has been incorporated into the appropriate host,
the host is
maintained under conditions suitable for high level expression of the
nucleotide sequences,
and the collection and purification of the crossreacting antibodies. The
vector or vectors
encoding the antibody chains can also contain a selectable gene, such as
dihydrofolate
reductase, to allow amplification of copy number of the nucleic acids encoding
the antibody
chains.
[0246] E. coil is a prokaryotic host particularly useful for expressing
antibodies,
particularly antibody fragments. Microbes, such as yeast are also useful for
expression.
Saccharomyces is an exemplary yeast host, with suitable vectors having
expression control
sequences, an origin of replication, termination sequences and the like as
desired. Typical
promoters include 3-phosphoglycerate kinase and other glycolytic enzymes.
Inducible yeast
promoters include, among others, promoters from alcohol dehydrogenase,
isocytochrome C,
and enzymes responsible for maltose and galactose utilizations.
[0247] Mammalian cells can be used for expressing nucleotide segments encoding
immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones,
(VCH
Publishers, NY, 1987). A number of suitable host cell lines capable of
secreting intact
heterologous proteins have been developed in the art, and include CHO cell
lines, various
COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing
myelomas
including Sp2/0 and NSO. It can be advantageous to use nonhuman cells.
Expression vectors
for these cells can include expression control sequences, such as an origin of
replication, a
promoter, an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and
necessary processing
information sites, such as ribosome binding sites, RNA splice sites,
polyadenylation sites, and
transcriptional terminator sequences. Suitable expression control sequences
are promoters
derived from endogenous genes, cytomegalovirus, 5V40, adenovirus, bovine
papillomavirus,
and the like. See Co et al., J. Immunol. 148:1149 (1992).
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[0248] Having introduced vector(s) encoding antibody heavy and light chains
into cell
culture, cell pools can be screened for growth productivity and product
quality in serum-free
media. Top-producing cell pools can then be subjected ot FACS-based single-
cell cloning to
generate monoclonal lines. Specific productivities above 50 pg or 100 pg per
cell per day,
which correspond to product titers of greater than 7.5 g/L culture, can be
advantageous.
Antibodies produced by single cell clones can also be tested for turbidity,
filtration
properties, PAGE, IEF, UV scan, HP¨SEC, carbohydrate-oligosaccharide mapping,
mass
spectrometery, and binding assay, such as ELISA or Biacore. A selected clone
can then be
banked in multiple vials and stored frozen for subsequent use.
[0249] Once expressed, antibodies can be purified according to standard
procedures of the
art, including protein A capture, column chromatography (e.g., hydrophobic
interaction or ion
exchange), low-pH for viral inactivation and the like (see generally, Scopes,
Protein
Purification (Springer-Verlag, NY, 1982)).
[0250] Methodology for commercial production of antibodies including codon
optimization, selection of promoters, transcription elements, and terminators,
serum-free
single cell cloning, cell banking, use of selection markers for amplification
of copy number,
CHO terminator, serum free single cell cloning, improvement of protein titers
(see, e.g., US
5,786,464, US 6,114,148, US 6,063,598, US 7,569,339, W02004/050884,
W02008/012142,
W02008/012142, W02005/019442, W02008/107388, and W02009/027471, and US
5,888,809).
V. FORMULATIONS
[0251] Formulations (also known as pharmaceutical compositions) of the
invention
comprise an antibody (e.g., a chimeric, veneered or humanized version of
murine 9E4
(ATCC Accession Number PTA-8221)) or antigen binding fragment thereof, a
buffer, one or
more sugars and/or polyols and a surfactant, and have a pH within the range
from about 5 to
about 7.5. The formulations can be prepared for storage in liquid form or in
lyophilized
form. When stored in lyophilized form, the formulations can be reconstituted
with a liquid
(e.g., sterile water) to the concentrations and properties described herein.
When a lyophilized
composition is said to be reconstitutable by adding water to generate a
formulation of
specified component concentrations and pH, it is meant that the lyophilized
formulation can
be so reconstituted simply by addition of water (i.e., without supplying
additional amounts of
components or adding acid or base to change the pH). The concentrations and
properties of a
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prelyophilized liquid formulation can also be in accordance with those
described below if the
lyophilized formulation is reconstituted to the same volume as the formulation
prelyophilization. If the volume is different, then concentrations of
formulations should be
adjusted proportionally. For example, if the reconstituted volume is half the
prelyophilization
volume, then the concentrations of components in the prelyophilization
formulation should be
half the concentrations in the reconstituted formulation.
[0252] Some formulations include a bulking agent, which may or may not be the
same as a
sugar/polyol component. Typically, the formulations are sterile, for example,
as
accomplished by sterile filtration using a 0.2 inn or a 0.22 inn filter. Some
formulations have
a bioburden of < about 3 CFU/30 mL. Some formulations contain < about 0.1
EU/mg of
bacterial endotwdns. The formulations of the invention are also generally
stable by low to
undetectable levels of fragmentation and/or aggregation as further defined
below on freezing
and thawing. Still other formulations are stable following reconstitution of a
lyophilized cake
for at least three months at 40 degrees Celsius. In some formulations, less
than about 10% of
the antibody is present as an aggregate in the formulation. In some
formulations, less than or
equal to about 5% of the antibody is present as an aggregate in the
formulation.
[0253] In some formulations, the antibody is present at a concentration within
the range
from about 5 mg/mL to about 100 mg/mL. In some formulations, the antibody is
present at a
concentration within the range from about 5 mg/mL to about 50 mg/mL. In some
formulations, the antibody is present at a concentration within the range from
about 25
mg/mL to about 50 mg/mL. For example, the antibody may be present at a
concentration of
about 35-45 mg/ml or about 40 mg/mL. The antibody may be present in a sterile
liquid
dosage form of about 50 mg/vial to about 500 mg/vial, or greater. The antibody
may be
present in a lyophilized dosage form of about 40 mg/vial to about 500 mg/vial.
For example,
the antibody may be present in a sterile liquid or lyophilized dosage form of
about 250-350
mg/vial or about 200 mg/vial.
[0254] Antibodies used in the disclosed formulations can be coupled with a
therapeutic
moiety, such as a cytotoxic agent, a radiotherapeutic agent, an
immunomodulator, a second
antibody (e.g., to form an antibody heteroconjugate), or any other
biologically active agent
that facilitates or enhances the activity of the formulated antibody. However,
antibodies are
usually used in naked form (i.e., not conjugated to any of these moieties).
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[0255] The formulated antibody can be any of the antibodies described above
including any
of the chimeric, veneered or humanized versions of antibody 9E4 described
above.
[0256] Buffers are used in the disclosed formulations to achieve a suitable pH
for the
antibody, such as, for example, histidine, succinate, and citrate buffers.
Some formulations
have a pH within the range from about 5.5 to about 7, for example, a pH of
5.5, 5.6, 5.7, 5.8,
5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7Ø Some
formulations have a pH of
between about 5.5 to about 6.5. Some formulations have a pH of about 6.0 and
other
formulations have a pH of about 6.5. In some formulations, citrate buffer or
succinate buffer
is present at a concentration within the range from about 10 mM to about 30
mM, for
example, at a concentration of about 15-25 mM or about 20 mM. Some citrate
buffers
comprise sodium citrate dehydrate and citric acid monohydrate at a
concentration within the
range from about 15 mM to about 20 mM and a range from about 2 mM to about 6
mM,
respectively.
[0257] Suitable sugars and/or polyols for the formulations include trehalose,
sucrose,
mannitol, or a combination thereof. Sugars/polyols serves as bulking agents,
lyoprotecting
agent, and/or tonicity adjusting agents. For example, some formulations
include trehalose
present at a concentration within the range from about 220 mM to about 260 mM,
sucrose
present at a concentration within the range from about 220 mM to about 260 mM,
or a
mixture of sucrose present at a concentration within the range from about 20
mM to about 40
mM and mannitol present at a concentration within the range from about 200 mM
to about
220 mM. Some formulations include trehalose present at a concentration of
about 230 mM
or 240 mM. Other formulations include sucrose present at a concentration of
about 230 mM
or 240 mM. Other formulations include a mixture of sucrose present at a
concentration of
about 50 mM and mannitol present at a concentration of about 200 mM. Another
formulation
includes a mixture of sucrose present at a concentration of about 28 mM and
mannitol present
at a concentration of about 212 mM. Some such formulations are characterized
by an
osmolality in the range of about 250-400, 300-400, or 300-350 mOsm/kg, such
as, for
example, 335 mOsm/kg.
[0258] Formulations can contain a surfactant to reduce antibody aggregation
and
absorption to surfaces. Suitable surfactants include polysorbate 20 (PS20)
present at a
concentration within the range from about 0.005% to about 0.05% by weight.
PS20 protects
against marked increases in aggregation or turbidity that would otherwise
occur in
formulations of 9E4 antibodies. The polysorbate 20 may be present at a
concentration within
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the range from about 0.01% to about 0.05%. For example, the concentration can
be 0.005%,
0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, or 0.05%.
Alternatively,
in some formulations, polysorbate 20 is present at a concentration within the
range of about
from about 0.05 g/L, 0.1 g/L, 0.15 g/L, 0.2 g/L, 0.25 g/L, 0.3 g/L, 0.35 g/L,
0.4 g/L, 0.45 g/L,
or 0.5 g/L. Some formulations include polysorbate 20 at a concentration of 0.2
g/L (i.e.,
0.163 mmol/L).
[0259] An exemplary formulation (liquid, prelyophilization or reconstituted
after
lyophilization) is characterized by a pH within the range from about 5.5 to
about 7 and
includes: (a) a chimeric, veneered, or humanized version of antibody 9E4, or a
fragment
thereof that specifically competes for binding to antigen with 9E4 at a
concentration within
the range from about 10 mg/ml to about 50 mg/ml; (b) a citrate buffer or
succinate buffer
present at a concentration within the range from about 10 mM to about 30 mM;
(c) one or
more sugars and polyols ("sugar/polyol") selected from trehalose present at a
concentration
within the range from about 220 mM to about 260 mM, sucrose present at a
concentration
within the range from about 220 mM to about 260 mM, and a mixture of sucrose
present at a
concentration within the range from about 20 mM to about 40 mM and mannitol
present at a
concentration within the range from about 200 mM to about 220 mM; and (d)
polysorbate 20
present at a concentration within the range from about 0.005% to about 0.05%
by weight.
For example, the formulation can include: (a) an antibody comprising a light
chain having the
amino acid sequence set forth as SEQ ID NO: 32 and a heavy chain comprising an
amino
acid sequence set forth as SEQ ID NO: 37, with or without the C-terminal
lysine, and which
is present at a concentration of about 40 mg/mL; (b) a citrate buffer at a
concentration of
about 20 mM; (c) trehalose at a concentration of about 230 mM; (d) polysorbate
20 at a
concentration of about 0.02%; and a pH of about 6Ø
[0260] Some lyophilized formulations include: (a) a humanized version of
antibody 9E4 or
an antigen binding fragment thereof; (b) citrate; (c) trehalose; and
polysorbate 20. The
lyophilized formulation can include about 200 mg of the antibody. Some
lyophilized
formulations are capable of being reconstituted with sterile water. Some
lyophilized
formulations include 100-300 or 150-250 mg 9E4 antibody, 15-35 or 20-25 mg
sodium
citrate dehydrate, 1.65-2.75 or 2-2.3 mg citric acid monohydrate, 360-500 or
400-470 mg
trehalose dehydrate, and 0.5 to 1.5 mg or 0.75 to1.25 mg polysorbate 20. An
exemplary
lyophilized formulation includes 200 mg of a 9E4 antibody (e.g., humanized 9E4
antibody),
25 mg of sodium citrate dehydrate, 2.15 mg citric acid monohydrate, 435 mg
trehalose
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dehydrate, and 1 mg polysorbate 20. Another exemplary lyophilized formulation
includes
200 mg of a 9E4 antibody (e.g., humanized 9E4 antibody), 25 mg of sodium
citrate
dehydrate, 3.15 mg citric acid monohydrate, 435 mg trehalose dehydrate, and 1
mg
polysorbate 20. Such formulations can be reconstituted to a volume of about 5
ml. Other
lyophilized formulations include the same components in the same proportions
as any
disclosed in this paragraph but in different amounts (e.g., 400 mg antibody,
50 mg sodium
citrate, 4.3 mg citric acid monohydrate, 870 mg Trehalose dehydrate, and 2 mg
polysorbate
20).
[0261] Lyophilized formulations can be reconstituted to an antibody
concentration of about
30-50 or 35-45 mg/mL, such as about 40 mg/mL; (b) a citrate buffer present at
a
concentration of about 10-30 or 15-25 mM, preferably about 20 mM; (c)
trehalose present at
a concentration of about 160-330 or 200-260 mM, such as about 230 mM; (d)
polysorbate 20
present at a concentration of about 0.1-0.3 or 0.15 to 0.25 g/L, such as about
0.2 g/L; and (e)
a pH of about 5.5-6.5, such as about 6Ø
[0262] Liquid or reconstituted lyophilized formulations can be substantially
isotonic,
implying an osmolality of about 250-350 mOsm/kg water. Some formulations have
an
osmolality of about 335 mOsm/kg. Some formulations have an osmolality of 270-
300
mOsm/kg. Liquid or reconstituted lyophilized formulations can also be
hypertonic > 350
mOsm/kg water or hypotonic (<250 mOsm/kg water).
[0263] Any of the formulations described can be made without pharmaceutical
excipients,
carriers or the like, other than those described as being components herein.
Such a
formulation can be described as consisting of the recited components, or
consisting
essentially of the recited components if insignificant amounts of other
components not
affecting the properties of the formulation are present. Formulations can be
made under good
manufacturing practices (GMP) approved or approvable by the FDA for
preparation of drugs
for administration to humans.
VI. DIAGNOSTIC CRITERIA FOR SYNUCLEINOPATHIES
[0264] The present methods are in general performed on subjects diagnosed with
a Lewy
body disease by a qualified health practitioner or are at elevated risk
thereof compared with
the general population as evidenced by genetic or biochemical markers, family
history or
prodromal symptoms of the disease. Such individuals include any who have
received a prior
prescription for treatment or prophylaxis of a Lewy body disease. Diagnosis of
the
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synucleinopathy can be based on art-recognized criteria for possible or
probable Lewy body
disease, such as those of DSM-V or DSM IV-TR, the Lewy Body dementia
association, the
Parkinson's disease society and the like. However, diagnosis can also be based
on presence
of any signs or symptoms of Lewy body disease that lead a treating physician
to conclude
that a subject probably has a Lewy body disease. Exemplary criteria for
diagnosing possible
or probable PD are shown below.
Group A: resting tremor, bradykinesia, rigidity and asymmetric onset
Group B features: suggestive of alternative diagnoses
Prominent postural instability in the first 3 years after symptom onset
Freezing phenomena in the first 3 years
Hallucinations unrelated to medications in the first 3 years
Dementia preceding motor symptoms or in the first year
Supranuclear gaze palsy (other than restriction of upward gaze) or slowing of
vertical
saccades
Severe symptomatic dysautonomia unrelated to medications
[0265] Documentation of a condition known to produce parkinsonism and
plausibly
connected to the subject's symptoms (such as suitably located focal brain
lesions or
neuroleptic use within the past 6 months).
[0266] Criteria for possible diagnosis of Parkinson disease:
At least 2 of the 4 features in Group A are present; at least 1 of these is
tremor or
bradykinesia and either none of the features in group B is present or symptoms
have been
present for less than 3 years and none of the features in group B is present
to date; and either
substantial and sustained response to levodopa or a dopamine agonist has been
documented,
or the subject has not had an adequate trial of levodopa or dopamine agonist.
[0267] Criteria for probable diagnosis of Parkinson disease:
At least 3 or the 4 features in Group A are present, and none of the features
in Group B is
present and substantial and sustained response to levodopa or a dopamine
agonist has been
documented.
[0268] Exemplary criteria for diagnosis of Lewy Body dementia are:
The presence of dementia
At least two of three core features:
fluctuating attention and concentration,
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recurrent well-formed visual hallucinations, and
spontaneous parkinsonian motor signs.
Suggestive clinical features include:
Rapid eye movement (REM) sleep behavior disorder
Severe neuroleptic sensitivity
Low dopamine transporter uptake in basal ganglia demonstrated by SPECT or PET
imaging
[0269] In the absence of two core features. the diagnosis of probable DLB can
also be
made if dementia plus at least one suggestive feature is present with one core
feature.
[0270] Possible DLB can be diagnosed with the presence of dementia plus one
core or
suggestive feature.
[0271] Early warning signs of Lewy Body disease include for example, EEG
slowing,
neuropsychiatric manifestations (depression, dementia, hallucinations,
anxiety, apathy,
anhedonia), autonomic changes (orthostatic hypotension, bladder disturbances,
constipation,
fecal incontinence, sialorrhea, dysphagia, sexual dysfunction, changes in
cerebral blood
flow), sensory changes (olfactory, pain, color discrimination abnormal
sensations), sleep
disorders (REM sleep behavior disorder (RBD), restless legs syndrome/periodic
extremity
movements, hypersomnia, insomnia) and miscellaneous other signs and symptoms
(fatigue,
diplopia, blurred vision, seborrhea, weight loss/gain). Genetic markers of
risk toward PD
include mutations in the alpha-synuclein or Parkin, UCHLI, and CYP2D6 genes;
particularly
mutations at positions 30 and 53 of the alpha-synuclein gene. None of these
genetic markers
or early warning sign is itself diagnostic of a Lewy body disease but they can
individually or
in combination contribute to a diagnosis of a Lewy body disease.
VII. THERAPEUTIC REGIMES
[0272] In therapeutic applications, an antibody is administered to a subject
diagnosed with
a synucleinopathy in a regime (dose, frequency and route of administration)
known or
suspected to be effective to ameliorate or at least inhibit further
deterioration of at least one
sign or symptom of the disease. In prophylactic applications, an antibody is
administered to a
subject at increased risk of a synucleinopathy but not yet having sufficient
symptoms to be
diagnosed with the disease in a regime known or suspected to be effective to
inhibit or delay
onset of at least one sign or symptom of the disease.
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[0273] A regime is considered therapeutically or prophylactically effective if
a more
favorable outcome is demonstrated in treated subjects versus control subjects
in a controlled
animal model or clinical trial (e.g., a phase II, phase II/III or phase III
trial) at the p <0.2, 0.1,
0.05 or 0.01 or even 0.001 level. However, due to variations in genetics,
subject
characteristic and environment and disease subtype between individual
subjects, a regime that
is effective in one subject may not be effective in another subject or may be
effective to
different extents.
[0274] An exemplary dosage range for antibodies is from 3000 to 5000 mg of an
antibody
against alpha-synuclein administered intravenously at intervals of 3-5 weeks,
such as every 4
weeks. In some subjects, the dosage is 3500-4500 mg every 3-5 weeks, such as
every 4
weeks. Subjects can receive the same or different dosages as each other (e.g.,
depending on
weight of the subject). In some methods, a subject receives one of two fixed
dosages. For
example, subjects with a weight less than 65 kg can receive 3500 mg and
subjects with a
weight greater or equal to 65 kg can receive 4500 mg. In some methods, the
dosage range for
at least some subjects lies within a range of 45-75 mg/kg, for example, 50-70
mg/kg, 45
mg/kg, 60 mg/kg or 65 mg/kg. Dosages are usually administered on multiple
occasions with
an interval of 3-5 weeks, such as every 28 days or four weeks, or every
calendar month.
Subjects can receive at least 6, 9, 12 or 18 dosages at such intervals, or can
be dosed while
symptoms of the conditions persist or for the remaining life of the subject.
In some regimes,
an initial loading dose of 2000 mg is administered followed by dosing within a
range of
greater or equal to 2000mg but less than the intended target dose until the
intended target
dose is reached. For example, a subject can receive an initial loading dose of
2000 mg,
followed by an up-titration to a 3500 mg dose or a 4500 mg dose. The up-
titration can occur
in a single subsequent dose or in gradual increases over several doses until a
target dose or
dose within a target range is reached. For example, a subject can receive an
initial dose of
2000 mg followed by subsequent doses of 3500 mg. Alternatively a subject can
receive an
initial dose of 2000 mg followed by one or more subsequent doses at greater or
equal to 2000
mg but less than 3500 mg, and subsequent doses at 3500 mg. Likewise a subject
can receive
an initial dose of 2000 mg followed by subsequent doses of 4500 mg.
Alternatively, a subject
can receive an initial dose of 2000 mg followed by subsequent doses at greater
or equal to
2000 mg but less than 4500 mg and subsequent doses at 4500 mg. In some regimes
a subject
receives a dose of 3000-5000 mg antibody intravenously every four weeks for at
least 52
weeks. In subjects receiving multi-dose regimes with the dose within a
specified range, such
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as 3500-5000 mg, the subject can receive the same or different dose within the
specified
range on each dosing. In some regimes, a subject receives the same dose within
a specified
range at each dosing.
[0275] In another exemplary regime, a dose of 1300-1700 mg antibody is
administered
intravenously to a subject at intervals of 3-5 weeks. An exemplary dose is
1500 mg.
Subjects can received a single fixed dose or two or more different dosages
within this range,
based on e.g., subject weight. Some subjects dosed within this range receive
18-25 mg/kg of
antibody, for example, 20 mg/kg. As in other methods, the intervals can be 3-5
weeks, such
as every 4 weeks or every calendar month. Subjects can receive at least 6, at
least 9, at least
12, or at least 18 dosages, or can be dosed at such intervals while symptoms
remain or for the
remaining life of a subject.
[0276] For any of the above described treatment regimes, an area under the
curve can be
calculated to indicate the amount of antibody delivered with time. Alternative
treatment
regimes can be devised (e.g., with a different route of administration,
frequency or dose) to
deliver substantially the same area under the curve (e.g., within 25, 20 or
15%). Preferably
such an alternative treatment regime does not substantially exceed the Cmax of
the specified
regime (e.g., by no more than 25, 20 or 15%). Other routes of administration
include topical,
oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal,
intranasal or
intramuscular.
[0277] Any of the treatment regimes can be accompanied by monitoring a subject
receiving
treatment for changes in movement and/or cognitive deficits. Preferably such
monitoring
includes at least one assessment before and after commencing treatment.
Preferably the
monitoring indicates reduced movement and/or cognitive deficits responsive to
treatment,
that is relative to before beginning treatment or at least indicates a reduced
rate of decline
relative to the previous rate of decline in the subject or the rate of decline
in control patients
not receiving any immunotherapy. Subjects can also be monitored for changes in
autonomic
dysfunction, gastrointestinal dysfunction, visual hallucination or one or more
psychological
symptom among other signs or symptoms.
[0278] Symptoms of subjects, for example motor symptoms such as tremor,
rigidity and
slowness of movement, may be monitored. Wearable systems or on-body sensors
may be
used to assess and quantifiy motor symptoms of subjects. "On-body sensors" may
be used in
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a laboratory setting or in free-living conditions [S. Del Din, et al., J. of
NeuroEngineering and
Rehabilitation, 2016 13:46].
[0279] Subjects may be monitored using mobile-device-based monitoring. The
mobile
device may be a smartphone, smartwatch, wearable sensor, portable multimedia
device or
tablet computer. Built-in mobile-device sensors may be used to record daily
activities of
subjects. Subjects may carry a mobile-device to record their daily activities.
Mobile-device-
based assessments and sensors may be used for remote, passive monitoring of
gait and
mobility in subjects receiving treatment, for example for Parkinson's disease.
(See e.g.,
Lipsmeier, F., et al.. Mov Disord. 2017; 32 (suppl 2); W. Y. Cheng et al.,
2017 IEEE/ACM
International Conference on Connected Health: Applications, Systems and
Engineering
Technologies (CHASE), Philadelphia, PA, 2017, pp. 249-250). Sensor data may be
analyzed
by machine learning-based activity profiling. Gait and mobility may be
correleated with the
MDS-UPDRS that is used in clinics to evaluate Parkinson's disease severity.
[0280] Some mobile-device-based monitoring may comprise (a) providing a
subject with a
mobile device programmed to receive and transmit data acquired from sensors
internal and/or
external to the device relating to movement deficits of a subject having or
suspected of
having a synucleinopathy, whereafter the subject undergoes a series of
movements to reveal
movement deficits, if present, and the internal or external sensors of the
device acquire data
relating to the movements; (b) collecting data transmitted from the mobile
device; and (c)
comparing the data acquired from the subjects with control data to assess
presence or extent
of movement deficits in the subject. In some mobile-device-based monitoring,
the mobile
device is programmed to receive and transmit data from at least two external
sensors attached
to upper and lower limbs of the subject. In some mobile-device-based
monitoring, the mobile
device acquires data from sensors on the upper and lower limbs of the subject.
In some
mobile-device-based monitoring, the mobile device is carried by the subject
and acquires data
from an internal sensor. In some mobile-device-based monitoring, the series of
movements
includes tapping the device, sitting and standing.
[0281] The present regimes can be administered concomitantly with another
agent effective
in treatment or prophylaxis of the disease being treated. The other agent can
be another
immunotherapeutic agent described herein or other agent for treating
Parkinson's disease
including levodopa, benzaseride, carbidopa, dopamine agonists, non-ergot
dopamine
agonists, catechol-O-methyl ("COMT") inhibitors such as, for example,
entacopone or
tolcopone, monoamine oxidase ("MAO") inhibitors, such as, for example,
rasagaline,
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amantadine, or anticholinergic agents can be used in combination with the
present regimes.
Some such other agents reduce one or more symptoms of the disease without
affecting
causative factors.
[0282] All publications (including GenBank Accession numbers, UniProtKB/Swiss-
Prot
accession numbers and the like), patents and patent applications cited are
herein incorporated
by reference in their entirety for all purposes to the same extent as if each
individual
publication, patent and patent application was specifically and individually
indicated to be
incorporated by reference in its entirety for all purposes. In the event of
any variance in
sequences associated with Genbank and UniProtKB/Swiss-Prot accession numbers
and the
like or in website, or disease criteria of an organization, the application
refers to those in
effect on its effective filing date.
VIII. Examples
[0283] Example 1. Phase II Clinical Trial for an Alpha-Synuclein Antibody
[0284] Trial Design: A phase II trial is conducted for an alpha-synuclein
antibody
with a heavy chain variable region designated SEQ ID NO:10 and a light chain
variable
region designated SEQ ID NO:5 on subjects with Parkinson's disease. The trial
has two
treatment arms and one control arm. Subjects are randomized 1:1:1 into the
arms, with
N=300. The initial phase of the trial is a 52-week double blind treatment.
During the initial
phase of the trial, subjects do not receive other treatments Parkinson's
disease (including
symptomatic treatment). The subjects in one treatment arm receive a fixed dose
of 1500 mg
antibody intravenously every four weeks. The subjects in the other treatment
arm receive
3500 mg or 4500 mg of antibody intravenously every four weeks depending on
weight with
subjects below 65 kg receiving the lower dosage and subjects at or above 65 kg
receiving the
higher dosage. The subjects in the second arm receive a loading dosage of 2000
mg and
optionally additional up titration dosages at 2000 mg or above until reaching
the target dose
of 3500 mg or 4500 mg. Dosing is continued for one year. The trial then has an
extension
period in which subjects initially in the placebo group receive one of the two
treatment
regimes from the initial phase, and subjects from the treatment arms in the
initial phase
continue to receive the same treatment as previously. During the extension
phase of the trial,
subjects may receive systematic treatment with levodopa as well as the
antibody subject of
the trial, but do not receive other treatments for Parkinson's disease.
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[0285] Objectives:
The primary objective is to evaluate efficacy of an alpha-synuclein antibody
with a heavy
chain variable region designated SEQ ID NO:10 and a light chain variable
region designated
SEQ ID NO:5 using the Movement Disorder Society (MDS)-sponsored revision of
the
Unified Parkinson's Disease Rating Scale (MDS-UPDRS).
Secondary objectives are to:
- evaluate the effects of the antibody on DaT-SPECT signal and signs and
symptoms of
Parkinson's disease including changes in movement and cognitive deficits
- evaluate safety and tolerability of the antibody for up to 104 weeks, and
- evaluate pharmacokinetics of the antibody.
[0286] Example 2. Passive monitoring of early-stage Parkinson's disease
patient mobility
in Phase I Alpha-Synuclein Antibody clinical trial with smartphone sensors
[0287] 1. Abstract ¨Gait and mobility in early-stage Parkinson's disease
(PD) patients
were measured using smartphone-based passive monitoring. In the Multiple
Ascending Dose
clinical trial of an alpha-synuclein antibody with a heavy chain variable
region designated
SEQ ID NO:10 and a light chain variable region designated SEQ ID NO:5, 44 PD
patients
and 35 age- and gender-matched healthy individuals performed smartphone-based
assessments for up to 24 weeks and up to 6 weeks respectively. (Lipsmeier, F.,
et al.. Mov
Disord. 2017; 32 (suppl 2); W. Y. Cheng et al., 2017 IEEE/ACM International
Conference on
Connected Health: Applications, Systems and Engineering Technologies (CHASE),
Philadelphia, PA, 2017, pp. 249-250).
[0288] For "passive monitoring", subjects carried the smartphone with them as
part of their
daily routine, while sensors in the smartphone recording movement data
continuously. In
total, over 30,000 hours of passive monitoring data were collected. To
classify the sensor
signal into activity profiles, a Human Activity Recognition (HAR) model was
built using
Deep Neural Networks (DNN) trained on previously published data. The activity
profiles of
the participants determined by the HAR model showed significant differences
between PD
patients and healthy controls in the percentage of time walking and frequency
in which
subjects changed positions (sitting and standing).
[0289] 2. METHODS
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[0290] A. Data Collection
The analysis solely focused on exploring differences between HC and PD
cohorts, and did
not look at antibody-related effects. In total, 24,104 hours of passive
monitoring data were
recorded for the PD cohort, and 8,614 hours for the HC cohort. In line with
the approach of
Rai, A. et at, (MobiCom'12, August 22-26, 2012], accelerometer data was
filtered out where
the standard deviation of Euclidean norm less than 0.03 m/s2 more than 30
minutes, as during
these spans smartphones were likely not carried by the subjects. This step
removed 14% of
the passive monitoring data.
[0291] B. HAR
A 9-layer neural network model structure was used. Similar structures have
been used
previously for HAR and have been shown to out-perform the traditional machine
learning
methods (F. J. Ordoliez and D. Roggen, Sensors 2016, 16, 115]. The HAR model
was trained
on two public data sets (G. M. Weiss and J. W. Lockhart, Proceedings of the
AAAI-12
Workshop on Activity Context Representation: Techniques and Languages,
Toronto, CA.
2012; A. Stisen, et al., 13th ACM Conference on Embedded Networked Sensor
Systems,
Seoul, Korea, 2015) to classify six activities: walking, stairs, jogging,
sitting, standing, and
lying down. The continuous accelerometer data were down-sampled into 20Hz and
segmented into 4-second windows with 75% overlapping with adjacent ones.
[0292] 3. RESULTS
[0293] A. Human Activity Recognition Performance Validation:
To ensure the HAR model can accurately translate the sensor data into activity
profile, the
performance of the model was first analyzed in the held-out validation set.
The HAR model
was able to correctly distinguish gait activities (walking, stairs, jogging)
from stationary
activities (sitting, standing, lying down) with more than 98% of accuracy.
Additional
validation on labeled Gait and Balance data from the trial data also showed
that the HAR
model was able to successfully profile the Gait segments with 96.9% of
accuracy, and
Balance segments with 99.5% accuracy.
[0294] B. Activity Profiles Comparison
The mobility of each subject was quantified by calculating the proportion of
time when the
subject engaged in gait activities (walking, stairs, jogging) over the total
passive monitoring
coverage time of the patient. The overall proportion of different gait
activities over the total
coverage for PD and HC cohorts was calculated. In the PD cohort a median was
detected of
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9.7% of gait spans over all coverage spans as opposed to HC cohort's 15.1%.
The HC cohort
had a significantly higher per-subject gait activity level than PD cohort,
with Mann-Whitney
test P value 2.43E-8.
[0295] C. Number of Sit-to-Stand and Stand-to-Sit Comparison
It has been observed that one manifestation of the functional impact of PD is
in the sit-to-
stand and stand-to-sit (STS) events (A. Zijlstra, et at, J. NeuroEngineering
and Rehabilitation
2012, 9:75]. From the activity profile, the coverage-normalized STS events
were calculated
for each subject. Median number of STS per hour of PD patient of 1.44 was
observed, which
was significantly lower than HC subject's 1.74. Mann-Whitney test P value
between two
groups was 1.60E-8.
[0296] 4. CONCLUSION
[0297] Results from this study show that it is feasible to measure gait and
mobility in early-
stage PD patients using smartphone- based passive monitoring. Sensor data
collected during
passive monitoring provides previously inaccessible, ecologically valid
insights into patients'
daily behavior and functioning. Significant differences were observed between
PD patients
and healthy controls (HC).
