NZ625217B2 - Use of an anti-?-synuclein antibody to diagnose an elevated level of ?-synuclein in the brain - Google Patents
Use of an anti-?-synuclein antibody to diagnose an elevated level of ?-synuclein in the brain Download PDFInfo
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- NZ625217B2 NZ625217B2 NZ625217A NZ62521712A NZ625217B2 NZ 625217 B2 NZ625217 B2 NZ 625217B2 NZ 625217 A NZ625217 A NZ 625217A NZ 62521712 A NZ62521712 A NZ 62521712A NZ 625217 B2 NZ625217 B2 NZ 625217B2
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Classifications
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2814—Dementia; Cognitive disorders
- G01N2800/2821—Alzheimer
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/38—Pediatrics
- G01N2800/385—Congenital anomalies
- G01N2800/387—Down syndrome; Trisomy 18; Trisomy 13
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
Abstract
Disclosed is amethod of diagnosing an elevated level of a-synuclein in the brain of a human subject, said method comprising: (a) assaying a level of a-synuclein in a blood plasma sample or cerebrospinal fluid (CSF) sample obtained from the human subject at a specified time interval following peripheral administration to the human subject of an anti-a-synuclein antibody or antigen-binding fragment thereof, wherein the antibody or fragment thereof can bind a-synuclein with sufficient affinity to alter a net efflux of a-synuclein from brain to blood or CSF; and (b) comparing the level of the a-synuclein in the blood plasma sample or CSF sample assayed to a reference standard, wherein a difference or similarity between the level of a-synuclein in the blood plasma sample or CSF sample and the reference standard correlates with the level of a-synuclein in the brain of the human subject. eral administration to the human subject of an anti-a-synuclein antibody or antigen-binding fragment thereof, wherein the antibody or fragment thereof can bind a-synuclein with sufficient affinity to alter a net efflux of a-synuclein from brain to blood or CSF; and (b) comparing the level of the a-synuclein in the blood plasma sample or CSF sample assayed to a reference standard, wherein a difference or similarity between the level of a-synuclein in the blood plasma sample or CSF sample and the reference standard correlates with the level of a-synuclein in the brain of the human subject.
Description
USE OF AN ANTI-(x-SYNUCLEIN ANTIBODY TO DIAGNOSE AN
ELEVATED LEVEL OF u-SYNUCLEIN IN THE BRAIN
REFERENCE TO SEQUENCE LISTING SUBMITTED
ELECTRONICALLY
The content of the electronically submitted sequence g in ASCII text file
(Name: sequencelistingPCT_ascii.txt; Size: 5,199 bytes; and Date of Creation:
October 18, 2012) filed with the application is incorporated herein by reference in its
entirety.
BACKGROUND
Field of the Disclosure
This disclosure s to the use of anti-u-synuclein antibody to se an
elevated level of u-synuclein in the brain. Specifically, the disclosure relates to the
method of ing the levels of u-synuclein in blood plasma or cerebrospinal fluid
(CSF) following administration to the test subject of an anti-(x-synuclein antibody or
antigen-binding fragment thereof, which can bind u-synuclein with sufficient
activity to alter the net efflux of u-synuclein from brain to blood or brain to CSF.
Background of the Disclosure
The mammalian brain is separated from blood by the blood-brain barrier (BBB)
localized to the brain capillaries and pia-subarachnoid membranes and the blood-
cerebrospinal fluid (CSF) barrier localized to the choriod plexi. u-synuclein is relatively
abundant in the brain under non-pathological conditions. It is a natively unfolded protein
present mostly in the cytosol. It plays an essential role in synaptic ission and
ic plasticity by augmenting transmitter release from the presynaptic terminal. (Liu
et al., EMBO J 23:4506—45 16 ). Mutations in u-synuclein are associated with rare
familial cases of early-onset Parkinson's disease, and conversion of u-synuclein from its
soluble into the aggregated insoluble form is one of key events in the pathogenesis of
neurodegenerative disorders, such as Parkinson's disease, dementia with Lewy bodies
(DLB), and several other egenerative illnesses. (Dawson et al., Science 302:819—
822 , Bennett et al., col Ther. 105:311—331 (2005), George, JM., Genome
Biol. 3(1): s3002.l—reviews3002.6 (2002)). Additionally, both monomeric and
oligomeric u-synuclein have been found in the cerebrospinal fluid (CSF) and serum of
Parkinson's disease patients, as apparently u-synuclein and even its aggregated species
can cross the blood-brain barrier. (El-Agnaf et al., FASEB J. 20:419—425 (2006), Tokuda
et (1]., Biochem s Res Commun. 349:162—166 (2006), Lee et al., JNeural Transm.
113:1435—1439 (2006), hauer et al., Exp Neurol. 213:315—325 (2008), El-Agnaf
et al., FASEB J. 17:1945—1947 (2003), Li et (11., Exp Neur01204: 583—588 .
Immunization studies in mouse models of Parkinson's disease show that mouse
onal antibodies against u-synuclein can reduce accumulation of intracellular 0L-
synuclein aggregates (Masliah et al., Neuron, 46: 857—868 ; Masliah et al., PLoS
One, 6(4): el9338 (2011) supporting the idea that antibodies that neutralize the
neurotoxic aggregates without interfering with beneficial fianctions of monomeric 0L-
synuclein can be useful therapeutics. However, the therapeutic and diagnostic utility of
murine based antibodies in human is hampered by the human anti-mouse antibody
(HAMA) response in view of their non-human origin.
Accordingly, there is a need to develop a method for assessing ts for
elevated levels of u-synuclein in the brain.
BRIEF SUMMARY
One embodiment is directed to a method of diagnosing an elevated level of
u-synuclein in the brain of a test subject comprising: (a) assaying the level of 0L—
synuclein in a blood plasma sample obtained from the test subject at a specified
al following peripheral administration to the test subject of an anti-(x-synuclein
antibody or antigen-binding fragment thereof, wherein the antibody or fragment
thereof can bind u-synuclein with ient ty to alter the net efflux of 0L-
synuclein from brain to blood; (b) comparing the assayed level of the u—synuclein in
the test subject to a reference standard; wherein the difference or rity n
the level of u-synuclein in the plasma sample and the reference standard correlates
with the level of u-synuclein in the brain of the test subject.
Also disclosed is a method of diagnosing an elevated level of u-synuclein in
the brain of a test subject comprising: (a) providing an anti-(x-synuclein antibody or
antigen-binding fragment f, wherein the antibody or fragment thereof can bind
u-synuclein with sufficient affinity to alter the net efflux of u-synuclein from brain
to blood; (b) directing a healthcare provider to peripherally administer the antibody
to the test subject and obtain a blood plasma sample from the subject at a specified
time interval following administration; (c) assaying the level of u—synuclein in the
blood plasma sample; (d) comparing the assayed level of u—synuclein in the test
t to a reference standard; wherein the difference or similarity n the level
of the u-synuclein in the plasma sample and the reference rd correlates with
the level of u-synuclein in the brain of the test subject.
Further disclosed is a method of diagnosing an ed level of d-synuclein
in the brain of a test subject comprising: (a) peripherally administering an anti-0L-
synuclein antibody or antigen-binding fragment thereof to the test subject, wherein
the antibody or fragment thereof can bind u-synuclein with sufficient y to alter
the net efflux of the u-synuclein from brain to blood; (b) ing a blood plasma
sample from the test subject at a specified time interval following administration,
and submitting the sample for determination of the level of the u—synuclein; (c)
ing the level of the u—synuclein in blood plasma sample to a reference
standard; wherein the difference or similarity between the level of the clein in
the plasma sample and the reference standard correlates with level of the u-synuclein
in the brain of the test subject.
Further disclosed is the method as bed herein, further comprising
comparing the level of the u—synuclein in the plasma sample to a plasma sample
obtained from the test subject prior to administration of the anti-(x-synuclein
antibody or antigen-binding nt thereof.
In specific embodiments, the reference standard in the above-described
method comprises measured levels of u—synuclein in one or more control subjects,
wherein the control subjects e normal healthy individuals, and individuals with
synucleinopathies of varying severity.
Further disclosed is a method of tracking the u-synuclein level in the brain of
a subject being treated for a einopathic disease, comprising assaying the level
of u—synuclein in the subject’s blood plasma at a specified time following peripheral
administration of an anti-(x-synuclein antibody or n-binding fragment thereof,
wherein the antibody or fragment thereof can bind u-synuclein with sufficient
affinity to alter the net effiux of the clein from brain to blood; and wherein the
u—synuclein level in the t’s blood plasma ates with the level in the
subject’s brain. In specific embodiments, the above-described method, r
comprises assaying the level of u—synuclein in the subject’s blood plasma at a
specified time following additional peripheral administrations of the x-synuclein
antibody or antigen-binding fragment thereof, thereby plotting the change in the ct—
synuclein level in the subject’s brain over time.
Some embodiments include the method as described herein, where the
method is directed to diagnosing an elevated level of u-synuclein in the brain of a
test subject by assaying the level of u-synuclein in a CSF sample obtained from the
test subject at a specified time intervals following administrations of an anti-0L-
synuclein antibody or n-binding fragment thereof, wherein the antibody or
fragment thereof can bind u-synuclein with sufficient affinity to alter the net effiux
of the u-synuclein from brain to CSF, and wherein the difference or similarity
between the level of the u-synuclein in the CSF sample and the reference standard
correlates with level of the clein in the brain of the test subject. Some
ments include the method as described herein, further comprising comparing
the level of the u—synuclein in the CSF sample to a CSF sample obtained from the
test subject prior to stration of the anti-(x-synuclein antibody or antigenbinding
fragment thereof.
Some embodiments include the method of tracking the clein level in
the brain of a subject being treated for a synucleinopathic disease, comprising
assaying the level of u—synuclein in the subject’s CSF sample at a specified time
following peripheral administration of an x-synuclein antibody or antigen-
binding fragment thereof, wherein the antibody or nt thereof can bind 0L-
synuclein with sufficient affinity to alter the net effiux of the u-synuclein from brain
to CSF; and wherein the u—synuclein level in the subject’s CSF correlates with the
level in the subject’s brain.
Some embodiments include the method as described herein, n the
antibody or antigen-binding fragment thereof specifically binds to the same 0L-
synuclein epitope a reference antibody comprising a VH and a VL, wherein the VH
comprises SEQ ID NO: 2 and the VL comprises SEQ ID NO: 3. In some
embodiments, the dy or antigen-binding fragment thereof competitively
inhibits a reference antibody comprising a VH and a VL, wherein the VH comprises
SEQ ID NO: 2 and the VL comprises SEQ ID NO: 3 from binding to u-synuclein.
Further provided is the method as described herein, wherein the antibody or
antigen-binding nt thereof comprises a heavy chain variable region (VH) and
a light chain variable region (VL), wherein the VH comprises a complementarity
determining region-1 (VHCDRI) amino acid sequence of SEQ ID NO: 4.
Also provided is the method as described herein, wherein the antibody or
antigen-binding nt thereof comprises a VH and a VL, wherein the VH
comprises a complementarity determining -2 (VHCDRZ) amino acid sequence
ofSEQ ID NO: 5.
Some embodiments include the method as described herein, wherein the
antibody or n-binding fragment thereof comprises a VH and a VL, wherein the
VH comprises a mentarity ining region-3 (VHCDR3) amino acid
sequence of SEQ ID NO: 6.
Also provided is the method as described herein, wherein the antibody or
antigen-binding fragment thereof comprises a VH and a VL, wherein the VL
comprises a complementarity determining region-l (VLCDRl) amino acid sequence
of SEQ ID NO: 7.
Some embodiments include the method as described herein, wherein the
antibody or antigen-binding fragment thereof comprises a VH and a VL, wherein the
VL comprises a complementarity determining region-2 (VLCDR2) amino acid
sequence of SEQ ID NO: 8.
Also disclosed is the method as described herein, wherein the dy or
antigen-binding fragment f comprises a VH and a VL, n the VL
comprises a complementarity determining region-3 3) amino acid sequence
of SEQ ID NO: 9.
Further provided is the method as described herein, wherein the antibody or
antigen-binding fragment thereof comprises n the antibody or antigen-binding
fragment thereof ses a VH and a VL, wherein the VH comprises VHCDRl,
VHCDRZ, and VHCDR3 amino acid sequences of SEQ ID NOs: 4, 5, 6.
Also disclosed is the method as described herein, wherein the antibody or
antigen-binding fragment thereof comprises a VH and a VL, wherein the VL
comprises VLCDRl, VLCDR2, and VLCDR3 amino acid ces of SEQ ID
NOs: 7, 8, 9.
Some embodiments include the method as described herein, wherein the
dy or antigen-binding fragment thereof comprises a VH and a VL, n the
VH comprises VHCDRl, , and VHCDR3 amino acid sequences of SEQ
ID NOs: 5, 6, 7, and the VL, comprises VLCDRl, VLCDR2, and VLCDR3 amino
acid sequences of SEQ ID NOs: 7, 8, 9.
Some embodiments include the method as described herein, wherein the
antibody or antigen binding fragment thereof comprises a VH amino acid sequence
of SEQ ID NO: 2 and a VL amino acid sequence of SEQ ID NO: 3.
Also disclosed is the method as described , wherein the antibody or
antigen binding nt thereof is a single chain Fv fragment (scFv), an F(ab')
fragment, an F(ab) fragment, or an F(ab')2 fragment.
Some ments include the method as described herein, wherein the
administering is by intravenous injection of the antibody. In one embodiment the
antibody is human.
Other embodiments include the method as described herein, wherein the
specified time interval is less than a week, or less than or equal to 24 hours, or less
than or equal to 3 hours.
Certain embodiments include the method as described herein, wherein the
synucleinopathic disease is selected from the group consisting of Parkinson's disease
(PD), Parkinson's disease dementia (PDD), ia with Lewy bodies (DLB), the
Lewy body variant of Alzheimer's e (LBVAD), multiple systems atrophy
(MSA), pure mic failure (PAF), neurodegeneration with brain iron
accumulation type-l (NBIA-I), Alzheimer’s disease, Pick disease, juvenile-onset
generalized neuroaxonal dystrophy (Hallervorden-Spatz disease), ophic
lateral sclerosis, traumatic brain injury and Down syndrome.
—6a-
[0028a] Definitions of specific embodiments of the invention as claimed herein follow.
[0028b] According to a first embodiment of the invention, there is provided a method of
diagnosing an elevated level of a-synuclein in the brain of a human subject, said method
comprising:
(a) assaying a level of a—synuclein in a blood plasma sample or
ospinal fluid (CSF) sample obtained from the human t at a specified time
interval following peripheral administration to the human subject of an anti-(x-synuclein
antibody or antigen-binding fragment thereof, n the antibody or fragment thereof
can bind clein with sufficient affinity to alter a net efflux of or-synuclein from
brain to blood or CSF; and
(b) ing the level of the a—synuclein in the blood plasma sample or
CSF sample assayed to a reference standard, wherein a difference or similarity between-
the level of oc-synuclein in the blood plasma sample or CSF sample and the reference
standard correlates with the level of d—synuclein in the brain of the human t.
[0028c] According to a second embodiment of the invention, there is provided a method
of tracking an uclein level in the brain of a human subject being treated for a
synucleinopathic disease, said method comprising assaying a level of a—synuclein in a
blood plasma sample or CSF sample obtained from the human subject at a specified
time interval following peripheral administration of an anti—or—synuclein antibody or
antigen-binding fragment thereof, wherein the antibody or fragment thereof can bind 0t-
synuclein with sufficient affinity to alter a net efflux of the a—synuclein from brain to
blood or CSF, and wherein the a—synuclein level in the blood plasma sample or CSF
sample correlates with the level in the brain of the human t.
[Text continues on page 7]
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
Figure l (A-B): Dose dependent human (x-synuclein plasma spike upon 12F4
antibody administration in transgenic mice overexpressing human (x-synuclein.
Figure 2: Time course of human u-synuclein plasma spike and plasma 12F4
antibody concentrations.
Figure 3 (A-C): Acute, high dose 12F4 antibody treatment of enic mice
pressing human u-synuclein reduces brain human u-synuclein levels.
Figure 4 (A-C): Human (x-synuclein plasma levels significantly reflect brain
0L-synuclein levels after 12F4 antibody injection.
Figure 5 (A-C): Plasma human (x-synuclein (A) and chimeric 12F4 antibody
levels (B) were determined by ELISA. (C) There was a significant correlation
between plasma and brain (x-synuclein levels after chronic treatment for six month
with chimeric 12F4 antibody of transgenic mice overexpressing human uclein.
Figure 6: u-synuclein cerebrospinal fluid (CSF) spike and CSF/serum 124F
ratio around 0.1% upon 12F4 administration in cynomolgus monkeys.
Figure 7: In vivo ialysis in transgenic u-synuclein mice shows drop of
brain interstitial fluid (ISF) uclein upon 12F4 administration.
DETAILED DESCRIPTION
I. DEFINITIONS
It is to be noted that the term "a" or "an" entity refers to one or more of that
entity; for example, "an anti-(x—synuclein antibody," is understood to represent one
or more antibodies which specifically bind to clein. As such, the terms "a"
(or "an"), "one or more," and "at least one" can be used interchangeably herein.
As used herein, the terms "synucleinopathic diseases" or "synucleinopathies"
are a diverse group of neurodegenerative disorders that share a common pathologic
lesion composed of ates of insoluble clein protein in selectively
vulnerable populations of neurons and glia. These disorders include Parkinson's
disease (PD), Parkinson's disease dementia (PDD), dementia with Lewy bodies
(DLB), the Lewy body variant of Alzheimer's disease (LBVAD), multiple systems
atrophy (MSA), pure mic failure (PAF), neurodegeneration with brain iron
accumulation type-l (NBIA-I), mer’s disease, Pick disease, juvenile-onset
generalized neuroaxonal dystrophy (Hallervorden-Spatz disease), amyotrophic
lateral sclerosis, traumatic brain injury and Down me. ally, they are
characterized by a chronic and progressive e in motor, cognitive, oral,
and autonomic functions, ing on the distribution of the lesions.
Unless stated otherwise, the terms "disorder" se" and "illness" are used
interchangeably herein.
As used herein, the terms “binding molecule” or en binding molecule"
refers in its broadest sense to a molecule that specifically binds an antigenic
determinant. Non-limiting examples of antigen binding molecules are antibodies and
fragments thereof that retain antigen-specific binding, as well as other non-antibody
les that bind to u-synuclein including but not limited to hormones, receptors,
ligands, major histocompatibility x (MHC) les, chaperones such as
heat shock proteins (HSPs) as well as cell-cell adhesion molecules such as members
of the cadherin, intergrin, C-type lectin and immunoglobulin (Ig) superfamilies.
Thus, for the sake of clarity only and without restricting the scope of the disclosure
most of the following embodiments are discussed with respect to antibodies and
antibody-like molecules which represent the g molecules for the development
of eutic and diagnostic agents. In another embodiment, a binding le
disclosed comprises at least one heavy or light chain CDR of an antibody molecule.
In another embodiment, a binding molecule disclosed comprises at least two CDRs
from one or more antibody molecules. In another embodiment, a binding molecule
disclosed comprises at least three CDRs from one or more antibody molecules. In
another embodiment, a binding molecule as disclosed comprises at least four CDRs
from one or more antibody molecules. In another embodiment, a binding molecule
as disclosed comprises at least five CDRs from one or more antibody molecules. In
r embodiment, a binding molecule as disclosed comprises at least six CDRs
from one or more antibody molecules.
Disclosed herein a method of diagnosing an elevated level of u-synuclein in
the brain of a test subject, comprising administering to the subject an anti-0t-
synuclein binding molecule, e.g,. an antibody, or antigen-binding fragment, variant,
or derivative thereof. Unless specifically referring to full-sized antibodies such as
naturally occurring antibodies, the term "anti-u-synuclein antibody" encompasses
full-sized antibodies as well as antigen-binding fragments, ts, analogs, or
derivatives of such antibodies, e. g., lly occurring antibody or immunoglobulin
molecules or engineered antibody molecules or fragments that bind antigen in a
manner r to dy molecules.
The terms "antibody" and "immunoglobulin" are used interchangeably
herein. An antibody or globulin comprises at least the variable domain of a
heavy chain, and normally comprises at least the variable domains of a heavy chain
and a light chain. Basic immunoglobulin structures in vertebrate systems are
relatively well understood. See, e.g., Harlow et al. (1988) Antibodies: A tory
Manual (2nd ed.; Cold Spring Harbor Laboratory Press).
As used herein, the term “immunoglobulin” comprises various broad classes
of polypeptides that can be distinguished biochemically. Those skilled in the art will
appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, (y,
u, 0t, 8, 8) with some subclasses among them (e. g., yl-y4). It is the nature of this
chain that determines the "class" of the antibody as IgG, IgM, IgA IgG, or IgE,
respectively. The immunoglobulin subclasses (isotypes) e.g., IgGl, IgG2, IgG3,
IgG4, IgAl, etc. are well characterized and are known to confer functional
specialization. Modified versions of each of these classes and isotypes are readily
discernable to the skilled artisan in view of the disclosure and, accordingly, are
within the scope of the disclosure. All immunoglobulin classes are clearly within the
scope of the disclosure. The following sion will generally be ed to the
IgG class of immunoglobulin molecules. With regard to IgG, a rd
immunoglobulin le comprises two identical light chain polypeptides of
molecular weight imately 23,000 Daltons, and two identical heavy chain
polypeptides of molecular weight 53,000-70,000. The four chains are typically
joined by disulfide bonds in a "Y" configuration wherein the light chains bracket the
heavy chains starting at the mouth of the "Y" and continuing through the variable
region.
Light chains are fied as either kappa or lambda (K, k). Each heavy
chain class can be bound with either a kappa or lambda light chain. In general, the
light and heavy chains are covalently bonded to each other, and the "tail" portions of
the two heavy chains are bonded to each other by covalent disulfide linkages or non-
covalent linkages when the immunoglobulins are generated either by hybridomas, B
cells or cally engineered host cells. In the heavy chain, the amino acid
sequences run from an N—terminus at the forked ends of the Y configuration to the
C-terminus at the bottom of each chain.
Both the light and heavy chains are divided into regions of structural and
functional homology. The terms "constan " and ble" are used functionally. In
this regard, it will be appreciated that the variable domains of both the light (VL or
VK) and heavy (VH) chain portions determine antigen recognition and city.
Conversely, the constant domains of the light chain (CL) and the heavy chain (CH1,
CH2 or CH3) confer important biological properties such as secretion, lacental
mobility, Fc receptor binding, complement binding, and the like. By convention the
numbering of the constant region domains increases as they become more distal
from the antigen binding site or amino-terminus of the antibody. The N—terminal
portion is a variable region and at the C-terminal portion is a constant region; the
CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light
chain, tively.
As indicated above, the variable region allows the antibody to selectively
recognize and specifically bind es on antigens. That is, the VL domain and
VH , or subset of the complementarity ining regions (CDRs) within
these variable domains, of an antibody combine to form the variable region that
defines a three dimensional antigen binding site. This quaternary antibody ure
forms the antigen binding site present at the end of each arm of the Y. More
specifically, the antigen binding site is defined by three CDRs on each of the VH and
VL chains. In some instances, e. g., certain immunoglobulin molecules d from
camelid species or engineered based on camelid immunoglobulins, a complete
immunoglobulin molecule can consist of heavy chains only, with no light chains.
See, e.g., -Casterman et al., Nature 363 :446-448 (1993).
In lly occurring antibodies, the six "complementarity determining
regions" or "CDRs" present in each antigen binding domain are short, non-
contiguous sequences of amino acids that are specifically positioned to form the
antigen binding domain as the dy assumes its three dimensional configuration
.11.
in an aqueous environment. The remainder of the amino acids in the antigen binding
domains, referred to as "framework" regions, show less inter-molecular variability.
The ork regions largely adopt a B-sheet conformation and the CDRs form
loops that connect, and in some cases form part of, the B-sheet structure. Thus,
framework regions act to form a scaffold that provides for positioning the CDRs in
correct orientation by chain, non-covalent interactions. The antigen binding
domain formed by the positioned CDRs defines a surface complementary to the
epitope on the immunoreactive antigen. This complementary surface promotes the
non-covalent g of the antibody to its cognate epitope. The amino acids
comprising the CDRs and the framework regions, respectively, can be readily
identified for any given heavy or light chain variable domain by one of ordinary skill
in the art, since they have been precisely defined (see below).
In the case where there are two or more definitions of a term that is used
and/or accepted within the art, the definition of the term as used herein is intended to
include all such meanings unless explicitly stated to the contrary. A specific
example is the use of the term "complementarity determining region" ("CDR") to
describe the non-contiguous antigen combining sites found within the le
region of both heavy and light chain polypeptides. This particular region has been
bed by Kabat et al. (1983) US. Dept. of Health and Human Services,
"Sequences of Proteins of Immunological Interest" and by Chothia and Lesk, J. M01.
Biol. 1-917 , which are incorporated herein by reference, where the
definitions include overlapping or subsets of amino acid residues when compared
against each other. Nevertheless, ation of either definition to refer to a CDR of
an antibody or variants f is intended to be within the scope of the term as
defined and used . The appropriate amino acid residues that encompass the
CDRs as defined by each of the above cited references are set forth below in Table 1
as a comparison. The exact residue numbers that encompass a particular CDR will
vary depending on the sequence and size of the CDR. Those skilled in the art can
routinely determine which residues se a particular CDR given the variable
region amino acid sequence of the antibody.
WO 66818
Table l. CDR Definitions1
——Chothia
VH CDRI 31—35 26-32
VH CDR2 50-65 52-58
VH CDR3 95—102
VL CDRI 26-32
VL CDR2 50-52
VL CDR3 91-96
ing of all CDR definitions in Table 1 is according to the
numbering conventions set forth by Kabat et al. (see below).
Kabat et al. also defined a numbering system for variable domain sequences
that is applicable to any dy. One of ordinary skill in the art can
unambiguously assign this system of "Kabat numbering" to any variable domain
sequence, without reliance on any experimental data beyond the sequence itself. As
used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et
al. (1983) US. Dept. of Health and Human Services, "Sequence of Proteins of
Immunological Interest." Unless otherwise specified, references to the numbering of
specific amino acid residue positions in an anti-(x—synuclein antibody or antigenbinding
fragment, variant, or derivative thereof of the present disclosure are
according to the Kabat numbering .
Antibodies or antigen-binding fragments, variants, or derivatives thereof of
the disclosure include, but are not d to, polyclonal, monoclonal, multispecific,
human, humanized, ized, or chimeric antibodies, single-chain antibodies,
epitope-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs
(scFv), de-linked Fvs (dev), fragments comprising either a VL or VH
domain, fragments produced by a Fab expression y, and anti-idiotypic (anti-Id)
antibodies. ScFv les are known in the art and are described, e.g., in US. Pat.
No. 5,892,019. Immunoglobulin or antibody molecules of the disclosure can be of
any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3,
IgG4, IgAl, and IgA2, etc.), or subclass of immunoglobulin molecule.
As used herein, the term "heavy chain portion" includes amino acid
sequences derived from an immunoglobulin heavy chain. In n embodiments, a
WO 66818
polypeptide comprising a heavy chain portion comprises at least one of: a VH
domain, a CHl domain, a hinge (e.g., upper, middle, and/or lower hinge )
domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For
example, a binding polypeptide for use in the disclosure can comprise a ptide
chain sing a CHl domain; a polypeptide chain sing a CHl domain, at
least a portion of a hinge domain, and a CH2 domain; a ptide chain
comprising a CHl domain and a CH3 domain; a polypeptide chain comprising a
CHl , at least a portion of a hinge domain, and a CH3 domain, or a
polypeptide chain comprising a CHl domain, at least a portion of a hinge domain, a
CH2 domain, and a CH3 . In another embodiment, a polypeptide of the
disclosure comprises a polypeptide chain comprising a CH3 domain. Further, a
binding polypeptide for use in the disclosure can lack at least a portion of a CH2
domain (e.g, all or part of a CH2 domain). As set forth above, it will be understood
by one of ordinary skill in the art that these domains (e.g., the heavy chain portions)
can be modified such that they vary in amino acid sequence from the naturally
occurring immunoglobulin molecule.
In n embodiments, anti-(x—synuclein antibodies, or antigen-binding
fragments, variants, or derivatives thereof disclosed herein, the heavy chain portions
of one polypeptide chain of a multimer are identical to those on a second polypeptide
chain of the multimer. Alternatively, heavy chain portion-containing monomers of
the disclosure are not identical. For example, each monomer can comprise a
different target binding site, forming, for e, a bispecif1c antibody.
The heavy chain portions of a binding molecule for use in the methods
disclosed herein can be derived from different immunoglobulin molecules. For
example, a heavy chain n of a polypeptide can comprise a CH1 domain derived
from an IgGl molecule and a hinge region derived from an IgG3 molecule. In
r example, a heavy chain n can comprise a hinge region derived, in part,
from an IgGl molecule and, in part, from an IgG3 molecule. In another example, a
heavy chain portion can comprise a chimeric hinge derived, in part, from an IgGl
molecule and, in part, from an IgG4 molecule.
As used herein, the term “light chain portion” includes amino acid sequences
derived from an immunoglobulin light chain, e.g., a kappa or lambda light chain.
Preferably, the light chain portion comprises at least one of a VL or CL domain.
As previously indicated, the subunit structures and three dimensional
configuration of the constant regions of the various immunoglobulin classes are well
known. As used herein, the term “VH domain” includes the amino terminal variable
domain of an immunoglobulin heavy chain and the term “CHl ” includes the
first (most amino terminal) constant region domain of an globulin heavy
chain. The CHl domain is adjacent to the VH domain and is amino terminal to the
hinge region of an globulin heavy chain le.
As used herein the term “CH2 domain” includes the portion of a heavy chain
molecule that extends, e.g., from about residue 244 to residue 360 of an antibody
using conventional numbering schemes ues 244 to 360, Kabat numbering
system; and residues 231-340, EU numbering ; see Kabat EA et al. op. cit.
The CH2 domain is unique in that it is not closely paired with another domain.
Rather, two N-linked branched carbohydrate chains are interposed between the two
CH2 domains of an intact native IgG molecule. It is also well documented that the
CH3 domain extends from the CH2 domain to the C-terminal of the IgG le
and comprises approximately 108 residues.
As used herein, the term “hinge region” includes the portion of a heavy chain
molecule that joins the CH1 domain to the CH2 domain. This hinge region
comprises approximately 25 residues and is flexible, thus allowing the two N-
terminal antigen binding regions to move independently. Hinge regions can be
subdivided into three distinct domains: upper, middle, and lower hinge domains
(Roux et al., J. Immunol. 83 (1998)).
As used herein the term “disulfide bond” includes the covalent bond formed
between two sulfur atoms. The amino acid cysteine comprises a thiol group that can
form a disulfide bond or bridge with a second thiol group. In most naturally
occurring IgG molecules, the CH1 and CL regions are linked by a disulfide bond and
the two heavy chains are linked by two disulfide bonds at positions ponding to
239 and 242 using the Kabat ing system (position 226 or 229, EU numbering
system).
2012/062430
Anti-u—synuclein antibodies, or antigen-binding fragments, variants, or
derivatives thereof disclosed herein can be described or specified in terms of the
epitope(s) or portion(s) of an antigen, e.g., a target polypeptide disclosed herein
(e.g., u—synuclein) that they recognize or specifically bind. The portion of a target
ptide that specifically interacts with the antigen binding domain of an
antibody is an "epitope," or an "antigenic determinan. A target polypeptide can
comprise a single e, but lly ses at least two epitopes, and can
include any number of epitopes, depending on the size, mation, and type of
antigen. Furthermore, it should be noted that an pe" on a target polypeptide can
be or can include lypeptide elements, e. g., an epitope can e a
carbohydrate side chain.
The minimum size of a peptide or polypeptide epitope for an antibody is
thought to be about four to five amino acids. Peptide or polypeptide epitopes
preferably contain at least seven, more preferably at least nine and most preferably
between at least about 15 to about 30 amino acids. Since a CDR can recognize an
antigenic peptide or polypeptide in its tertiary form, the amino acids comprising an
epitope need not be contiguous, and in some cases, can not even be on the same
peptide chain. A peptide or polypeptide epitope recognized by anti-u—synuclein.
antibodies of the disclosure can contain a sequence of at least 4, at least 5, at least 6,
at least 7, more preferably at least 8, at least 9, at least 10, at least 15, at least 20, at
least 25, or between about 15 to about 30 contiguous or non-contiguous amino acids
of u—synuclein.
By "specifically binds," it is generally meant that an antibody binds to an
epitope via its antigen binding domain, and that the binding entails some
mentarity between the antigen binding domain and the epitope. According to
this definition, an antibody is said to "specifically bind" to an epitope when it binds
to that epitope, via its n binding domain more readily than it would bind to a
random, unrelated epitope. The term "specificity" is used herein to qualify the
relative affinity by which a certain antibody binds to a n epitope. For example,
antibody "A" can be deemed to have a higher specificity for a given epitope than
dy "B," or antibody "A" can be said to bind to epitope "C" with a higher
specificity than it has for related epitope "D."
By "preferentially binds," it is meant that the antibody specifically binds to
an epitope more readily than it would bind to a d, similar, homologous, or
analogous epitope. Thus, an antibody that rentially binds" to a given epitope
would more likely bind to that epitope than to a related e, even though such an
dy can cross-react with the related epitope.
By way of non-limiting example, an antibody can be ered to bind a
first epitope preferentially if it binds said first epitope with a dissociation constant
(KD) that is less than the dy's KD for the second epitope. In another non-
limiting example, an antibody can be considered to bind a first antigen preferentially
if it binds the first epitope with an affinity that is at least one order of magnitude less
than the dy's KD for the second epitope. In another non-limiting example, an
antibody can be considered to bind a first epitope preferentially if it binds the first
epitope with an y that is at least two orders of magnitude less than the
antibody's KD for the second epitope.
In another non-limiting example, an antibody can be considered to bind a
first epitope preferentially if it binds the first epitope with an off rate (k(off)) that is
less than the antibody's k(off) for the second epitope. In another non-limiting
example, an antibody can be considered to bind a first epitope preferentially if it
binds the first e with an affinity that is at least one order of magnitude less
than the antibody's k(off) for the second epitope. In another non-limiting e,
an antibody can be considered to bind a first epitope preferentially if it binds the first
epitope with an affinity that is at least two orders of magnitude less than the
antibody's k(off) for the second epitope.
An antibody or antigen-binding fragment, variant, or derivative disclosed
herein can be said to bind a target polypeptide disclosed herein (e.g., human 0L—
synuclein) or a fragment or variant thereof with an off rate (k(off)) of less than or
equal to 5 X 10'2 sec'l, 10'2 sec'l, 5 X 10'3 sec"1 or 10'3 sec'l. More preferably, an
antibody of the disclosure can be said to bind a target polypeptide sed herein
(e.g., human u—synuclein) or a fragment or variant thereof with an off rate (k(off))
less than or equal to 5 X 10'4 sec'l, 10'4 sec'l, 5 X 10'5 sec'l, or 10'5 sec'l, 5 X 10'6
sec'l, 10'6 sec'l, 5 X 10'7 sec"1 or 10'7 sec'l.
An antibody or antigen-binding fragment, variant, or tive disclosed
herein can be said to bind a target polypeptide disclosed herein (e.g., human 0L—
synuclein) or a fragment or variant thereof with an on rate (k(on)) of greater than or
equal to 103 M"1 sec'l, 5 X 103 M"1 sec'l, 104 M"1 sec"1 or 5 X 104 M"1 sec'l. More
preferably, an antibody of the disclosure can be said to bind a target ptide
disclosed herein (e.g., human (it—synuclein) or a fragment or variant thereof with an
on rate (k(on)) r than or equal to 105 M"1 sec'l, 5 X 105 M"1 sec'l, 106 M"1 sec'l,
or 5 X 106 M"1 sec"1 or 107 M"1 sec'l.
An antibody is said to competitively inhibit binding of a reference antibody
to a given epitope if it preferentially binds to that epitope to the extent that it blocks,
to some , binding of the reference antibody to the epitope. Competitive
inhibition can be determined by any method known in the art, for example,
ition ELISA assays. An antibody can be said to competitively inhibit binding
of the reference antibody to a given epitope by at least 90%, at least 80%, at least
70%, at least 60%, or at least 50%.
As used herein, the term "affinity" refers to a measure of the strength of the
binding of an dual epitope with the CDR of an immunoglobulin molecule.
See, e.g., Harlow et al. (1988) Antibodies: A Laboratory Manual (Cold Spring
Harbor Laboratory Press, 2nd ed.) pages 27-28 The term "sufficient affinity" as used
herein, refers to a sufficient strength of binding of an anti-(x—synuclein antibody or
antigen binding fragment thereof to (it—synuclein or an epitope thereof, to alter the
net efflux of alpha synuclein from brain to blood, or from brain to CSF. As used
herein, the term "net efflux" refers to the total flow of (it—synuclein from brain to
blood or brain to CSF.
As used herein, the term "avidity" refers to the overall stability of the
complex between a population of immunoglobulins and an n, that is, the
onal combining strength of an immunoglobulin mixture with the antigen. See,
e.g., Harlow at pages 29-34. Avidity is related to both the affinity of individual
immunoglobulin molecules in the population with specific es, and also the
valencies of the immunoglobulins and the antigen. For e, the interaction
between a bivalent monoclonal antibody and an antigen with a highly ing
epitope structure, such as a r, would be one of high avidity.
-l8-
Anti-(x—synuclein antibodies or antigen-binding fragments, variants, or
derivatives thereof as disclosed herein can also be described or specified in terms of
their cross-reactivity. As used herein, the term "cross-reactivity" refers to the ability
of an antibody, specific for one n, to react with a second antigen; a measure of
relatedness between two different antigenic substances. Thus, an antibody is cross
reactive if it binds to an epitope other than the one that induced its formation. The
cross reactive epitope generally contains many of the same complementary ural
features as the inducing epitope, and in some cases, can actually fit better than the
original.
For example, certain antibodies have some degree of cross-reactivity, in that
they bind related, but non-identical epitopes, e.g., epitopes with at least 95%, at least
90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least
60%, at least 55%, and at least 50% identity (as ated using methods known in
the art and described herein) to a reference epitope. An antibody can be said to have
little or no cross-reactivity if it does not bind epitopes with less than 95%, less than
90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less
than 60%, less than 55%, and less than 50% identity (as calculated using methods
known in the art and described herein) to a reference epitope. An antibody can be
deemed "highly specific" for a certain epitope, if it does not bind any other ,
ortholog, or homolog of that epitope.
Anti-(x—synuclein g molecules, e.g., antibodies or antigen-binding
fragments, variants or derivatives f, as described herein can also be bed
or specified in terms of their binding affinity to a polypeptide of the disclosure, e. g.,
human u—synuclein. red binding affinities e those with a dissociation
constant or Kd less than 5 x 10'2 M, 10'2 M, 5 x 10'3 M, 10'3 M, 5 x 10'4 M, 10'4 M, 5
x 10‘5 M, 10‘5 M, 5 x106 M, 10‘6 M, 5 X10'7 M, 10‘7 M, 5 X108 M, 10‘8 M, 5 x10-9
M, 10‘9 M, 5 x 10‘10 M,10'10 M, 5 x , 10'11M,5 x 10‘12 2 M, 5 x10—13
M, 10‘13 M, 5 x 10‘14 M, 10‘14 M, 5 x 10‘15 M, or 10‘15 M.
Antibody fragments including single-chain antibodies can comprise the
variable region(s) alone or in combination with the entirety or a portion of the
following: hinge region, CH1, CH2, and CH3 domains. Also included are antigenbinding
fragments also comprising any combination of variable region(s) with a
2012/062430
hinge region, CH1, CH2, and CH3 domains. Binding molecules, e.g., dies, or
antigen-binding fragments thereof disclosed herein can be from any animal origin
including birds and mammals. The dies can be human, murine, donkey,
rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another
embodiment, the variable region can be condricthoid in origin (6.g. , from sharks).
As used herein, the term "chimeric dy" will be held to mean any
antibody n the immunoreactive region or site is obtained or derived from a
first species and the constant region (which can be intact, partial or modified in
accordance with the instant disclosure) is obtained from a second species. For
example, the target binding region or site can be from a non-human source (e. g.,
mouse or primate) and the constant region can be human. Alternatively, a fillly
human binding region can be combined with a non-human (e.g., mouse) constant
region.
As used herein, the term "murinized antibody" or "murinized
immunoglobulin" refers to an antibody comprising one or more CDRs from a human
antibody of the present disclosure; and a human framework region that contains
amino acid substitutions and/or deletions and/or insertions that are based on a mouse
antibody sequence. The human immunoglobulin providing the CDRs is called the
"parent" or "acceptor" and the mouse antibody providing the framework changes is
called the "donor". Constant regions need not be present, but if they are, they are
y substantially identical to mouse dy constant regions, z'.e. at least about
85- 90%, preferably about 95% or more identical. Hence, in some embodiments, a
full length murinized human heavy or light chain immunoglobulin contains a mouse
constant , human CDRs, and a substantially human framework that has a
number of "murinizing" amino acid substitutions. Typically, a "murinized antibody"
is an antibody comprising a zed variable light chain and/or a zed
variable heavy chain. For e, a murinized antibody would not encompass a
typical chimeric antibody, e.g., because the entire variable region of a chimeric
antibody is non-mouse. A modified antibody that has been "murinized" by the
process of "murinization" binds to the same n as the parent antibody that
provides the CDRs and is usually less immunogenic in mice, as compared to the
parent antibody.
As used , the term "engineered antibody" refers to an antibody in
which the variable domain in either the heavy or light chain or both is altered by at
least partial replacement of one or more CDRs from an antibody of known
specificity and, if necessary, by partial framework region replacement and sequence
changing. Although the CDRs can be derived from an antibody of the same class or
even subclass as the antibody from which the framework regions are derived, it is
envisaged that the CDRs will be derived from an antibody of different class and
preferably from an antibody from a different species. An engineered dy in
which one or more " CDRs from a non-human antibody of known specificity
is grafted into a human heavy or light chain framework region is referred to herein as
a ized antibody." It can not be necessary to e all of the CDRs with the
complete CDRs from the donor variable domain to transfer the antigen binding
capacity of one variable domain to another. Rather, it can only be necessary to
transfer those residues that are ary to maintain the ty of the target
binding site.
As used herein, "human" or "fully human" dies include antibodies
having the amino acid sequence of a human immunoglobulin and include antibodies
isolated from human immunoglobulin libraries or from animals transgenic for one or
more human immunoglobulins and that do not express nous
immunoglobulins, as described infra and, for example, in US. Pat. No. 5,939,598 by
Kucherlapati et a]. " or "fillly human" antibodies also include antibodies
comprising at least the variable domain of a heavy chain, or at least the variable
domains of a heavy chain and a light chain, where the variable domain(s) have the
amino acid sequence of human immunoglobulin variable domain(s).
"Human" or "fully human" antibodies also include "human" or "fillly human"
antibodies, as described herein, that comprise, consist essentially of, or consist of,
variants (including tives) of antibody molecules (e. g., the VH regions and/or
VL regions) bed herein, which antibodies or fragments f
immunospecifically bind to an u—synuclein polypeptide or fragment or variant
thereof. Standard techniques known to those of skill in the art can be used to
introduce mutations in the tide sequence encoding a human anti-(x—synuclein
antibody, including, but not limited to, site-directed mutagenesis and PCR-mediated
.21.
mutagenesis which result in amino acid substitutions. Preferably, the variants
(including derivatives) encode less than 50 amino acid substitutions, less than 40
amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino
acid tutions, less than 20 amino acid tutions, less than 15 amino acid
substitutions, less than 10 amino acid substitutions, less than 5 amino acid
substitutions, less than 4 amino acid substitutions, less than 3 amino acid
substitutions, or less than 2 amino acid substitutions relative to the reference VH
region, VHCDRl, , VHCDR3, VL region, VLCDRl, VLCDRZ, or
VLCDR3.
In one aspect, the antibody of the disclosure is a human monoclonal antibody
isolated from a human. Optionally, the framework region of the human antibody is
aligned and adopted in accordance with the pertinent human germ line variable
region sequences in the database; see, e.g., Vbase (http://vbase.mrc-cpe.cam.ac.uk/)
hosted by the MRC Centre for Protein Engineering (Cambridge, UK). For example,
amino acids considered to potentially deviate from the true germ line sequence could
be due to the PCR primer sequences incorporated during the g process.
Compared to artificially ted human-like antibodies such as single chain
antibody fragments (scFvs) from a phage displayed dy library or xenogeneic
mice the human monoclonal antibody of the present disclosure is characterized by (i)
being obtained using the human immune response rather than that of animal
surrogates, z'.e., the antibody has been ted in response to natural u-synuclein in
its relevant conformation in the human body, (ii) haVing ted the indiVidual or
is at least significant for the presence of u-synuclein, and (iii) since the dy is of
human origin the risks of reactivity against self-antigens is minimized. Thus,
in accordance with the disclosure the terms "human monoclonal antibody", "human
monoclonal autoantibody", "human antibody" and the like are used to denote an 0L-
synuclein binding molecule which is of human origin, z'.e. which has been isolated
from a human cell such as a B cell or hybridoma thereof or the cDNA of which has
been directly cloned from mRNA of a human cell, for example a human memory B
cell. A human antibody is still "human" even if amino acid substitutions are made in
the antibody, e.g., to improve binding characteristics.
Antibodies derived from human immunoglobulin libraries or from s
transgenic for one or more human immunoglobulins and that do not s
endogenous immunoglobulins, as described infra and, for example in, US patent no
,939,598 by Kucherlapati et al., are denoted human-like antibodies in order
distinguish them from truly human antibodies of the present disclosure.
As used herein, the term "sample" refers to any biological material obtained
from a t or t. In one aspect, a sample can comprise blood, cerebrospinal
fluid ("CSF"), or urine. In other aspects, a sample can comprise whole blood,
plasma, B cells enriched from blood samples, and cultured cells (e.g., B cells from a
subject). A sample can also include a biopsy or tissue sample including neural .
In still other aspects, a sample can se whole cells and/or a lysate of the cells.
Blood samples can be ted by methods known in the art. In one aspect, the
pellet can be resuspended by vortexing at 4°C in 200 ul buffer (20 mM Tris, pH. 7.5,
0.5% Nonidet, 1 mM EDTA, 1 mM PMSF, 0.1M NaCl, IX Sigma Protease Inhibitor,
and IX Sigma Phosphatase Inhibitors l and 2). The suspension can be kept on ice for
minutes with intermittent vortexing. After spinning at 15,000 x g for 5 minutes at
about 4°C, aliquots of supernatant can be stored at about -70°C.
As used herein, the terms "treat" or "treatment" refer to both therapeutic
treatment and prophylactic or preventative measures, wherein the object is to prevent
or slow down (lessen) an undesired physiological change, infection, or disorder.
Beneficial or desired clinical results e, but are not limited to, alleviation of
symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of
disease, clearance or reduction of an infectious agent in a subject, a delay or slowing
of disease progression, amelioration or palliation of the disease state, and ion
(whether partial or total), whether detectable or undetectable. "Treatment" can also
mean prolonging survival as compared to expected survival if not receiving
ent. Those in need of treatment e those already with the infection,
condition, or disorder as well as those prone to have the condition or disorder or
those in which the condition or disorder is to be prevented.
By "test subject" or "individual" or l" or "patient" or “mammal,” is
meant any subject, particularly a mammalian subject, for whom diagnosis,
sis, or therapy is d. Mammalian subjects include humans, domestic
animals, farm animals, and zoo, , or pet animals such as dogs, cats, guinea
pigs, rabbits, rats, mice, horses, cattle, cows, bears, and so on.
II. TARGET POLYPEPTIDE DESCRIPTION
As used herein, the terms "u-synuclein
, synuclein , a-synuclein"
and "aSyn" are used interchangeably to specifically refer to the native monomer
form of u-synuclein. The term "(x-synuclein" is also used to generally identify other
conformers of u-synuclein, for e, u-synuclein bonded to dopamine-quinone
(DAQ) and oligomers or aggregates of u-synuclein. The term "(x-synuclein" is also
used to refer collectively to all types and forms of u-synuclein. The protein sequence
for human u-synuclein is:
MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVV
HGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKD
QLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA (SEQ ID NO:
The amino acid sequence of u-synuclein can be ved from the literature
and pertinent databases; see, e.g., Ueda et al., PNAS 90: 1282-11286 (1993);
GenBank swissprot: locus SYUA_HUMAN, accession number P37840. u-synuclein
was originally identified in human brains as the precursor protein of the non-B-
amyloid component of (NAC) of mer’s disease (AD) plaques; see, e.g., Ueda
et al., ibid. clein, is a protein of 140 amino acids and exists in its native form
as a random coil. However, changes in pH, molecular crowding, heavy metal
content, and dopamine levels all affect protein conformation. Changes in
conformation to oligomeric, proto-fibrillar, fibrillar, and aggregate moieties are
thought to regulate protein toxicity. Increasing evidence tes that dopamine-
adducted u-synuclein has a faster time course to fibril formation compared to non-
adducted protein. Furthermore, dopamine in the background of u-synuclein
overexpression is toxic.
NAC, a highly hobic domain within u-synuclein, is a peptide
consisting of at least 28 amino acids residues ues 60-87) and ally 35
amino acid residues (residues 61-95). NAC displays a tendency to form a beta-sheet
structure (Iwai et al., Biochemistry, 34: 10139-10145 (1995)). The amino acid
sequences of NAC are described in Jensen et al., Biochem. J. 310: 91-94 (1995);
GenBank accession number $56746 and Ueda et al., ibid.
Disaggregated (x-synuclein or fragments thereof, including NAC, means
ric peptide units. Disaggregated (x-synuclein or fragments f are
lly soluble, and are capable of self-aggregating to form soluble oligomers.
Oligomers of (x-synuclein and fragments thereof are usually soluble and exist
predominantly as u-helices. Monomeric (x-synuclein can be prepared in vitro by
dissolving lyophilized peptide in neat DMSO with sonication. The resulting solution
is centrifuged to remove any insoluble particulates. Aggregated (x-synuclein or
fragments thereof, including NAC, means oligomers of uclein or fragments
thereof which have ate into insoluble B-sheet assemblies. ated 0L-
synuclein or fragments thereof, including NAC, means also means f1brillar
polymers. Fibrils are usually insoluble. Some dies bind either soluble 0L-
synuclein or fragments thereof or aggregated (x-synuclein or fragments thereof. Some
antibodies bind to oligomers of (x-synuclein more strongly than to monomeric forms
or f1brillar forms. Some dies bind both soluble and aggregated (x-synuclein or
fragments thereof, and optionally oligomeric forms as well.
III. x-SYNUCLEIN ANTIBODIES
Antibodies that bind (x-synuclein have been described in the art. See, for
example, International Patent Publication WO 2010/069603, which is herein
incorporated in its entirety by reference.
The human anti-(x-synuclein antibodies described herein specifically bind to
(x-synuclein and epitopes thereof and to various conformations of u-synuclein and
es thereof. For example, disclosed herein are antibodies that specifically bind
(x-synuclein, uclein in its native r form, full-length and truncated 0t-
synuclein and (x-synuclein aggregates. For example, 12F4 dy, as described
herein, binds to full length uclein and to (x-synuclein truncations containing
amino acids (aa) l-60 in as tested by direct ELISA, pointing to an epitope of 12F4
within the N-terminal amphipathic repeat region of alpha synuclein. (See WO
2010/069603).
As used herein, reference to an antibody that "specifically binds",
tively binds", or "preferentially binds" (x-synuclein refers to an antibody that
does not bind other unrelated ns. In one example, an (x-synuclein antibody
disclosed herein can bind u-synuclein or an epitope thereof and show no binding
above about 1.5 times background for other proteins. An antibody that "specifically
binds" or "selectively binds" u-synuclein conformer refers to an antibody that does
not bind all conformations of clein, z'.e., does not bind at least one other 0L-
synuclein conformer. For example, disclosed herein are antibodies that can
distinguish among ric and aggregated forms of (x-synuclein, human and
mouse u-synuclein; fiJll-length u-synuclein and truncated forms as well as human 0t-
synuclein versus [3- and y—synuclein. Since the human anti-u-synuclein antibodies of
the present disclosure have been isolated from a pool of elderly subjects with no
signs of Parkinsonism and exhibiting an (x-synuclein-specific immune response the
anti-(x-synuclein antibodies disclosed herein can also be called "human auto-
antibodies" in order to emphasize that those antibodies were indeed expressed by the
subjects and have not been isolated from, for example a human immunoglobulin
expressing phage library, which hitherto represented one common method for trying
to provide human-like antibodies.
The sure generally relates to a method of diagnosing an ed level
of (x-synuclein in the brain of a test t, comprising administration of an
antibody which specifically binds to (x-synuclein, or an antigen-binding fragment,
variant, or derivative thereof. Anti-u-synuclein antibodies can be used in the
s provided herein. dies that can be used include, but are not limited to
recombinant human uclein antibodies NI-202.3Gl2, 12F4, or NI-202.3D8 and
antigen-binding fragments, variants, or derivatives thereof which are fully described
in International Patent Publication WC 2010/069603.
In certain ments, an x-synuclein antibody or antigen-binding
fragment, variant, or derivative thereof useful in the methods provided herein has an
amino acid sequence that has at least about 80%, about 85%, about 88%, about 89%,
about 90%, about 91%, about 92%, about 93%, about 94%, or about 95% sequence
identity to the amino acid sequence for a reference anti-(x-synuclein antibody
molecule, for example those described herein. In a r embodiment, the binding
molecule shares at least about 96%, about 97%, about 98%, about 99%, or 100%
sequence identity to a reference antibody. In certain embodiments, the antibody or
antigen-binding fragment thereof specifically binds to the same (x-synuclein epitope
as a reference antibody comprising an immunoglobulin heavy chain variable region
(VH) and an immunoglobulin light chain le region (VL), wherein the VH
ses amino acid sequence at least 80%, 85%, 90% 95% or 100% cal to
SEQ ID NO: 2 and the VL comprises amino acid sequence at least 80%, 85%, 90%
95% or 100% identical to SEQ ID NO: 3, as shown in Table 2.
Further disclosed is the antibody or antigen-binding fragment, variant, or
tive thereof useful in the methods ed herein which specifically binds to
the same ct-synuclein epitope as a reference antibody comprising VH and a VL,
wherein the VH comprises amino acid sequence cal to, or cal except for
one, two, three, four, five, or more amino acid substitutions to SEQ ID NO: 2, and
the VL comprises amino acid sequence identical to, or identical except for one, two,
three, four, five, or more amino acid substitutions to SEQ ID NO: 3, as shown in
Table 2.
Some embodiments include an anti-u-synuclein antibody or antigen-binding
fragment, variant, or derivative thereof useful in the methods provided herein
comprising a VH, where one or more of the VHCDRl, VHCDR2 or VHCDR3
regions of the VH are at least 80%, 85%, 90%, 95% or 100% identical to one or
more reference heavy chain VHCDRl, VHCDR2 and/or VHCDR3 amino acid
sequences of one or more of: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, as
shown in Table 3.
Further disclosed is an anti-(x-synuclein antibody or antigen-binding
fragment, variant, or derivative thereof useful in the methods provided herein
comprising a VH, where one or more of the , VHCDR2 or VHCDR3
regions of the VH are identical to, or identical except for four, three, two, or one
amino acid substitutions, to one or more reference heavy chain , VHCDR2
or VHCDR3 amino acid ces of one or more of: SEQ ID NO: 4, SEQ ID NO:
, SEQ ID NO: 6, as shown in Table 3.
Also disclosed is an anti-(x-synuclein antibody or antigen-binding fragment,
variant, or derivative thereof useful in the methods provided herein comprising a VL,
where one or more of the , VLCDR2 or VLCDR3 regions of the VL are at
least 80%, 85%, 90%, 95% or 100% identical to one or more reference heavy chain
VLCDRl, VLCDR2 or VLCDR3 amino acid ces of one or more of: SEQ ID
NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, as shown in Table 3.
Some ments disclose an anti-u-synuclein antibody or antigen-binding
fragment, variant, or derivative thereof useful in the s provided herein
comprising a VL, where one or more of the VLCDRl, VLCDR2 or VLCDR3
s of the VL are cal to, or identical except for four, three, two, or one
amino acid substitutions, to one or more reference heavy chain VLCDRl, VLCDR2
or VLCDR3 amino acid sequences of one or more of: SEQ ID NO: 7, SEQ ID NO:
8, SEQ ID NO: 9, as shown in Table 3.
In other embodiments, an anti-(x-synuclein antibody or antigen-binding
fragment, variant, or derivative thereof useful in the methods provided herein
comprises, consists essentially of, or consists ofVH and VL amino acid sequences at
least 80%, 85%, 90% 95% or 100% identical to: SEQ ID NO: 2 and SEQ ID NO: 3,
as shown in Table 2.
Table 2: Reference VH and VL amino acid sequences*
EVQLVQSGGGLVEPGGSLRLSCAVSGFDFEE QSVLTQPPSVSVSPGQTARITCSGEAL
AWMSWVRQAPGQGLQWVARIKSTADGGT PMQFAHWYQQRPGKAPVIVVYKDSE
TSYAAPVEGRFIISRDDSRNMLYLQMNSLKT RPSGVPERFSGSSSGTTATLTITGVQA
EDTAVYYCTSA_HWGQGTLVTVSS EDEADYYCQSPDSTNTYEVFGGGTK
LTVL
SEQ ID NO: 2 SEQ ID NO: 3
*VH and VL CDRl, CDR2, and CDR3 amino acid sequences are underlined
Table 3: Reference VH and VL CDRl, CDR2, and CDR3 amino acid sequences
VHCDRl VHCDR2 VHCDR3 VLCDRl VLCDR2 VLCDR3
RIKSTADGGTTS AH SGEALPMQF KDSERPS QSPDSTNTYEV
YAAPVEG SEQ ID NO: 9
SEQ ID NO: 5 SEQ ID NO: 7 NO: 8
2012/062430
Also included for use in the methods described herein are polypeptides
encoding anti-(x-synuclein antibodies, or antigen-binding fragments, variants, or
derivatives thereof as described herein, polynucleotides encoding such polypeptides,
vectors comprising such polynucleotides, and host cells comprising such vectors or
polynucleotides, all for producing anti-(x-synuclein antibodies, or antigen-binding
fragments, variants, or tives f for use in the s described herein.
Suitable biologically active variants of anti-(x-synuclein antibodies as
described herein can be used in the methods of the disclosure. Such variants will
retain the desired binding properties of the parent x-synuclein antibody.
Methods for making antibody variants are generally ble in the art.
Methods for mutagenesis and nucleotide sequence alterations are well known
in the art. See, for example, Walker and a, eds. (1983) Techniques in
Molecular y llan Publishing Company, New York); Kunkel, Proc.
Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel et al., Methods Enzymol. [54:367-
382 (1987); Sambrook et al. (1989) Molecular Cloning: A tory Manual (Cold
Spring Harbor, NY); US. Pat. No. 4,873,192; and the references cited therein;
herein incorporated by reference. Guidance as to appropriate amino acid
substitutions that do not affect biological activity of the polypeptide of st can
be found in the model of Dayhoff et al. in Atlas of Protein Sequence and Structure
(Natl. Biomed. Res. Found., Washington, DC), pp. 345-352 (1978), herein
incorporated by nce in its entirety. The model of Dayhoff et al. uses the Point
Accepted Mutation (PAM) amino acid similarity matrix (PAM 250 matrix) to
determine suitable conservative amino acid substitutions. Conservative substitutions,
such as exchanging one amino acid with another having similar properties, can be
preferred. Examples of conservative amino acid substitutions as taught by the PAM
250 matrix of the Dayhoff et al. model include, but are not limited to, Gly<—>Ala,
Val<—>Ile<—>Leu, Asp<—>Glu, Lys<—>Arg, Asn<—>Gln, and Phe<—>Trp<—>Tyr.
Methods for measuring an anti-(x-synuclein antibody or antigen-binding
fragment, variant, or derivative thereof, binding icity include, but are not
limited to, standard competitive binding assays, assays for monitoring
immunoglobulin secretion by T cells or B cells, T cell proliferation assays, apoptosis
assays, ELISA assays, and the like. See, for e, such assays disclosed in WO
93/14125; Shi et al., Immunity 13:633-642 (2000); Kumanogoh et al., J Immunol
[69:1175-1181 (2002); Watanabe et al., Jlmmunol [67:4321-4328 (2001); Wang et
al., Blood 97:3498-3504 (2001); and Giraudon et al., Jlmmunol I72(2):1246-1255
(2004), all of which are herein incorporated by reference.
When discussed herein whether any particular ptide, including the
nt s, CDRs, VH domain or VL domains disclosed herein, is at least
about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%,
about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,
about 99%, or even about 100% identical to another polypeptide, the % identity can
be determined using methods and computer programs/software known in the art such
as, but not limited to, the BESTFIT program nsin ce Analysis
Package, Version 8 for Unix, Genetics Computer Group, University Research Park,
575 Science Drive, Madison, Wis. 53711). BESTFIT uses the local homology
algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482-489, to find the
best segment of homology between two sequences. When using BESTFIT or any
other sequence ent program to determine r a particular sequence is, for
example, 95% identical to a reference sequence according to the present disclosure,
the parameters are set, of course, such that the percentage of identity is calculated
over the filll length of the reference polypeptide sequence and that gaps in homology
of up to 5% of the total number of amino acids in the reference sequence are
allowed.
For purposes of the disclosure, percent sequence identity can be determined
using the Smith-Waterman homology search algorithm using an affine gap search
with a gap open y of 12 and a gap extension penalty of 2, BLOSUM matrix of
62. The Smith-Waterman homology search algorithm is taught in Smith and
Waterman (1981) Adv. Appl. Math. 2:482-489. A variant can, for example, differ
from a reference -synuclein antibody by as few as 1 to 15 amino acid residues,
as few as 1 to 10 amino acid residues, such as 6-10, as few as 5, as few as 4, 3, 2, or
even 1 amino acid residue. A "conservative amino acid substitution" is one in which
the amino acid e is replaced with an amino acid residue having a side chain
with a similar . Families of amino acid residues having side chains with
r charges have been defined in the art. These families include amino acids
with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,
aspartic acid, ic acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, ine, tyrosine, cysteine), nonpolar side chains (e.g., alanine,
, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-
branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be
introduced randomly along all or part of the coding ce, such as by saturation
mutagenesis, and the resultant mutants can be screened for biological activity to
identify mutants that retain activity (e.g., the ability to bind an u-synuclein
polypeptide).
For example, it is possible to introduce mutations only in framework regions
or only in CDR regions of an antibody molecule. uced mutations can be silent
or neutral missense mutations, z'.e. have no, or , effect on an antibody's ability to
bind antigen. These types of mutations can be useful to optimize codon usage, or
improve a hybridoma's antibody production. Alternatively, non-neutral missense
mutations can alter an antibody's y to bind antigen. One of skill in the art
would be able to design and test mutant molecules with desired properties such as no
alteration in n binding activity or alteration in binding activity (e.g.,
improvements in antigen binding activity or change in antibody icity).
Following mutagenesis, the encoded protein can routinely be expressed and the
onal and/or biological activity of the encoded protein, (e.g., ability to
immunospecifically bind at least one epitope of an u-synuclein polypeptide) can be
determined using techniques described herein or by routinely modifying techniques
known in the art.
IV. DIAGNOSING OR NG METHODS USING ANTI-0L-
SYNUCLEIN DIES
The present disclosure relates to the use of an anti-u-synuclein binding
molecule, e.g., antibody or antigen-binding nt thereof, for diagnosing an
elevated level of u-synuclein in the brain of a test subject (e.g., for determining a
subject's risk for developing a synucleinopathic disease), or for monitoring the
progression of a synucleinopathic disease or a response to a synucleinopathic disease
treatment in a test subject.
In certain embodiments, the methods as bed herein are directed to the
use of anti-(x-synuclein antibodies, including antigen-binding nts, variants,
and derivatives thereof, described herein, to diagnose an elevated level of 0L—
synuclein in the brain of a test subject by: (a) assaying the level of u—synuclein in a
blood plasma, or a CSF sample obtained from the test subject at a specified interval
following peripheral administration to the test subject of an anti-(x-synuclein
antibody or n-binding fragment thereof, wherein the anti-(x-synuclein antibody
or fragment thereof can bind u-synuclein with sufficient affinity to alter the net
effluX of clein from brain to blood, or from brain to CSF; (b) ing the
assayed level of the u—synuclein in the test subject to a reference standard; wherein
the difference or rity between the level of u-synuclein in the plasma sample, or
the CSF sample and the reference standard ates with the level of u-synuclein in
the brain of the test subject.
In other embodiments, the methods as described herein are directed to the use
of anti-(x-synuclein antibodies, including antigen-binding fragments, variants, and
derivatives thereof, described herein, to diagnose an elevated level of u—synuclein in
the brain of a test subject by: (a) assaying the level of u—synuclein in a blood plasma,
or a CSF sample ed from the test subject at a specified interval following
peripheral stration to the test subject of an anti-(x-synuclein antibody or
antigen-binding fragment thereof, wherein the anti-(x-synuclein antibody or fragment
thereof stabilizes or sequesters u-synuclein in blood or CSF; (b) ing the
assayed level of the u—synuclein in the test subject to a reference standard; wherein
the difference or similarity between the level of u-synuclein in the plasma sample, or
the CSF sample and the reference standard correlates with the level of u-synuclein in
the brain of the test subject.
In some embodiments, the s as described herein are directed to the
use of anti-(x-synuclein antibodies, including antigen-binding fragments, variants,
and derivatives thereof, described herein, to diagnose an elevated level of 0L—
synuclein in the brain of a test subject by: (a) providing an anti-(x-synuclein antibody
or antigen-binding fragment f, wherein the antibody or fragment thereof can
bind u-synuclein with sufficient ty to alter the net efflux of u-synuclein from
brain to blood, or from brain to CSF; (b) ing a healthcare provider to
peripherally ster the antibody to the test t and obtain a blood plasma
sample, or a CSF sample from the subject at a specified time interval following
administration; (c) assaying the level of clein in the blood plasma sample, or
the CSF sample; (d) comparing the assayed level of clein in the test subject to
a reference standard; n the difference or similarity between the level of the ct-
synuclein in the plasma sample, or the CSF sample and the reference rd
correlates with the level of u-synuclein in the brain of the test subject.
In other embodiments, the methods as described herein are directed to the use
of anti-(x-synuclein antibodies, including antigen-binding fragments, variants, and
derivatives f, described herein, to diagnose an elevated level of u—synuclein in
the brain of a test subject by: (a) providing an anti-(x-synuclein antibody or antigen-
binding fragment thereof, wherein the antibody or fragment thereof, wherein the
x-synuclein antibody or fragment thereof stabilizes or sequesters u-synuclein in
blood or CSF; (b) directing a healthcare provider to erally administer the
antibody to the test subject and obtain a blood plasma sample, or a CSF sample from
the subject at a specified time interval following stration; (c) assaying the
level of u—synuclein in the blood plasma sample, or the CSF sample; (d) comparing
the assayed level of u—synuclein in the test subject to a reference standard; wherein
the difference or similarity between the level of the u-synuclein in the plasma
sample, or the CSF sample and the reference standard correlates with the level of 0L-
synuclein in the brain of the test subject.
In order to apply the methods and systems of the disclosure, samples from a
patient can be obtained before or after the administration of an anti-u—synuclein
dy or antigen-binding nt thereof. Samples can, for example, be
requested by a care provider (e.g., a doctor) or healthcare benefits provider,
obtained and/or processed by the same or a different healthcare provider (e.g., a
nurse, a hospital) or a clinical laboratory, and after processing, the results can be
forwarded to yet another healthcare provider, healthcare benefits provider or the
patient. Similarly, assaying the level of u—synuclein in the sample, comparing the
assayed level of the u—synuclein in the test subject to the reference rd,
evaluation of the results can be med by one or more healthcare providers,
healthcare benefits providers, and/or clinical laboratories.
As used herein, the term “healthcare provider” refers to individuals or
institutions which directly interact and administer to living subjects, e.g., human
patients. Non-limiting examples of healthcare providers include doctors, nurses,
technicians, therapist, pharmacists, counselors, alternative medicine practitioners,
medical facilities, doctor’s offices, hospitals, emergency rooms, clinics, urgent care
centers, alternative medicine clinics/facilities, and any other entity providing general
and/or specialized treatment, assessment, maintenance, therapy, medication, and/or
adVice relating to all, or any n of, a patient’s state of health, ing but not
limited to general medical, lized medical, surgical, and/or any other type of
treatment, assessment, maintenance, therapy, medication and/or adVice.
As used herein, the term “clinical laboratory” refers to a facility for the
examination or processing of materials d from a liVing t, e.g., a human
being. miting examples of processing include biological, biochemical,
serological, chemical, immunohematological, hematological, sical,
cytological, pathological, genetic, or other examination of materials derived from the
human body for the e of providing ation, e.g., for the diagnosis,
prevention, or treatment of any disease or impairment of, or the ment of the
health of liVing subjects, e.g., human beings. These examinations can also include
procedures to collect or otherwise obtain a sample, e, determine, measure, or
otherwise describe the presence or absence of s substances in the body of a
liVing subject, e.g., a human being, or a sample ed from the body of a liVing
subject, e.g., a human being. In certain aspects a clinical laboratory can be
"centralized" or "local", meaning that a small number or a single laboratory makes
all measurements of samples ted from all outside sources. In other aspects,
multiple al laboratories, also referred to as "satellite" or "global" laboratories,
can be validated to all provide standard, reliable results that can be easily compared.
As used herein, the term “healthcare benefits provider” encompasses
indiVidual parties, organizations, or groups providing, presenting, offering, paying
for in whole or in part, or being otherwise associated with giVing a patient access to
one or more healthcare benefits, benefit plans, health insurance, and/or healthcare
expense account programs.
In some aspects, a healthcare er can administer or instruct another
healthcare provider to administer an anti-(x—synuclein antibody or n-binding
fragment thereof. A healthcare er can implement or instruct another healthcare
er or patient to perform the following actions: obtain a sample, process a
sample, submit a sample, receive a sample, transfer a sample, analyze or measure a
sample, quantify a sample, e the results ed after
analyzing/measuring/quantifying a sample, e the results obtained after
analyzing/measuring/quantifying a sample, compare/score the results obtained after
analyzing/measuring/quantifying one or more samples, e the comparison/score
from one or more samples, obtain the comparison/score from one or more samples,
administer a therapy or eutic agent (e.g., an anti-(x—synuclein antibody or
antigen-binding fragment thereof), commence the stration of a therapy, cease
the administration of a y, continue the administration of a therapy, temporarily
interrupt the administration of a therapy, increase the amount of an administered
therapeutic agent, decrease the amount of an administered therapeutic agent,
continue the administration of an amount of a therapeutic agent, increase the
frequency of administration of a therapeutic agent, decrease the frequency of
administration of a therapeutic agent, maintain the same dosing ncy on a
therapeutic agent, replace a therapy or therapeutic agent by at least another therapy
or eutic agent, combine a therapy or therapeutic agent with at least another
therapy or additional therapeutic agent.
In some aspects, a healthcare benefits provider can authorize or deny, for
example, collection of a sample, processing of a sample, submission of a sample,
receipt of a sample, transfer of a sample, analysis or measurement a sample,
quantification a sample, provision of results ed after
analyzing/measuring/quantifying a sample, er of results obtained after
analyzing/measuring/quantifying a sample, comparison/scoring of results obtained
after analyzing/measuring/quantifying one or more samples, transfer of the
comparison/score from one or more samples, administration of a therapy or
therapeutic agent, commencement of the administration of a y or therapeutic
agent, cessation of the administration of a therapy or therapeutic agent, continuation
of the administration of a therapy or therapeutic agent, temporary interruption of the
administration of a therapy or eutic agent, increase of the amount of
stered eutic agent, decrease of the amount of administered therapeutic
agent, continuation of the administration of an amount of a therapeutic agent,
increase in the frequency of administration of a therapeutic agent, decrease in the
frequency of administration of a therapeutic agent, in the same dosing
frequency on a therapeutic agent, replace a therapy or therapeutic agent by at least
another therapy or therapeutic agent, or combine a therapy or therapeutic agent with
at least another therapy or additional therapeutic agent.
In addition a healthcare s providers can, e.g., authorize or deny the
prescription of a therapy, authorize or deny coverage for y, authorize or deny
reimbursement for the cost of therapy, determine or deny eligibility for y, etc.
In some aspects, a clinical laboratory can, for example, collect or obtain a
sample, process a sample, submit a sample, receive a sample, transfer a sample,
analyze or measure a , quantify a sample, provide the results ed after
analyzing/measuring/quantifying a sample, receive the results obtained after
analyzing/measuring/quantifying a sample, e/score the results obtained after
analyzing/measuring/quantifying one or more samples, provide the comparison/score
from one or more samples, obtain the comparison/score from one or more samples,
The above enumerated actions can be performed by a healthcare provider,
healthcare benefits provider, or patient automatically using a er-implemented
method (e.g., via a web service or stand-alone computer system).
As used herein the term "directing a healthcare provider" includes orally
directing a healthcare provider, or directing a healthcare provider by using a written
order, or both.
In some embodiments the methods as described herein are directed to the use
of x-synuclein antibodies, including antigen-binding fragments, variants, and
derivatives thereof, described herein, to diagnose an elevated level of u—synuclein in
the brain of a test subject by: (a) peripherally administering an anti-(x-synuclein
antibody or antigen-binding fragment thereof to the test t, wherein the
antibody or fragment f can bind u-synuclein with sufficient affinity to alter the
net efflux of the u-synuclein from brain to blood, or from brain to CSF; (b) obtaining
a blood plasma sample, or a CSF sample from the test t at a specified time
interval following administration, and submitting the plasma , or the CSF
sample for determination of the level of the u—synuclein; (c) comparing the level of
the u—synuclein in blood plasma sample to a reference standard; n the
ence or rity between the level of the u-synuclein in the plasma sample, or
the CSF sample and the nce standard correlates with level of the u-synuclein in
the brain of the test subject.
In other embodiments the methods as described herein are directed to the use
of anti-(x-synuclein antibodies, including antigen-binding fragments, variants, and
derivatives thereof, described herein, to diagnose an elevated level of u—synuclein in
the brain of a test subject by: (a) peripherally stering an anti-(x-synuclein
antibody or antigen-binding fragment f to the test subject, wherein the
antibody or fragment thereof stabilizes or sequesters u-synuclein in blood or CSF;
(b) obtaining a blood plasma sample, or a CSF sample from the test subject at a
specified time interval following stration, and submitting the plasma sample,
or the CSF sample for determination of the level of the u—synuclein; (c) comparing
the level of the u—synuclein in blood plasma sample to a reference standard; wherein
the difference or similarity between the level of the u-synuclein in the plasma
sample, or the CSF sample and the reference standard correlates with level of the OL-
synuclein in the brain of the test subject. In some embodiments, anti-(x-synuclein
antibodies, including antigen-binding fragments, variants, and derivatives thereof,
described herein, can bind u-synuclein with sufficient affinity to alter the net efflux
of the u-synuclein from brain to blood, or from brain to CSF and stabilize or
sequester u-synuclein in blood or CSF.
The test subject to be diagnosed can be asymptomatic or preclinical for the
disease. In specific ments test subjects include individuals who are pre-
symptomatic or have preclinical synucleopathic disease.
In specific embodiments, the "reference standard" in the method described
herein comprises measured levels of u—synuclein in one or more control subjects,
n the control subjects include normal healthy indiViduals, and indiViduals with
synucleinopathies of varying severity. For e, the control subject has a
synucleinopathic disease, for example Parkinson's disease (PD), dementia with Lewy
bodies (DLB) or the Lewy body variant of Alzheimer's disease (LBVAD), wherein a
similarity between the level of u-synuclein and the reference standard indicates that
the t to be sed has a synucleinopathic disease. Alternatively, or in
addition as a second control the l subject does not have a synucleinopathic
disease, wherein a difference between the level of clein and the reference
standard indicates that the subject to be diagnosed has a synucleinopathic disease.
Preferably, the subject to be diagnosed and the control subject(s) are age-matched.
In some embodiments the s as described herein, further comprise
comparing the level of the u—synuclein in the plasma sample (i.e., test sample) to a
plasma sample (z'.e., baseline sample) obtained from the test subject prior to
administration of the anti-(x-synuclein antibody including antigen-binding fragments,
variants, and derivatives thereof In other embodiments the methods as described
herein, further comprise comparing the level of the u—synuclein in the CSF sample
(i.e., test sample) to a CSF sample (i.e., baseline sample) obtained from the test
t prior to administration of the anti-(x-synuclein antibody including antigenbinding
fragments, variants, and derivatives thereof. For example, the comparison
can be made to a ne sample instead of or in addition to comparison with a
reference standard. In this respect, the baseline sample can be used to calibrate the
test samples to the reference rd (e.g., the measurement is a ence or a ratio
rather than an absolute value).
By a further embodiment, the anti-(x-synuclein binding molecules, in
particular anti-u-synuclein antibodies, as bed herein, can also be used in a
method for the diagnosis of a disorder in an individual by obtaining a body fluid
sample from the tested individual which may be a blood sample, a lymph , a
CSF sample, or any other body fluid sample, and contacting the body fluid sample
with an anti-(x-synuclein antibody as described herein, under conditions enabling the
formation of antibody-antigen complexes. The level of such complexes is then
determined by methods known in the art, a level significantly higher than that
formed in a control sample indicating the disease in the tested individual. In the
same manner, the specific antigen bound by the x-synuclein antibodies as
described herein can also be used. Thus, the disclosure relates to an in vitro
immunoassay comprising an anti-(x-synuclein binding molecule, e.g., antibody or
antigen-binding nt thereof of the disclosure.
The level of u-synuclein can be assessed by any suitable method known in
the art comprising, e. g., analyzing u-synuclein by one or more techniques chosen
from Western blot, immunoprecipitation, enzyme-linked immunosorbent assay
(ELISA), radioimmunoassay (RIA), cent activated cell sorting (FACS), two-
dimensional gel ophoresis, mass spectroscopy (MS), matrix-assisted laser
desorption/ionization-time of flight-MS (MALDI-TOF), surface-enhanced laser
desorption ionization-time of flight (SELDI-TOF), high performance liquid
tography (HPLC), fast protein liquid chromatography (FPLC),
multidimensional liquid chromatography (LC) followed by tandem mass
ometry (MS/MS), and laser densitometry. Preferably, said in vivo imaging of
u-synuclein comprises on emission tomography (PET), single photon emission
tomography (SPECT), near infrared (NIR) optical imaging or magnetic resonance
imaging (MRI).
As is well known in the medical arts, dosages for any one patient depends
upon many factors, including the patient's size, body surface area, age, the particular
compound to be administered, sex, time and route of stration, general ,
and other drugs being administered concurrently. Generally, the dosage can range,
e. g., from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg (e.g., 0.02
mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.), of the host body
weight. For e dosages can be 1 mg/kg body weight or 10 mg/kg body weight
or within the range of 1-10 mg/kg. Doses intermediate in the above ranges are also
intended to be within the scope of the disclosure. The term "peripheral
administration" is described elsewhere herein.
In certain embodiments, an antibody-based array can be used, which is for
example loaded with anti-(x-synuclein dies or equivalent antigen-binding
molecules of the disclosure which specifically recognize u-synuclein. Design of
microarray immunoassays is summarized in Kusnezow et al., Mol. Cell Proteomics
-1696 (2006). Accordingly, the disclosure also relates to microarrays loaded
with anti-(x-synuclein binding molecules identified in accordance with the present
disclosure.
In some embodiments, the methods as bed herein are also ed to
the use of anti-(x-synuclein antibodies, including antigen-binding fragments, variants,
and derivatives thereof, to track the u-synuclein level in the brain of a subject being
treated for a synucleinopathic disease, comprising assaying the level of clein
in the t’s blood plasma, or the subject’s CSF at a specified time following
peripheral administration of an anti-(x-synuclein antibody or antigen-binding
fragment thereof, wherein the antibody or fragment thereof can bind u-synuclein
with sufficient affinity to alter the net efflux of the u-synuclein from brain to blood,
or brain to CSF; and wherein the clein level in the subject’s blood , or
the t's CSF correlates with the level in the subject’s brain. In specific
embodiments, the method as described herein, further comprises assaying the level
of u—synuclein in the subject’s blood plasma, or the subject’s CSF at a specified time
following additional peripheral administrations of the anti-(x-synuclein antibody or
n-binding fragment thereof, y plotting the change in the u—synuclein
level in the subject’s brain over time.
In some embodiments, the methods as bed herein are also directed to
the use of anti-(x-synuclein antibodies, including antigen-binding fragments, variants,
and tives thereof, to track the u-synuclein level in the brain of a subject being
treated for a synucleinopathic disease, comprising assaying the level of u—synuclein
in the subject’s blood plasma, or the t’s CSF at a specified time following
peripheral administration of an anti-(x-synuclein antibody or antigen-binding
fragment thereof, wherein the antibody or fragment thereof izes or sequesters
u-synuclein in blood or CSF; and wherein the u—synuclein level in the subject’s
blood plasma, or the subject's CSF correlates with the level in the subject’s brain. In
specific embodiments, the method as described herein, further comprises assaying
the level of u—synuclein in the subject’s blood plasma, or the t’s CSF at a
specified time following additional peripheral administrations of the anti-(x-synuclein
antibody or n-binding fragment thereof, thereby plotting the change in the ct—
synuclein level in the subject’s brain over time.
Some embodiments include methods as described herein, where the specified
time interval is less than 12 months, less than 11 months, less than 10 months, less
than 9 months, less than 8 months, less than 7 months, less than 6 months, less than
months, less than 4 months, less than 3 , less than 2 months, less than a
month, less than a week, or less than or equal to 24 hours, or less than or equal to 3
hours.
V. ITIONS AND ADMINISTRATION METHODS
The s of ing and stering anti-(x—synuclein dies, or
antigen-binding fragments, variants, or derivatives thereof to a subject in need
thereof are well known to or are readily determined by those skilled in the art. The
route of administration of an anti-(x—synuclein antibody, or antigen-binding
fragment, variant, or derivative thereof, can be, for example, oral, parenteral, by
inhalation or topical. The term "peripheral administration" as used herein includes,
e.g., intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or
vaginal administration. While all these forms of administration are clearly
contemplated as being within the scope of the disclosure, an example of a form for
administration would be a solution for injection, in particular for intravenous or
intraarterial injection or drip. A suitable pharmaceutical composition for injection
can comprise a buffer (6. g. acetate, phosphate or citrate buffer), a surfactant (6. g.
polysorbate), optionally a stabilizer agent (6.g. human albumin), etc.
As sed herein, anti-u—synuclein dies, or antigen-binding
fragments, ts, or derivatives thereof can be formulated so as to facilitate
administration and promote stability of the active agent. In certain embodiments,
pharmaceutical compositions in accordance with the present sure comprise a
pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline,
non-toxic buffers, preservatives and the like. For the es of the instant
application, a pharmaceutically effective amount of an anti-(x—synuclein antibody, or
antigen-binding fragment, variant, or derivative thereof, shall be held to mean an
amount sufficient to achieve effective g to a target and to e a benefit,
e.g., to alter the net efflux of u-synuclein from brain to blood, or to alter the net
efflux of clein from brain to CSF.
The pharmaceutical compositions used in this disclosure comprise
pharmaceutically acceptable carriers, including, e.g., ion exchangers, alumina,
aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer
substances such as phosphates, glycine, sorbic acid, potassium e, partial
glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes,
such as protamine sulfate, disodium hydrogen ate, potassium hydrogen
phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,
polyvinyl pyrrolidone, cellulose-based substances, polyethylene , sodium
carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-
block polymers, polyethylene glycol, and wool fat.
Preparations for peripheral administration include sterile aqueous or non-
aqueous ons, suspensions, and ons. Examples of non-aqueous solvents
are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and
injectable organic esters such as ethyl oleate. Aqueous carriers include, e. g., water,
alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered
media. In the subject disclosure, pharmaceutically acceptable carriers include, but
are not limited to, 0.0l-O.l M phosphate buffer or 0.8% saline. Other common
parenteral vehicles include sodium phosphate solutions, Ringer's dextrose, se
and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include
fluid and nt replenishers, electrolyte replenishers, such as those based on
Ringer's dextrose, and the like. Preservatives and other additives can also be present
such as, for example, antimicrobials, idants, chelating agents, and inert gases
and the like.
More particularly, pharmaceutical compositions suitable for injectable use
include sterile aqueous ons (where water soluble) or sions and sterile
powders for the extemporaneous preparation of sterile injectable solutions or
dispersions. In such cases, the composition must be sterile and should be fluid to the
extent that easy syringability exists. It should be stable under the conditions of
cture and storage and will preferably be preserved against the contaminating
action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or
dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol,
propylene glycol, and liquid hylene glycol, and the like), and le es
thereof. The proper fluidity can be maintained, for e, by the use of a coating
such as lecithin, by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. Suitable formulations for use in the
therapeutic methods disclosed herein are described in Remington's ceutical
Sciences (Mack Publishing Co.) 16th ed. (1980).
Prevention of the action of microorganisms can be achieved by various
antibacterial and antifilngal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal and the like. In many cases, it will be preferable to include
isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or
sodium chloride in the composition. Prolonged absorption of the injectable
compositions can be brought about by including in the composition an agent which
delays tion, for example, aluminum monostearate and gelatin.
In any case, sterile injectable solutions can be prepared by incorporating an
active compound (e.g., an anti-(x-antibody, or antigen-binding fragment, variant, or
derivative thereof, by itself or in combination with other active agents) in the
required amount in an appropriate solvent with one or a combination of ingredients
enumerated herein, as required, followed by filtered sterilization. lly,
dispersions are prepared by orating the active compound into a sterile e,
which ns a basic sion medium and the ed other ingredients from
those ated above. In the case of sterile powders for the preparation of sterile
injectable solutions, the preferred methods of preparation are vacuum drying and
freeze-drying, which yields a powder of an active ingredient plus any additional
desired ingredient from a previously sterile-filtered solution thereof. The
preparations for ions are processed, filled into containers such as ampoules,
bags, bottles, syringes or vials, and sealed under aseptic ions according to
methods known in the art. Further, the preparations can be ed and sold in the
form of a kit. Such articles of manufacture can have labels or package inserts
indicating that the associated compositions are useful for treating a subject suffering
from, or predisposed to a disease or disorder.
Parenteral formulations can be a single bolus dose, an infilsion or a loading
bolus dose followed with a nance dose. These compositions can be
administered at specific fixed or variable intervals, e.g., once a day, or on an "as
" basis.
n pharmaceutical compositions, as disclosed herein, can be orally
administered in an acceptable dosage form including, e.g., capsules, tablets, aqueous
suspensions or solutions. Certain pharmaceutical itions also can be
administered by nasal aerosol or inhalation. Such compositions can be prepared as
solutions in saline, employing benzyl alcohol or other suitable preservatives,
absorption promoters to e bioavailability, and/or other conventional
solubilizing or dispersing agents.
The amount of an anti-(x—synuclein antibody, or fragment, variant, or
derivative thereof, to be combined with the carrier als to produce a single
dosage form will vary depending upon the host treated and the particular mode of
administration. The ition can be administered as a single dose, multiple
doses or over an ished period of time in an on. Dosage regimens also can
be adjusted to provide the optimum d response (e. g., a therapeutic or
prophylactic response).
The practice of the disclosure will , unless otherwise indicated,
conventional techniques of cell biology, cell culture, lar biology, transgenic
biology, microbiology, recombinant DNA, and immunology, which are within the
skill of the art. Such techniques are explained fillly in the literature. See, for
example, Molecular Cloning A Laboratory Manual, 2nd Ed., Sambrook et al., ed.,
Cold Spring Harbor Laboratory Press: (1989); Molecular Cloning: A Laboratory
Manual, Sambrook et al., ed., Cold s Harbor Laboratory, New York (1992),
DNA Cloning, D. N. Glover ed., Volumes I and II (1985); Oligonucleotide Synthesis,
M. J. Gait ed., (1984); Mullis et al. US. Pat. No: 195; Nucleic Acid
Hybridization, B. D. Hames & S. J. Higgins eds. (1984); Transcription And
Translation, B. D. Hames & S. J. Higgins eds. (1984); Culture OfAnimal Cells, R. I.
Freshney, Alan R. Liss, Inc., (1987); Immobilized Cells And Enzymes, IRL Press,
(1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise,
Methods In Enzymology, Academic Press, Inc., N.Y.; Gene Transfer Vectors For
Mammalian Cells, J. H. Miller and M. P. Calos eds., Cold Spring Harbor Laboratory
(1987); s In Enzymology, Vols. 154 and 155 (Wu et al. eds.);
Immunochemical Methods In Cell And Molecular Biology, Caner and Walker, eds.,
Academic Press, London (1987); Handbook 0fExperimental Immunology, Volumes
I-IV, D. M. Weir and C. C. Blackwell, eds., ; Manipulating the Mouse
WO 66818
Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1986);
and in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and
Sons, Baltimore, Maryland (1989).
General principles of antibody engineering are set forth in Antibody
Engineering, 2nd edition, C.A.K. Borrebaeck, Ed., Oxford UniV. Press (1995).
General principles of protein engineering are set forth in Protein Engineering, A
Practical Approach, Rickwood, D., et al., Eds., IRL Press at Oxford UniV. Press,
Oxford, Eng. . General principles of dies and antibody-hapten binding
are set forth in: Nisonoff, A., Molecular Immunology, 2nd ed., Sinauer Associates,
Sunderland, MA ; and Steward, M.W., Antibodies, Their Structure and
Function, Chapman and Hall, New York, NY (1984). Additionally, standard
methods in logy known in the art and not specifically described are
generally followed as in Current ols in Immunology, John Wiley & Sons,
New York; Stites et al. (eds), Basic and Clinical -Immunology (8th ed.), Appleton &
Lange, Norwalk, CT (1994) and l and Shiigi (eds), Selected Methods in
Cellular Immunology, W.H. Freeman and Co., New York (1980).
Standard reference works setting forth general principles of logy
include Current Protocols in Immunology, John Wiley & Sons, New York; Klein, J
Immunology: The Science ofSelf-NonselfDiscrimination, John Wiley & Sons, New
York (1982); Kennett, R., et al., eds., Monoclonal Antibodies, I-beridoma: A New
Dimension in Biological Analyses, Plenum Press, New York (1980); Campbell, A.,
“Monoclonal Antibody Technology” in Burden, R., et al., eds., Laboratory
Techniques in Biochemistry and Molecular Biology, Vol. 13, Elsevere, dam
(1984), Kuby Immunnology 4th ed. Ed. Richard A. Goldsby, Thomas J. Kindt and
a A. Osborne, H. Freemand & Co. (2000); Roitt, 1., Brostoff, J. and Male D.,
Immunology 6th ed. London: Mosby (2001); Abbas A., Abul, A. and an, A.,
Cellular and Molecular Immunology Ed. 5, ElseVier Health es Division
(2005); Kontermann and Dubel, Antibody Engineering, Springer Verlan (2001);
Sambrook and Russell, Molecular Cloning: A Laboratory Manual. Cold Spring
Harbor Press (2001); Lewin, Genes VIII, Prentice Hall ; Harlow and Lane,
dies: A Laboratory Manual, Cold Spring Harbor Press (1988); Dieffenbach
and Dveksler, PCR Primer Cold Spring Harbor Press (2003).
EXAMPLES
ed ptions of conventional methods, such as those employed
herein can be found in the cited literature. Unless indicated otherwise below,
identification of (x-synuclein-specific B cells and molecular cloning of (x-synuclein
antibodies displaying specificity of interest as well as their recombinant expression
and functional characterization has been or can be performed as described in the
Examples and Supplementary Methods section of international applications
published as W02008/081008, and international ations
hed as W02010/069603, the disclosure content of which
is orated herein by nce in its ty.
Example 1: Dose dependent human (x-synuclein plasma spike upon 12F4 administration
in transgenic mice overexpressing human (x-synuclein
This example describes ination of human u-synuclein levels in mouse
plasma in transgenic mice overexpressing human (x-synuclein after ion of 12F4
antibody. Three and half months old transgenic mice overexpressing human wild-
type (wt) u-synuclein (PDGFB-h[wt] u-synuclein; z'.e., D-line; Masliah et al.,
Science, 287(5456):1265-9 (2000) were intraperitoneally injected with a single dose
of 0, 0.3, l, 3, 10 or 30 mg/kg 12F4 antibody. Functional recombinant monoclonal
antibodies 12F4 and chimeric 12F4 were obtained upon co-transfection into CHO
cells (or any other appropriate recipient cell line of human or mouse origin) of an Ig-
heavy-chain expression vector and a kappa or lambda Ig-light-chain expression
. Recombinant monoclonal antibody was subsequently purified from the
conditioned medium using a standard Protein A column purification as described in
W02008/081008. Recombinant human monoclonal antibody can be produced in
unlimited quantities using either transiently or stably transfected cells. Chimeric
12F4 antibody had primer induced mutations at the ini of the Ig-variable
regions being adjusted to the germ line (GL) sequences of human variable heavy and
light chains (see W02010/069603), and was expressed as a chimeric molecule where
the adjusted human le domains were fused to mouse IgG2a constant regions.
2012/062430
D-line transgenic (x-synuclein mice were kept under standard housing
conditions on a reversed h dark cycle with free access to food and water.
The treatment groups were balanced for age and gender. 24 hrs after the injection,
plasma samples were prepared and plasma trations of human (x-synuclein
were determined by a sandwich ELISA (Invitrogen, USA). Plasma samples were
diluted 1:4 and standard was prepared in dilution buffer with 1:4 plasma of wt mice.
Results were controlled for influence of l2F4 antibody on ELISA readings. l2F4
antibody plasma levels were determined by a human ch capture ELISA using
recombinant l2F4 of known concentration as standard. Standard was prepared in
PBS containing diluted plasma from the wild-type mice.
Plasma levels of human (x-synuclein were significantly increased after a
single dose of l, 3, 10 or 30 mg/kg l2F4 antibody when compared to vehicle control.
The increase in plasma human (x-synuclein is significantly dose dependent (Figures 1
A-B). No significant levels of human u-synuclein were detected in mice that were
treated with vehicle only (Figure 1B).
Example 2: Time course of (x-synuclein plasma spike and l2F4 plasma concentration
This example bes the time course of changes in human u-synuclein
levels and l2F4 antibody levels in mouse plasma in transgenic mice overexpressing
human uclein, measured over time. Eight month old transgenic mice
overexpressing human clein A53T (Prp-h[A53T] u-synuclein] (Giasson et al.,
Neuron, 34: 521-533 ) were intraperitoneally injected with a single dose of 5
mg/kg l2F4 antibody and plasma samples were collected at time points 0, l, 24, 72
and 168 hrs post injection. Plasma human (x-synuclein was quantified by ELISA as
described in Example 1. Figure 2 shows that plasma human (x-synuclein peaks
already in 1 hr time point and then declines over time. On the other hand, highest
levels of l2F4 antibody were found at 24 hrs time point and l2F4 plasma levels
ed to decline more slowly than human u-synuclein levels.
Example 3: Acute high dose l2F4 treatment of transgenic mice overexpressing human 0L-
synuclein reduces brain human u-synuclein levels that correlate with plasma human (1-
synuclein levels
This e describes the ination of human (x-synuclein levels in
brain samples after injection with 12F4 antibody. Three and half months old
transgenic mice overexpressing human ype (x-synuclein (PDGFB-h[wt] 0L-
synuclein; D-line] were intraperitoneally injected with four 50 mg/kg doses of 12F4
antibody within 8 days (72, 144 and 192 hrs post first injection). 24 hrs after the last
injection animals were sacrificed and perfused with PBS. Cortex and hippocampus
were homogenized in PBS and soluble (PBS-soluble) and insoluble (PBS-insoluble)
brain fractions were prepared by differential centrifugation. Specifically, brains were
removed, dissected and frozen at -80°C. Frozen brain tissues were homogenized in
volumes (v/w) of PBS using a dounce nizer (500 rpm, 30 strokes) and
ation for 1 min. Cell debris was removed by centrifugation at 5000 g for 5
min (4°C). Supernatant (SN) was centrifuged for 1 hr (4°C) at 35000 rpm (Ti51
rotor; Beckman-Coulter). Resulting SN was designated soluble fraction. The pellet
was resuspended in 1% Trition PBS and sonicated (3x 1min). This fraction was
designated insoluble fraction.
Human (x-synuclein levels in both fractions were quantified by a sandwich
ELISA (Invitrogen, USA) and normalized to protein content. As shown in Figure 3A
cortical soluble human (x-synuclein levels of 12F4 treated mice were significantly
reduced by 34% (190 :: 29 ug/g for 12F4 vs. 288 :: 36 ug/g for vehicle l,
n=10, p<0.05, Student’s Test). Similarly, a 33% reduction of e hippocampal
human uclein (Figure 3B)-(119 :: 22 ug/g for l2F4 vs. 178 :: 30 ug/g for
vehicle control, n=9-10, p=0.l4, Student’s Test) and a 26% ion of insoluble
hippocampal human (x-synuclein (Figure 3 :: 3 ug/g for l2F4 vs. 31 :: 6 ug/g
for vehicle control, n=9-10, p=0.29, Student’s Test) after acute 12F4 treatment was
observed. These s show that a short treatment with 12F4 leads to a reduction of
brain (x-synuclein pathology in transgenic mice overexpressing human u-synuclein.
In order to see if the observed human uclein plasma rise was linked to
brain (x-synuclein ogy, plasma human (x-synuclein levels were plotted against
brain human (x-synuclein levels. There was a highly significant correlation between
plasma human u-synuclein levels and soluble cortical human u-synuclein levels
(Figure 4A), with soluble hippocampal human (x-synuclein levels (Figure 4B), and
insoluble hippocampal human (x-synuclein levels (Figure 4C) after acute 12F4
2012/062430
treatment. No correlation between plasma and brain (x-synuclein was observed upon
vehicle treatment.
Example 4: Correlation n plasma and brain human (x-synuclein levels after chronic
12F4 treatment of transgenic mice overexpressing human (x-synuclein
This example describes the determination of human (x-synuclein levels in
brain samples after weekly injections with 12F4 antibody for six months.
Six month old transgenic mice overexpressing human wt (x-synuclein A30P
(Thyl-h[A30P]-0t-synuclein) (Kahle et al., Am J Pathol., 159(6): 2215-2225 (2001))
were intraperitoneally injected weekly with 10 mg/kg chimeric 12F4 for 6 months.
Plasma and brain samples were prepared 24 hrs after last ion.
Cortex/hippocampus were homogenized together in PBS and e (PBS-soluble)
and insoluble (PBS-insoluble) brains fractions were prepared by ential
centrifilgation as described in Example 3. Human (x-synuclein levels in both
fractions were quantified by ELISA and normalized to protein content as described
in Example 3. Plasma human (x-synuclein levels (Figure 5A) and chimeric 12F4
levels e 5B) were determined by ELISA. Plasma human u-synuclein levels
were determined as bed in Example 1, and chimeric 12F4 plasma levels were
determined using a direct (x-synuclein ELISA using recombinant chimeric 12F4 of
known concentration as standard.
In order to see if plasma and brain (x-synuclein correlate upon chronic
treatment with chimeric 12F4, plasma human (x-synuclein levels were plotted against
human brain uclein levels. There was a significant correlation between plasma
and brain a-synuclein levels after chronic treatment for six month with chimeric
12F4 of transgenic mice pressing human u-synuclein. (Figure 5C).
Example 5: (x-synuclein cerebrospinal fluid (CSF) spike upon 12F4 stration in
cynomolgus monkeys
This example describes determination of 12F4 levels in serum and
cerebrospinal fluid (CSF), as well as endogenous (x-synuclein levels in CSF of
cynomolgus monkeys upon 12F4 administration. Three male na'ive cynomolgus
monkeys were intravenously injected with a single dose of 10 mg/kg 12F4. Animals
were fasted 1 to 12 minutes prior to the 12F4 administration and for the 2 hours post-
dose CSF sample collection. Blood samples (approximately 0.5 ml/sample) were
collected from femoral vein/artery at 0.5, 2, 5, 24, 48, 72, 96, 168, 240, 336, 408,
504, 672 and 840 hours post-dose. Samples were allowed to clot for at least 30
minutes and fuged under ambient conditions following completion of the
sample collection at each intervall. The resulting serum was separated and stored
fozen at -50 to -900 until d on dry ice for analysis. 12F4 serum levels were
determined by a sandwich ELISA (Covance).
12F4 and endogenous (x-synuclein concentrations were also determined by
ELISA (Covance) in the CSF samples collected from the cistema magna at various
time points post-dose. Prior to the CSF sample collection animals were sedated with
an intramuscular (1M) injection of 0.1 mg/kg acepromazine maleate, with additional
maintenance doses as necessary. Anesthesia was d with an IM injection of 20
mg/kg Ketamine. The back of the head was shaved for access to the cistema magna
and the access site was prepared with chlorhexidine scrub and chlorhexidine solution
within a sterile field. The animal was placed in a lateral recumbent on and the
head was brought forward until the chin rested on the chest. Using aseptic technique,
an over the needle catheter was used to access the a magna. The animals were
stered 0.01 mg/kg buprenorphine 1M three times daily, approximately every 6
to 9 hours (beginning prior to CSF collection) on each day of the sample collection.
ing anesthesia and CSF sampling, the animals were closely monitored during
ry for physiological disturbances including cardiovascular/respiratory
depression, hypothermia, and excessive bleeding from the surgical site. The CSF
samples (approximately 0.2 mL/sample) were collected at the 2, 24, 72, 168, 336,
504 and 672 hours post-dose from the cistema magna and placed on ice. The
samples were stored frozen at -50 to -90°C until shipped on dry ice for analysis.
CSF/serum 12F4 ratio was around 0.1 % as expected for a human IgG
antibody. CSF clein sed about 5-fold upon 12F4 treatment. (Figure 6).
Example 6: In vivo microdialysis in transgenic (x-synuclein mice
This example describes determination of u-synuclein levels in plasma and
brain interstitial fluid (ISF) upon 12F4 administration in transgenic (x-synuclein
A53T mice. In vivo ialysis in transgenic (x-synuclein mouse ISF was
med upon administration of 12F4 or vehicle control (control IgG antibody).
Specifically, guide as were stereotaxically implanted in the striatum of 6-9
months old A53T (x-synuclein transgenic mice (B6;C3-Tg(PrP-
SNCA*A53T)83Vle/J) under isoflurane anesthesia %). The head was shaved
and the skin was cut with a sterile scalpel to expose the skull. Bore holes were made
above the right striatum according to the atlas of Paxinos and Franklin (The Mouse
Brain in Stereotaxic Coordinates, Second Edition (2004)) (coordinates, AP = +0.5
mm, ML = -2.2 mm, DV = -2.4 mm). CMA-12 guide cannulas (CMA Microdialysis
AB, Sweden) were inserted and fixed to the skull with stainless steel screws and
dental cement. Mice were removed from the stereotaxic device and allowed to
recover in individual cages. Five days after surgery, mice were removed to the
microdialysis cage(s). CMA-12 custom made probes (2 mm, 100 kDa cut-off) were
inserted and connected to a CMA pump with a constant flow rate of 0.6 ul/min.
ion was performed in artificial CSF containing BSA as an osmotic agent. Prior
to sample collection, the probe was allowed to equilibrate for 4-20 hours with the
same flow rate. Baseline samples were collected mostly bihourly for 2 hrs. 12F4 or
e l, were intraperitoneally injected in a single dose of 30 mg/kg. Upon
injection s were collected hourly for approximately 24 hours. All samples
were collected using a refrigerated fraction collector and stored at -80°C until
ed by an in house ultra-sensitive (x-synuclein sandwich ELISA
(Emmanouilidou et al., PLOS ONE 6(7): e22225 (2011)). Plasma samples were also
collected pre-dose and at 2 and 24 hours post-dose, and then were analyzed by an
human (x-synuclein specific sandwich ELISA (Invitrogen, Carlsbad CA).
There was an approximately 60% reduction in extracellular, ISF (x-synuclein,
2-3 hours post 12F4 administration. Vehicle control did not alter ISF (x-synuclein
levels in the microdialysate. (Figure 7).
Example 7: Effect of anti-(x-synuclein antibody on cerebrospinal fluid (CSF) (x-synuclein
concentration in an AAV-(x-synuclein rat model
The effect of x-synuclein antibody on CSF (x-synuclein concentration
will be evaluated in an Adeno-associated viral (AAV)-0t-synuclein rat model. AAV
2012/062430
vectors will be created that express either the wild-type human u-synuclein or one of
the human u-synuclein sequence variants that are associated with familial
Parkinson’s disease (e. g., A53T or A30P). The AAV-(x-synuclein vector will be
injected into a specific region of the adult rat brain (e.g., striatum, cortex or
hippocampus) or into the lateral ventricle of a neonatal rat. A period of one to
several months will be allowed for the concentration of human u-synuclein to build
up in the brain. At that time, rats will be treated with an anti-(x-synuclein dy
by intraperitoneal or intravenous administration and samples of CSF will be taken at
various times. The concentration of u-synuclein will be measured in the CSF
samples by ELISA.
The disclosure is not to be limited in scope by the specific embodiments
described which are intended as single illustrations of individual aspects of the
sure, and any compositions or methods which are functionally equivalent are
within the scope of this sure. Indeed, various modifications of the sure
in addition to those shown and described herein will become nt to those
d in the art from the foregoing description and accompanying drawings. Such
modifications are intended to fall within the scope of the appended claims.
All publications and patent applications mentioned in this specification are
herein incorporated by reference to the same extent as if each individual publication
or patent application was specifically and individually indicated to be incorporated
by reference.
Claims (17)
1. A method of diagnosing an elevated level of oc-synuclein in the brain of a human subj ect, said method comprising: (a) assaying a level of a—synuclein in a blood plasma sample or ospinal fluid (CSF) sample obtained from the human subject at a specified time interval following peripheral administration to the human subject of an anti-OL—synuclein antibody or antigen-binding fragment thereof, wherein the antibody or fragment thereof can bind a-synuclein with sufficient affinity to alter a net efflux of (x—synuclein from brain to blood or CSF; and (b) ing the level of the a—synuclein in the blood plasma sample or CSF sample assayed to a reference rd, wherein a difference or similarity between the level of uclein in the blood plasma sample or CSF sample and the reference standard correlates with the level of a—synuclein in the brain of the human subject.
2. The method of claim 1, r comprising ing the level of the 0t~synuclein in the blood plasma sample to a blood plasma sample obtained from the human subject prior to the administration of the L-synuclein antibody or n—binding fragment thereof.
3. The method of claim 1, further comprising comparing the level of the a—synuclein in the CSF sample to a CSF sample obtained from the human subject prior to the administration of the anti—a—synuclein antibody or antigen—binding fragment thereof.
4. The method of any one of claims 1 to 3, wherein the reference standard comprises ed levels of u—synuclein in one or more control subjects, wherein the control subjects are normal healthy individuals.
5. The method of any one of claims 1 to 3, n the reference standard comprises measured levels of (x—synuclein in one or more control subjects, wherein the control subjects are individuals with synucleinopathies of varying severity.
6. A method of tracking an a—synuclein level in the brain of a human subject being treated for a synucleinopathic disease, said method comprising assaying a level of a—synuclein in a blood plasma sample or CSF sample obtained from the human subject at a specified time interval following peripheral administration of an anti—or—synuclein antibody or antigen-binding fragment thereof, wherein the antibody or fragment thereof can bind (x-synuclein with ent affinity to alter a net efflux of the a-synuclein from brain to blood or CSF, and wherein the or— -5.3- synuclein level in the blood plasma sample or CSF sample correlates with the level in the brain of the human subject.
7. The method of claim 6, further comprising assaying the level of or—synuclein in a blood plasma sample or CSF sample obtained from the human subject at a specified time following additional peripheral strations of the anti-a-synuclein antibody or n—binding fragment thereof, thereby plotting a change in the a—synuclein level in the brain of the human subject over time.
8. The method of any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof specifically binds to the same a—synuclein epitope as a reference antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH ses SEQ ID NO: 2 and the VL comprises SEQ ID NO: 3.
9. The method of any one of claims 1 to 7, wherein the dy or antigen—binding fragment thereof competitively inhibits a reference antibody comprising a VH and a VL, wherein the VH comprises SEQ ID NO: 2 and the VL comprises SEQ ID NO: 3, from binding to or-synuclein.
10. The method of any one of claims 1 to 7, wherein the antibody or antigen—binding fragment f comprises a heavy chain le region (VH) and a light chain variable region (VL), and wherein the VH and/or the VL comprise one or more of the following sequences: (a) the VH comprises a mentarity determining region-1 (VHCDRI) amino acid sequence of SEQ ID NO: 4; (b) the VH comprises a complementarity determining region—2 (VHCDRZ) amino acid sequence of SEQ ID NO: 5; (c) the VH comprises a complementarity determining region—3 (VHCDR3) amino acid ce of SEQ ID NO: 6; (d) the VL comprises a complementarity determining region-l (VLCDRI) amino acid sequence of SEQ ID NO: 7; (e) the VL comprises a mentarity determining region-2 Z) amino acid sequence of SEQ ID NO: 8; and/or (1) the VL comprises a complementarity determining region-3 (VLCDR3) amino acid sequence of SEQ ID NO: 9.
11. The method of any one of claims 1 to 7, wherein the dy or antigen-binding nt thereof comprises a VH and a VL, wherein the VH comprises VHCDR1,'VHCDR2 and VHCDR3 amino acid sequences of SEQ ID NOs: 4, 5 and 6 respectively, and n the VL comprises VLCDRI, VLCDR2 and VLCDR3 amino acid ces of SEQ ID NOs: 7, 8 and 9', respectively.
12. The method of any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof comprises a VH amino acid sequence of SEQ ID NO: 2 and a VL amino acid ce of SEQ ID NO: 3.
13. The method of any one of claims 1 to 12, wherein the antibody or antigen-binding fragment thereof is a single chain FV fragment (scFv), an F(ab’) fragment, an F(ab) fragment or an F(ab'); fragment.
14. The method of any one of claims 1 to 13, wherein said antibody or antigen-binding fragment f is formulated for administration by intravenous injection.
15. The method of any one of claims 1 to 14, wherein the antibody or antigen—binding fragment thereof is human.
16. The method of any one of claims 1 to 15, wherein the specified time interval is less than a week.
17. The method of any one of claims 6 and 7 to 16 when dependent from claim 6, wherein the synucleinopathic disease is selected from the group consisting of Parkinson's disease (PD), Parkinson’s disease dementia (PDD), ia with Lewy bodies (DLB), the Lewy body variant of Alzheimer's disease (LBVAD), multiple systems atrophy (MSA), pure autonomic failure (PAF), neurodegeneration with brain iron accumulation type-1 (NBIA-I), Alzheimer’s e, Pick disease, le-onset generalized neuroaxonal dystrophy (Hallervorden—Spatz disease), amyotrophic lateral sclerosis, traumatic brain injury and Down syndrome. Biogen International Neuroscience GmbH by the patent attorneys for the applicant CULLENS WO 66818 in C) (:3 5.0 CI) N N \“" V“ £323 iiw/Su} EWSEECf uAg—n FEG. C) D O O C3 C) O O O V? (‘0 N \— <2E [Iw/Bfi] 175a ewsmd WO 66818 1 w““M.wm wcwmmuxcmmaa m93 L “Wwas wmmw gs; L? frztciziiifffr [amgfifi uzfi‘sm [6/5“] 0 ugelon elqn|osug oddgq |€101J8d uAs-D elqnlosu! ledweooddgH E W F c ~ A ................................ g g a fix 250 200 C3 Li) (3? L0 V... {@5141 ugegmd m agqngas aecfiwmaddgq gem: Jad ufiwo egqngas gedmmaddgg 400 300 D N 100 [5/5“] <1: ugelon e|qn|os leomoo mo; Jed uAs—n elqnlos memos EN? m_o_;m> IT IT imam E303 mmnfiew "$53333 “mmEmuennf $533 cm 33 O C) O (0 V N m u] uAS-ao ewsmd V“— N ®_0_£®> Emw fiva) IOI. III + é: cam 8me cow Eflofi E imam \\ imé EfiEm \\.\ . g \\. 3333:“ \V, fitti,~ltr31111 mew $533 “3&8 “mmamuammfi . . ,\ \k mew ”3350 @323 Xxx “ES ”$53333 @533ch .\ o oow Em “£3 :5 0v ON O wow E < Whm.w. U,nAb _b 2wDuH (.3 wfl.gm,mmm 8wSBM WO 66818 12F4 VeHicle O O O O O (I) (.0 V N m [
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