NZ625217B2 - Use of an anti-?-synuclein antibody to diagnose an elevated level of ?-synuclein in the brain - Google Patents

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)

What we claim is:

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 [