Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02643633 2008-10-17
Attorney Docket No. CF50072.
TREATMENT OF AMYLOIDOGENIC DISEASES
Backsround of the Invention
[0001] Alzheimer's disease (AD) is a progressive disease resulting in senile
dementia.
See generally Selkoe, TINS 16:403 (1993); Hardy et ar., WO 92113069; Selkoe,
J.
Neuropathol. Exp. Neurol. 53:438 (1994); Duff et al., Nature 373:476 (1995);
Games et al.,
Nature 373:523 (1995). Broadly speaking, the disease falls into two
categories: late onset,
which occurs in old age (65 + years) and early onset, which develops well
before the senile
period, i.e., between 35 and 60 years. In both types of disease, the pathology
is the same but
the abnormalities tend to be more severe and widespread in cases beginning at
an earlier age.
The disease is characterized by at least two types of lesions in the brain,
neurofibrillary
tangles and senile plaques. Neurofibrillary tangles are intracellular deposits
of microtubule
associated tau protein consisdng of two filaments twisted about each other in
pairs. Senile
plaques (i.e., amyloid plaques) are areas of disorganized neuropil up to 150
m across with
extracellular amyloid deposits at the center which are visible by nricroscopic
analysis of
sections of brain tissue. The accumulation of amyloid plaques within the brain
is also
associated with Down's syndrome and other cognitive disorders.
[0002] The principal constituent of the plaques is a peptide te,rmed 4 or f~-
amyloid
peptide. A(3 peptide is a 4-kDa internal fragment of 39-43 amino acids of a
larger
transmembrane glycoprotein named protein termed amyloid precursor protein
(APP). As a
result of proteolytic processing of APP by different secretase enzymes, AJS is
primarily found
in both a short form, 40 amino acids in length, and a long form, ranging from
42-43 amino
acids in length. Part of the hydrophobic transmembrane domain of APP is found
at the
carboxy end of Ap, and may account for the ability of A(3 to aggregate into
plaques,
particularly in the case of the long form. Accumulation of amyloid plaques in
the btain
eventually leads to neuronal cell death. The physical symptoms associated with
this type of
neural deterioration characteaize Alzheimer's disease.
[0003] Several mutations within the APP protein have been correlated with the
presence of Alzheimer's disease. See, e.g., Goate et al., Nature 349:704
(1991) (valine717 to
isoleucine); Chartier Harlan et al. Nature 353:844 (1991)) (valine"'to
glycine);1Vlurrell et
al., Scfence 254:97 (1991) (valine7i7 to phenylalanine); MuIlan et al., Nature
Genet. 1:345
(1992) (a double mutation changing lysineS95-methionine-96 to asparagine~-
leucinesn.
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Such mutations are thought to cause Alzheimer's disease by increased or
altered processing of
APP to Ap, particularly processing of APP to increased amounts of the long
form of Ap (i.e.,
AP1-42 and A(3143). Mutations in other genes, such as the presenilin genes,
PS1 and PS2,
are thought indirectly to affect processing of APP to generate increased
amounts of long form
Ap (see Hardy, TINS 20: 154 (1997)).
[00041 Mouse models have been used successfully to determine the significance
of
amyloid plaques in Alzheimer's (Ganxs et al., supra, Johnson-Wood et al.,
Proc. Natl. Acad
Scf. USA 94:1550 (1997)). In particular, when PDAPP transgenic mice, (which
express a
mutant form of human APP and develop Alzheimer's disease at a young age), are
injected
with the long form of A¾, they display both a decrease in the prognession of
Alzheimer's and
an increase in antibody titers to the Aft peptide (Schenk et al., Nature
400,173 (1999)). The
observations discussed above indicate that A,6, particularly in its long form,
is a causative
element in Alzheimer's disease.
100051 McMichaei, EP 526,511, proposes administration of homeopathic dosages
(less than or equal to 10'2 mg/day) of AQ to patients with preestablished AD.
In a typical
human with about 5 liters of plasma, even the upper linut of this dosage would
be expected to
generate a concentration of no more than 2 pg/ml. The normal concentration of
A43 in human
plasma is typically in the range of 50-200 pg/ml (Seubert et al., Nature
359:325 (1992)).
Because EP 526,511's proposed dosage would barely alter the level of
endogenous circulating
Ap and because EP 526,511 does not recommend use of an adjuvant, as an
inununostimulant,
it seems implausible that any therapeutic benefit would result.
[00061 Accordingly, there exists the need for new therapies and reagents for
the
treatment of Alzheimer's disease, in particular, therapies and reagents
capable of effecting a
therapeutic benefit at physiologic (e.g., non-toxic) doses.
Cross-Reference to Related Anatications
[00071 U.S. Application No. 60/648,631 filed on January 28, 2005; U.S.
Publication
No. US 20060193850 Al published on August 31, 2006; International Publication
No.
WO 06/083689 published on August 10, 2006; U.S. Application No. 607622,525
filed on
October 26, 2004 and, U.S. Publication No. US 20060160161 Al published on Jnly
20, 2006
are related applications, all of which are incorporated by herein reference in
their entirety for
all puiposes.
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Attorney Docket No. CF50072: _
Sununarv of the Invention
100081 The invention provides methods of therapeutically treating Alzheimer's
disease. The methods comprise administering by intravenous infusion to a
patient suffering
from the disease a dosage of an antibody within a range of about 0.5 mg/kg to
less than 5
mg/kg. The antibody specificaily binds to an N-terminal fragment of beta
amyloid peptide
(AP) with a binding affinity of at least 107 M'1, and thereby therapeuticatly
treats the patient.
Optionally, the antibody is a humanized antibody. Optionally, the humanized
antibody is a
humanized version of mouse antibody 3D6 expressed by the hybridoma deposited
under
ATCC under No. PTA-5130. Optionally, the humanized antibody eoniprises (i) a
light chain
comprising three complementarity deternuning regions (CDRs) from the
immunological light
chain variable region of the mouse antibody 3D6; and (ii) a heavy chain
comprising three
complementarity detenaining regions (CDRs) from the immunological heavy chain
variable
region of mouse antibody 3D6. Optionally, the humanized antibody comprises (i)
a variable
light chain region having the sequence as set forth in SEQ ID NO:1, SEQ ID
N0:3 or SEQ
ID N0:5; and (ii) a variable heavy chain region having the sequence as set
forth in SEQ ID
N0:2, SEQ ID N0:4 or SEQ ID N0:6. Optionally, the humanized antibody comprises
(i) a
variable light chain region having the sequence as set forth in SEQ ID NO:1 or
SEQ ID
N0:5; and (ii) a variable heavy chain region having the sequence as set forth
in SEQ ID
N0:2 or SEQ ID N0:6. Optionally, the humanized antibody is bapineuzumab.
Optionally,
the antibody is a humanized version of mouse antibody lOD5 expressed by the
hybridoma
deposited under ATCC under No. PTA-5129. Optionally, the humanized antibody
comprises
(i) a light chain comprising three complementarity determining regions (CDRs)
from the
immunological light chain variable region of the mouse antibody lOD5; and (ii)
a heavy
chain comprising three complementarity determining regions (CDRs) from the
immunological heavy chain variable region of mouse antibody 1OD5. Optionally,
the
humanized antibody comprises (i) a variable light chain region having the
sequence as set
forth in SEQ ID N0:7 or SEQ ID N0:7 as set forth in US Patent Publication No.
20050142131; and (ii) a variable heavy chain region having the sequence as set
forth in SEQ
ID N0:8 or SEQ ID N0:8 as set fotth in US Patent Publication No. 20050142131.
Optionally, the humanized antibody is a humanized version of mouse antibody
12A11
expressed by the hybridoma deposited under with the American Type Cult.ure
Collection
(ATCC), Manassas, VA 20108, USA on Apri18, 2003 under the tenms of the
Budapest
Treaty and has deposit number PTA-7271.
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[0009] Optionally, the humanized antibody comprises (i) a light chain
comprising
three complementarity determining regions (CDRs) from the immunological light
chain
vartable region of the mouse antibody 12A11; and (ii) a heavy chain comprising
thre.e
complementarity determining regions (CDRs) from the immunological heavy chain
variable
region of mouse antibody 12A11. Optionally, the humanized antibody comptises
(i) a
variabte light chain region having the sequence as set forth in SEQ ID N0:2 as
set foith in
US Patent Publication No. 20050118651; and (ii) a variable heavy chain region
having the
sequence as set forth in SEQ ID N0:4 as set forth in US Patent Publication No.
20050118651.In sotne methods, the dosage is about 0.5 mg/kg. In some methods,
the dosage
is about 1.5 mg/kg. In some methods, the dosage is 0.5 to 3 mg/kg. In some
methods, the
dosage is 0.5 to 1.5 mg/kg. In some methods, the dosage is administered on
multiple
occasions, such as every 13 weeks.
[OOlOj Some methods further comprise monitoring the patient by at least one
type of
assessment selected from the group of consisting of Mini-Mental State Exam
(M1vISE),
Alzheimer's Disease Assessment Scale - cognitive (ADAS-COG), Clinician
Interview-Based
Impression (CIBI), Neurological Test Battery (NTB), Disability Assessment for
Dementia
(DAD), Clinical Dementia Rating-sum of boxes (CDR-SOB), Neuropsychiatric
Inventory
(NPI), Positron Emission Tomography (PET Imaging) scan, Magnetic Resonance
Imaging
(MRI) scan, and measurement of blood pressure. In some methods, the type of
assessment is
an MMSE, and the MMSE is administered on multiple occasions, such as before
administering the dosage, and at week 4, week 16, 6 months, and 1 year after
administering
the dosage. In some methods, the MMSE score measured after administration is
higher than
a previously assessed MMSE score.
[OOllj The invention further provides methods of therapeutically ueating
Alzheimer's
disease, comprising administering by intravenous infusion to a patient
suffering from the
disease a dosage of an antibody within a range of about 0.5 mg/kg to less than
5 mg/kg,
wherein the antibody specifically binds to beta amyloid peptide (Ap) with a
binding affinity
of at least 10' M-1, and monitoring the patient for posterior reversible
encephalopathy
syndrome (PRES) or vascular edema. Optionally, the monitoring comprises
performing an
MRI scan, optionally with a FT,AIR (Fluid Attenuated Inversion Recovery)
sequence
imaging. In some methods, the monitoring identifies of at least one clinical
symptom
associated with PRES, such as headache, nausea, vomiting, confusion, seizures,
visual
abnormalities, altered mental functioning, ataxia, frontal symptoms, parietal
symptoms,
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stupor, or focal neurological signs. In some methods, the dosage is reduced or
suspended
based on an outcome of the MRI scan that is indicative of PRES or vascular
edema. In some
mtthods, the dosage is reduced or suspended based on an outcome of the FLAIR
sequence
imaging that is indicative of PRES or vascuiar edema. In some methods, the
dosage is
reduced or suspended based on an identification of at least one clinical
symptom associated
with PRES. In some methods, the MRI scan is every 3 months, eve.ry 6 months,
or every
year. In some methods, the PT,AIR sequence imaging is every 3 months, every 6
months, or
every year.
[00121 Some of the above methods further comprise determining presence or
absence
of hypertension in the patient, wherein if the patient has hypertension, the
method further
comprises administering an antihypertensive. Optionally, the antihypertensive
is selected
from the group consisting of hydroclorothiazide, angiotensin-converting enzyme
(ACfi)
inhibitors, angiotensin II-receptor blockers (ABB), beta blockers, and calcium
channel
blockers.
[0013] Some methods further comprise administering a steroid to the patient to
treat
the PRES or vascular edema. Optionally, the steroid is dexamethasone or
methyprednisolone.
[0014] Some methods further comprise reducing or suspending the dosage based
on
an outcome of the MRI scan and that is indicative of PRBS or vascular and
identifying at
least one clinical symptom associated with PRES or vascular edema, such as
headache,
nausea, vomiting, confusion, seizures, visual abnormalities, altered mental
functioning,
ataxia, frontal syrnptom.s, parietal symptoms, stupor, or focal neurological
signs. Some
methods further comprise reducing or suspending the dosage based on an outcome
of the
FLAIR sequence imaging that is indicative of PRES or vascular edema and
identifying of at
least one clinical symptom assoeiated with PRES or vascular edema, such as
headache,
nausea, vomiting, confusion, seizures, visual abnormalities, altered mental
functioning,
ataxia, frontal symptoms, parietal symptoms, stupor, or focal neurological
sigas.
[0015) In some methods, the monitoring indieates presence of PRES or vascular
edema at a first time point after administration, and absence of PRES or
vascular edema at a
second time point after the first point, and the patient is administered a
fust dosage before the
monitoring indicates presence of PRES or vascular edema, a second dosage or no
dosage
after the monitoring detects presence of PRES or vascular edema, and a third
dosage after the
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monitoring detects absence of PRES or vascular edema, wherein the first and
third dosage are
higher than the second dosage.
[0016] In some of the above methods, the antibody is a bumanized antibody.
Optionally, the humanized antibody is a humanized version of mouse antibody
3D6
expressed by the hybridoma deposited with the American Type Culture Collection
(ATCC),
Manassas, VA 20108, USA on Apri18, 2003 under the tenns of the Budapest Treaty
and has
deposit number PTA-5130.
[0017] Optionally, the humanized antibody comprises (i) a light chain
comprising
three complementarity detennining regions (CDRs) from the immunological light
chain
variable region of the mouse antibody 3D6; and (ii) a heavy chain comprising
three
complementarity determining regions (CDRs) from the immunological heavy chain
variable
region of mouse antibody 3D6. Optionally, the humanized antibody comprises (i)
a variable
light chain region having the sequence as set forth in SEQ ID N0:1, SEQ ID
N0:3 or SEQ
ID N0:5; and (ii) a variable heavy chain region having the sequence as set
forth in SEQ ID
N0:2, SEQ ID N0:4 or SEQ ID N0:6. Optionally, the humanized antibody comprises
(i) a
variable light chain region having the sequence as set forth in SEQ ID NO:1 or
SEQ ID
N0:5; and (ii) a variable heavy chain region having the sequence as set forth
in SEQ ID
N0:2 or SEQ ID N0:6. Optionally, the humanized antibody is bapineuzumab.
[0018] In some of the above ttethods, the dosage is about 0.5 mg/kg. In some
methods, the dosage is about 1.5 mg/kg. In some methods, the dosage is 0.5 to
3 mg/kg. In
some methods, the dosage is 0.5 to 1.5 mg/kg. In some methods, the dosage is
administered
on multiple occasions, such as every 13 weeks.
[0019] In some of the above methods, Bapineuzumab is administered at a first
dosage
before PRES or vascular edema is determined from the MRI scan and a second
dosage after
PRES or vascular edema is determined from the MRI scan, and the second dosage
is less then
the first dosage. Optionally, the first dosage is 3-5 mg/kg and the second
dosage is 0.5 to 3
mg/kg. Optionally, the second dosage is half of the first dosage. Optionally,
the
Bapineuzumab is administered at a first frequency before the MRI shows PRES or
vascular
edema and a second frequency after the MRI shows PRES or vascular edema, and
the second
frequency is less than the f'n'st frequency.
[0020] In some of the above methods, the type of assessment is blood pressure,
and
the presence or absence of hypertension is determined. Optionally, if the
patient has
hypertension, the method further comprises administering an antihypertensive.
Optionally,
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the antihypertensive is selected from the group consisting of
hydroclorothiazide,
Angiotensin-converting Enzyme (ACE) Inhibitors, angiotensin II-teceptor
blockers (ARB),
beta blockers, and calcium channel blockers.
[0021] The invention further provides therapeutic products. The products
comprise a
glass vial and instructions. The glass vial contains a fotmulation comprising
about 10 mg to
about 250 mg of a humanized anti Ap antibody, about 496 mannitol or about 150
ni1v1 NaCI,
about 5 mM to about 10 mM histidine, and about 10 niIvl methionine. The
instructions to
monitor a patient to whom the formulation is administered for PRES and or
vascular edema
are included with the products.
[0022] The invention provides methods rnethod of treating Alzheimer disease
comprising subcutaneously administering to a patient having the disease an
antibody that
specifically binds to an N-terminal fragment of Aft, wherein the antibody is
adniinistered at a
dose of 0.01-0.6 mg/kg and a frequency of between weekly and monthly.
Optionally, the
antibody is administered at a dose of 0.05-0.5 mg/kg. Optionally, the antibody
is
administered at a dose of 0.05-0.25 mg/kg. Optionally, the antibody is
administered at a dose
of 0.015-0.2 mg/kg weekly to biweekly. Optionally, the antibody is
administered at a dose of
0.05-0.15 mg/kg weekly to biweekly. Optionally, the antibody is administered
at a dose of
0.05-0.07 mg/kg weekly. Optionally, the antibody is administered at a dose of
0.06 mg/kg
weekly. Optionally, the antibody is administered at a dose of 0.1 to 0.15
mg/kg biweekly.
Optionally, the antibody is administeted at a dose of 0.1 to 0.3 mg/kg
monthly. Optionally,
the antibody is administered at a dose of 0.2 mg/kg monthly.
[0023] The invention provides methods of treating Alzheimer disease comprising
subcutaneously administering to a patient having the disease an antibody that
specifically
binds to an N-terntinal fragment of Ap, wherein the antibody is administered
at a dose of 1-
40 mg and a frequency of between weekly and monthly. Optionally, the antibody
is
administered at a dose of 5-25 mg. Optionally, the antibody is adn-inistered
at a dose of 2.5-
15 mg. Optionally, the antibody is administered at a dose of 1-12 mg weekly to
biweekly.
Optionally, the antibody is administered at a dose of 2.5-10 mg weekly to
biweekly.
Optionally, the antibody is administered at a dose of 2.5-5 mg weekly.
Optionally, the
antibody is administered at a dose of 4-5 mg weekly. Optionally, the antibody
is
administered at a dose of 7-10 mg biweekly.
[0024] The invention provides methods of treating Alzheimer disease,
comprising
administering to a patient having the disease an antibody that specifically
binds to an N-
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Attorney Docket No. CF50072,
terminal fragment of Ap in a regitne sufficient to maintain a maximum serum
concentration
of the antibody in the patient less than about 28 g antibody/mi serum and
thereby treating
the patient. Optionally, the maximum serum concentration is within a range of
about 4-28 g
antibody/mi serunti Optionally, the maximum serum concentration is within a
range of about
4-18 g antibody/nil serum. Optionally, the average serum concentration of the
antibody in
the patient is below about 7 g antibody/mi serum. Optionally, the average
serum
concentration is within a range of about 2-7 g antibody/mi serum. Optionally,
the average
serum concentration is about 5 g antibody/mi serum. Optionally, the antibody
is
administered intravenously. Optionally, the antibody is administered
subcutaneously.
Optionally, a dose of 0.1-1.0 mg/kg is administered monthly. Optionally, a
dose of 0.5-1.0
mg/kg is administered monthly. Optionally, the antibody is administered at a
frequency
between weekly and monthly. Optionally, the antibody is administered weekly or
biweekly.
Some methods further comprise measuring the concentration of antibody in the
serum and
adjusting the regime if the measured concentration falls outside the range.
Optionally, the
antibody is a humanized antibody. Optionally, the humanized antibody is a
humanized
version of mouse antibody 3D6 expressed by the hybridoma deposited under ATCC
under
No. PTA-5130. Optionally, the humanized antibody is bapineuzumab. Optionally,
the
humanized antibody is a humanized version of mouse antibody lOD5 expressed by
the
hybridoma deposited under ATCC under No. PTA-5129. Optionally, the humanized
antibody is a humanized version of mouse antibody 12A11 expressed by the
hybridoma
deposited under ATCC under No. PTA-7271.
[00251 The invention provides methods of treating Alzheimer disease,
comprising
administering to a patient having the disease an antibody that specificaily
binds to an N-
temtinal fragment of AQ in a regime sufficient to maintain an average serum
concentration of
the antibody in the patient below about 7 g antibody/mi serum and thereby
treat ihe patient.
Optionally, the average serum concentration is within a range of about 2-7 g
antibody/ml
serum. Optionally, the average serum concentration is about 5 g antibody/mi
serum.
Optionally,,the antibody is administered intravenously. Optionally, the
antibody is
administered subcutaneously. Optionally, a dose of 0.1-1.0 mg/kg is
administered monthly.
Optionally, a dose of 0.5-1.0 mg/kg is administered monthly. Optionally, the
antibody is
administered at a frequency between weekly and monthly. Optionally, the
antibody is
administered weekly or biweekly. Some methods further comprise measuring the
concentration of ant'body in the serum and adjusting the regime if the
measured
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concentration falls outside the range. Optionally, the antibody is a humanized
antibody.
Optionally, the humanized antibody is a humanized version of mouse antibody
3D6
expressed by the hybridoma deposited under ATCC under No. PTA-5130.
Optionally, the
humanized antibody is bapineuzumab. Optionally, the humanized antibody is a
humanized
version of mouse antibody lOD5 expressed by the hybridoma deposited under ATCC
under
No. PTA-5129. Optionally, the humanized antibody is a humanized version of
mouse
antibody 12A11 expressed by the hybridoma de.posited under ATCC under No. PTA-
7271.
Brief Descrintion of the Drawines
[0026] Figure 1 shows light and heavy chain amino acid sequence of
Bapineuzumab
predicted from the expression construct DNA sequences.
[0027] Figure 2 shows the average MIvISE change from baseline.
[00281 Figure 3 shows the change from baseline MMSE by cohort at month 4.
[0029] Figure 4 shows the mean, median and standard deviation MMSE change from
baseline at month four.
[0030] Figure 5 shows the results of statistical testing of the MMSE change
from
baseline at month four.
[0031] Figure 6 shows simulated steady state serum concentrations of antibody
from
various subcutaneous regimes assuming bioavailability of 70%.
[00321 Figure 7 shows steady state concentrations of antibody following
subcutaneous administration of AAB-001 at doses of 0.05 or 0.06 mg/kg assuming
70% or
100% bioavailability.
[0033] Figure 8 shows plasma A(3levels following administration of AAB-001 at
doses of 0.15, 0.5, or 1.0 mg/kg.
[00341 Figure 9 shows pharmacokinetic parameters following intravenous
administration of AAB-001 at doses of 0.15, 0.5, 1.0, and 2.0 mg/kg.
[00351 Figure 10 shows the mean serum AAB-001 concentration vs. time profiles
fallowing intravenous administration of AAB-001 at doses of 0.15, 0.5,1.0, and
2.0 mg/kg.
[0036] Figure 11 shows plasma A(i leveis following intravenous administration
of
AAB-001 at doses of 0.15, 0.5, and 1.0 mg/kg.
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Detailed DescriuHon of the Invention
[0037] The application provides preferred dosages and frequencies of
administration
of antibodies to an N-terminal fragment of M to maximize therapeutic benefit
relative to
occurrence of side effects, particularly vasogenic edema. A series of
experiments using
different regimes of the mouse 3136 antibody in transgenic PDAPP mouse have
identified a
steady state average serum concentration of antibody of about 3.7 g/ml for
reducing amyloid
accumulation. The present data provide evidence that doses of 0.5 mg/kg and
1.5 mg/kg
administered intravenously every 13 weeks were effective in inhibiting
cognitive decline in
Alzheimer's patients. These regimes give rise to average serum concentrations
of antibody
that bracket the effective dose of 3.7 g/ml in mice. For example, a dose of
0.5 mg/kg
administered every 13 weeks was found to give an average serum concentration
of about 3
g/ml. A dose of 1 mg/kg administered every 13 weeks was found to give an
average serum
concentration of about 5.5 g/ml and a dose of 2 mg/kg administered every 13
weeks was
predicted to give an average seram concentration of 9.4 g/ml. Thus, the
present data
indicate that the same order of magnitude of serum concentrations of antibody
that are
effective in mice are effective in humans. These data are further supported by
clinical trials
using immunotherapy with full-length A(31-42. In these trials, antibody
responders were
found to have a statistically significant inhibition of cognitive decline. The
antibody
responders had an ELISA titer of antibody of at least 1 in 2200, which
corresponds to a serum
titer of about 1 g/ml.
[0038] The present data also provide evidence that higher doses of antibody
particularly 5 mg/kg achieve no greater (and possibly less) therapeutic
benefit than lower
doses in the 0.5-1.5 mg/kg range but also produce significant side effects,
particularly
vasogenic edema, in some patients. Although practice of the present invention
is not
dependent on an understanding of inechanism, it is believed that the side
effects result from
high maximum concentrations of antibody following its administration.
[0039] In the aggregate, these data indicate that therapeutic benefit can be
obtained
with relatively modest doses of ants'body designed to give similar average
serum
concentrations to the 3.7 g/ml found effective in mice or the values
bracketing this figure
which appear to be effective in humans. The present data also indicate that
for regimes
deiivering equivalent areas under the curve in terms of serum concentration of
antibody as a
CA 02643633 2008-10-17
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function of time that smaller dosages administered more frequently have a
better efficacy to
side effects profile than large dosages administered less frequently because
the former
regimes avoid the spikes in antibody concentration attendant to administering
larger doses in
the latter regimes. In the study in the present egamples, doses were
administered
intravenously every 13 weeks. Although the interval for doses can be reduced
to about
monthly with commensurate reductions in individual doses, fiuther increase in
the frequency
to weekly or biweekly (biweekly) has a high risk of noncompliance due to the
inconvenience
of intravenous administration, which usually requires a visit to an infusion
oenter. However,
weekly or biweekly dosing is practical by subcutaneous administration, which
can easily be
self-administered or administered by a caregiver without medical training.
Subcutaneous
delivery also results in more gradual delivery to the blood avoiding spikes in
concentration.
The bioavailability measured by area under the curve of antibody in plasma of
subcutaneous
delivery relative to intravenous delivery is about 70-100%.
[0040] Thus, preferred regimes for administering antibodies specifically
binding to an
N-terminal fragment of Ap achieves an average serum concentration of
administered
antibody of 1-15 g/ml in a patient. This range brackets the demonstrated
effective
concentrations in mice and humans allowing some margin for error in
measurement and
individual patient variation. The serum concentration can be determined by
actual
measurement or predicted from standard pharmacokinetics (e.g., WinNonline
Version 4Ø1
(Pharsight Corporation, Cary, USA)) based on the amount of antibody
administered,
frequency of administration, route of administration and antibody half-life.
The average
antibody concentration in the serum is preferably within a range of 1-10,1-5
or 2-4 g/ml.
[0041] The present data also provide evidence that administering an antibody
that
specifically binds to an N-temiinal fragment of A43 in a regime sufficient to
maintain a
magimum serum concentration of the antibody in the patient less than about 28
g
antibody/mi serum maximizes therapeutic benefit relative to the occurrence of
possible side
effects, particularly vascular edema. A preferred maaimum serum concentration
is within a
range of about 4-28 g antibody/mi serum. The combination of magimum serum
less than
about 28 g antibody/mi serum and an average serum concentration of the
antibody in the
patient is below about 7 g antibody/mi serum is particularly beneficial. See
Fgures 9 and
10.
[0042] The present data also provide evidence that adminisbering an antibody
that
specifically binds to an N-terminal fragment of Aj3 in a regime sufficient to
maintain an
11
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average serum concentration of the antibody below about 7 g antibody/mi serum
maximizes
therapeutic benefit relative to the occurrence of possible side effects,
particularly vascular
edema. A preferred average wncentration is within a range of about 2-7 g
antibody/ml
serum.
[0043] If the antibody is administered intravenously it is as discussed above
inconvenient to have to administer it more frequently than about monthly.
Preferred doses of
antibody for monthly intravenous administration occur in the range of 0.1-1.0
mg/kg antibody
or preferably 0.5-1.0 mg/kg antibody.
[0044] For more frequent dosing, e.g., from weekly to monthly dosing,
subcutaneous
administration is preferaed. The doses used for subcutaneous dosing are
usually in the range
of 0.1 to 0.6 mg/kg or 0.01-0.35 mg/kg, preferably, 0.05-0.25 mg/kg. For
weekly or
biweekly dosing, the dose is preferably in the range of 0.015-0.2 mg/kg, or
0.05-0.15 mg/kg.
For weekly dosing, the dose is preferably 0.05 to 0.07 mg/kg, e.g., about 0.06
mg/kg. For
biweekly dosing, the dose is preferably 0.1 to 0.15 mg/kg. For monthly dosing,
the dose is
preferably 0.1 to 0.3 mg/kg or about 2 mg/kg. Monthly dosing includes dosing
by the
calendar month or lunar month (i.e., every four weeks).
[0045] Fig. 6 shows simulated steady state serum concentrations of antibody
from
various subcutaneous regimes assuming bioavailability of 70%. It can be seen
that a dose of
0.1 mg/kg gives an average serum concentration very close to the 3.7 g/ml
found effective
in mice with little peak to trough variation. Fig. 7 shows steady state
concentrations of
antibody following subcutaneous administration at doses of 0.05 and 0.06 mg/kg
assuming
70% or 100% bioavailability. It can be seen that at 70% bioavailability, the
0.05 and 0.06
mg/kg doses lie just below and above the 3.7 g/ml dose found effective in
mice with little
peak to trough variation.
[0046] The treatment regime is usually continued so that the average serum
concentrations of antibody described above are maintained for at least six
months or a year,
and sometimes for life. The serum concentration can be measured at any time
during
treatment and the dose and/or frequency of administration increased if the
average
concentration f.alls beneath a target range or the dose and/or frequency
decreased if the
average concentration falls above a target range.
[0047] Although determining optimal plasma concentrations of antibody is
useful in
determining a dosage regime or optimizing dosage in an individual patient, in
practice once
an effective dosage regime in terms of mg/kg or mg and frequency of
administration has
12
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been detemiined, the same dosage regime can be used on many other patients
without the
need for detailed calculation or measurement of patient titers. Thus, any of
the above
mentioned dosages and treatment regimes can be used irrespective whether a
titer is
measured or predicted in a particular patient. For example, one suitable
regime is intravenous
administration at monthly intervals with a dose in range of 0.1-1.0 mg/kg
antibody or
preferably 0.5-1.0 mg/kg antibody. For subcutaneous dosing the dose used is
usually in the
range of 0.01-0.6 mg/kg or 0.01-0.35 mg/kg, preferably, 0.05-0.25 mg/kg. For
weekly or
biweekly dosing, the dose is preferably in the range of 0.015-0.2 mg/kg, or
0.05-0.15 mg/kg.
For weekly dosing, the dose is preferably 0.05 to 0.07 mg/kg, e.g., 0.06
mg/kg. For biweekly
dosing, the dose is preferably 0.1 to 0.15 mg/kg. For monthly dosing, the dose
is preferably
0.1 to 0.3 mg/kg or 2 mg/kg.
[0048] Here as elsewhere in the application, dosages expressed in mg/kg can be
converted to absolute mass dosages by multiplying by the mass of a typical
patient (e.g., 70 ~
or 75 kg) typically rounding to a whole number. Expressed in terms of absolute
mass,
antibodies are usually administered at a dose of 1-40 mg at a frequency of
between weekly
and monthly. Preferred ranges are 5-25 mg or 2.5-15 mg at a frequency of
weekly to
monthly. For weekly to biweekly administration, the dose is often 1-12 mg or
2.5 to 10 mg.
For weekly administration, the dose is often 2.5 to 5 mg or 4-5 mg. For
biweekly
administration, the dose can be 7-10 mg. The mass of antibody packaged for
administration
in unit doses is usually round to whole number, such as 1, 5, 10, 20, 30, 40,
50, 75 or 100 mg.
[0049] The invention provides preferred dosage ranges and monitoring regimes
for
use in treatment of Alzheimer's disease using antibodies to Ap. The methods
are premised in
part on results of a clinical trial described in the Examples. A preferred
dosage range for
antibodies that bind to an N-terminal fragment of Ap is from about 0.5 to 5 mg
antibody per
kg patient body weight. Preferred dosages are less than 5 mg/kg. Dosages from
0.5 to 3
mg/kg, 0.5 to 1.5 mg/kg and 1.5 mg/kg are particularly preferred.
[0050] The invention also provides monitoring regimes that can assess changes
in
symptoms or signs of the patient following treatment. The symptoms or signs
can relate to
Alzheimer's disease itself and/or side effects of the treatment. The dosage of
drug or its
frequency of administration can be adjusted based on the outcome of the
monitoring.
Alternatively or additionally, additional drugs can be administered to treat
any side effects.
For example, monitoring by MRI and/or FLAIR sequence imaging can be used to
detect
PRES or vascular edema or signs oi symptoms thereof. Presence of PRF.S or
vascular edema
13
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Attomey Docket No. CF500722
is an indication that the dosage should be reduced or suspended, or the
interval between
administration of dosages increased. Alternatively, or additaonally, the
patient can be
administered a steroid to treat the PRES or vascular edema. After reducing or
suspending
dosage or increasing the interval between dosages, and/or administering the
steroid,
continued monitoring can indica.te disappearance of PRES or vascular edema, in
which case
the original amount and/or interval of dosing can be resumed. Administration
of the steroid
may or may not be continued as a prophylactic measure at this point. As
another example,
monitoring of blood pressure can indicate development of hypertension. In
analogous
fashion, the dosage can be reduced in amount or suspended, and/or intervals
between dosage
increased and/or an antthypertensive administered. If and when further
monitoring indicates
the hypertension has disappeared, the original amount and/or interval of
dosing can be
resumed. Administration of antihypertensive may or may not be continued as a
prophylactic
measure at this point.
[0051] Prior to describing the invention, it may be helpful to an
understanding thereof
to set forth definitions of certain terms to be used hereinafter.
[0052] The term "immunoglobulin" or "antibody" (used interchangeably herein)
refers to an antigen-binding protein having a basic four-polypeptide chain
structure consisting
of two heavy and two light chains, said chains being stabilized, for example,
by interchain
disulfide bonds, which has the ability to specifically bind antigen. Both
heavy and light
chains are folded into domains. The term "domain" refers to a globular region
of a heavy or
light chain polypeptide comprising peptide loops (e.g., comprising 3 to 4
peptide loops)
stabilized, for example, by P-pleated sheet and/or intrachain disulfide bond.
Domains are
further referred to herein as "constant" or "variable", based on the relative
lack of sequence
variation within the domains of various class members in the case of
a"constant" domain, or
the signi.f'icant variation within the domains of various class members in the
case of a
"variable" domain. "Constant" domains on the light chain are referred to
interchangeably as
"light chain constant regions", "light chain constant domains", "CL" regions
or "CL"
domains). "Constant" domains on the heavy chain are referred to
interchangeably as "heavy
chain constant regions", "heavy chain c.onst.ant domains", "CH" regions or
"CH" domains).
"Variable" domains on the light chain are referred to interchangeably as
"light chain variable
regions", "light chain variable domains ;"VL" regions or "VL" domains).
"Variable"
domains on the heavy chain are referred to interchangeably as "heavy chain
constant
regions", "heavy chain constant domains", "CH" regions or "CH" domains).
14
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Attorney Docket No. CF500722
[0053] The term "region" refers to a part or portion of an antibody chain and
includes
constant or variable domains as defined herein, as well as more discrete parts
or portions of
said domains. For example, light chain variable domains or regions include
"complementarity determining regions" or "CDRs" interspersed among "framework
regions"
or "FRs", as defined herein.
[0054] Immunoglobulins or antibodies can exist in monomeric or polymeric form.
The term "antigen-binding fragment" refers to a polypeptide fragment of an
immunoglobulin
or antibody binds antigen or competes with intact antibody (i.e., with the
intact antibody from
which they were derived) for antigen binding (i.e., specific binding). The
term
"conformation" refers to the ter4iary structure of a protein or polypeptide
(e.g., an antibody,
antibody chain, domain or region thereof). For example, the phrase "light (or
heavy) chain
conformation" refers to the tertiary structare of a light (or heavy) chain
variable region, and
the phrase "antrbody conformation" or "antibody fragment confomiation" refers
to the
tertiary structure of an antibody or fragment thereof.
[0055] "Specific binding" of an anti'body mean that the antibody exhibits
appreciable
affinity for antigen or a preferred epitope and, preferably, does not exhibit
significant cross
reactivity. "Appreciable" or preferred binding include binding with an af6nity
of at least 106,
107,108,109 M"1, or 10101V1'1. Aftinities greater than 107 M"1, preferably
greater than 10g M"1
are more preferred. Values intermediate of those set forth herein are also
intended to be
within the srnope of the present invention and a preferred binding affinity
can be indicated as a
range of affmities, for example,106 to 1010 Nfl, preferably W to 1010 M-',
more preferably
108 to 1010 M4. An antibody that "does not exhibit significant cross
reactivity" is one that
will not appreciably bind to an undesirable entity (e.g., an undesirable
proteinaceous entity).
For example, an antibody that specifically binds to A(3 will appreciably bind
Ap but will not
significantiy react with non-A43 proteins or peptides (e-g., non-Ap proteins
or peptides
included in plaques). An antibody specific for a preferred epitope will, for
example, not
significantly cross react with remote epitopes on the same protein or peptide.
Specific
bindiag can be determined acxording to any art recognized means for
determining such
binding. Preferably, specific binding is determined a :ording to Scatchard
analysis and/or
competitive binding assays.
[0056] Binding fragments are produced by recombinant DNA techniques, or by
enzymatic or chemical cleavage of intact immunoglobulins. Binding fragments
include Fab,
Fab', F(ab)2, Fabc, Fv, single chains, and single-chain antibodies. Other than
"bispecific" or
CA 02643633 2008-10-17
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"bifunctional" immunoglobulins or antibodies, an immunoglobulin or antibody is
understood
to have each of its binding sites identical. A"bispecific" or "bifunctional
antibody" is an
artificial hybrid antibody having two different heavy/light chain pairs and
two different
binding sites. Bi.specific antibodies can be produced by a variety of inethods
including fusion
of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai 8c
Lachmann, Clfn. Exp.
InununoL 79:315-321(1990); Kostelny et al., J. ImmunoL 148,1547-1553 (1992).
[0057] The term "humanized immunoglobulin" or "humanized antibody" refers to
an
immunoglobulin or antibody that includes at least one humanized immunoglobulin
or
antibody chain (i.e., at least one humanized light or heavy chain). 'Ihe term
"humanized
immunoglobulin chain".or "humanized antibody chain" (i.e., a"humanized
immunoglobulin
light chain" or "humanized immunoglobulin heavy chain') refers to an
immunoglobulin or
antibody chain (i.e., a light or heavy chain, respectively) having a variable
region that
includes a variable framework region substantially from a human immunoglobulin
or
antibody and c:omplementarity determining regions (CDRs) (e.g., at least one
CDR,
preferably two CDRs, more preferably three CDRs) substantially from a non-
human
immunoglobulin or antibody, and further includes constant regions (e.g., at
least one constant
region or portion thereof; in the case of a light chain, and preferably three
constant regions in
the c ase of a heavy chain). The term "humanized variable region" (e-g.,
"humanized light
chain variable region" or "humaniz.ed heavy chain variable region') refers to
a variable
region that includes a variable framework region substantially from a human
immunoglobulin
or anribody and complementarity determining regions (CDRs) substantially from
a non-
human immunoglobulin or antibody.
[0058] The phrase "substantially from a human immunoglobulin or antibody" or
"substantially human" means that, when aligned to a human immunoglobulin or
antibody
amino sequence for comparison purposes, the region shares at least 80-90%,
preferably 90-
95%, more preferably 95-99% identity (i.a, local sequence identity) with the
human
fiamework or constant region sequenve, allowing, for example, for conservative
substitutions,
c onsensus sequence substitutions, germline substitutions, backmutations, and
the like. The
introduction of conservative substitutions, consensus sequence substitutions,
germline
substitutions, backmutations, and the like, is often referred to as
"optimization" of a
humanized antibody or chain. The phrase "substantially from a non human
immunoglobulin
or antibody" or "substantially non human" means having an immunoglobulin or
antibody
16
CA 02643633 2008-10-17
Attomey Docket No. CF500722
sequence at least 80-95%, preferably 90-95%, more preferably, 96%, 97%, 98%,
or 99%
identical to that of a non-human organism, e.g., a non-human mammal.
[0059] Accordingly, all regions or residues of a humanized immunoglobulin or
antibody, or of a humanized immunoglobulin or antibody chain, except possibly
the CDRs,
are substantially identical to the corresponding regions or residues of one or
more native
human immunoglobulin sequences. The term "corresponding region" or
"corresponding
residue" refers to a region or residue on a second amino acid or nucleotide
sequence which
occupies the same (i.e., equivalent) position as a region or residue on a
first amino acid or
nucleotide sequence, when the first and second sequences are optimally aligned
for
comparison purposes.
[0060] The terms "humanized immunoglobulin" or "humanized antibody" are not
intended to encompass chimeric immunoglobulins or antibodies, as defined
infra. Although
humanized immunoglobulins or antibodies are chimeric in their constraction
(i.e., comprise
regions from more than one species of protein), they include additional
features (i.e., variable
regions comprising donor CDR residues and acceptor framework residues) not
found in
chimeric immunoglobulins or antibodies, as defined herein.
[0061] The term "chimeric immunoglobulin" or antibody refers to an
immunoglobulin or antibody whose variable regions derive from a first species
and whose
constant regions derive from a second species. Chimeric immunoglobulins or
antibodies can
be constructed, for example by genetic engineering, from immunoglobulin gene
segments
belonging to different species.
[0062] An "antigen" is an entity (e.g., a protenaceous entity or peptide) to
which an
antibody specifically binds.
[0063] The term "epitope" or "antigenic determinant" refers to a site on an
antigen to
which an immunoglobulin or antibody (or antigen binding fragment thereof)
specifically
binds. Epitopes can be formed both from contiguous amino acids or
noncontiguous amino
acids juxtaposed by tertiary folding of a protein. Epitopes form.ed from
contiguous amino
acids are typically retained on exposure to denaturing solvents whereas
epitopes formed by
tertiary folding are typically lost on treatment with denaturing solvents. An
epitope typically
includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in
a unique spatial
conformation. Methods of detemiining spatial conformation of epitopes include,
for
example, x-ray crystallography and 2-dimensional nuclear magnetic resonance.
See, e.g.,
17
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Attomey Docket No. CF500722
Epitope Mapping ProtocolS in Methods in Molecular Biology, Vol. 66, G. E.
Morris, Ed.
(1996).
[0064] Antibodies that recognize the same epitope can be identified in a
simple
immunoassay showing the ability of one antibody to block the binding of
another antibody to
a target antigen, i.e., a competitive binding assay. Competitive binding is
determined in an
assay in which the immunoglobulin under test inhibits specific binding of a
refeience
antibody to a common antigen, such as A(3. Numerous types of competitive
binding assays
are known, for example: solid phase d'uect or indirect radioimmunoassay (RIA),
solid phase
direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see
Stahli et al.,
Methods in Enrymology 9:242 (1983)); solid phase d'uect biotin-avidin EIA (see
Kirkland et
al., J. Immunol.137:3614 (1986)); solid phase direct labeled assay, solid
phase direct labeled
sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring
Harbor Press (1988)); solid phase direct label RIA using I-125 label (see
Morel et al., Mol.
Immunol. 25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et al.,
Virology
176:546 (1990)); and direct labeled RIA. (Moldenhauer et al., Scand J.
Immunol. 32:77
(1990)). Typically, such an assay involves the use of purified antigen bound
to a solid
surface or cells bearing either of these, an unlabeled test immunoglobulin and
a labeled
reference immunoglobulin. Competitive inhibition is measured by determining
the amount of
label bound to the solid surface or cells in the presence of the test
immunoglobulin. Usually
the test immunoglobulin is present in excess. Usually, when a competing
antibody is present
in excess, it will inhibit specific binding of a reference antibody to a
common antigen by at
least 50-55%, 55-60%, 60-65%, 65-70% 70-75% or more.
[0065] An epitope is also recognized by immunologic cells, for example, B
cells
and/or T cells. Cellular recognition of an epitope can be detemiined by in
vitro assays that
measure antigen-dependent proliferation, as determined by 3H-thymidine
incorporation, by
cytokine secretion, by antibody secretion, or by antigen-dependent killing
(cytotoxic T
lymphocyte assay).
[0066] Exemplary epitopes or antigenic determinants can be found within the
human
amyloid precursor protein (APP), but are preferably found within the A(3
peptide of APP.
Multiple isoforms of APP exist, for example APP69s, APP7sI and APP77O. Amino
acids
within APP are assigned numbers according to the sequence of the APP770
isoform (see e.g.,
GenBank Accession No. P05067, also set forth as SEO ID N0:38). A(3 (also
referred to
herein as beta amyloid peptide and A-beta) peptide is a-4-kDa internal
fragment of 39-43
18
CA 02643633 2008-10-17
Attomey Docket No. CF500722
amino acids of APP (A(339, AP40, A041, A042 and A(343). A(340, for example,
consists of
residues 672-711 of APP and A(342 consists of residues 673-713 of APP. As a
result of
proteolytic processing of APP by different secretase enzymes iv vivo or in
situ, A(3 is found in
both a"short form", 40 amino acids in length, and a"long form", ranging from
42-43 amino
acids in length. Prefened epitopes or antigenic determinants, as described
herein, are located
within the N-terminus of the AR peptide and include residues within amino
acids 1-10 of A(3,
preferably from residues 1-3, 1-4, 1-5, 1-6, 1-7 or 3-7 of A(342. Additional
referred epitopes
or antigenic determinants include residues 2-4, 5, 6, 7 or 8 of A(3, residues
3-5, 6, 7, 8 or 9 of
A(3, or residues 4-7, 8, 9 or 10 of A042.
[00671 The term "amyloidogenic disease" includes any disease associated with
(or
caused by) the formation or deposition of insoluble amyloid fibriLs. Exemplary
amyloidogenic diseases include, but are not limited to systemic amyloidosis,
Alzheimer's
disease, mature onset diabetes, Parkinson's disease, Huntington's disease,
fronto-temporal
dementia, and the prion-related transmissible spongiform encephalopathies
(kuru and
Creutzfeldt-Jacob disease in humans and scrapie and BSE in sheep and ca.ttle,
respectively).
Different amyloidogenic diseases are defined or characterized by the nature of
the
polypeptide component of the fibrils deposited. For example, in subjects or
patients having
Alzheimer's disease, 0-amyloid protein (e.g., wild-type, variant, or truncated
0-amyloid
protein) is the characterizing polypeptide component of the amyloid deposit.
Accordingly,
Alzheimer's disease is an example of a"disease characterized by deposits of
Ap" or a
"disease associated with deposits of A(3", e.g., in the brain of a subject or
patient. The terms
"(3-amyloid protein", "(3-amyloid peptide", "(3-amyloid", "A(3" and "A(3
peptide" are used
interchangeably herein.
[0068] The term "effective dose" or "effective dosage" is defined as an amount
sufficient to achieve or at least partially achieve the desired effect. The
term "therapeutically
effective dose" is defined as an amount sufficient to cure or at least
partially arrest the disease
and its complications in a patient already suffering from the disease. Amounts
effective for
thi.s use will depend upon the severity of the infection and the general state
of the patient's
own immune system.
[00691 The term "patient" includes human and other mammalian subjects that
receive
either prophylactic or therapeutic tr.eatment.
19
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Attorney Docket No. CF500722
[0070] "Soluble" or "dissociated" A(3 refers to non-aggregating or
disaggregated AR
polypeptide. "Insoluble" A43 refers to aggregating A(3 polypeptide, for
example, A(3 held
together by noncovalent bonds. A(3 (e.g., A(342) is believed to aggregate, at
least in part, due
to the presence of hydrophobic residues at the C-terminus of the peptide (part
of the
transmembrane domain of APP). One method to prepare soluble A(3 is to dissolve
lyophilized peptide in neat DMSO with sonication. The resulting solution is
centrifuged to
remove any insoluble particulates.
[0071] The term "effector function" refers to an activity that resides in the
Fc region
of an antibody (e.g., an IgG antibody) and includes, for example, the ability
of the antibody to
bind effector molecules such as complement and/or Fc receptors, which can
control several
inunune functions of the antibody such as effector cell activity, lysis,
complement-mediated
activity, antibody clearance, and antibody half-life.
[0072] The term "effector molecule" refers to a molecule that is capable of
binding to
the Fc region of an antibody (e.g., an IgG antibody) including, but not
limited to, a
complement protein or a Fc receptor.
[0073] The term "effector cell" refers to a cell capable of binding to the Fc
portion of
an antibody (e.g., an IgG antibody) typically via an Fc receptor expressed on
the surface of
the effector cell including, but not limited to, lymphocytes, e.g., antigen
presenting cells and
T cells.
[0074] The term "Fc region" refers to a C-terminal region of an IgG antibody,
in
particular, the C-terminal region of the heavy chain(s) of said IgG antibody.
Although the
boundaries of the Fc region of an IgG heavy chain can vary slightly, a Fc
region is typically
defined as spanning from about amino acid residue Cys226 to the carbogyl-
terminus of an
IgG heavy chain(s).
[0075] The term "Kabat numbering" unless otherwise stated, is defined as the
numbering of the residues in, e.g., an IgG heavy chain antibody using the EU
indeg as in
Kabat et al. (Sequences of Proteins of Immunological Interest, 5th Ed. Public
Heaith Service,
National Institutes of Health, Bethesda, Md. (1991)), expressly incorporated
herein by
reference.
[00761 The term "Fc receptor" or "FcR" refers to a receptor that binds to the
Fc
region of an antibody. Typical Fc receptors which bind to an Fc region of an
antibody (e-g.,
an IgG antibody) include, but are not limited to, receptors of the FcyRI,
FcrRII, and FcyRIII
siibclasses, including allelic variants and altematively spliced forms of
these receptors. Fc
CA 02643633 2008-10-17
Attomey Docket No. CF500722
receptors are reviewed in Ravetch and Kinet, Annu. Rev. Immuno19:457-92
(1991); Capel et
al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.
126:330-41
(1995).
Inununological and Therapeutic Rea ents
[0077] Immunological and therapeutic reagents of the invention comprise or
consist
of immunogens or antibodies, or functional or antigen binding fragments
thereof, as defined
herein. The basic antibody struchuml unit is known to comprise a tetramer of
subunits. Each
tetramer is composed of two identical pairs of polypeptide chains, each pair
having one
"light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-
tenninal
portion of each chain includes a variable region of about 100 to 110 or more
amino acids
primarily responsible for antigen recognition. The carboxy-temiinal portion of
each chain
defines a constant region primarily responsible for effector fundion.
Antibodies
[0078] The term "antibody" as used herein refers to immunoglobulin molecules
and
immunologicaily adive portions of immunoglobulin moleeules, i.e, molecules
that contain
an antigen binding site which specifically binds (recognizes) an antigen.
Ezamples of
immunologically active portions of immunoglobulin molecules include F(ab) and
F(ab')2
fragments which can be generated by treating the antibody with an enzyme such
as pepsin or
produced by art-recognized recombinant engineering techniques. Aspects of the
invention
also relevant for the stabilization of antibodies include, for egample,
polyclonal and
monoclonal antibodies that bind an antigen, for eaample a therapeutic target
antigen, such as,
Ap. The term "monoclonal anh'body" or "monoclonal antibody oomposition", as
used herein,
refers to a population of antibody molecules that contain only one species of
an antigen
binding site capable of recognizing and binding to a particular epitope of a
target antigen, for
example, an epitope(s) of Ap. A monoclonal anfibody composition thus typically
displays a
single binding specificity and affinity for a particular target antigen with
which it
immunoreacts.
PolYclonal Antibodies
[0079] Polyclonal ants'bodies can be prepared as described above by immunizing
a
suitable subject with an immunogen. The antibody titer in the immunized
subject can be
monitored over time by standard techniques, such as with an enzyme linked
immunosorbent
21
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Attorney Docket No. CF500722
assay (ELISA) using immobilized target antigen. If desired, the antibody
molecules direcxed
against the target antigen can be isolated from the mammal (e.g., from the
blood) and further
purified by well lmown techniques, such as protein A Sepharose chromatography
to obtain
the antibody, e-g., IgG, fraction. At an appropriate tune after immunization,
e.g., when the
anti-antigen antibody titers are highest, antibody-producing cells can be
obtained from the
subject and used to prepare monoclonal antibodies by standard techniques, such
as the
hybridoma technique originally described by Kohler and Milstein (1975) Nature
256:495-
497) (see also, Brown et al. (1981) J. Immunol. 127:539-46; Brown et al.
(1980) J. Biol.
Chem.255:4980-83; Yeh et al. (1976) Proc. Natl: Acad Sci. USA 76:2927-31; and
Yeh et al.
(1982) Int. J. Cancer 29:269-75). For the preparation of chimeric polyclonal
antibodies, see
Buechler et al: U.S. Patent No. 6,420,113.
Monoclonal Aritibodies
[ooso] Any of the many well known protocols used for fusing lymphocytes and
immortalized cell lines can be applied for the purpose of generating a
monoclonal antibody
(see, e.g., G. Galfre et aL (1977) Nature 26655052; Gefter et al: Somatic Cell
Genet., cited
supra; Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal
Antibodies, cited supra).
Moreover, the ordinarily skilled worker will appreciate that there are many
variations of such
methods which aiso would be useful. Typically, the immortal cell line (e.g., a
myeloma cell
line) is derived from the same mammalian species as the lymphocytes. For
example, murine
hybridomas can be made by fusing lymphocytes from a mouse immunized with an
immunogenic preparation of the present invention with an immortalized mouse
cell line.
Preferred immortal cell lines are mouse myeloma cell lines that are sensitive
to culture
medium containing hypoganthine, aminopterin and thymidine ("HAT medium"). Any
of a
number of myeloma cell lines can be used as a fusion partner according to
standard
techniques, e.g., the P3-NSl/1-Ag4-1, P3-a63 Ag8.653 or Sp2/O-Ag14 myeloma
lines.
These myeloma lines are available from ATCC. Typically, HAT-sensitive mouse
myeloma
cells are fused to mouse splenocytes using polyethylene glycol ("PBG").
Hybridoma cells
resulting from the fusion are then selected ueing HAT medium, which ldll.s
unfused and
unproductively fused myeloma cells (unfused splenocytes die after several days
because they
are not transformed). Hybridoma cells producing a monoclonal antibody of the
invention are
detectei by screening the hybridoma culture supernatants for antz'bodies that
bind a target
antigen, e.g., AP, using a standard ELISA assay.
22
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Recombinant Antibodies
[0081] Altemative to preparing monoclonal antibody-secreting hybridomas, a
monoclonal antibody can be identified and isolated by screening a recombinant
combinatorial
immunoglobulin library (e.g., an antibody phage display library) with a target
antigen to
thereby isolate immunoglobulin library members that bind the target antigen.
Kits for
generating and screening phage display libraries are commercially available
(e.g., the
Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the
Stratagene
SurfZAPTM Phage Display Kit, Catalog No. 240612). Additionally, examples of
inethods and
reagents partlcularly amenable for use in generating and screening antibody
display library
can be found in, for example, Ladner et aL U.S. Patent No. 5,223,409; Kang et
aL PCT
International Publication No. WO 92/18619; Dower et al. PCT Intemational
Publication No.
WO 91/17271; Winter et al. PCT International Publication WO 92/20791; Markland
et al.
PCT Intemational Publication No. WO 92/15679; Breitling et al. PCT
Intemational
Publication WO 93/01288; McCafferty et al. PCT Intemational Publication No. WO
92/01047; Garrard et al. PCT International Publication No. WO 92/09690; Ladner
et al. PCT
Intemational Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology
9:1370-
1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989)
Science
246:1275-1281; Griffiths et al (1993) E.MBO J 12:725-734; Hawkins et al.
(1992) J. Mol.
Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram et aL
(1992) Proc. Natl.
Acad. Sci. USA 89:3576-3580; Garrad et at (1991) Bio/Technology 9:1373-1377;
Hoogenboom et al. (1991) Nuc. Acid Res. 19:4133-4137; Barbas et al. (1991)
Proc. NatL
Acad Sci. USA 88:7978-7982; and McCafferry et al. Nature (1990) 348:552-554.
Chimeric and Humanized Antibodies
[0082] Additionally, recombinant antibodies, such as chimeric and humanized
monoclonal antibodies, comprising both human and non-human porrions, which can
be made
using standard recombinant DNA techniques, are within the scope of the
invention.
[0083] The term "humanized immunoglobulin" or "humanized antibody" refers to
an
immunoglobulin or antibody that includes at least one humanized immunoglobulin
or
antibody chain (i.e., at least one humanized light or heavy chain). The term
"humanized
immunoglobulin chain" or "humanized antibody chain" (i.e., a"humanized
immunoglobulin
23
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Attomey Docket No. CF500722
light chain" or "humanized immunoglobulin heavy chain") refers to an
immunoglobulin or
antibody chain (i.e., a light or heavy chain, respectively) having a variable
region that
includes a variable framework region substantially from a human immunoglobulin
or
antibody and complementarity determining regions (CDRs) (e.g., at least one
CDR,
preferably two CDRs, more preferably three CDRs) substantially from a non-
human
inununoglobulin or antibody, and further includes constant regions (e.g., at
least one constant
region or portion thereof, in the case of a light chain, and three constant
regions in the case of
a heavy chain). The term "humanized variable region" (e.g., "humanized light
chain variable
region" or "humanized heavy chain variable region') refers to a variable
region that includes
a variable framework region substantially from a human immunoglobulin or
antibody and
complementarity determining regions (CDRs) substantially from a non-human
immunoglobulin or antibody.
[0084] The phrase "substantially from a human immunoglobulin or antibody" or
"substantially human" means that, when aligned to a human immunoglobulin or
antibody
amino sequence for comparison purposes, the region shares at least 80-90%, 90-
95%, or 95-
99% identity (i.e., local sequence identity) with the human framework or
constant region
sequence, allowing, for example, for conservative substitutions, consensus
sequence
substitutions, germline substitutions, backmutations, and the like. The
introduction of
conservative substitutions, consensus sequence substitutions, gemiline
substitutions,
backmutations, and the like, is often referred to as "optimization" of a
humanized antibody or
chain. The phrase "substantially from a non-human immunoglobulin or antibody"
or
"substantially non-human" means having an immunoglobulin or antibody sequence
at least
80-95%, preferably at least 90-95%, more preferably, 96%, 97%, 98%, or 99%
identical to
that of a non-human organism, e.g., a non-human mammal.
[0085] Accordingly, all regions or residues of a humanized immunoglobulin or
antibody, or of a humanized immunoglobulin or antibody chain, except the CDRs,
are
substantially identical to the corresponding regions or residues of one or
more native human
immunoglobulin sequences. The tenn "corresponding region" or "corresponding
residue"
refers to a region or residue on a second amino acid or nucleotide sequence
which occupies
the same (i.e., equivalent) position as a region or residue on a first amino
acid or nucleotide
sequence, when the first and second sequences are optimally aligned for
comparison
purposes.
24
CA 02643633 2008-10-17
Attorney Docket No. CF500722
[00861 The term "significant identity" means that two polypeptide sequences,
when
optimally aligned, such as by the programs GAP or BESTFIT using default gap
weights,
share at least 50-60% sequence identity, preferably at least 60-70% sequence
identity, more
preferably at least 70-80% sequence identity, more preferably at least 80-90%
sequence
identity, even more preferably at least 90-95% sequence identity, and even
more preferably at
least 95% sequence identity or more (e.g., 99% sequence identity or more). The
tenn
"substantial identity" means that two polypeptide sequences, when optimally
aligned, such as
by the programs GAP or BBSTFIT using default gap weights, share at least 80-
90% sequence
identity, preferably at least 90-95% sequence identity, and more preferably at
least 95%
sequence identity or more (e.g., 99% sequence identity or more). For sequence
comparison,
typically one sequence acas as a reference sequence, to which test sequences
are compared.
When using a sequence comparison algorithm, test and reference sequences are
input into a
computer, subsequence coordinates are designated, if necessary, and sequence
algorithm
program parameters are designated. The sequence comparison algorithm then
calculates the
percent sequence identity for the test sequence(s) relative to the reference
sequence, based on
the designated program parameters.
[00871 Optimal alignment of sequences for comparison can be conducted, ag., by
the
local homology algorithm of Smith & Waterman, Adv. Appl Math. 2:482 (1981), by
the
homology alignment algorithm of Needleman & Wunsch, J. Mol. BioL 48:443
(1970), by the
search for similarity method of Pearson & Lipman, Proc. Nat'1. Acad Sci. iTSA
85:2444
(1988), by computerized implementations of these algorithms (GAP, BESTFIT,
FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group,
575
Science Dr., Madison, WI), or by visual inspection (see generally Ausubel et
al., Current
Protocols in Molecular Biology). One example of algorithm that is suitable for
detennining
percent sequence identity and sequence similarity is the BLAST algorithm,
which is
described in Altschul et aL, J. MoL Biol. 215:403 (1990). Software for
performing BI.AST
anaiyses is publicdy available through the National Center for Biotechnology
Information
(publicly acce.ssible through the National Institutes of Health NCBI internet
server).
Typically, default program parameters can be used to perform the sequence
comparison,
although customized parameters can also be used. For amino acid sequences, the
BLASTP
program uses as defaults a word length (W) of 3, an expectation (E) of 10, and
the
BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. iTS4
89:10915
(1989)).
CA 02643633 2008-10-17
Attorney Docket No. CF500722
[0088] Preferably, residue positions which are not identical differ by
conservative
amino acid substitutions. For purposes of classifying amino acids
substitutions as
conservative or nonconservative, amino acids are grouped as follows: Group
I(hydrophobic
sidechains): leu, 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.
[0089] Preferably, humanized immunoglobulins or antibodies bind antigen with
an
aff'inity that is within a factor of three, four, or five of that of the
corresponding non-
humanized antibody. For example, if the nonhumanized antibody has a binding
affinity of
10-9 M, humanized antibodies will have a binding affinity of at least 3 x 10$
M, 4 x 10$ M, 5
x 10$ M, or 10-9 M. When describing the binding properties of an
immunoglobulin or
antibody chain, the chain can be described based on its ability to "direct
antigen (e.g., AP)
binding". A chain is said to "direct antigen binding" when it confers upon an
intact
immunoglobulin or antibody (or antigen binding fragment thereof) a specific
binding
property or binding affinity. A mutation (e.g., a backmutation) is said to
substantially affect
the ability of a heavy or light chain to direct antigen binding if it affects
(e.g., decreases) the
binding affinity of an intact immunoglobuli.n or antibody (or antigen binding
fragment
thereof) comprising said chain by at least an order of magnitude compared to
that of the
antibody (or antigen binding fragment thereof) comprising an equivalent chain
lacking said
mutation. A mutation "does not substantially affect (e.g., decrease) the
ability of a chain to
direct antigen binding" if it affects (e.g., decreases) the binding affinity
of an intact
immunoglobulin or antibody (or antigen binding fragment thereof) c:omprising
said chain by
only a factor of two, three, or four of that of the antibody (or antigen
binding fragment
thereof) comprising an equivalent chain lacking said mutation.
[0090] The term "chimeric immunoglobulin" or antibody refers to an
immunoglobulin or antibody whose variable regions derive from a first species
and whose
constant regions derive from a second species. Chimeric immunoglobulins or
antibodies can
be constructed, for example by genetic engineering, from immunoglobutin gene
segments
belonging to different species. The terms "humanized immunoglobulin" or
"humanized
antibody" are not intended to encompass chimeric immunoglobulins or
antibodies, as defined
26
CA 02643633 2008-10-17
Attomey Docket No. CF500722
infra. Although humanized immunoglobulins or antibodies are chimeric in their
construction
(i.e., comprise regions from more than one species of protein), they include
additional
features (i.e., variable regions comprising donor CDR residues and acceptor
framework
residues) not found in chimeric immunoglobulins or antibodies, as defined
herein.
[0091] Such chimeric and humanized monoclonal antibodies can be produced by
recombinant DNA techniques known in the art, for example using methods
described in
Robinson et al. International Applica.tion No. PCT/US86/02269; Akira, et al.
European
Patent Application 184,187; Taniguchi, M., European Patent Application
171,496; Morrison
et al. European Patent Application 173,494; Neuberger et al. PCT Intemational
Publication
No. WO 86/01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al.
European Patent
Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al.
(1987) Proc.
Natl. Acad Sci. USA 84:3439-3443; Liu et aL (1987) J. Immunol. 139:3521-3526;
Sun et al.
(1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987) Canc.
Res. 47:999-
1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl.
Cancer Inst.
80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al. (1986)
BioTechniques 4:214; Winter U.S. Patent 5,225,539; Jones et al. (1986) Nature
321:552-525;
Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J.
Immunol. 141:4053-
4060.
Human Antibodies from Transgenic Animals and Phage Disalav
[0092] Alternatively, it i.s now possible to produce transgenic animals (e.g.,
mice) that
are capable, upon immunization, of producing a full repertoire of human
antibodies in the
absence of endogenous immunoglobulin production. For example, it has been
described that
the homozygous deletion of the antibody heavy-chain joining region (JH) gene
in chimeric
and germ-line mutant mice results in complete inhibition of endogenous
antibody production.
Transfer of the human germ-line immunoglobulin gene array in such germ-line
mutant mice
results in the production of human antibodies upon antigen challenge. See,
e.g., U.S. Patent
Nos. 6,150,584; 6,114,598; and'5,770,429.
[0093] Fully humuan antibodies can also be derived from phage-display
libraries
(Fioogenboom et aL, J. Mol. Biol., 227:381(1991); Marks et af, J. Mol. Biol.,
222:581-597
(1991)). Chimeric polyclonal antibodies can also be obtained from phage
display libraries
(Buechler et aL U.S. Patent No. 6,420,113).
27
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Attomey Docket No. CF500722
Bisneciic Antibodies Antibody Fusion Polypeptides, and Single-Chain
Antibodies
[0094] Bispecific antibodies (BsAbs) are antibodies that have binding
specificities for
at least two different epitopes. Such antibodies can be derived from full
length antibodies or
antibody fragments (e.g. F(ab)'2 bispecific antibodies). Methods for making
bispecific
antibodies are known in the art. Traditional production of full length
bispecific antibodies is
based on the coexpression of two immunoglobulin heavy chain-light chain pairs,
where the
two chains have different specificities (Millstein et al., Nature, 305:537-539
(1983)).
Because of the random assortment of immunoglobulin heavy and light chains,
these
hybridomas (quadromas) produce a potential mixture of different antibody
molecules (see,
WO 93/08829 and in Traunecker et aL, EMBO J., 10:3655-3659 (1991)).
[0095] Bispecific antibodies also include cross-linked or "heteroconjugate"
antibodies. For example, one of the antibodies in the heteroconjugate can be
coupled to
avidin, the other to biotin or other payload. Heteroconjugate antibodies may
be made using
any convenient cross-linking methods. Suitable cross-linking agents are well
known in the
art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of
cross-linking
techniques.
[0096] In yet another aspect, the antibody can be fused, chemically or
genetically, to a
payload such as a reactive, detectable, or functional moiety, for example, an
immunotoxin to
produce an antibody fusion polypeptide. Such payloads include, for example,
immunotoxins,
chemotherapeutics, and radioisotopes, all of which are well-known in the art.
[0097] Single chain antibodies are also suitable for stabilization according
to the
invention. The fragments comprise a heavy-chain variable domain (VH) connected
to a light-
chain variable domain (VL) with a linker, which allows each variable region to
interface with
each other and recreate the antigen binding pocket of the parent antibody from
which the VL
and VH regions are derived. See Gruber et aL, J. Immuno1.,152:5368 (1994).
NANOBODIES
[0098] Nanobodies are anti'body-derived therapeutic proteins that contain the
properties of naturally-occurring heavy chain antibodies. Nanobodies can
function as a
single, relatively small, functional antigen-binding structural unit, domain
or protein. The
Nanobody7m technology (Ablynx N.V.) was originally developed following the
discovery
28
CA 02643633 2008-10-17
Attorney Docket No. CF500722
that camelidae (camels and llamas) possess fuily functional antibodies that
lack light chains.
These heavy-chain antibodies contain a single variable domain (VHFI) and two
constant
domains (CH2 and CH3). VHIH is used to distinguish them from the heavy chain
variable
domains that are present in conventional 4-chain antibodies (which are
referred to as "VH
domains"). The cloned and isolated VHH domain is a stable polypeptide
harboring the full
antigen-binding capacity of the original heavy-chain antibody. VHH domains and
nanobodies can also be engineered into multivalent and multispecific formats.
Nanobodies
with an amino acid sequence that corresponds to the amino acid sequence of a
naturally
occurring VHH domain can be humanized, i.e. by replacing one or more amino
acid residues
in the amino acid sequence of the naturally occurring VHi sequence (and in
particular in the
framework sequences) by one or more of the amino acid residues that occur at
the
corresponding position(s) in a VH domain from a conventional4-chain antibody
from a
human being. For details, see e.g., US 20050130266, US 20040253638,
WO/2006/040153,
US 20050214857, WO/2006/079372, or WO/2006/122825, each of which is
incorporated
herein by reference for all purposes. Antibodies against A(i can also be
produced via the
NanobodyTm methods.
[0099] It is understood that any of the foregoing polypeptide molecules, alone
or in
combination, are suitable for preparation as stabilized fonnulations according
to the
invention.
Anti A#Antibodies
[00100] Generally, the formulations of the present invention include a variety
of
antibodies for treating amyloidogenic diseases, in particular, Alzheimer's
Disease, by
targeting A(3 peptide.
[00101] The tenns "Ap antibody", "anti Ap antibody" and "anti A(3" are used
interchangeably herein to refer to an antibody that binds to one or more
epitopes or antigenic
determinants of the human amyloid precursor protein (APP), Ap protein, or
both. Exemplary
epitopes or antigenic determinants can be found within APP, but are preferably
found within
the A(3 peptide of APP. Multiple isoforms of APP egist, for egample APP'05,
APPMi and
APP70. Amino acids within APP are assigned numbers according to the sequence
of the
APP70 isoform (see e.g., GenBank.Accession No. P05067). Examples of specific
isotypes of
APP which are currently known to egist in humans are the 695 amino acid
polypeptide
descn'bed by Kang et. al. (1987) Nature 325:733-736 which is designated as the
"normal"
29
CA 02643633 2008-10-17
Attorney Docket No. CF500722
APP; the 751 amino acid polypeptide described by Ponte et al. (1988) Nature
331:525-527
(1988) and Tanzi et al. (1988) Nature 331:528-530; and the 770-amino acid
polypeptide
described by Kitaguchi ez al. (1988) Nature 331:530-532. As a result of
proteolytic
prooessing of APP by different secretase enzymes in vivov or in situ, M is
found in both a
"short form", 40 amino acids in length, and a"long form", ranging from 42-43
amino acids in
length. The short form, AP40, consists of residues 672-711 of APP. The long
form, e.g.,
A(342 or A043, consists of residues 672-713 or 672-714, respectively. Part of
the hydrophobic
domain of APP is found at the carboxy end of AR, and may account for the
ability of Ap to
aggregate, particularly ,in the case of the long form. A(3 peptide can be
found in, or purified
from, the body fluids of humans and other mammals, e.g. cerebrospinal fluid,
including both
normal individuals and individuals suffering from amyloidogenic disorders.
[00102] The tenvs "P-amyloid protein", "P-amyloid peptide", "p-amyloid", "Ap"
and
"Ap Peptide" are used interchangeably herein. Ap peptide (eg., A039, A040,
A041, A(342
and A(i43) is a-4-kDa intemal fragment of 39-43 amino acids of APP. A(340, for
example,
consists of residues 672-711 of APP and AP42 consists of residues 672-713 of
APP. Ap
peptides include peptides resulting from secretase cleavage of APP and
synthetic peptides
having the same or essentially the same sequence as the cleavage products. A(3
peptides can
be derived from a variety of sources, for example, tissues, cell lines, or
body fluids (e.g. sera
or cerebrospinal fluid). For example, an AR can be derived from APP-expressing
cells such
as Chinese hamster ovary (CHO) cells stably transfected with APP717v~F, as
described, for
example, in Walsh et aL, (2002), Nature, 416, pp 535-539. An A.p preparation
can be
derived from tissue sources using methods previously described (see, e-g.,
Johnson-Wood et
al., (1997), Proc. Natl. Acad Sci. USA 94:1550). Altematively, A(3 peptides
can be
synthesized using methods which are well known to those in the art. See, for
example, Fields
et a1, Synthetic Peptides: A User's Guide, ed. Grant, W.H. Freeman & Co., New
York, NY,
1992, p 77). Hence, peptides can be synthesized using the automated Merrifield
techniques
of solid phase synthesis with the a-amino group protected by either t-Boc or F-
moc chemistry
using side chain protected amino acids on, for example, an Applied Biosystems
Peptide
Synthesizer Mode1430A or 431. Longer peptide antigens can be synthesized using
well
known recombinant DNA techniques. For example, a polynucleotide encoding the
peptide or
fusion peptide can be synthesized or molecalarly cloned and inserted in a
suitable expression
vector for the transfection and heterologous expression by a suitable host
cell. Ap peptide
CA 02643633 2008-10-17
Attomey Docket No. CF500722
also refers to related A(3 sequences that results from mutations in the AR
region of the normal
gene.
[00103] The terms "Ap-derived diffusible ligand" and "ADDL" are small, soluble
A042 oligomers, predominantly trimers and tetramers but also higher-onier
species (See e.g.,
Lambert, M. P. et al. (1998) Proc. Natl. Acad. Sci. USA, vol. 95, pp. 6448-
6453; Chromy, B.
A. et al. (2000) Soc. Neurosci. Abstr., vol. 26, p.1284, WO 2004/031400, each
of which is
incorporated by reference in its entirety for all purposes.)
[00104] The term "anti-ADDL anhbody" refers to an antibody that has been
generated
and selected for the ab~ity to bind ADDLs spec'if'icaIly, without binding to
A(3 monomer or
amyloid fibrils. See e.g.; WO 2004/031400, incorporated by reference in its
entirety for all
purposes.
[00105] The term "epitope" or "antigenic determinant" refers to a site on an
antigen to
which an immunoglobulin or antibody (or antigen binding fragment thereof)
specifically
binds. Exemplary epitopes or antigenic det.erminants to wluch an 4 antibody
binds can be
found within the human amyloid precarsor protein (APP), but are preferably
found within the
Ap peptide of APP. Exemplary epitopes or antigenic determinants within Ap are
located
within the N-terminus, central region, or C-tenninus of 4. An "N-terminal
epitope", is an
epitope or antigenic detenminant located within the N-tenaiinus of the Aft
peptide. Exemplary
N-tenninal epitopes include residues within amino acids 1-10 or 1-12 of Ap,
preferably from
residues 1-3, 1-4, 1-5, 1-6, 1-7, 2-6, 2-7, 3-6, or 3-7 of 442. Other
exemplary N-ternninal
epitopes start at residues 1-3 and end at residues 7-11 of Ap. Additional
exemplary N-
terminal epitopes include residues 2-4, 5, 6, 7 or 8 of Ap, residues 3-5, 6,
7, 8 or 9 of AP, or
residues 4-7, 8, 9 or 10 of A042. "Central epitopes" are epitopes or antigenic
determinants
comprising residues located within the central or mid-portion of the AS
peptide. Exemplary
centtal epitopes include residues within amino acids 13-28 of A¾, preferably
fcom residues
14-27, 15-26, 16-25, 17-24, 18-23, or 19-22 of A(3. Other exemplary central
epitopes include
residues within amino acids 16-24, 16-23, 16-22, 16-21, 18-21, 19-21, 19-22,
19-23, or 19-24
of A(3. "C-termirial" epitopes or antigenic determinants are located within
the C-terminus of
the A(3 peptide and include residues within amino acids 33-40, 33-41, or 33-42
of M. "C-
terminal epitopes" are epitopes or antigenic determinants comprising residues
located within
the C-terminus of the Ap peptide (e.g., within about amino acids 30-40 or 30-
42 of Ap.
31
CA 02643633 2008-10-17
Attorney Docket No. CF500722
Additional exemplary C-terminal epitopes or antigenic determinants include
residues 33-40
or 33-42 of AR.
[00106} When an antibody is said to bind to an epitope within specified
residues, such
as AR 3-7, what is meant is that the antibody specificalIy binds to a
polypeptide containing
the specified residues (i.e., A(3 3-7 in this an example). Such an antibody
does not
necessarily contact every residue within A(3 3-7. Nor does every single amino
acid
substitution or deletion within A(3 3-7 necessarily significantly affect
binding af6nity.
[001071 In various aspects, an Aj3 antibody i.s end-specific. As used herein,
the term
"end-specific" refers tcZ an antibody which specifically binds to the N-
terminal or C-terminal
residues of an Ap peptide but that does not recognize the same residues when
present in a
longer 4 species comprising the residues or in APP. In various aspects, an A(3
antibody is
"C-terminus-specific." As used herein, the term "C terminus-specific" means
that the
antibody specifically recognizes a free C-terminus of an Ap peptide. Examples
of C
terminus-specific Ap anhbodies include those that: recognize an 4 peptide
ending at residue
40 but do not recognize an A(i peptide ending at residue 41, 42, and/or 43;
recognize an AB
peptide ending at residue 42 but do not recognize an Ap peptide ending at
residue 40, 41,
and/or 43; etc.
[00108] In one aspect, the Ap antibody may be a 3D6 antibody or variant
thereof, or a
lOD5 antibody or variant thereof, both of which are described in U.S. Patent
Publication No.
20030165496A1, U.S. Patent Publication No. 20040087777A1, International Patent
Publication No. WO 02/46237A3 and Interaational Pabent Publication No.
W004/080419A2.
Description of 3D6 and lOD5 antibodies can aLso be found, for egample, ia
lnternational
Patent Publication No. W002/088306A2 and International Patent Publication No.
W002/088307A2. lOD5 antibodies are also described in U.S. Patent Publication
No.
20050142131. Additiona13D6 antibodies are described in U.S. Patent Application
No.
11/303,478 and International Application No. PCT/US05/45614. 3D6 is a
monoclonal
antibody (mAb) that specifically binds to an N-terminal epitope located in the
human P-
amyloid peptide, specifically, residues 1-5. By comparison, 1OD5 is a mAb that
specifically
binds to an N-teaninal epitope located in the human (3-amyloid peptide,
speci.fically, residues
3-6. A cell line producing the 3D6 monoclonal antibody (RB96 3D6.32.2.4) was
deposited
with the American Type Culture Collection (ATCC), Manassas, VA 20108, USA on
Apri18,
2003 under the terms of the Budapest Treaty and has deposit number PTA-5130. A
cell line
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producing the lOD5 monoclonal antibody (RB4410D5.19.21) was deposited with the
ATCC
on Apri18, 2003 under the terms of the Budapest Treaty and has deposit number
PTA-5129.
[00109] Bapineuzumab means a humanized 3D6 antibody comprising a light chain
having a mature variable region having the amino acid sequence designated SEQ
ID NO:1
and a heavy chain having a mature variable region having the amino acid
secluence
designated SEQ ID NO: 2.
Humanized 3D6 Light Chain Variable Region
Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro
Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser
Leu Leu Asp Ser Asp Gl.y Lys Thr Tyr Leu Asn Trp I.eu Leu Gln Lys
Pro Gly Gln Ser Pro Gln Arg Leu lle Tyr Leu Val Ser Lys Leu Asp
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
Cys Trp Gln Gly Thr His Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys (SEQ ID NO: 1)
Humanized 3D6 Heavy Chain Variable Region
Glu Val Gln Leu I.eu Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly Ser I.eu Arg Leu Ser Cys Ala Ala Ser Gly Phe 'Ihr Phe
Ser Asn Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val Ala Ser Ile Arg Ser Gly Gly Gly Arg Thr Tyr Tpr Ser
Asp Asn Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr A1a Val
Tyr Tyr Cys Val 'Arg Tyr Asp His Tyr Ser Gly Ser Ser .Asp Tyr Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser (SEQ ID NO: 2)
Bapineuzumab is also known as AAB-001. Figure 1 shows light and heavy chain
mature
variable region amino acid sequences of Bapineuzumab predicted from the
expression
construct DNA sequenc es. Amino acid sequence of the AAB-001 light and heavy
chains
predicted from the expression cbnstract DNA sequences. CDR regions are
underlined.
Cysteines expected to form intermolecular disulfide bonds are underlined and
the
connectivity indicated. The N-linked glycosylation consensus site is in bold
italics. The
predicted heavy chain COOH terminal lysine is shown in parenthesis.
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[0100] A second version of humanized 3D6 anti'body comprising a light chain
having
a mature variable region having the amino acid sequence designated SEQ ID NO:
3 and a
heavy chain having a mature variable region having the amino acid sequence
designated SEQ
ID NO: 4 is shown below.
Humanized 3D6 Light Chain Variable Region
Tyr Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro
Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser
Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Lys
Pro Gly Gln Ser Pro Gln Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile Ser Arg Vai Glu Ala Glu Asp Val Gly Val Tyr Tyr
Cys Trp Gln Gly Thr Ii'is Phe -Pro Arg Thr Phe Gly G1n Gly Thr Lys
Val Glu Ile Lys (SEQ ID N0:3)
H m n ed 3D6 Heavy Chain Variable Region
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly Ser Leu Arg Leu Ser Cys A1a Ala Ser Gly Phe Thr Phe
Ser Asn Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val Ala Ser Ile Arg Ser Gly Gly Gly Arg Thr Tyr Tyr Ser
Asp Asn Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Ser Leu Tyr I.eu Gln Met Asn Ser Leu Arg Ala Glu Asp Tfir Ala Leu
Tyr Tyr Cys Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp
Gly Gln Gly Thr Leu Val Tbr Vai Ser Ser (SEQ ID NO:4)
[0101] A third version of humanized 3D6 antibody comprising a light chain
having the
amino acid sequence designated SEQ ID NO: 5 and a heavy chain having the amino
acid
sequence designated SEQ ID NO: 6 is describe in US 2005/0090649 Al published
on April
28, 2005, which is incorporated herein by reference for all purposes.
Humanized 3D6 Light Chain
Asp Val Val Met Tbr Gln Ser Pro L=eu Ser Leu Pro Val Thr Leu Gly
Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser
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Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser
Pro Arg Arg Leu Ile Tyr Leu Vai Ser Lys Leu Asp Ser Gly Val Pro
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly
Thr His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val Tyr A1a Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser Phe Asn Arg Giy Glu Cys (SEQ ID NO: 5)
Humanized 3D6 Heavy Chain
Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gin Pro Gly Gly
Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Asn Tyr
Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
Ala Ser Ile Arg Ser Gly Gly Gly Arg Thr Tyr Tyr Ser Asp Asn Val
Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser I.eu Tyr
Leu Gln Met Asn Ser I.eu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pno Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Gin Pro Val Thr Val Ser Trp
Asn Ser Gly A1a Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn Hfs Lys Pro
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Gln Leu Leu Gly Gly Pro
Ser Val Phe I.eu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
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Ala Lys Thr Lys Pro Arg G1u Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Uu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Tlu Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Uu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Uu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys (SEQ ID NO: 6)
[0102] A version of humanized lOD5 antibody comprising a Iight chain having a
mature variable region having the amino acid sequence designated SEQ ID NO: 7
and a
heavy chain having a mature variable region having the amino acid sequence
designated
SEQ ID NO: 8 is shown below.
Humanized lOD5 Lsght Chain Variable Region
Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val
Ser Uu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln A,sn Ile
Ile His Ser Asn Gly Asn Thr Tyr Uu Glu Trp Tyr Leu Gln Lys Pro
Gly Gin Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile Lys Lys Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys
Phe Gln Gly Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
Glu Leu Glu (SEQ ID N0:7)
Human ed lODS Heavy Chain Variable Region
Gln Ala Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln
Ser Ser Gln Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu
Ser Thr Ser Gly Met Gly Val Ser Trp Ile Arg Gln Pro Ser Gly Lys
Gly Uu Glu Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr
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Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Arg
Lys Gln Val Phe Leu Lys Ile Thr Ser Val Asp Pro Ala Asp Thr Aia
Thr Tyr Tyr Cys Val Arg Arg Pro Ile Thr Pro Val Leu Val Asp Ala
Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser (SEQ ID NO: 8)
[01031 In another aspect, the antibody may be a 12B4 antibody or variant
thereof, as
described in U.S. Patent Publication No. 20040082762A1 and International
Patent
Publication No. WO 03/077858A2. 12B4 is a mAb that specifically binds to an N-
terminal
epitope located in the human f~-amyloid peptide, specifically, residues 3-7.
[0104] 12A11 or a chimeric or humanized or nanobody form thereof is a
preferred
antibody. The 12A11 antibody or a variant thereof, is deseribed in U.S. Patent
Publication
No. 20050118651, U.S. Patent Publication No. 20060198851, International Patent
Publication No: WO 04/108895, and Intemational Patent Publication No. WO
06/066089, all
of which are incorporated by reference in their entirety herein for all
purposes. 12A11 is a
mAb that specifically binds to an N-terminal epitope located in the human 0-
amyloid peptide,
specifically, residues 3-7. A cell line producing the 12A11 monoclonal
antibody was
deposited at the ATCC (American Type Culture Collection, 10801 University
Boulevard,
Manassas, VA 20110-2209) on December 12, 2005 and has the ATCC accession
number
PTA-7271.
[0105] A fust version of the humanized 12A11 antibody comprising a light chain
having the amino acid sequence designated SEQ ID N0: 7 and a heavy chain
having the
amino acid sequence designated SEQ ID NO: 8(version 1) is descn'bed in US
20050118651
Al published on June 2, 2005, which is incorporated herein by reference for
all purposes.
[0106] Humanized 12A11 Light Chain
Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Tlu Pro Gly Glu Pro Ala
Ser Ile Ser
Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr
Leu G1n Lys
Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val
Pro Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu A1a
Glu Asp Val
Gly Val Tyr Tyr Cys Phe Gln Ser Ser His Val Pro Leu Thr Phe Gly Gln Gly Thr
Lys Leu Glu
Ile Lys (SEQ ID NO: 7)
[0107] Humanized 12A11 Heavy Chain (version 1)
[0108] Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu
Arg Leu Ser Cys Ala Phe Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Ser Val Gly
Trp Ile Arg
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Gln Ala Pro Gly Lys Gly Leu Glu Trp I.eu Ala His Ile Trp Trp Asp Asp Asp Lys
Tyr Tyr
Asn Pro Ser Leu Lys Ser Arg Leu Thr lle Ser Lys Asp Thr Ser Lys Asn Thr Val
Tyr Leu G)n
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Thr Thr
Thr Ala
Asp Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO: 8)
[0109] A second version of the humanized 12A11 antibody comprising a li.ght
chain
having the amino acid sequence designated SEQ ID NO: 7 and a heavy chain
having the
amino acid sequence designated SEQ ID NO: 9(version 2) is described in US
20050118651
A1 published on June 2, 2005, which is incorporated herein by reference for
all purposes.
[0110] HiLanized 12A11 Heavy Chain (version 2)
[Olll] Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu
Arg Leu Ser Cys Ala Phe Ser Gly Phe Thr Leu Ser Thr Ser Gly Met Ser Val Gly
Trp Ile Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala His Ile Trp Trp Asp Asp Asp Lys
Tyr Tyr
Asn Pro Ser Leu Lys Ser Arg -Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr I.eu
Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Thr Thr
Thr Ala
Asp Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser (SBQ ID No: 9)
[0112] A third version of the humanized 12A11 antibody comprising a light
chain
having the amino acid sequence designated SEQ ID NO: 7 and a heavy chain
having the
amino acid sequence designated SEQ ID NO: 10 (version 2.1) is descnbed in US
20050118651 A1 published on June 2, 2005, which is incorporated herein by
reference for all
purposes.
[0113] Human ed 12A11 Heavy Chain (version 2.1)
[0114] Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu
Arg Leu Ser Cys Ala Phe Ser Gly Phe Zfir Leu Ser Tfir Ser Gly Met Ser Val Gly
Trp Ile Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ii"is Ile Trp Trp Asp Asp Asp Lys
Tyr Tyr
Asn Pro Ser Leu Lys Ser Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu
Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Thr Thr
Thr Ala
Asp Tyr Phe Ala Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO:10)
[0115] A fourth version of the humanized 12A11 antibody comprising a light
chain
having the amino acid sequence designated SEQ ID NO: 7 and a heavy chain
having the
amino acid sequence designated SEQ ID NO: l i(version 3) is descnbed in US
20050118651
Al published on June 2, 2005, which is incorporated herein by reference for
all purposes.
[0116] Humanized 12A11 Heavy Chain (version 3)
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Attorney Docket No. CF500722
[0117] Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Vai Gln Pro Gly Arg Ser Leu
Arg Leu Ser Cys Ala Phe Ser Gly Phe Tlu Leu Ser Thr Ser Gly Met Ser Val Gly
Trp Ile Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala His Ile Trp Trp Asp Asp Asp Lys
Tyr Tyr
Asn Pro Ser Leu Lys Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Thr Thr
Thr Ala
Asp Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser (SEC ID NO:11)
[0118] A fifth version of the humanized 12A11 antibody comprising a light
chain
having the amino acid sequence designated SEQ ID NO: 7 and a heavy chain
having the
amino acid sequence designated SEQ ID NO: 12 (version 4.1) is described in US
20050118651 Al published on June 2, 2005, which is incorporated herein by
reference for all
purposes.
[0119] Humanized 12A11 Heavy Chain (version 4.1)
[0120] Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu
Arg Leu Ser Cys Ala Phe Ser Gly Phe Thr Leu Ser Thr Ser Gly Met Ser Val Gly
Trp Ile Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu Ala I~'is Ile Trp Trp Asp Asp Asp Lys
Tyr Tyr
Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val
Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Thr Thr
Thr Ala
Asp Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO:
12)
[01211 A sixth version of the humanized 12A1 1.antibody comprising a light
chain
having the amino acid sequence designated SEQ ID NO: 7 and a heavy chain
having the
amino acid sequence designated SEQ ID N0:13 (version 4.2) is described in US
20050118651 Al published on June 2, 2005, which is incorporated herein by
reference for all
purposes.
[0122] Humanized 12A11 Heavy Chain (version 4.2)
[0123] Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu
Arg Leu Ser Cys Ala Phe Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Ser Val Gly
Trp Ile Arg
Gln Ala Pro Gly Lys Gly Lsu Glu Trp Val Ala His Ile Trp Trp Asp Asp Asp Lys
Tyr Tyr
Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val
Tyr Leu Gln
Met Asn Ser LeirArg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Thr Thr
Thr Ala
Asp Tyr Phe Ata Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser (SEQ ID N0:13)
[0124] An seventh version of the humanized 12A11 antibody comprising a light
chain having the amino acid sequence designated SEQ ID NO: 7 and a heavy chain
having
the amino acid sequence designated SEQ ID NO: 14 (version 4.3) is described in
US
39
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20050118651 Al published on June 2, 2005, which is incorporated herein by
reference for all
purposes.
[0125] Humanized 12A11 Heavy Chain (version 4.3)
[0126] Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu
Arg Leu Ser Cys Ala Phe Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Ser Val Gly
Trp Ile Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys
Tyr Tyr
Asn Pro Ser Leu Lys Ser Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val
Tyr Leu Gln
Met Asn Ser I.eu Arg A1a Glu Asp Thr Ala Val Tyr Tyr Cys A1a Arg Arg Thr Thr
Thr Ala
Asp Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO:
14)
[0127] A eighth.version of the humanized 12A11 antibody comprising a light
chain
having the amino acid sequence designated SEO ID NO: 7 and a heavy chain
having the
amino acid sequence designated SEQ ID NO: 15 (version 4.4) is described in US
20050118651 Al published on June 2, 2005, which is incorporated herein by
reference for all
purposes.
[0128] Humanized 12A11 Heavy Chain (version 4.4)
[0129] Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Grn Pro Gly Arg Ser Leu
Arg Leu Ser Cys Ala Phe Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Ser Val Gly
Trp Ile Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu Ala Hi.s Ile Trp Trp Asp Asp Asp Lys
Tyr Tyr
Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn 1fir Leu
Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Thr Thr
Thr Ala
Asp Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser (SEQ ID N0:15)
[0130] A ninth version of the humanized 12A11 antibody comprising a light
chain
having the amino acid sequence designated SEO ID NO: 7 and a heavy chain
having the
amino acid sequence designated SEQ ID N0:16 (version 5.1) is described in US
20050118651 Al published on June 2, 2005, which is incorporated herein by
reference for all
purposes=
[0131] Humanized 12A11 Heavy Chain (version 5.1)
[0132] Gln Val G1n- Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser
Leu
Arg Leu Ser Cys Ala Phe Ser Gly Phe Thr Leu Ser Thr Ser Gly Met Ser Val Gly
Trp Ile Arg
Gln Ala Pro Gly Lys Gly Leu Glu Tirp Val Ala His Ile Trp Trp Asp Asp Asp Lys
Tyr Tyr
Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val
Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Thr Thr
Thr Ala
Asp Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser (SEQ ID N0:16)
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[0133] A tenth version of the humanized 12A11 antibody comprising a light
chain
having the amino acid sequence designated SEQ ID NO: 7 and a heavy chain
having the
amino acid sequence designated SEQ ID N0:17 (version 5.2) is descn'bed in US
20050118651 Al published on June 2, 2005, which is incorporated herein by
reference for aIl
purposes.
[0134] Humanized 12A11 Heavy Chain (version 5.2)
[0135] Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu
Arg Leu Ser Cys Ala Phe Ser GIy Phe Thr Leu Ser 1hr Ser G1y Met Ser Val Gly
Trp Ile Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu Ala His Ile Trp Trp Asp .Asp Asp Lys
Tyr Tyr
Asn Pro Ser Leu Lys Ser Arg Phe Thr Ile Ser Lys Asp 'Ifir Ser Lys Asn Thr Val
Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Thr Thr
Thr Ala
Asp Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO:
17)
[0136] An eleventh version of the humanized 12A11 antibody comprising a light
chain having the amino acid sequence designated SEQ ID NO: 7 and a heavy chain
having
the amino acid sequence designated SEQ ID NO: 18 (version 5.3) is described in
US
20050118651 Al published on June 2, 2005, which is incorporated herein by
reference for all
purposes.
[0137] Humanized 12A11 Heavy Chain (version 5.3)
[0138] Gln Val Gln I.eu Val Glu Ser GIy Gly GIy Val Val Gln Pro Gly Arg Ser
Leu
Arg Leu Ser Cys Ala Phe Ser Gly Phe Thr Leu Ser Thr Ser Gly Met Ser Val Gly
Trp Ile Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys
Tyr Tyr
Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Tlu Leu
Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Thr Thr
Thr Ala
Asp Tyr Phe Ala Tyr Trp Gly GIn Gly Thr Thr Val Thr Val Ser Ser Val (SEQ ID
N0:18)
[0139] A twelfth version of the humanized 12A11 antibody comprising a light
chain
having the amino acid sequence designated SEQ ID NO: 7 and a heavy chain
having the
amino acid sequence designated SEQ ID NO: 19 (version 5.4) is descn'bed in US
20050118651 A1 published on June 2, 2005, which is incorporated herein by
reference for all
purposes.
[0140] Humanized 12A11 Heavy Chain (version 5.4)
[0141] Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu
Arg Leu Ser Cys Ala Phe Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Ser Val Gly
Trp Ile Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala His Ile Trp Trp Asp Asp Asp Lys
Tyr Tyr
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Asn Pro Ser Leu Lys Ser Arg Phe Tlu Ile Ser Lys Asp Thr Ser Lys Asn Thr Val
Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala A.rg Arg Thr Thr
Thr Ala
Asp Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Tht Val Thr Vai Ser Ser Val (SEQ ID
NO: 19)
[0142] A thirteenth version of the h manizPd 12A11 antibody comprising a light
chain having the amino acid sequence designated SEQ ID NO: 7 and a heavy chain
having
the amino acid sequence designated SEQ ID NO: 20 (version 5.5) is described in
US
20050118651 Al published on June 2, 2005, which is incorporated herein by
reference for all
purposes.
[0143] Humanized 12A11 Heavy Chain (version 5.5)
[0144] Gln Val_Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu
Arg Leu Ser Cys Ala Phe Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Ser Val Gly
Trp Ile Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Vai Ala His Ile Trp Trp Asp Asp Asp Lys
Tyr Tyr
Asn Pro Ser I.eu Lys Ser Arg'Lcu Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr I.eu
Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Thr Thr
Thr Ala
Asp Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO:
20)
[0145] A fourteenth version of the humanized 12A11 antibody comprising a light
chain having the amino acid sequence designated SEQ ID NO: 7 and a heavy chain
having
the amino acid sequence designated SEQ ID NO: 21(version 5.6) is described in
US
20050118651 Al published on June 2, 2005, which is incorporated herein by
reference for all
purposes.
[0146] Humanized 12A11 Heavy Chain (version 5.6)
[0147] Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu
Arg Leu Ser Cys Ala Phe Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Ser Val Gly
Trp Ile Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys
Tyr Tyr
Asn Pro Ser Leu Lys Ser Arg Phe Tlu Ile Ser Lys Asp Thr Ser Lys Asn Thr Leu
Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Gys Ala Arg Arg Thr Thr
Thr Ala
Asp Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO:
21)
[0148] A fifteenth version of the humanized 12A11 antibody comprising a light
chain having the amino acid sequence designated SEQ ID NO: 7 and a heavy chain
having
the amino acid sequence desigpated SEQ ID NO: 22 (version 6.1) is described in
US
20050118651 Al published on June 2, 2005, which is incorporated herein by
reference for all
purposes.
[0149] Humanized 12A11 Heavy Chain (version 6.1)
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[0150] Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu
Arg Leu Ser Cys Ala Phe Ser Gly Phe Thr Leu Ser Thr Ser Gly Met Ser Val Gly
Trp Ile Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala His Ile Trp Trp Asp Asp Asp Lys
Tyr Tyr
Asn Pro Ser Leu Lys Ser Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val
Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Thr Thr
Thr Ala
Asp Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO:
22)
[0151] A sixteenth version of the humanized 12A11 anti'body comprising a light
chain having the amino acid sequence designated SEQ ID NO: 7 and a heavy chain
having
the amino acid sequence designated SEQ ID NO: 23 (version 6.2) is described in
US
20050118651 AI published on June 2, 2005, which is incorporated herein by
reference for all
purposes.
[0152] Humanized 12A11 Heavy Chain (version 6.2)
[0153] Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu
Arg Leu Ser Cys Ala Phe Ser Gly Phe Thr Leu Ser Thr Ser Gly Met Ser Val Gly
Trp Ile Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala His Ile Trp Trp Asp Asp Asp Lys
Tyr Tyr
Asn Pro Ser Leu Lys Ser Arg Leu Tbr Ile Ser Lys Asp Thr Ser Lys Asn Thr Leu
Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Thr Thr
Thr Ala
Asp Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO:
23)
[0154] A seventeenth version of the humanized 12A11 antibody comprising a
light
chain having the amino acid sequence designated SEQ ID NO: 7 and a heavy chain
having
the am.ino acid sequence desigaated SEQ ID NO: 24 (version 6.3) is described
in US
20050118651 AI published on June 2, 2005, which is incorporated herein by
reference for all
purposes.
[0155] Humanized 12A11 Heavy Chain (version 6.3)
[0156] Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu
Arg Leu Ser Cys Ala Phe Ser Gly Phe Thr Leu Ser Thr Ser Gly Met Ser Val Gly
Trp Ile Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys
Tyr Tyr
Asn Pro Ser Leu Lys Ser Arg Phe Thr Ile Ser Lys Asp T7u Ser Lys Asn Thr Leu
Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Thr Thr
Thr Ala
Asp Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Thr Va1 Thr Val Ser Ser (SEQ ID NO:
24)
[01571 A eighteenth version of the humanized 12A11 antibody comprising a light
chain having the amino acid sequence designated SEQ ID NO: 7 and a heavy chain
having
the amino acid sequence designated SEQ ID NO: 25 (version 6.4) is described in
US
43
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Attorney Docket No. CF500722
20050118651 Al published on June 2, 2005, which is incorporated herein by
reference for all
purposes.
[0158] Humanized 12A11 Heavy Chain (version 6.4)
[0159] Gln Val Gln Leu Val Glu Ser Gly G1y Gly Val Val Gln Pro Gly Arg Ser Leu
Arg Leu Ser Cys Ala Phe Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Ser Val Gly
Trp Ile Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala H'is Ile Trp Trp Asp Asp Asp Lys
Tyr Tyr
Asn Pro Ser Leu Lys Ser Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Leu
Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Thr Thr
Thr Ala
Asp Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO:
25)
[0160] A nineteenth version of the humanized 12A11 antibody comprising a light
chain having the amino acid sequence designated SEQ ID NO: 7 and a heavy chain
having
the amino acid sequence designated SEQ ID NO: 26 (version 7) is descn'bed in
US
20050118651 Al published ot, June 2, 2005, which is incorporated herein by
reference for all
purposes=
(0161] Humanized 12A11 Heavy Chain (version 7)
[0162] Gln Val Gln Leu Val GIu Ser Gly Gly Gly Vai Val Gln Pro Gly Arg Ser Leu
Arg Leu Ser Cys A1a Phe Ser Gly Phe Thr Leu Ser Tlu Ser Gly Met Ser Val Gly
Trp Val Arg
Gln A1a Pro Gly Lys Gly Leu Glu Trp Leu Ala Ms Ile Trp Trp Asp Asp Asp Lys Tyr
Tyr
Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val
Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Thr Thr
Thr Ala
A,sp Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Thr Va1 Thr Val Ser Ser (SEQ ID NO:
26)
[0163] A twentieth ver3ion of the humanized 12A11 antibody comprising a light
cliain having the amino acid sequence designated SEQ ID NO: 7 and a heavy
chain having
the amino acid sequence designated SEQ ID NO: 27 (version 8) is descn'bed in
US
20050118651 Al published on June 2, 2005, which is incorporated herein by
reference for all
purposes-
[0164] Humanized 12A11 Heavy Chain (version 8)
[0165] Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu
Arg l:eu Ser Cys Ala Phe Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Ser Val Gly
Trp Ile Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu Ala I~i'is Ile Trp Trp Asp Asp Asp Lys
Tyr Tyr
Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Val
Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Thr Thr
Thr Ala
Asp Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser (SEQ ID N0: 8)
44
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[01667 In yet another aspect, the antibody may be a 6C6 antibody, or a variant
thereof, as described in a U.S. Patent Publication No. US 20060165682 and
intemational
Patent Publication No. WO 06/06604 entitled "Humanized Antibodies that
Recognize Beta
Amyloid Peptide" 6C6 is a mAb that specifically binds to an N-tenninal epitope
located in
the human (3-amyloid peptide, specifically, residues 3-7. A cell line
producing the antibody
6C6 was deposited on November 1, 2005, with the ATCC under the terms of the
Budapest
Treaty and assigned accession number PTA 7200.
[0167] ln yet another aspect, the antibody may be a 2H3 antibody as described
in U.S.
Patent Publication US 20060257396. 2113 is a mAb that specifically binds to an
N temiinal
epitope located in the humanan 0-amyloid peptide, specifically, residues 2-7.
A cell line
producing the antibody 2H3 was deposited on December 13, 2005, with the ATCC
under the
terms of the Budapest Treaty and assigned accession number PTA-7267.
[0168] Tn yet another aspect, the antibody may be a 3A3 antibody as described
in U.S.
Patent Publication US 20060257396. 3A3 is a mAb that specifically binds to an
N-temiinal
epitope located in the human fi-amyloid peptide, speci5cally, residues 3-7. A
cell line
producing the antibody 3A3 was deposited on December 13, 2005, with the ATCC
under the
terms of the Budapest Treaty and assigned accession number PTA 7269.
[0169] In yet another aspect, the antibody may be 2B1,1C2 or 9G8. Cell lines
producing the antibodies 2B1, 1C2 and 9G8 were deposited on November 1, 2005,
with the
ATCC under the terms of the Budapest Treaty and were assigned accession
numbers PTA-
7202, PTA 7199 and PTA-7201, respectively.
[0170] Antibodies for use in the present invention may be recombinantly or
synthetically produced. For example, the antibody may be produced by a
recombinant cell
culture process, using, e.g., CHO eells, NIH 3T3 cells, PER.C68 cells, NSO
cells, VERO
cells, chick embryo fibroblasts, or BHK cells. In addition, antibodies with
minor
modifications that retain the primary functional property of binding 4 peptide
are
contemplated by the present invention. In a particalar aspect, the antibody is
a humanized
anti Ap peptide 3D6 antibody that selectively binds Ap pelptide. More
specifically, the
humanized anti 4 pepti.de 3D6 antibody is designed to specifically bind to an
NHZ-terminal
epitope, for example, amino acid residues 1-5, located in the human fl-amyloid
1-40 or 1-42
peptide found in plaque deposits in the brain (e.g., in patients suffering
from Alzheimer's
disease)-
CA 02643633 2008-10-17
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Pronhvlactic and Theraneutic Methods
[0171] The present invention is directed inter alia to treatment of
Alzheimer's and
other amyloidogenic diseases by admi.nistration of therapeutic immunological
reagents (e.g.,
humanized irnmunoglobulins) to specific epitopes within A.P to a patient under
conditions
that generate a beneficial therapeutic response in a patient (e.g., induction
of phagocytosis of
A(3, reduction of plaque burden, inhibition of piaque formation, reduction of
neuritic
dystrophy, improving cognitive function, and/or reversing, treating or
preventing wgnitive
decline) in the patient, for egample, for the prevention or treatment of an
amyloidogenic
disease. The invention i.s also directed to use of the disclosed immunologicai
reagents (e.g.,
humanized immunoglobulins) in the manufacture of a medicament for the
treatment or
prevention of an amyloidogenic disease.
[0172] The term "treatment" as used herein, is defined as the application or
administration of a therapeutic agent to a patient, or application or
administtation of a
therapeutic agent to an isolated tissue or cell line from a paxient, who has a
disease, a
symptom of disease or a predisposition toward a disease, with the putpose to
cure, heal,
alieviate, relieve, alter, remedy, ameliorate, improve or affect the disease,
the symptoms of
disease or the predisposition toward disease.
[0173) In one aspect, the invention provides methods of preventing or treating
a
disease associated with amyloid deposits of Ap in the brain of a patient. Such
diseases
include Alzheimer's disease, Down's syndrome and cognitive impairment. The
latter can
occur with or without other characteristics of an amyloidogenic disease. Some
methods of
the invention entail administering an effective dosage of an antibody that
specifically binds to
a component of an amyloid deposit to the patient. Such methods are
particularly useful for
preventing or treating Alzheimer's disease in human patients. Exemplary
methods entail
administering an effective dosage of an antibody that binds to A(3. Preferred
methods entail
administ.ering an effective dosage of an antibody that specifically binds to
an epitope within
residues 1-10 of Ap, for example, antt'bodies that specifically bind to an
epitope within
residues 1-3 of A(3, antibodies that specifically bind to an epitope within
residues 1-4 of Ap,
antz'bodies that specificaIIy bind to an epitope within residues 1-5 of Ap,
antibodies that
speci5cally bind to an epitope within residues 1-6 of A(3, antibodies that
specificaIIy bind to
an epitope within residues 1-7 of M, or antibodies that specifically bind to
an epitope within
residues 3-7 of Ap. In yet another aspect, the invention features
administering antibodies
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that bind to an epitope comprising a free N terminal residue of A43. In yet
another aspect, the
invention features administering antibodies that bind to an epitope within
residues of 1-10 of
AB wherein residue 1 and/or residue 7 of Ap is aspartic acid. In yet another
aspect, the
invention features administering antibodies that specifically bind to Ap
peptide without
binding to full-length amyloid precursor protein (APP). In yet another aspect,
the isotype of
the antibody is human IgGl.
[0174] In yet another aspect, the invention features administering antibodies
that bind
to an amyloid deposit in the patient and induce a clearing response against
the amyloid
deposit. For example, such a clearing response can be effected by Fc receptor
mediated
phagocytosis.
[0175] Therapeutic agents of the invention are typically substantially pure
from
undesired contaminant. This means that an agent is typically at least about
50% w/w
(weight/weight) purity, as well as being substantially free from interfering
proteins and
contaminants. Sometimes the agents are at least about 80% w/w and, more
preferably at least
90 or about 95% w/w purity. However, using conventional protein purification
techniques,
homogeneous peptides of at least 99% w/w can be obtained.
[0176] The methods can be used on both asymptomatic patients and those
currently
showing symptoms of disease. The antibodies used in such methods can be human,
humanized, chimeric or nonhuman antibodies, or fragments thereof (e.g.,
antigen binding
fragments) and can be monoclonal or polyclonal, as described herein. In yet
another aspect,
the invention features administering antibodies prepared from a human
immunized with A(3
peptide, which human can be the patient to be treated with antibody.
[01771 In another aspect, the invention features administering an antibody
with a
pharmaceutical carrier as a pharmaceutical composition. Alternatively, the
antibody can be
administered to a patient by administering a polynucleotide encoding at least
one antibody
chain. The polynucleotide is egpressed to produce the antibody chain in the
patient.
Optionally, the polynucleotide encodes heavy and light chains of the antibody.
The
polynucleotide is;expressed to produce the heavy and light chains in the
patient. In other
aspects, the patient i.s monitored for level of administered antibody in the
blood of the patient.
[0178] The invention thus fulfills a longstanding need for therapeutic regimes
for
preventing or ameliorating the neuropathology and, in some patients, the
cognitive
impairment associated with Alzheimer's disease.
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Patients Amenable to Treatment
[0179] Patients amenable to treatment include individuals at risk of disease
but not
showing symptoms, as well as patients presently showing symptoms. 1n the case
of
Alzheimer's disease, virtually anyone is at risk of suffering from Alzheimer's
disease if he or
she lives long enough. Therefore, the present methods can be administered
prophylactically
to the general population without the need for any assessment of the risk of
the subject
patient. The present methods are especiaUy useful for individuals who have a
known genetic
risk of Alzheimer's disease. Such individuals include those having relatives
who have
egperienced this diseas6, and those whose risk is determined by analysis of
genetic or
biochemical markers. Genetic markers of risk toward Alzheimer's disease
include mutations
in the APP gene, particularly mutations at position 717 and positions 670 and
671 referred to
as the Hardy and Swedish mutations respectively (see Hardy, supra). Other
markers of risk
are mutations in the presenilin genes, PS1 and PS2, and ApoE4, family history
of AD,
hypercholesterolemia or atherosclerosis. Individuals presently suffering from
Alzheimer's
disease can be recognized from characteristic dementia, as well as the
presence of risk factors
described above. In addition, a number of diagnostic tests axe available for
identifying
individuais who have AD. These include measurement of CSF tau and 4421evels.
IIevated tau and decreased AP421evels signify the presence of AD. Individuals
suffering
from Alzheimer's disease can aiso be diagnosed by ADRDA criteria as discussed
in the
Egamples secti.on.
[0180] In asymptomatic patients, treatment can begin at any age (eg.,10, 20,
30).
Usually, however, it is not necessary to begin treatment until a patient
reaches 40, 50, 60 or
70. Treatment typically entails multiple dosages over a period of time.
Treatment can be
monitored by assaying antibody levels over time. If the response falls, a
booster dosage is
indicated. In the case of potential Down's syndrome patients, treatment can
begin antenatally
by administering therapeutic agent to the mother or shortly after birth.
[01811 Patients amenable to treatment include patients 50 to 87 years of age,
patients
suffering from mild to moderate Alzheimer's disease, patients having an MMSE
score of 14-
26, patients having a diagnosis of probable Alzheimer's disease based on
Neurological and
Communicative Disorders and Stroke-Alzheimer's disease Related Disorders
(NINCDS-
ADRDA) criteria, attd/or patients having an Rosen Modified Hachinski rschemic
score less
than or equal to 4. Patients with 1VfltI an scan consistent with the diagnosis
of Alzheimer's
disease, i.e., that there are no other abnormalities present on the MRI that
could be attn"buted
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CA 02643633 2008-10-17
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to other diseases, e.g. stroke, traumatic brain injury, arachnoid cysts,
tumors, etc are also
amendable to treatment.
[0182] The methods of the invention are particular amendable for patients that
have
no history or evidence of any of the following: encephalitis; clinically
evident stroke;
clinically signfficant carotid or vertebrobasi'lar stenosis or plaque;
seizures, excluding febrile
seizures in childhood; any significant autoinunune disease or disorder of the
immune system;
and/or clinically significant renal disorder. The methods of the invention are
particular
amendable for patients that have had no clinically significant infection
within the 30 days
before treatment commences, e.g., a chronic persistent or acute infection. The
methods of the
invention are particular amendable for patients that have not been treated
with
immunosuppressive medication (e.g., systemic corticosteroids) within 90 days
before
treatment commences (topical and nasal corticosteroid,s and inhaled
corticosteroids for
asthma are permiteed) or chemotherapeutic agents for malignancy within 3 years
before
treatment commences. The methods of the invention are also particular
amendable for
patients that have no clinically significant abnormality on physical,
neurological, laboratory,
or EKG examination (e.g. atrial fibrillation) if the abnonnality could be
detrimental to the
patient. The methods of the invention are also particular amendable for
patients that do not
use anticonvulsants for seizure, anti-Parldnson's, anticoagulant (excluding
the use of aspirin
325 mg/day or less), or narcotic medications.
Treatment Regimes and Dosa¾es
[0183] In prophylactic applications, pharmaceutical compositions or
medicaments are
administered to a patient susceptible to, or otherwise at risk of, Alzheimer's
disease in an
amount sufficient to eliminate or reduce the risk, lessen the severity, or
delay the outset of the
disease, including biochemical, histologic and/or behavioral symptoms of the
disease, its
complications and intermediate pathological phenotypes presenting during
development of
the disease. In therapeutic applications, compositions or medicants are
administered to a
patient suspected 'of, or already suffering from such a disease in an amount
sufficient to cure,
or at least partially arrest, the symptoms of the disease (biochemical,
histologic and/or
behavioral), including its complications and intermediate pathological
phenotypes in
development of the disease.
[0184] Fn some methods, administration of agent reduces or eliminates
myocognitive
impairment in patients that have not yet developed characteristic Alzheimer's
pathology. An
49
CA 02643633 2008-10-17
Attorney Docket No. CF500722
amount adequate to accomplish therapeutic or prophylactic treatment is defined
as a
therapeuticaily- or prophylacticaily-effective dose. In both prophylactic and
therapeutic
regimes, agents are usually administered in several dosages until a sufficient
immune
response has been achieved. The term "immune response" or "immunological
response"
includes the development of a humoral (antibody mediated) and/or a cellular
(mediated by
antigen-specific T cells or their secretion products) response directed
against an antigen in a
recipient subject. Such a response can be an active response, i. e., induoed
by administration
of immunogen, or a passive response, i.e-, induced by administration of
immunoglobulin or
anti'body or primed T-cells.
[0185] An "immunogenic agent" or "immunogen" is capable of inducing an
immunological response against itself on administration to a mammal,
optionally in
conjunction with an adjuvant. Typically, the immune response is monitored and
repeated
dosages are given if the immune response starts to wane.
[0186] Effective doses of the compositions of the present invention, for the
treatment
of the above described conditions vary depending upon many different factors,
including
means of administration, target site, physiological state of the patient,
whether the patient is
human or an animal, other medications administered, and whether treatment is
prophylactic
or therapeutic. Usually, the patient is a human but non-human mammals
including transgenic
mammals can also be treated. Treatment dosages need to be titrated to optimize
safety and
efficacy.
[0187] For passive innnunization, the dosage ranges from about 0.0001 to 100,
and
more usually 0.01 to 5 mg/kg of the host body weight. For example, dosage
ranges can be
less than 20 mg/kg body weight or 10 mg/kg body weight or within the range of
1.0 to 7
mg/lrg. For passive immunization, the preferred dosage ranges from about 0.5
to less than 5
mg/kg, and more usually 0.5 to 3 mg/kg, of the host body weight. For example
dosages can
be less than 5 mg/kg body weight or 1.5 mg/kg body weight or within the range
of 0.5 to 1.5
mg/kg, preferably at least 1.5 mg/kg. Subjects can be administered such doses
daily, on
alternative days, weekly or acxording to any other scliedule determined by
empirical analysis.
An exemplary treatment entails administration in multiple dosages over a
prolonged period,
for example, of at least six months. Additional exemplary treatment regimes
entail
administration once per every two weeks or once a month or once every 3 to 6
months.
[0188] Exemplary passive dosage schednles include 1.5-3 mg/kg or 1.5 mg/kg
every
thirteen weeks. Agents of the invention are usually administered on multiple
occasions.
CA 02643633 2008-10-17
Attomey Docket No. CF500722
Intervals between single dosages can be weekly, monthly, every thirteen weeks,
or yearly.
Intervals can also be irregular as indicated by measuring blood levels of
antibody to Ap in the
patient.
[0189] In some methods, dosage is adjusted to achieve a plasma antibody
concentration of 1-1000 g/ml and in some methods 25-300 g/ml. Altematively,
antibody
can be administered as a sustained release formulation, in which case less
frequent
administration is required. Dosage and frequency vary depending on the half-
life of the
antibody in the patient. In general, human antibodies show the longest half-
life, followed by
humanized antibodies, chimeric antibodies, and nonhuman antibodies.
[0190] The dosage and frequency of admuiistration can vary depending on
whether
the treatment is prophylactic or therapeutia ln prophylactic applications,
compositions
containing the present antibodies or a cocktail thereof are administered to a
patient not
already in the disease state to enhance the patient's resistance. Such an
amount is defined to
be a"prophylactic effedive dose." In this use, the precise amounts again
depend upon the
patient's state of health and general immunity, but generally range from 0.1
to 25 mg per
dose, especially 0.5 to 2.5 nig per dose. A relatively low dosage is
administered at relatively
infrequent intervals over a long period of time. Some patients continue to
receive treatment
for the rest of their lives.
[0191] In therapeutic applications, a relatively high dosage (e.g., from about
10 to 250
mg of antibody per dose, with dosages of from 5 to 25 mg being more commonly
used) at
relafively short intervals is sometimes required until progression of the
disease is reduced or
temiinated, and preferably until the patient shows partial or complete
amelioration of
symptoms of disease. Thereafter, the patent can be administered a prophylactic
regime.
[0192] Therapeutic agents can be administered by parenteral, topical,
intravenous,
oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal
or intramuscular
means for prophylactic and/or therapeutic treatment. The most typical route of
administration of a passive immunogenic agent is intravenous infusion although
other routes
can be equally effeWve. The next most common route is intramuscolar injedion.
This type
of injection is most typically performed in the arm or leg muscles. In some
methods, agents
are injected d"uectly into a particular tissue where deposits have
accumulated, for example
intracranial injecxion. Intramusc.ular injection or intravenous infiusion are
preferred for
administration of antibody. In some methods, particular therapeutic antibodies
are injecxed
51
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directly into the cranium. In some methods, antibodies are administered as a
sustained
release composition or device, such as a Medipad71*1 device.
[0193] Agents of the invention can optionally be administered in combination
with
other agents that are at least partly effective in treatment of amyloidogenic
disease. In the
case of Alzheimer's and Down's syndrome, in which amyloid deposits occur in
the brain,
agents of the invention can also be administered in conjunction with other
agents that
increase passage of the agents of the invention across the blood-brain
barrier.
Phamiaceutical Compositions
[0194] Agents of the invention are often administered as pharmaceutical
compositions comprising an active therapeutic agent, i.e., and a variety of
other
pharmaceutically acceptable components_ See Remington's Pharmaceutical Science
(15th ed.,
Mack Publishing Company, Easton, Pennsylvania (1980)). The preferred form
depends on
the intended mode of administration and therapeutic application. The
compositions can also
include, depending on the formulation desired, pharmaceutically-acceptable,
non-toxic
carriers or diluents, which are defined as vehicles commonly used to formulate
pharmaceutical compositions for animal or human administration. The diluent is
selected so
as not to affect the biological activity of the combination. Egamples of such
diluents are
distilled water, physiological phosphate-buffered saline, Ringer's solutions,
dextrose solution,
and Hank's solution. In addition, the pharmaceutical composition or
formulation may also
include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic
stabilizers
and the like.
[0195] Pharinaceutical compositi.ons can also include large, slowly
metabolized
macromotecules such as proteins, polysaccharides such as chitosan, polylactic
acids,
polyglycolic acids and copolymers (such as latex functionalized sepharose(TM),
agarose,
cellulose, and the like), polymeric amino acids, amino acid copolymers, and
lipid aggregates
(such as oil droplets or liposomes). Additionally, these carriers can function
as
immunostimulatitig agents (i.e., adjuvants).
[0296] For parenteral administration, agents of the invention can be
administered as
injectable dosages of a solution or suspension of the substance in a
physiologically acceptable
diluent with a pharmaceutical carrier that can be a sterile liquid such as
water oils, saline,
glyceml, or ethanol. Additionally, auxiliary substances, such as wetting or
emulsifying
agents, surfactants, pH buffering substances and the like can be present in
compositions.
52
CA 02643633 2008-10-17
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Other components of pharmaceutical compositions are those of petroleum,
animal, vegetable,
or synthetic origin, for example, peanut oil, soybean oil, and mineral oil. In
general, glycols
such as propylene glycol or polyethylene glycol are preferred liquid carriers,
particularly for
injectable solutions. Antibodies can be administered in the form of a depot
injection or
implant preparation, which can be formulated in such a manner as to permit a
sustained
release of the active ingredient. An exemplary composition comprises
monoclonal antibody
at 5 mg/mL, formulated in aqueous buffer consisting of 50 mM Uhistidine,150 mM
NaCl,
adjusted to pH 6.0 with HCI.
[0197] Typically, compositions are prepared as injectables, either as liquid
solutions
or suspensions; solid forms suitable for solution in, or suspension in, liquid
vehicles prior to
injection can also be prepared. The preparation also can be emulsif ed or
encapsulated in
liposomes or micro particles such as polylactide, polyglycolide, or copolymer
for enhanced
adjuvant effect, as discussed above (see Langer, Science 249:1527 (1990) and
Hanes,
Advanced Drug Delivery Reviews 28:97 (1997)). The agents of this invention can
be
administered in the form of a depot injection or implant preparation, which
can be formulated
in such a manner as to permit a sustained or pulsatile release of the active
ingredient.
[0198] Additional formulations suitable for other modes of administration
include
oral, intranasal, and pulmonary formulations, suppositories, and transdermal
applications.
For suppositories, binders and carriers include, for example, polyalkylene
glycols or
triglycerides; such suppositories can be formed fiom mixtures containing the
active
ingredient in the range of 0.5% to 10%, preferably 1%-2%. Oral formulations
include
excipients, such as pharmaceutical grades of mannitol, lactose, starch,
magnesium stearate,
sodium saccharine, cellulose, and magnesium carbonate. These compositions take
the form
of solutions, suspensions, tablets, pills, capsules, sustained release
formulations or powders
and oontain 10%-95% of active ingredient, preferably 25%-70%.
[0199] Topical application can result in twLsdennal or intradermal delivery.
Topical
administration can be facilitated by co-administration of the agent with
cholera toxin or
detoxified derivatives or subunits thereof or other similar bacterial toxins
(See Glenn et al.,
Nature 391, 851(1998)). Co-administration can be achieved by using the
components as a
mixture or as linked molecules.obtained by chemical crosslinking or expression
as a fusion
protein.
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CA 02643633 2008-10-17
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[0200] Aitematively, transdermal delivery can be achieved using a slan path or
using
transferosomes (Paul et al., Eur. J. Immunol. 25:3521(1995); Cevic et aL,
Biochem. Biophys.
Acta 1368:201-15 (1998)).
Monitoringthe Course of Treatment
[0201] The invention provides methods of monitoring treatment in a patient
suffering
from or susceptible to Alzheimer's, i.e., for monitoring a course of treatment
being
administered to a patient. The methods can be used to monitor both therapeutic
treatment on
symptomatic patients and prophylactic treatment on asymptomatic patients. ln
particular, the
methods are useful for monitoring passive immunization (e.g., measuring level
of
administered antibody).
[0202] Some methods entail determining a baseline value, for example, of an
antibody level or profile in a patient, before administering a dosage of
agent, and comparing
this with a value for the profile or level after treatment. A significant
increase (i.e., greater
than the typical margin of experimentai error in repeat measurements of the
same sample,
expressed as one standard deviation from the mean of such measurements) in
value of the
level or profile signals a positive treatment outcome (i.e., that
administration of the agent has
achieved a desired response). If the value for immune response does not change
significantly, or decreases, a negative treatment outcome is indicated.
[0203] In other methods, a control value (i.e., a mean and standard deviation)
of level
or profile is determined for a control population. Typically the individuals
in the control
populatfon have not received prior treatment. Measured values of the level or
profile in a
patient after administering a therapeutic agent are then compared with the
control value. A
significant increase relative to the control value (e.g., greater than one
standard deviation
from the mean) signats a positive or sufflcient treatment outcome. A lack of
significant
increase or a decrease signals a negative or insufficient treatment outcome.
Administration of
agent is generally continued while the level is increasing relative to the
control vatue. As
before, attainment of a plateau relative to control values is an indicator
that the admini.stration
of breatment can be discont'nnued or reduced in dosage and/or frequency.
[0204] In other methods, a control value of the level or profile (e.g., a mean
and
standard deviation) is determined from a control population of individuals who
have
undergone treatment with a therapeutic agent and whose Ievels or profiles have
reached a
plateau in response to treatment. Measured values of levels or profiles in a
patient are
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Attorney Docket No. CF500722
compared with the control value. If the measured level in a patient is not
significantly
different (e.g., more than one standard deviation) from the control value,
treatment can be
discontinued. If the level in a patient is signi.ficantly below the control
value, continued
administration of agent is warranted. If the level in the patient persists
below the control
value, then a change in treatment may be indicated.
[0205] Tn other methods, a patient who is not presently receiving treatment
but has
undergone a previous course of treataient is monitored for antibody levels or
profiles to
determine whether a resumption of treatment is required. The measured level or
profile in the
patient can be compared with a value previously achieved in the patient after
a previous
course of treatment. A signi.ficant decrease relative to the previous
measurement (i.e., greater
than a typical margin of error in repeat measurements of the same sample) is
an indication
that treatment can be resumed. Alternatively, the value measured in a patient
can be
compared with a control value (mean plus standard deviation) detemiined in a
population of
patients after undergoing a course of treatment. Altematively, the measured
value in a patient
can be compared with a control value in populations of prophylactically
treated patients who
remain free of symptoms of disease, or populations of therapeutically treated
patients who
show amelioration of disease characxeristics. In all of these cases, a
significant decrease
relative to the control level (i.e., more than a standard deviation) is an
indicator that treatment
should be resumed in a patient.
[0206J The tissue sample for analysis is typically blood, plasma, seram,
mucous fluid
or cerebrospinal fluid from the patient. The sample is analyzed, for example,
for levels or
profiles of anti'bodies to A(3 peptide, ag., levels or profiles of humanized
antibodies. ELISA
methods of detecting antibodies specific to A(3 are described in the Bxamples
section. In
some methods, the level or profile of an administered antibody is determined
using a clearing
assay, for example, in an in vitro phagocytosis assay, as described herein. ln
such methods, a
tissue sample from a patient being tested is contacted with amyloid deposits
(e.g., from a
PDAPP mouse) and phagocytic cells bearing Fc receptors. Subsequent clearing of
the
amyloid deposit is then monitored. The egistence and extent of clearing
response provides an
indication of the existencE and level of antibodies effective to clear Aj3 in
the tissue sample of
the patient under test.
[02071 The andbody profile following passive immunization typically shows an
imtnediate peak in antibody concentration followed by an exponential decay.
Without a
further dosage, the decay approaches pretreatment levels within a period of
days to months
CA 02643633 2008-10-17
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depending on the half-life of the antibody administered. For example the half-
life of some
human antibodies is of the order of 20 days.
[0208] In some methods, a baseline measurement of antibody to Ap in the
patient is
made before administration, a second measurement is made soon thereafter to
determine the
peak antibody level, and one or more further measurements are made at
intervals to monitor
decay of antibody levels. When the level of antibody has declined to baseline
or a
predetermined percentage of the peak less baseline (e.g., 50 b, 25% or 10%),
administration
of a further dosage of antibody is administered. In some methods, peak or
subsequent
measured levels less background are compared with reference levels previously
determined
to constitute a beneficial prophylactic or therapeutic treatment regime in
other patients. lf the
measured antibody level is significantly less than a reference level (e.g.,
less than the mean
minus one standard deviation of the reference value in population of patients
benefiting from
treatment) aditinistration of an additional dosage of antibody is indicated.
[0209] Additional methods include monitoring, over the course of treatment,
any art
recognized physiologic symptom (e.g., physical or mental symptom) routinely
relied on by
researchers or physicians to diagnose or monitor amyloidogenic diseases (e.g.,
Alzheimer's
disease). For example, one can monitor cognitive impairment. The latter is a
symptom of
Alzheimer's disease and Down's syndrome but can also occur without other
characteristics of
either of these diseases. For example, cognitive impairment can be monitored
by determining
a patient's score on the Mini-Mental State Exam in accordance with convention
throughout
the course of treatment.
[0210] The patient may be monitored by at least one type of assessment
selected from
the group of consisting of Mini-Mental State Exam (MMSE), A zheimer's Disease
Assessment Scale - cognitive (ADAS-COG), Clinician Interview-Based Impression
(CIBl),
Neurological Test Battery (NTB), Disability Assessment for Dementia (DAD),
Clinical
Dementia Rating-sum of boxes (CDR-SOB), Neuropsychiatric lnventory (NPI),
Positron
Emission Tomography (PET Imaging) scan, Magnetic Resonance Imaging (11V.i)
scan, an
EKG and measurement of blood pressure. The type of assessment may be
administered on
multiple occa.sions. For example, an MMSE may be performed before
administering a
dosage of the immunogenic agent, and at week 4, week 6, week 16, 6 months, and
1 year
after administering the dosage of the immunogenic agent. In some patients an
MMSE may
be performed before admuustering a dosage of the immunogenic agent, and at
week 6, and
week 16. An MRI scan may be performed every 3 months, every 6 months, or every
year.
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[0211] Patients may be monitored for posterior reversible encephalopathy
syndrome
(PRES) or vascular edema after administration of an antibody within a range of
about 0.5
mg/kg to less than 5 mg/kg, wherein the antibody specifi'ically binds to beta
amyloid peptide
(AP) with a binding affinity of at least 10' 1VL'1. PRES classically consists
of reversible
vasogenic edema in the posterior circulation territories, however, conversion
to irreversible
cytoxic edema has been described. PRES is typically characterized by headache,
nausea,
vomiting, wnfusion, seizures, visual abnormalities, altered mental
functioning, ataxia, frontal
symptoms, parietal symptoms, stupor, and focal neurologic signs. In addition
to the
foregoing clinical symptoms, MRI scans or Fluid Attenuated Inversion Recovery
(FLA1R)
sequence imaging can be used to indicate the presence on PRES. (See Pediatric
Neurology,
20(3):241-243; AJ1VA% 26:825-830; NEJM, 334(8):494-500; Pediatr Nephro4
18:1161-1166;
Internal Medicine Journa4 35:83-90; JNNP, 68:790-791; AJNR, 23:1038-1048; Pak
J Med
Sc4 21(2):149=154 and, AJ1VA% 21:1199-1209.)
[0212) Patients may be monitored for PRES or vascular edema monthly, every
four
months, every six months, or yearly. The patient may be monitored for at least
one clinical
symptom associated with PRES or vascular edema. The monitoring may comprise
perfonming an MRI scan. The monitoring may further comprise perfomung FI.AIR
sequence
imaging. The results of the monitoring may impact the dosing regime. For
example, if PRF.S
or vascular edema is detected, dosing may be suspended or dosages may be
reduced or the
intervals between dosages may be increased.
[0213] Patients with PRES or vascular edema may have their blood pressure
measured for hypertension. If hypertension is detected in the patient, the
patient may be
treated for the hypertension by administration of an antihypertensive. The
antihypertensive
may be selected from the group consisting of hydrociorothiazide, angiotensin-
converting
enzyme (ACE) inhibitors, angiotensin II-receptor blockers (ARB), beta
blockers, and calcium
channel blockers. Patient with PRES or vaswlar edema may be treated with
steroids such as
dexamethasone or methyprednisol.
C. Kits
[0214] The invention further provides therapeutic products. The products
comprise a
glass vial and instrudions. The glass viai contains a formulation comprising
about 10 mg to
about 250 mg of a humanized anti A43 antibody, about 4% mannitol or about 150
mM NaCI,
about 5 mM to about 10 mM histidine, and about 10 mM methionine. The
instructions to
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monitor a patient to whom the formulation is administered for PRF.S and or
vascular edema
are included with the products. In some therapeutic products the glass vial
contains a
formulation comprising about 10 mg of a humanized anti Ap antibody in about 10
mM
histidine, about 10 mM methionine, about 4% mannitol, and about 0.005%
polysorbate-80
(vegetable derived), with a pH of about 6Ø The instructions to monitor a
patient to whom
the formulation is administered for PRES or vascular edema are included with
the products.
Egample I. Prevention and Treatment of Human Suti-jects
[0215] Bapineiizumab (A.AB-001) is a humanized monoclonal antibody to A(3. The
objective of this study is to determine the safety and tolerability of single
doses of
bapineuzumab in AD.
[0216] Methods: Randomized, double-blind, placebo-controlled single ascending
dose trial of bapineuzumab infusion in patients with mild to moderate AD.
Patients enrolied
in the trial met all of the following criteria:
1. Diagnosis of probable Alzheimer's disease (AD) according to the National
institute of Neurologicai and Communicative Disorders and Stroke-Alzheimer's
disease and Related Disorders (NINCDS-ADRDA) criteria.
2. Age from 50 to 87 years, inclusive.
3. Mini-Mental Status Egamination (1VIlVISE) score of 14-26.
4. Rosen Mod'if'ied Hachinski Ischemic score <4.
5. Lives at home with appropriate caregiver capable of aocompanying the
patient on
all clinic visits, or oommunity dwelling with caregiver ca,pable of
accompanying
the patient on all clinic visits and visiting with the patient appros.imately
5 times
per week for the duration of the study.
6. Screening visit brain magnetic resonance imaging (MRI) scan consistent with
the
diagnosis of AD, i.e., that there are no other abnormalities present on the
MRI that
can be attributed to other diseases (e.g., stroke, traumatic brain injury,
arachnoid
cysts, tumors, etc).
7. Surgically sterile or 2 years post-menopausal.
8. Fluent in English and evidences adequate premorbid intellectual
functioning.
Patient must have adequate visual and auditory abilities to perfonm all
aspects of
the oognitive and functionai assessments.
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9. Receiving stable doses of inedication(s) for the treatment of non-excluded
medical
condition(s) for at least 30 days prior to screening. If a patient is taking
acetylcholinesterase inhibitors or memantine, then these medication(s) must be
maintained on a stable dose regimen for at least 60 days prior to screening
evaluations.
Anyone of the following criteria excluded a patient from being enrolled in the
trial:
1. Significant neurological disease, other than AD, that may affect cognition.
2. Current presence of a clinically significant major psychiatric disorder
(e.g., major
Depressive Disorder) according to the criteria of the Diagnostic and
Statistical
Manual of Mental Disorders, Fourth Edition (DSM-1V), or symptom (e.g.,
hallucinations), that could affed the patient's ability to complete the study.
3. current clinicaIIy significant systemic illness that is li.kely to result
in
deterioration of the patient's condition or affect the patient's safety during
the
study.
4. I~'istory or evidence of any of the following: encephaiitis; clinically
evident stroke;
clinicaIly significant carotid or vertebrobasilar stenosis or plaque;
seizures,
excluding febrile seizures in childhood; any clinically significant autoimmune
disease or disorder of the immune system; clinically significant renai
disorder.
5. Clinically significant infection with the last 3.0 days (e.g., chronic
persistent or
acute infection).
6. Treatment with immunosuppressive medications (e.g., systemic
corticosteroids)
within the last 90 days (topical and nasal corticosteroids and inhaled
corticosteroids for asthma are permitted) or chemotherapeutic agents for
malignancy within the last 3 years.
7. Myocardial infarction within the last 2 years.
8. History of cancer within the last 5 years, with the exception of non-
metastatic
basal cell carci.noma and squamous cell carcinoma of the skin.
9. Other clinically significant abnormality on physicai, neurological,
laboratory, or
ECG examination (e.g., atrial fibrfflation) that could compromise the study or
be
detrimental to the patient.
10. Hemoglobin less than 11g/dL.
i l. Ii'istory of alcohol or drug dependence or abuse within the last 2 years.
12. Hamilton Psychiatric Rating Scale for Depression (HAM-D) (17-item) score
>12.
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13. Current use of anticonvulsants for seizure, anti-Parldnson's,
anticoagulant
(excluding the use of aspirin 325 mg/day or less), or narcotic medications.
14. Gurrent use of prescription or nonprescription medication for cognitive
enhancement other than cholinesterase inlubitors and memantine. Carrent
cholinesterase inhibitor and memantine use is prohibit:ed unless the following
conditions are met: (a) maintained on a stable dose regimen for at least 60
days
prior to screening; (b) patient is free of any clinically significant side
effects
attributable to the drug; and (c) patient and caregiver agree that, barring
unforeseen circumstances, they will continue the same regimen for the duration
of
the trial.
15. Unless maintained on a stable dose regimen for at least 30 days prior to
screening,
any other medications with the potential to affect cognition other than those
mentioned in #18 (including, but not limited to, anxiolytics, sedatives,
hypnotics,
antipsychotics, antidepressants, over-the-counter (OTC) sleeping aids,
sedating
anti-allergy medications, vitamin E, thyroid supplements, and vitamin B12
supplements by injection).
16. Patients who have discontinued cholinesterase inhibitors, memantine,
cognitive
enhancing agents, or drugs that potentially affect cognition in the 60 days
prior to
scxeening.
17. Use of an egperimental medication (chemical compound) or device for AD or
any
other investigational medica.tion or device for indication other than
treatment for
AD within 30 days prior to screening or within 5 half-lives of use of such a
medication prior to screening, whichever is longer.
18. Any prior experimental treatment with AN1792, AAB-001, ACC-001, or other
experimental immunotherapeutic or vaccine for AD.
19. Any prior treatment with a biologic product other than those mentioned in
#18 for
the treatment of AD within the last 3 years.
20. Patients who have donated blood (routine blood donation) in the 90 days
prior to
screening.
21. Any known hypersensitivity to any of the excipients contained in the study
drug
formulation.
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22. Presence of pacemakers, aneurysm clips, artificial heart valves, ear
implants,
metal ftagments of foreign objects in the eyes, skin, or body that would
contraindicate a brain MRI scan.
23. Weight greater than 120 kg (2641bs).
[0157] Results: 30 patients received bapineuzumab at doses of 0.5 mg/kg (6
active, 2
placebo) 1.5 mg/kg (6 active, 2 placebo) and 5 mg/kg (10 active, 4 placebo).
3/10 patients at
mg/kg developed MRI abnormalities, consisting predominantly of high signal
abnormalities
on FLAIR sequences, and the study did not continue past that dose. In two
patients these
were seen on routine surveillance scans without clinical symptoms, however a
third patient
experienced increased confusion. The MRI FLA IR abnonmlities resolved in all
three cases
by 12 weeks post-dose. As part of the safety assessments MMSE was performed at
baseline,
week 4, week .16, 6 months and at 1 year. . Figure 2 shows the average MMSE
change from
baseline. Figure 3 shows the change from baseline MMSE by cohort at month 4.
At week
16, the prespecified primary time point for analysis, the treatment difference
relative to
placebo favored the treated group at the 0S mglkg dose (treatment vs. placebo
difference of
2.0, p=.152) and reached statistical significance at the 1.5 mg/kg dose
(treatment vs. placebo
difference of 2.5, p=.047). There was no significant difference in MMSE change
relative to
placebo for the 5.Omg/kg group. Figure 4 shows the mean, median and standard
deviation
MMSE change from baseline at month four. Figure 5 shows the results of
statistical testing
of the MMSE change from baseline at month four. No correlation was found
between the
MRI FLAIR abnormalities and the differenoe in 1V1MSE change.
[0158] Plasma A beta was elevated from baseline levels in a dose dependent
fashion,
peaking approximately 24 hours after the infusion. Pharmacokinetic analysis
showed a half
life of 22 - 28 days and was supportive of a 13-week dosing interval in
multiple dosing.
[0159] Conclusion: In this small study, MMSE was statistically significantly
improved
compared with placebo at the 1.5mg/kg dose of bapineuzamab. The highest single
infiision
dose of 5mg/kg was associated with MRI FLAIR abnonnalities which resolved.
[0160] Although the foregoing invention has been descn'bed in detail for
purposes of
clarity of understanding, it will be obvious that certain modifications may be
practiced withfn
the scope of the appended claims. All publications and patent documents cited
herein, as
well as text appearing in the figures, are hereby incorporated by reference in
their entirety for
all purposes to the same extent as if each were so individually denoted.
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Example II. Pharmacokinetic Study of AAB-001
[0161] The objective of this study is to determine human pharmacokinetics (PK)
after
intravenous administration of AAB-001.
[0162] Methods: Randomized, double-blind, placebo-controlled multiple
ascending
dose trial of AAB-001 administered intravenously. 6 doses of AAB-001 were
administered
intravenously q13wk. There were four dose cohorts were 0.15, 0.5,1.0 and 2.0
mg/kg.
[0163] Results: PK was predictable and dose-independent. PK - CL 0.05-0.06
mL/h/kg across all dose levels. Dose-proporkional exposure. Very little
accumulation with
q13wk IV dosing, alttiough quantifiable pre-infusion concentrations at all
dose levels. tl/z
ranged from 21-34 hours. At steady-state, Cavg after 0.5 mg/kg AAB-001(i.e., -
35 mg)
-3.3 g/mL See Figure 9. Cavg after 0.5 mg/kg AAB-001 of -3.3 g/mL is close
to the 3.7
g/mL concentration found to be efficacious in PDAPP mice. Figure 10 shows mean
serum
AAB-001 concentration vs. time profiles following intravenous administration
of AAB-001
at doses of 0.15, 0.5,1.0, and 2.0 mg/kg.
[0164] Plasma 4 concentrations tend to mirror AAB-001 concentrations. At end
of
13-week dosing interval, plasma A13levels above baseline levels and above
placebo group at
?0.5 mg/kg AAB-001 dose levels. See Figure 11. tmax ranged from 14-48 hours.
[0165] Serum anti-AAB-001 antibody levels have been undetectable in any of the
samples (up to pre-Infusion #6 for the 0.5 mg/kg AAB-001 cohort).
[0166] From the foregoing it will be apparent that the invention provides for
a number
of uses. For example, the invention provides for the use of any of the
antibodies to A(3
described above in the treatment, prophylaxis or diagnosis of amyloidogenic
disease, or in the
manufacture of a medicament or diagnostic composition for use in the same.
62
