NZ618914B2 - Anti-alpha synuclein binding molecules - Google Patents

Anti-alpha synuclein binding molecules Download PDF

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NZ618914B2
NZ618914B2 NZ618914A NZ61891412A NZ618914B2 NZ 618914 B2 NZ618914 B2 NZ 618914B2 NZ 618914 A NZ618914 A NZ 618914A NZ 61891412 A NZ61891412 A NZ 61891412A NZ 618914 B2 NZ618914 B2 NZ 618914B2
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antibody
seq
antigen
synuclein
amino acid
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NZ618914A
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NZ618914A (en
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Jan Grimm
Christoph Hock
Roger Nitsch
Lihe Su
Andreas Weihofen
Paul Weinreb
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Biogen International Neuroscience Gmbh
University Of Zurich
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Priority claimed from PCT/US2012/043701 external-priority patent/WO2012177972A1/en
Publication of NZ618914A publication Critical patent/NZ618914A/en
Publication of NZ618914B2 publication Critical patent/NZ618914B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2828—Prion diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease

Abstract

Disclosed is an isolated antibody or antigen-binding fragment thereof that specifically binds to human ?-synuclein (SEQ ID NO: 1), said antibody or antigen-binding fragment comprising: -a VH CDR1 region comprising the amino acid sequence of NYAMH or a sequence that differs from NYAMH by one amino acid; -a VH CDR2 region comprising the amino acid sequence of WINAGNGKRKYSQKFQD or a sequence that differs from WINAGNGKRKYSQKFQD by one amino acid; -a VH CDR3 region comprising the amino acid sequence of EEDHAGSGSYLSMDV or a sequence that differs from EEDHAGSGSYLSMDV by one amino acid; -a VL CDR1 region comprising the amino acid sequence of KSSQNVLYSSNNKNYLA or a sequence that differs from KSSQNVLYSSNNKNYLA by one amino acid; -a VL CDR2 region comprising the amino acid sequence of WASTRES or a sequence that differs from WASTRES by one amino acid; and -a VL CDR3 region comprising the amino acid sequence of QQYYSSPLT or a sequence that differs from QQYYSSPLT by one amino acid. cid; -a VH CDR2 region comprising the amino acid sequence of WINAGNGKRKYSQKFQD or a sequence that differs from WINAGNGKRKYSQKFQD by one amino acid; -a VH CDR3 region comprising the amino acid sequence of EEDHAGSGSYLSMDV or a sequence that differs from EEDHAGSGSYLSMDV by one amino acid; -a VL CDR1 region comprising the amino acid sequence of KSSQNVLYSSNNKNYLA or a sequence that differs from KSSQNVLYSSNNKNYLA by one amino acid; -a VL CDR2 region comprising the amino acid sequence of WASTRES or a sequence that differs from WASTRES by one amino acid; and -a VL CDR3 region comprising the amino acid sequence of QQYYSSPLT or a sequence that differs from QQYYSSPLT by one amino acid.

Description

ANTI-ALPHA SYNUCLEIN BINDING LES REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY The content of the electronically submitted sequence listing in ASCII text file (Name: celisting_ascii.txt; Size: 23KB; and Date of Creation: June 23, 2011) filed with the application is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION The present invention generally s to novel uclein-specific binding molecules, particularly human antibodies as well as fragments, derivatives and variants thereof that ize u-synuclein and aggregated forms of u-synuclein, respectively. In addition, the present invention relates to ceutical and diagnostic compositions sing such binding molecules, antibodies and mimics thereof valuable both as a diagnostic tool to identify toxic species of (x-synuclein in plasma and CSF and also in passive vaccination strategies for treating ers related to aggregates of (x-synuclein such as Parkinson’s disease (PD), ia with Lewy bodies (DLB) and Lewy body variant of Alzheimer’s disease (AD) and other synucleinopathic es.
Protein misfolding and aggregation are pathological aspects of numerous neurodegenerative diseases. Aggregates of (x-synuclein are major components of the Lewy bodies and Lewy neurites associated with Parkinson's disease (PD). A natively unfolded protein, (x-synuclein can adopt different aggregated morphologies, including oligomers, protofibrils and fibrils. The small oligomeric aggregates have been shown to be particularly toxic.
Naturally occurring autoantibodies against (x-synuclein have been ed in healthy persons and altered levels in patients were associated with particular neurodegenerative ers; see for review Neff et al., Autoimmun. Rev. 7 (2008), 501- 507. Thus, naturally occurring antibodies in patients suffering from son’s disease, either spontaneously or upon vaccination, in particular in healthy patients can serve a protective role with respect to (x-synuclein aggregation; see, e. g., Woulfe et al., Neurology 58 (2002), 1435-1436 and Papachroni et al., J. Neurochem. 101 (2007), 749- 756. Hitherto, the therapeutic significance of autoantibodies had been difficult to assess.
This is mostly due to the lack of ht-forward experimental approaches for their isolation and subsequent characterization in vitro.
Recently, oligomeric species of u-synuclein have been reported ellularly in plasma and CSF (El-Agnaf et al., FASEB J. 20 (2006), 5) and immunization studies in mouse models of PD show that extracellular mouse monoclonal antibodies against u-synuclein can reduce accumulation of intracellular u-synuclein ates (Masliah et al., Neuron, 46 (2005), 857—868) supporting the idea that dies that neutralize the neurotoxic aggregates without ering with beneficial functions of monomeric u-synuclein can be useful therapeutics. However, the therapeutic utility of murine based dies in human is ed by the human anti-mouse antibody (HAMA) response in view of their non-human origin.
Emadi et al. in J. Mol. Biol. 368 (2007), 1132—1144, describe the isolation of single chain antibody fragments (scFvs) from a phage displayed antibody library based on human sequences against u-synuclein, which bind only to an oligomeric form of 0L- synuclein and inhibit both aggregation and toxicity of u-synuclein in vitro. However, although the generation of scFvs from phage display is rather simple, this technique has severe drawbacks since the antibodies so produced bear the risk of undesired crossreactivity against self-antigens and lack the characteristics of evolutionary optimized natural human antibodies produced by the human immune system. Furthermore, such antibodies may not be specific enough because of cross-reactivity with other proteins and/or with the target protein in context with normal logical environment and function. In case of Parkinson’s disease, for example, antibodies that also cross-react with physiological derivatives of u-synuclein bear the ial to cause side effects related to the normal functions of the physiologic target structures. In this respect, an undesired autoimmune disease would downrightly be d — a hardly calculable risk also in the conceptual design of active immunization ments employing protein structures that, in variant form, also occur physiologically.
More recently, Seitz et al. (81. Kongress der Deutschen Gesellschaft fur Neurologie mit ldungsakademie Hamburg lO.-l3.09.2008), reported on the isolation of anti-(x-synuclein polyclonal autoantibody from different immunoglobulin solutions and s of single blood donors h affinity chromatography. However, besides the fact that this approach provides mere limited amounts of the desired antibody, W0 2012/177972 2012/043701 _ 3 _ polyclonal antibodies are of only limited use for therapeutic application, for example because of their geneity and the risk of being contaminated with other (x-synuclein associated les which have undesired side effects. Likewise, the diagnostic value of polyclonal antibodies is reduced since the variability of the composition of the antibodies will influence the overall specificity and reactivity. This is all the more true for antibodies against proteins subject of aggregation and deposition due to misfolding.
Thus, there is a need to overcome the above-described limitations and to provide a therapeutic and diagnostic human antibody against uclein.
SUMMARY OF THE INVENTION In one embodiment, the present invention provides an isolated binding molecule which specifically binds to an epitope within amino acids 4 to 15 of human (x-synuclein (SEQ ID NO:1). In certain aspects the binding molecule itively inhibits the binding of reference monoclonal antibody NI-202.12F4 to (x-synuclein. A binding molecule of the invention can be an antibody, or an antigen-binding fragment thereof In particular embodiments the binding molecule is not human monoclonal antibody NI- 202.12F4, or an antigen-binding nt, t, or tive thereof.
Another embodiment es an isolated binding molecule which specifically binds to an epitope within amino acids 113 to 123 or within amino acids 117 to 123 of 0L- synuclein (SEQ ID NO:1). In certain aspects the binding molecule specifically binds to the same human (x-synuclein epitope as the reference monoclonal antibody NI- 202.22D11, or competitively inhibits the binding reference monoclonal antibody NI- 202.22D11 to human uclein. A binding molecule of the invention can be an antibody, or an antigen-binding fragment thereof. A particular binding molecule of the invention is the human monoclonal antibody NI-202.22D11, or an antigen-binding fragment, variant, or derivative f.
The invention further provides an isolated antibody or n binding fragment thereof that specifically binds to human uclein, comprising an globulin heavy chain le region (VH) and an immunoglobulin light chain variable region (VL), where the VH comprises a polypeptide sequence at least 90%, or 100% identical to SEQ ID NO:15 or SEQ ID NO:20. Also provided is an isolated antibody or antigen binding fragment thereof that specifically binds to human (x-synuclein, comprising a VH 2012/043701 and a VL, where the VL comprises a polypeptide sequence at least 90%, or 100% identical to SEQ ID NO:22 or SEQ ID NO:26. Similarly, the invention provides an isolated antibody or antigen binding fragment thereof that cally binds to human or- synuclein, comprising a VH and a VL where the VH and VL comprise, respectively, polypeptide sequences at least 90%, or 100% identical to the reference ptides SEQ ID NO:lS and SEQ ID NO:22, SEQ ID NO:lS and SEQ ID NO:26, SEQ ID N020 and SEQ ID NO:22, or SEQ ID N020 and SEQ ID NO:26.
Further ed are isolated polypeptides, including an isolated ptide comprising a VH, where the CDRl region of the VH is identical, or identical except for less than 3 conservative amino acid substitutions, to reference heavy chain CDRl sequence SEQ ID NO:l6, an isolated polypeptide comprising a VH, where the CDR2 region of the VH is identical, or identical except for less than 5 conservative amino acid substitutions, to reference heavy chain CDR2 sequence SEQ ID NO:l7, an isolated ptide comprising a VH, where the CDR3 region of the VH is identical, or identical except for less than 5 conservative amino acid tutions, to reference heavy chain CDR3 sequence SEQ ID NO:18, an isolated polypeptide comprising a VL, where the CDRl region of the VL is identical, or identical except for less than 5 conservative amino acid substitutions, to reference light chain CDRl sequence SEQ ID NO:23, an isolated polypeptide comprising a VL, where the CDR2 region of the VL is identical, or identical except for less than 3 conservative amino acid substitutions, to reference heavy chain CDR2 sequence SEQ ID N024, and an isolated polypeptide comprising a VL, where the CDR3 region of the VL is identical, or identical except for less than 3 conservative amino acid substitutions, to reference heavy chain CDR3 sequence SEQ ID NO:25. In each of the above stated polypeptides, an antibody or antigen-binding fragment thereof comprising the polypeptide specifically binds to human clein.
In certain embodiments the invention the isolated dy or fragment f preferentially binds to a non-linear mational epitope of human (x-synuclein. In other embodiments the isolated antibody or fragment f preferentially binds human (x-synuclein in the oligomeric or aggregated form. In filrther embodiments the isolated antibody or fragment thereof does not specifically bind to human B-synuclein or human y- synuclein, and/or does not specifically bind to murine u-synuclein.
Also provided is a composition comprising an antibody or fragment thereof as described above, and a carrier. The composition may be a therapeutic or a diagnostic composition.
Further provide are one or more isolated polynucleotides encoding a polypeptide or binding molecule as described herein, and vectors and host cells for expressing such binding molecules.
It is a particular object of the present ion to provide methods for treating or preventing a einopathic disease such as, but not limited to Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple systems atrophy (MSA). The methods comprise administering an effective concentration of anti-human a-synuclein binding molecule, e.g., an antibody or antigen-binding nt, variant, or derivative to the subject where the antibody targets a-synuclein.
It is also an object of the invention to provide a method of diagnosing a synucleinopathic disease in a subject, sing assessing the level, zation, conformation or a combination thereof of a-synuclein in a subject to be diagnosed with an antibody or fragment thereof of the invention and comparing the level, localization, conformation or ation thereof of a-synuclein in the subject to one or more reference standards derived from one or more control samples, where a difference or similarity between the level, localization, conformation or combination thereof of asynuclein in the t and the nce standard indicates whether the subject has a synucleinopathic disease.
Diagnostic methods of the invention can be through in vitro assay of patient samples, or by in vivo imaging techniques. [0018a] Definitions of specific embodiments of the ion as claimed herein follow. [0018b] According to a first embodiment of the invention, there is provided an isolated antibody or antigen-binding fragment thereof that specifically binds to human αsynuclein (SEQ ID NO:1), said dy or antigen-binding fragment comprising: a VH CDR1 region comprising the amino acid ce of SEQ ID NO:16 or a ce that differs from SEQ ID NO:16 by one amino acid; a VH CDR2 region comprising the amino acid ce of SEQ ID NO:17 or a ce that differs from SEQ ID NO:17 by one amino acid; a VH CDR3 region sing the amino acid sequence of SEQ ID NO:18 or a sequence that differs from SEQ ID NO:18 by one amino acid; - 5a - a VL CDR1 region comprising the amino acid sequence of SEQ ID NO:23 or a sequence that differs from SEQ ID NO:23 by one amino acid; a VL CDR2 region comprising the amino acid sequence of SEQ ID NO:24 or a ce that differs from SEQ ID NO:24 by one amino acid; and a VL CDR3 region comprising the amino acid sequence of SEQ ID NO:25 or a sequence that differs from SEQ ID NO:25 by one amino acid. [0018c] According to a second ment of the invention, there is provided an isolated dy or antigen-binding fragment thereof that specifically binds to human α-synuclein (SEQ ID NO:1) comprising: a VH CDR1 region comprising the amino acid sequence of SEQ ID NO:16; a VH CDR2 region comprising the amino acid sequence of SEQ ID NO:17; a VH CDR3 region comprising the amino acid sequence of SEQ ID NO:18; a VL CDR1 region comprising the amino acid sequence of SEQ ID NO:23; a VL CDR2 region comprising the amino acid sequence of SEQ ID NO:24; and a VL CDR3 region comprising the amino acid sequence of SEQ ID NO:25. [0018d] According to a third embodiment of the invention, there is provided a composition comprising the antibody or antigen-binding fragment of the first or second embodiments and a carrier. [0018e] According to a fourth embodiment of the invention, there is provided an isolated polynucleotide comprising a nucleic acid encoding the antibody or antigen-binding fragment thereof of the first or second ments. [0018f] According to a fifth embodiment of the invention, there is provided a composition sing the cleotide of the fourth ment and a carrier. [0018g] According to a sixth embodiment of the invention, there is provided a vector comprising the polynucleotide of the fourth embodiment. [0018h] According to a seventh embodiment of the invention, there is provided an isolated host cell comprising the polynucleotide of the fourth embodiment or the vector of the sixth embodiment.
] According to an eighth embodiment of the invention, there is provided a method of producing an anti-human clein antibody or antigen-binding fragment f, said method comprising culturing the host cell of the seventh embodiment and recovering the antibody or antigen-binding nt thereof. - 5b - [0018j] According to a ninth embodiment of the invention, there is provided an anti- human α-synuclein antibody or antigen-binding fragment thereof produced by the method of the eighth embodiment. [0018k] According to a tenth embodiment of the invention, there is provided use of the antibody or antigen-binding nt of the first or ninth embodiments, the composition of the third or fifth embodiments, the polynucleotide of the fourth embodiment, the vector of the sixth embodiment or the host cell of the seventh embodiment in the manufacture of a medicament for ng or preventing a synucleinopathic disease in a subject. [0018l] According to an eleventh embodiment of the invention, there is provided use of the antibody or antigen-binding fragment thereof of the first or ninth embodiments in the cture of a medicament for diagnosing whether a subject has a synucleinopathic disease, wherein said diagnosing ses: (a) assessing a level, localization, conformation or a combination thereof of clein in the subject to be diagnosed with the medicament; and (b) comparing the level, localization, mation or combination thereof of said α-synuclein in the subject to one or more reference rds derived from one or more control samples, wherein a difference or similarity between the level, localization, conformation or combination thereof of said α-synuclein in the subject and the one or more reference standards indicates r the subject has a synucleinopathic e.
Further embodiments of the present invention will be apparent from the description and Examples that .
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Amino acid and nucleotide sequences of the variable region, i.e. heavy chain and kappa light chains of human antibody .21D11. Framework (FR) and complementarity determining regions (CDRs) are indicated with the CDRs being underlined. Due to the cloning strategy the amino acid sequence at the N-terminus of the heavy chain and light chain may ially contain primer-induced alterations in FR1, [Text continues on page 6] W0 2012/177972 PCT/U82012/043701 _ 6 _ which however do not substantially affect the biological activity of the antibody. In order to provide a consensus human antibody, the nucleotide and amino acid sequences of the original clone were aligned with and tuned in accordance with the pertinent human germ line variable region sequences in the database; see e.g., Vbase (vbase.mrc-cpe.cam.ac.uk) hosted by the MRC Centre for Protein Engineering (Cambridge, UK). Those amino acids, which are ered to potentially deviate from the consensus germ line sequence and thus could be due to the PCR , are indicated in bold.
Figure 2 Recombinant human NI-202.21Dll selectively binds human (x-synuclein over [3-, y-synuclein and murine u-synuclein in a direct ELISA. Recombinant human u-,B- ,y- synuclein and recombinant human and murine His-tagged (it-synuclein were coated onto ELISA plates at equal concentration (2 ug/ml). Plates were then probed with recombinant human NI-202.21Dl 1, human NI-202.12F4 and with a nuclein antibody. (A) Recombinant NI-202.21D11 selectively binds u-synuclein whereas pan — synuclein antibody binds to all three synuclein proteins ing equal coating of inant proteins. (B) Recombinant NI-202.21Dll is selective for human vs murine (x-synuclein. On the other hand NI-202.12F4 binds to both human and murine (x-synuclein in a direct ELISA.
Figure 3 Recombinant .21Dll entially binds to high density coated (it-synuclein. Recombinant human u-synuclein was coated onto ELISA plates at indicated concentrations and probed with various concentrations of .21Dll by direct ELISA (El 20 ug/ml;A Sug/mlg91 ug/ml;l 0.25 ug/ml; V 0.1 ug/ml). The half maximal effective concentration (ECSO) indicating the potency of the antibody was determined for each coating concentration.
Figure 4 Immunohistochemical binding analysis of NI-202.21Dll showed prominent staining of (x-synuclein pathology including Lewy body and Lewy neurite like inclusions in paraffin sections from (A) transgenic mice overexpressing human 0L- synuclein A53T and (C) from human brain tissue of a Dementia with Lewy Bodies patient. (B) No staining was observed in wild-type mouse tissue and (bottom right) in a secondary dy only control. HC=Hippocampus, CTX=Cortex, instem.
Figure 5 Epitope mapping revealed a C-terminal g epitope within human 0L- synuclein (aa 117-123) for NI-202.21Dll. (A) Recombinant .21Dll bound to the C-terminal domain of human (x-synuclein in a direct ELISA. (x-synuclein truncations were coated onto ELISA plates at equal trations (2ug/ml). NI-202.2lDll bound only to truncated (x-synuclein aa 61-140 and 95-140 but not to truncations aa l-60, l-95. (B) Pepscan analysis showed binding of NI-202.21Dll to overlapping peptides B08 (aa 109- 123), B09 (aa 113-127) and B10 (aa 117-131) of human (x-synuclein ting that the l sequence required for NI-202.21Dll binding is PVDPDNE (aa 117-123) within human (x-synuclein. (C) Recombinant NI-202.21Dll showed reduced binding to human (x-synuclein DlZlG/N122S in a direct ELISA. Recombinant wt and mutated (x-synuclein proteins were coated at equal concentration (2ug/ml) onto ELISA plates and tested for recombinant NI-202.2lDl 1 g.
Figure 6 NI-202.12F4 ively binds to very N-terminus of (x-synuclein. (A) Pepscan analysis shows binding of NI-202. 12F4 to peptide AOl showing that the minimal recognition sequence is within residue l-lS of (x-synuclein. (B) Synthetic (x-synuclein peptides from residue 1-30, 4-30 and 5-30 were tested for NI-202.12F4 binding in an in- solution ELISA. NI-202.12F4 bound aa l-30 and 4-30 but not 5-30. This showed that N1202.12F4 epitope sequence starts at residue 4 of (x-synuclein. (C) Residue K10 within NI-202.12F4 epitope is a key amino acid for selectivity of NI-202.12F4 for (x-synuclein over B-synuclein. Recombinant wt and mutant (x- and B-synuclein proteins were tested by direct ELISA for .12F4 binding. NI-202.12F4 bound to wt (x-synuclein and mutant B-synuclein MlOK but not to wt B-synuclein and mutant (x-synuclein KlOM. This shows that residue K10 is responsible for NI-202.12F4 u-synuclein selectivity.
DETAILED DESCRIPTION OF THE ION I. ions einopathic es or synucleinopathies are a diverse group of neurodegenerative disorders that share a common pathologic lesion composed of aggregates of insoluble u-synuclein protein in selectively vulnerable populations of neurons and glia. These disorders include Parkinson's disease (PD), son’s Disease Dementia (PDD), dementia with Lewy bodies (DLB), juvenile-onset generalized neuroaxonal dystrophy (Hallervorden-Spatz disease), pure autonomic failure (PAF), le system atrophy (MSA) and neurodegeneration with brain iron accumulation type-l (NBIA-I). Clinically, they are characterized by a chronic and progressive decline in motor, ive, behavioral, and autonomic fianctions, depending on the distribution of the lesions.
Parkinson's disease is an age-dependent neurodegenerative disease with unknown etiology. It is believed that ic Parkinson's disease results from a combination of genetic vulnerability and nmental insults. It is r ed that Parkinson's disease (PD) while triggered by disparate mechanisms follows a shared hysiologic pathway. One shared node is the involvement of u-synuclein. Linkage of this protein with Parkinson's disease pathogenesis has been ished by the identification of both point mutations and triplication of the gene in familial cases, the localization of u-synuclein to Lewy bodies, one of the hallmark pathological features of Parkinson's disease, and the correlation of u-synuclein expression and disease pathology in neurotoxic models of Parkinson's disease. r ce indicates that particular forms of u-synuclein (e.g., misfolded and clein bonded dopamine) are involved in sporadic disease.
Synucleins are small, soluble proteins expressed primarily in neural tissue and in certain tumors. The family includes three known proteins: u-synuclein, B-synuclein, and y-synuclein. All synucleins have in common a highly conserved u-helical lipid-binding motif with similarity to the class-A2 lipid-binding domains of the exchangeable apolipoproteins. Synuclein family members are not found outside vertebrates, although they have some conserved structural similarity with plant 'late-embryo-abundant' ns.
The 0L- and B-synuclein proteins are found primarily in brain tissue, where they are seen mainly in presynaptic terminals. The y-synuclein protein is found primarily in the peripheral nervous system and retina, but its expression in breast tumors is a marker for tumor progression. Normal cellular fianctions have not been determined for any of the ein proteins, although some data suggest a role in the regulation of membrane stability and/or turnover. Mutations in u-synuclein are associated with rare familial cases of early-onset son's disease, and the protein accumulates ally in Parkinson's disease, Alzheimer's disease, and several other egenerative illnesses. For review see, e. g., George, Genome Biol. 3 (2002), reviews3002.l—reviews3002.6 published online December 20, 2001, in which Table l catalogs the unique members of the synuclein family that are currently listed in k, the disclosure content of which is incorporated herein by reference. (x-synuclein was originally identified in human brains as the sor protein of the amyloid component of (NAC) of Alzheimer’s disease (AD) plaques; see, e. g., Ueda et al, Proc. Natl. Acad. Sci. USA. 90 (1993), 1282-1286. u-synuclein, also termed the precursor of the non-AB component of AD amyloid (NACP), is a protein of 140 amino acids. (x-synuclein 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 eric, proto-fibrillar, fibrillar, and aggregate moieties are t to regulate protein toxicity. Increasing evidence indicates that dopamine-adducted (x-synuclein has a faster time course to fibril formation compared to non-adducted n. Furthermore, dopamine in the background of (x-synuclein overexpression is toxic.
In this specification, the terms "(x-synuclein", "alpha-synuclein", uclein" and "aSyn" are used interchangeable to specifically refer to the native monomer form of 0L- ein. The term "(x-synuclein" is also used to generally identify other conformers of u-synuclein, for example, (x-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 0L- synuclein is MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHG VATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKN EEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA (SEQ ID NO: 1). The amino acid ce of (x-synuclein can be retrieved from the literature and pertinent databases; see, e.g., Ueda et al., ibid.; GenBank swissprot: locus SYUA_HUMAN, accession number P37840. The non-AB component of AD d (NAC) is derived from 0L- synuclein. NAC, a highly hydrophobic domain within (x-synuclein, is a peptide consisting of at least 28 amino acids residues (residues 60-87) and optionally 35 amino acid residues ues 61-95). NAC displays a tendency to form a beta-sheet ure (Iwai, et al., Biochemistry, 34 (1995) 10139-10145). The amino acid sequences ofNAC are described in Jensen et al., Biochem. J. 310 (1995), 91-94; k accession number S56746 and Ueda et al., PNAS USA 90 (1993), 1282-11286.
Disaggregated (x-synuclein or fragments thereof, including NAC, means monomeric peptide units. Disaggregated (x-synuclein or fragments thereof are generally PCT/U82012/043701 _ 10 _ soluble, and are capable of self-aggregating to form soluble oligomers. Oligomers of 0L- ein and fragments thereof are usually soluble and exist predominantly as ces.
Monomeric clein can be prepared in vitro by dissolving lyophilized e in neat DMSO with sonication. The resulting solution is centrifuged to remove any insoluble particulates. Aggregated u-synuclein or fragments thereof, including NAC, means oligomers of u-synuclein or fragments thereof which have associate into insoluble B-sheet lies. Aggregated u-synuclein or fragments thereof, including NAC, means also means fibrillar polymers. Fibrils are usually insoluble. Some dies bind either soluble u-synuclein or fragments thereof or aggregated u-synuclein or fragments thereof.
Some antibodies bind to oligomers of u-synuclein more strongly than to monomeric forms or fibrillar forms. Some antibodies bind both soluble and ated u-synuclein or fragments thereof, and optionally oligomeric forms as well.
The human anti-u-synuclein antibodies disclosed herein cally bind 0L- ein and epitopes thereof and to various conformations of u-synuclein and epitopes thereof. For e, disclosed herein are antibodies that specifically bind u-synuclein, 0L- synuclein in its native monomer form, fiJll-length and truncated u-synuclein and 0t- synuclein aggregates. As used herein, reference to an antibody that "specifically binds", "selectively binds", or "preferentially binds" u-synuclein refers to an antibody that does not bind other unrelated proteins. In one example, an u-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 dy that "specifically binds" or "selectively binds" u-synuclein conformer refers to an antibody that does not bind all mations of u-synuclein, z'.e., does not bind at least one other u-synuclein conformer. For example, disclosed herein are antibodies that can distinguish among monomeric and ated forms of u-synuclein, human and mouse u-synuclein; full-length u-synuclein and truncated forms as well as human u-synuclein versus [3- and y-synuclein. Since the human anti-(x-synuclein antibodies of the present invention have been isolated from a pool of elderly subjects with no signs of Parkinsonism and exhibiting an u-synuclein-specific immune response the x-synuclein antibodies of the present invention are also referred to as "human ntibodies" in order to emphasize that those antibodies were indeed expressed by the subjects and have not been isolated from, for example a human -1]- immunoglobulin expressing phage library, which hitherto represented one common method for trying to provide human-like antibodies.
It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "an antibody," is understood to represent one or more antibodies. As such, the terms "a" (or "an" "one or more," and "at least one" can be used interchangeably herein.
As used herein, the term "polypeptide" is intended to encompass a singular "polypeptide" as well as plural "polypeptides," and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds).
The term eptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, eptides, "protein, amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" can be used instead of, or hangeably with any of these terms.
The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, ation, phosphorylation, ion, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not arily translated from a ated nucleic acid sequence. It an be generated in any manner, including by chemical synthesis.
A ptide of the invention can be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides can have a defined three-dimensional structure, although they do not arily have such structure. ptides with a defined three-dimensional structure are referred to as folded, and polypeptides which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded. As used herein, the term glycoprotein refers to a protein coupled to at least one carbohydrate moiety that is attached to the protein via an oxygen-containing or a nitrogen-containing side chain of an amino acid residue, e.g., a serine residue or an asparagine residue.
PCT/U82012/043701 _ 12 _ By an "isolated" polypeptide or a fragment, t, or derivative f is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purposed of the invention, as are native or inant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.
Also ed as polypeptides of the present invention are fragments, derivatives, analogs, or ts of the foregoing polypeptides, and any combination thereof. The terms "fragment," "variant," "derivative" and "analog" when referring to antibodies or antibody polypeptides of the present invention include any polypeptides which retain at least some of the antigen-binding properties of the corresponding native binding molecule, antibody, or polypeptide. Fragments of polypeptides of the present ion include proteolytic fragments, as well as deletion fragments, in addition to specific antibody fragments discussed elsewhere herein. Variants of antibodies and antibody polypeptides of the t invention include nts as described above, and also polypeptides with altered amino acid ces due to amino acid substitutions, deletions, or insertions. Variants can occur naturally or be non-naturally occurring. Non- naturally ing variants can be produced using own mutagenesis techniques.
Variant polypeptides can comprise conservative or non-conservative amino acid substitutions, deletions or additions. Derivatives of u-synuclein specific binding molecules, e. g., dies and antibody polypeptides of the present invention, are polypeptides which have been altered so as to exhibit onal features not found on the native polypeptide. Examples include fusion proteins. Variant polypeptides are also referred to herein as "polypeptide analogs". As used herein a "derivative" of a binding le or fragment thereof, an antibody, or an dy polypeptide refers to a subject ptide having one or more residues chemically derivatized by reaction of a functional side group. Also included as "derivatives" are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids.
For example, oxyproline can be substituted for proline; 5-hydroxylysine can be substituted for lysine; 3-methylhistidine can be substituted for histidine; homoserine can be substituted for serine; and omithine can be substituted for lysine.
The term "polynucleotide" is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e. g., messenger RNA (mRNA) or plasmid DNA . A polynucleotide can comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)). The term "nucleic acid" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.
By ted" nucleic acid or cleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, a recombinant polynucleotide encoding an dy contained in a vector is considered isolated for the purposes of the present invention. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides of the present invention. Isolated polynucleotides or nucleic acids according to the present invention r include such les produced synthetically. In on, a polynucleotide or a nucleic acid can be or can include a regulatory element such as a er, ribosome binding site, or a transcription terminator.
As used herein, a "coding region" is a portion of nucleic acid which ts of codons ated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it can be considered to be part of a coding , but any flanking ces, for example ers, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. Two or more coding s of the present invention can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. Furthermore, any vector can contain a single coding region, or comprise two or more coding regions, e.g., a single vector can separately encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. In addition, a vector, polynucleotide, or nucleic acid of the invention can encode heterologous coding regions, either fused or unfiJsed to a nucleic acid encoding a binding molecule, an antibody, or nt, variant, or derivative thereof. Heterologous coding regions include without tion specialized elements or motifs, such as a secretory signal peptide or a heterologous fianctional domain.
In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid which encodes a polypeptide can include a promoter and/or other transcription or translation control elements operably associated with one or more coding regions. An operable association is when a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the ce or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and a promoter ated therewith) are "operably associated" or "operably linked" if induction of promoter function results in the transcription of mRNA encoding the d gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be ly associated with a nucleic acid encoding a polypeptide if the er was capable of effecting transcription of that nucleic acid. The promoter can be a pecif1c promoter that s substantial transcription of the DNA only in predetermined cells. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription ation signals, can be operably associated with the polynucleotide to direct pecific transcription. Suitable promoters and other transcription control regions are disclosed herein.
A variety of transcription control regions are known to those skilled in the art.
These include, without limitation, transcription control regions which function in vertebrate cells, such as, but not d to, promoter and er segments from galoviruses (the immediate early promoter, in ction with -A), simian Virus 40 (the early promoter), and retroviruses (such as Rous sarcoma Virus). Other transcription control regions include those d from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit B-globin, as well as other sequences capable of controlling gene sion in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).
W0 2012/177972 PCT/U82012/043701 _ 15 _ Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picomaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).
In other embodiments, a polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA). cleotide and nucleic acid coding regions of the present invention can be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present ion. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is d from the mature protein once export of the growing protein chain across the rough asmic reticulum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells lly have a signal peptide fused to the N—terminus of the ptide, which is cleaved from the complete or "full length" polypeptide to produce a secreted or e" form of the polypeptide. In certain embodiments, the native signal peptide, e.g., an immunoglobulin heavy chain or light chain signal e is used, or a functional derivative of that sequence that s the ability to direct the secretion of the polypeptide that is operably associated with it. atively, a heterologous mammalian signal peptide, or a functional derivative thereof, can be used. For example, the wild-type leader sequence can be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse B-glucuronidase.
Unless stated otherwise, the terms "disorder" and "disease" are used interchangeably herein.
A "binding molecule" as used in the context of the present invention relates primarily to antibodies, and nts f, but can also refer to other non-antibody molecules that bind to u-synuclein including but not limited to hormones, receptors, ligands, major ompatibility complex (MHC) molecules, chaperones such as heat shock proteins (HSPs) as well as cell-cell adhesion molecules such as members of the in, intergrin, C-type lectin, immunoglobulin (Ig) superfamilies, and synthetic binding molecules. Thus, for the sake of clarity only and without restricting the scope of the present invention most of the ing embodiments are discussed with respect to PCT/U82012/043701 _ 16 _ antibodies and antibody-like molecules which represent exemplary binding molecules for the development of therapeutic and diagnostic agents.
The terms "antibody" and "immunoglobulin" are used interchangeably herein. An antibody or immunoglobulin is an u-synuclein-binding molecule which comprises at least the variable domain of a heavy chain, and ly comprises at least the variable domains of a heavy chain and a light chain. Basic globulin structures in rate systems are relatively well understood; see, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).
As will be discussed in more detail below, the term "immunoglobulin" ses various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will iate 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 onal specialization.
Modified versions of each of these classes and es are readily discemable to the skilled artisan in view of the t disclosure and, ingly, are within the scope of the instant invention. All immunoglobulin classes are clearly within the scope of the present invention, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With regard to IgG, a rd immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 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 h the variable region.
Light chains are classified as either kappa or lambda (K, k). Each heavy chain class can be bound with either a kappa or lambda light chain. In l, 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 genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N- W0 2012/177972 PCT/U82012/043701 _ 17 _ terminus at the forked ends of the Y ration to the C-terminus at the bottom of each chain.
Both the light and heavy chains are d into regions of structural and functional homology. The terms "constant" and "variable" are used filnctionally. In this regard, it will be appreciated that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental 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 terminus of the antibody. The N-terminal portion is a variable region and at the C- terminal n is a constant ; 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 dy to selectively recognize and specifically bind epitopes on antigens. That is, the VL domain and VH domain, or subset of the mentarity determining regions , of an antibody e to form the variable region that defines a three dimensional antigen-binding site.
This quaternary antibody structure forms the antigen-binding site t 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. Any antibody or immunoglobulin fragment which contains ent structure to specifically bind to u-synuclein is denoted herein hangeably as a "binding fragment" or an "immunospecific fragment." In naturally occurring antibodies, an antibody comprises six hypervariable regions, sometimes called "complementarity determining regions" or "CDRs" present in each antigen-binding domain, which are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain as the antibody assumes its three dimensional configuration in an aqueous environment. The "CDRs" are flanked by four relatively conserved "framework" regions or "FRs" which show less inter-molecular variability. The framework regions largely adopt a [3-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the B-sheet ure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen- _ 18 _ binding domain formed by the oned CDRs defines a e complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non- covalent binding 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 region by one of ordinary skill in the art, since they have been precisely defined; see, "Sequences of Proteins of Immunological Interest," Kabat, E., et al., US. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196 (1987), 901-917, which are incorporated herein by reference in their entireties.
In the case where there are two or more definitions of a term which 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 ntiguous antigen ing sites found within the variable region of both heavy and light chain polypeptides. This particular region has been described by Kabat et al., US. Dept. of Health and Human Services, "Sequences of Proteins of logical Interest" (1983) and by Chothia and Lesk, J. Mol. Biol., 196 (1987), 901-917, which are incorporated herein by reference, where the definitions include overlapping or s of amino acid residues when compared against each other. Nevertheless, application of either ion to refer to a CDR of an antibody or ts thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison. The exact e numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely ine which residues comprise a particular hypervariable region or CDR of the human IgG subtype of antibody given the le region amino acid sequence of the antibody.
Table 1: CDR Definitions1 VH CDRl VH CDR2 VH CDR3 VL CDRl 24-34 26-32 PCT/U82012/043701 _ 19 _ VL CDR2 50-56 50-52 VL CDR3 89-97 91-96 1Numbering of all CDR definitions in Table l 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 antibody. One of ordinary skill in the art can unambiguously assign this system of "Kabat numbering" to any variable domain sequence, without reliance on any mental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al., US. Dept. of Health and Human Services, nce of Proteins of Immunological Interest" (1983). Unless otherwise specified, references to the numbering of specific amino acid residue positions in an antibody or antigen-binding fragment, variant, or tive thereof of the present invention are according to the Kabat numbering system. dies or antigen-binding nts, immunospecific fragments, variants, or derivatives thereof of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, murinized or chimeric antibodies, single chain dies, epitope-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (dev), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies disclosed herein). ScFv molecules are known in the art and are described, e. g., in US patent 5,892,019. Immunoglobulin or dy molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
In one embodiment, the antibody of the present invention is not IgM or a derivative thereof with a pentavalent structure. Particular, in c applications of the present invention, especially eutic use, Ing are less useful than IgG and other bivalent antibodies or ponding binding les since Ing due to their pentavalent structure and lack of affinity maturation often show unspecific cross- reactivities and very low affinity.
In one embodiment, the antibody of the present invention is not a onal dy, z'.e. it substantially consists of one particular dy species rather than being a mixture obtained from a plasma immunoglobulin sample.
PCT/U82012/043701 _ 20 _ Antibody fragments, including single-chain antibodies, can comprise the le region(s) alone or in combination with the ty or a portion of the following: hinge , CH1, CH2, and CH3 domains. Also included in the invention are u-synuclein- binding fragments also comprising any combination of variable region(s) with a hinge region, CH1, CH2, and CH3 domains. Antibodies or specif1c fragments thereof of the present invention can be from any animal origin ing birds and mammals. In certain embodiments, the dies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or n antibodies. In another embodiment, the variable region can be condricthoid in origin (6.g. from sharks).
In one aspect, the antibody of the present invention 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 (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 cloning process. Compared to artificially generated like antibodies such as single chain antibody fragments ) from a phage displayed antibody library or xenogeneic mice the human monoclonal dy of the present invention is characterized by (i) being obtained using the human immune response rather than that of animal surrogates, z'.e. the antibody has been generated in response to natural u-synuclein in its relevant conformation in the human body, (ii) having protected the individual or is at least icant for the presence of u-synuclein, and (iii) since the antibody is of human origin the risks of cross-reactivity against self- antigens is minimized. Thus, in accordance with the present invention the terms "human monoclonal antibody", "human monoclonal autoantibody", "human antibody" and the like are used to denote an u-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 tutions are made in the antibody, 6.g. to e binding characteristics.
Antibodies d from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and that do not express endogenous PCT/U82012/043701 _ 21 _ immunoglobulins, as described infra and, for example in, US patent no 5,939,598 by Kucherlapati et al., are denoted human-like antibodies in order distinguish them from truly human antibodies of the present invention.
As used herein, the term ized antibody" or "murinized immunoglobulin" refers to an dy comprising one or more CDRs from a human antibody of the present invention; 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 "paren " or "acceptor" and the mouse antibody ing the framework changes is called the "donor". nt regions need not be present, but if they are, they are usually substantially identical to mouse antibody constant regions, z'.e. at least about 85- 90%, or about 95% or more identical. Hence, in some embodiments, a filll length murinized human heavy or light chain immunoglobulin contains a mouse constant region, human CDRs, and a substantially human framework that has a number of "murinizing" amino acid substitutions. Typically, a ized antibody" is an antibody comprising a zed variable light chain and/or a zed variable heavy chain. For example, 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 s of "murinization" binds to the same antigen as the parent antibody that provides the CDRs and is usually less immunogenic in mice, as compared to the parent antibody.
As used herein, the term "heavy chain n" includes amino acid sequences derived from an immunoglobulin heavy chain. A polypeptide sing a heavy chain portion comprises at least one of: a CHl domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 , or a variant or nt thereof. For example, a binding polypeptide for use in the invention can comprise a polypeptide chain comprising a CHl domain; a polypeptide chain comprising a CHl domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide 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 domain. In another embodiment, a polypeptide of the invention comprises a polypeptide chain comprising a CH3 domain. Further, a g polypeptide for use in PCT/U82012/043701 _ 22 _ the invention can lack at least a portion of a CH2 domain (e.g., all or part of a CH2 ). 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 certain antibodies, or antigen-binding fragments, variants, or derivatives thereof disclosed herein, the heavy chain portions of one polypeptide chain of a er are identical to those on a second polypeptide chain of the multimer. Alternatively, heavy chain portion-containing monomers of the invention are not identical. For e, each monomer can comprise a different target binding site, forming, for example, a bispecific antibody or diabody.
In another embodiment, the antibodies, or antigen-binding nts, variants, or derivatives thereof disclosed herein are composed of a single polypeptide chain such as scFvs and are to be expressed intracellularly (intrabodies) for potential in viva therapeutic and stic applications.
The heavy chain portions of a binding polypeptide for use in the diagnostic and ent methods disclosed herein can be derived from different immunoglobulin molecules. For e, a heavy chain portion of a ptide can comprise a CHl domain derived from an IgGl molecule and a hinge region derived from an IgG3 molecule. In another example, a heavy chain portion can se 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. In one embodiment, the light chain portion comprises at least one of a VL or CL domain.
The m size of a e or ptide epitope for an antibody is thought to be about four to five amino acids. Peptide or polypeptide epitopes can contain, for e, at least seven, at least nine, or 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, may not even be on the same peptide chain. In the present invention, a peptide or polypeptide epitope recognized by antibodies of the present invention contains a sequence of at least PCT/U82012/043701 _ 23 _ 4, at least 5, at least 6, at least 7, 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 0L- synuclein. As used herein, when an antibody is said to bind "within" a given range of amino acids, e.g., bind to an epitope "within amino acids 4 to 15 of u-synuclein," it is meant that the epitope encompasses the full range of stated amino acids or is smaller. In other words, for an e "within amino acids 4 to 15 of u-synuclein," the epitope can include the entire 12-amino acid peptide chain of 4 to 15, but can also be smaller, e.g., amino acids 4 to 12, amino acids 4 to 10 or amino acids 4 to 8. The person of ordinary skill in the art will also recognize that amino acids outside of the stated range may contribute to better binding affinity or increased recognition of a conformational epitope, but are not required for binding.
By "specifically binding", or "specifically recognizing", used interchangeably herein, it is generally meant that a binding molecule, e.g., an antibody binds to an epitope via its n-binding domain, and that the binding entails some complementarity 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 antigen-binding domain more readily than it would bind to a , unrelated e.
The term "specificity" is used herein to qualify the relative affinity by which a certain antibody binds to a certain e. For example, antibody "A" can be deemed to have a higher specificity for a given epitope than antibody "B," or antibody "A" can be said to bind to e "C" with a higher specificity than it has for related epitope "D".
Where present, the term ological g characteristics," or other binding characteristics of an antibody with an n, in all of its grammatical forms, refers to the specificity, affinity, cross-reactivity, and other binding characteristics of an antibody.
By "preferentially binding", it is meant that the binding molecule, e.g., antibody specifically binds to an epitope more readily than it would bind to a related, r, homologous, or analogous epitope. Thus, an dy which "preferentially binds" to a given epitope would more likely bind to that epitope than to a related epitope, even though such an antibody can cross-react with the related epitope.
By way of non-limiting example, a binding molecule, e.g., an antibody binds a first epitope preferentially if it binds said first epitope with a dissociation nt (KD) that is less than the antibody’s KD for the second epitope. In another non-limiting e, an antibody binds a first antigen entially if it binds the first epitope with an affinity that is at least one order of magnitude less than the antibody’s KD for the second e. In another miting example, an antibody binds 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 KD for the second epitope.
In another miting example, a binding molecule, e.g., an antibody binds 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 binds a first epitope preferentially if it binds the first epitope with an y that is at least one order of magnitude less than the antibody’s k(off) for the second epitope. In another non-limiting example, an antibody binds 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.
In some embodiments binding molecule, e.g, an antibody or antigen-binding nt, variant, or derivative disclosed herein can bind a u-synuclein or a fragment or variant f 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. In some ments, an antibody of the invention can bind a- synuclein or a fragment or t 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"1 5 X 10'6 sec'l, 10'6 sec'l, 5 X 10'7 sec"1 or 10'7 sec'l.
In certain embodiments, a g molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative disclosed herein can bind u-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. In some embodiments, an antibody of the invention can bind u-synuclein or a fragment or t thereof with an on rate (k(on)) greater 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.
A binding molecule, 6.g. an antibody is said to itively 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 degree, binding of the reference antibody to the epitope.
Competitive inhibition can be determined by any method known in the art, for example, competition ELISA assays. As an example, an antibody can competitively inhibit binding W0 2012/177972 2012/043701 _ 25 _ of the reference dy 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 individual epitope with the CDR of a binding molecule, e.g., an immunoglobulin molecule; see, e.g., Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988) at pages 27-28. As used herein, the term "avidity" refers to the overall stability of the complex between a population of immunoglobulins and an antigen, that is, the onal combining th of an immunoglobulin mixture with the antigen; see, e.g., Harlow at pages 29-34. Avidity is related to both the affinity of dual immunoglobulin molecules in the tion with specific epitopes, and also the valencies of the immunoglobulins and the n. For example, the ction between a bivalent monoclonal antibody and an n with a highly repeating epitope structure, such as a polymer, would be one of high avidity. The affinity or avidity of an antibody for an n can be determined experimentally using any suitable method; see, for example, Berzofsky et al., "Antibody-Antigen Interactions" In Fundamental Immunology, Paul, W. E., Ed., Raven Press New York, N Y (1984), Kuby, Janis Immunology, W. H. Freeman and Company New York, N Y (1992), and s described herein. General techniques for measuring the affinity of an antibody for an antigen include ELISA, RIA, and surface plasmon resonance. The measured affinity of a particular antibody-antigen interaction can vary if ed under different conditions, e.g., salt concentration, pH. Thus, measurements of affinity and other n- binding parameters, e.g., KD, IC50, can be made with standardized solutions of antibody and antigen, and a standardized buffer.
Binding molecules, e.g., antibodies or antigen-binding fragments, variants or derivatives thereof of the invention 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 antigen, 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 e generally contains many of the same complementary structural features as the inducing epitope, and in some cases, can actually fit better than the original. _ 26 _ 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 calculated using methods known in the art and described herein) to a nce epitope. In some ments, 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 analog, ortholog, or homolog of that epitope.
Binding molecules, e.g., dies or antigen-binding fragments, variants or derivatives thereof of the invention can also be described or specified in terms of their binding affinity to u-synuclein. Binding affinities include 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 10'4M, 10‘4 M, 5 x 10‘5 M, 10‘5 M, 5 x 10'6M, 10‘6 M, 5 X10'7M,10'7M,5 x 10‘8 M, 10'8M, 5 x 10'9M, 10‘9 M, 5 x 10'10M, 10'10M, 5 x 10‘11 M, 10‘11 M, 5 x10'12M,10'12M,5 x 10‘13 M, 10'13M, 5 x10— 14M, 10'14M, 5 x 10‘15 M, or 10‘15 M.
As previously indicated, the subunit structures and three dimensional configuration of the nt 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 domain" includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain. The CHl domain is nt to the VH domain and is amino terminal to the hinge region of an immunoglobulin heavy chain molecule.
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 s (residues 244 to 360, Kabat ing system; and es 231-340, EU numbering system; see Kabat EA et al. op. cit). The CH2 domain is unique in that it is not closely paired with another . Rather, two N—linked branched ydrate chains are interposed n the two CH2 domains of an intact native IgG W0 2012/177972 _ 27 _ molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminal of the IgG molecule and ses approximately 108 residues.
As used herein, the term "hinge region" includes the n of a heavy chain le 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, , and lower hinge domains; see Roux et al., J. Immunol. 161 (1998), 4083.
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 de 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 numbering system (position 226 or 229, EU numbering system).
As used herein, the terms "linked," "fused" or n" are used interchangeably.
These terms refer to the joining together of two more elements or components, by whatever means ing chemical conjugation or recombinant means. An "in-frame fusion" refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a uous longer ORF, in a manner that maintains the correct translational reading frame of the original ORFs. Thus, a recombinant fusion n is a single protein containing two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature). gh the reading frame is thus made continuous throughout the fused segments, the segments can be physically or spatially separated by, for e, in-frame linker sequence. For example, polynucleotides encoding the CDRs of an immunoglobulin variable region can be filSGd, in-frame, but be separated by a polynucleotide encoding at least one immunoglobulin framework region or additional CDR regions, as long as the "filSGd" CDRs are co- translated as part of a continuous polypeptide.
The term "expression" as used herein refers to a process by which a gene produces a biochemical, for example, an RNA or polypeptide. The process includes any manifestation of the fianctional presence of the gene within the cell including, without limitation, gene knockdown as well as both transient sion and stable expression. It PCT/U82012/043701 _ 28 _ includes t limitation transcription of the gene into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA ) or any other RNA product, and the translation of such mRNA into polypeptide(s). If the final desired product is a biochemical, expression includes the creation of that biochemical and any precursors. Expression of a gene produces a "gene product." As used herein, a gene product can be either a nucleic acid, e. g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript. Gene products described herein fiarther include nucleic acids with post riptional modifications, e.g., enylation, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.
As used herein, the term "sample" refers to any biological material obtained from a subject or patient. 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 t). A sample can also include a biopsy or tissue sample including neural . In still other aspects, a sample can comprise whole cells and/or a lysate of the cells. Blood samples can be collected 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 atase Inhibitors l and 2). The suspension can be kept on ice for 20 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 " 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 or disorder, such as the development of sonism. ial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or g of disease ssion, ration or palliation of the disease state, and ion (whether partial or total), whether able or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with PCT/U82012/043701 _ 29 _ the condition or disorder as well as those prone to have the condition or disorder or those in which the manifestation of the condition or disorder is to be prevented.
By ct" or "individual" or "animal" or nt" or l,” is meant any subject, particularly a mammalian subject, e.g., a human patient, for whom diagnosis, prognosis, prevention, or therapy is desired. 11. Antibodies The present ion generally relates to human anti-(x-synuclein antibodies and n-binding fragments thereof, which can demonstrate the immunological g characteristics and/or biological properties as outlined for the dies illustrated in the Examples. In accordance with the present invention human monoclonal dies specific for u-synuclein were cloned from a pool of aged subjects.
In the course of the experiments performed in accordance with the present invention initial attempts failed to clone u-synuclein specific antibodies but almost always resulted in false-positive clones. Further investigation of these clones revealed that they produced antibodies recognizing proteins of E. 0012'. In order to circumvent this problem, antibodies in conditioned media of human memory B cell cultures were screened in parallel for binding to coated fiJll-length alpha synuclein monomer and e of g to E. 0011'. proteins and bovine serum albumin (BSA). In particular, B cell conditioned medium was preabsorbed with E. coli proteins prior to subjecting the medium to an ELISA assay for screening of u-synuclein binding human antibodies.
Initial attempts at isolating specific antibodies were focused at pools of human subjects with high plasma binding activity to u-synuclein, tive of elevated levels of circulating u-synuclein antibodies plasma. These attempts failed to produce u-synuclein specific human memory B cells and the antibodies described in the current invention were isolated from pools of subjects with low plasma reactivity to u-synuclein.
Due to this measure, several antibodies were isolated. Selected dies were further analyzed for class and light chain subclass determination. Selected relevant antibody messages from memory B cell cultures are then transcribed by RT-PCR, cloned and combined into expression vectors for recombinant production; see PCT Publication No. Al. Exemplary anti-human clein antibodies NI-202.12F4, NI-202.3G12, and NI-202.3D8 are disclosed in PCT Publication No.
W0 2012/177972 Disclosed herein is human monoclonal antibody NI-202.22Dll. Recombinant expression of NI-202.22Dll in HEK293 or CHO cells and subsequent characterization of its binding specificity for human u-synuclein (Fig. 2A-B) was determined. Thus, one aspect of the present invention relates to the isolated human monoclonal anti-(x-synuclein antibody NI-202.22Dll and n-binding fragments, derivatives and variants thereof.
The present invention is also drawn to a binding molecule such as an antibody, or antigen-binding fragment, variant or derivatives thereof, where the antibody comprises a VH with the amino acid sequence of SEQ ID N014 or SEQ ID N020, and a VL with the amino acid sequence of SEQ ID N022 or SEQ ID NO:26, or n-binding fragments, ts or derivatives f. In one embodiment, NI-202.22Dl l, as well as variants, fragments, or derivatives thereof are terized as specifically binding human 0L- synuclein compared to human B-synuclein and human y-synuclein, and to human (1- synuclein as compared to murine u-synuclein. .22D11 preferentially binds to usynuclein in the oligomeric or aggregated form.
In one ment, the present invention is directed to an anti-(x-synuclein antibody, or antigen-binding fragment, variant or derivatives thereof, where the antibody specifically binds to the same epitope of u-synuclein as the reference antibody NI- 202.22Dl 1. As illustrated in the Examples, antibody .22Dll binds to u-synuclein truncations containing the inal acidic region (amino acids 96-140) in a direct ELISA assay, e.g., within amino acids 113 to 123 of SEQ ID NO:l, and specifically binds to an epitope within the amino acids PVDPDNE (amino acids 117-123 of SEQ ID NO: 1).
Antibody NI-202.22Dll preferentially binds to u-synuclein aggregates or fibrils over the monomeric form of u-synuclein as shown in Example 2. Furthermore, antibody NI-202.22Dll binds to pathological forms of u-synuclein in brain, e.g. pathological aggregates of u-synuclein as exemplified by histochemical staining described in Example 3. Hence, the present ion provides a new human anti-(x-synuclein antibody useful for diagnostic and therapeutic purposes.
In one embodiment, the present invention provides binding les, e.g., antibodies or antigen-binding nts, variants, or derivatives thereof which exhibit the precise binding ties of the ary NI-202.l2F4 antibody as described in PCT Publication No. Al. The present invention provides binding molecules which bind to an epitope at the N-terminus of u-synuclein, e. g., binding molecules which PCT/U82012/043701 _ 31 _ bind within amino acids 4 to 15 of SEQ ID NO:1. n embodiments provide binding molecules, e.g., dies or antigen-binding fragments, variants or derivatives thereof, which bind within amino acids 4 to 15 of SEQ ID NO:1, but excluding antibodies comprising a VH (SEQ ID N05 or SEQ ID NO:9), VL (SEQ ID NO:lO or SEQ ID NO:14), VHCDRI (SEQ ID NO:6), VHCDR2 (SEQ ID NO:7), VHCDR3 (SEQ IDNO:8), VLCDRI (SEQ ID NO:11), VLCDR2 (SEQ ID NO:12) and/or VLCDR3 (SEQ ID NO: 13) ofNI-202. 12F4, or fragments, ts, or derivatives thereof The present invention fithher provides binding molecules, e. g. antibodies and antigen-binding fragments, variants, or derivatives thereof, which ses at least one, two, three, four, five, or six mentarity determining regions (CDRs) of a NI- 202.22Dll VH and/or VL variable region comprising any one of the amino acid sequences depicted in Fig. l. The corresponding nucleotide ces encoding the above-identified variable regions are set forth in the attached sequence listing. An exemplary set of CDRs of the above amino acid sequences of the VH and/or VL region as depicted in Fig. 1 is also indicated in the appended sequence listing. However, as discussed in the following the person skilled in the art is well aware of the fact that in addition or alternatively CDRs can be used, which differ in their amino acid sequence from those set forth in Fig. l by one, two, three, four, five, or more amino acids. The VH of NI-202.22D11 is ented by amino acid sequence SEQ ID NO:15 and DNA ce SEQ ID NO:l9, and its GL-corrected form is represented as amino acid sequence SEQ ID N020 and DNA sequence SEQ ID NO:21. The VL of NI-202.22D11 is represented by amino acid ce SEQ ID N022 and DNA sequence SEQ ID N028, and its GL-corrected form is represented as amino acid sequence SEQ ID N026 and DNA sequence SEQ ID NO:27. The heavy chain CDR amino acid sequences of VH- CDRl, VH-CDR2 and VH-CDR3 are represented by SEQ ID NOl6, SEQ ID NO:l7, and SEQ ID NO:18, respectively. The light chain CDR amino acid sequences of VL-CDRl, VL-CDR2 and VL-CDR3 are represented by SEQ ID N023, SEQ ID N024, and SEQ ID NO:25, respectively.
In one embodiment, a binding molecule, e.g., an antibody or antigen g nt, variant, or derivative thereof of the present invention is any one of the antibodies comprising an amino acid sequence of the VH and/or VL region as depicted in Fig. 1. Alternatively, the antibody of the present invention is a binding molecule, e.g., an antibody or antigen binding fragment, variant, or derivative thereof which competes for binding to (x-synuclein with an antibody having a VH and/or VL region as depicted in Fig. 1. Those antibodies can be human as well, in particular for therapeutic applications.
Alternatively, the antibody is a murine, murinized and chimeric murine-human antibody, which are particularly useful for diagnostic methods and studies in animals.
As mentioned above, due to its generation upon a human immune response the human monoclonal antibody of the present invention will recognize epitopes which are of particular physiological relevance and which might not be accessible or less genic in case of immunization processes for the generation of for example mouse monoclonal antibodies and in in vitro screening of phage display libraries, respectively.
Accordingly, an epitope of a human anti-(x-synuclein antibody of the present invention can be unique. Therefore, the present invention also extends lly to anti-(x-synuclein antibodies and (x-synuclein binding molecules which compete with the human monoclonal dy of the present invention for specific binding to (x-synuclein. The present invention is more specifically directed to a binding molecule, e.g., an dy, or antigen-binding fragment, variant or tives f, where the antibody specifically binds to the same e of (x-synuclein as the reference antibody NI-202.22G11. ition between antibodies can be determined, for e, by an assay in which the immunoglobulin under test ts specific binding of a reference antibody to a common antigen, such as u-synuclein. Numerous types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich ition assay; see Stahli et al., Methods in Enzymology 9 (1983), 242-253; solid phase direct biotin-avidin EIA; see Kirkland et al., J. Immunol. 137 , 3614-3619 and Cheung et al., Virology 176 , 546-552; solid phase direct labeled assay, solid phase direct labeled sandwich assay; see Harlow and Lane, dies, A Laboratory Manual, Cold Spring Harbor Press (1988); solid phase direct label RIA using I125 label; see Morel et a], Molec. Immunol. 25 (1988), 7-15 and Moldenhauer et al., Scand. J. Immunol. 32 , 77-82. Typically, such an assay es the use of purified (x-synuclein or aggregates thereof bound to a solid surface or cells bearing either of these, an unlabelled test immunoglobulin and a labeled reference immunoglobulin, z'.e. a human monoclonal antibody of the present invention. Competitive inhibition is measured by determining the amount of label bound PCT/U82012/043701 _ 33 _ to the solid surface or cells in the presence of the test immunoglobulin. Usually the test immunoglobulin is present in excess. A itive binding assay can be performed under conditions as described for the ELISA assay in the appended es. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur. Usually, when a competing antibody is present in excess, it will inhibit specific g of a reference antibody to a common antigen by at least 50% or 75%. Hence, the present invention is further drawn to a binding molecule, e.g., an antibody, or antigen- binding fragment, variant or derivatives f, where the antibody competitively inhibits the reference antibody NI-202.22Gll from binding to (x-synuclein.
Also disclosed is an isolated binding molecule, e.g., an dy or antigen- binding fragment thereof which specifically binds to human (x-synuclein, comprising an immunoglobulin heavy chain variable region (VH) amino acid ce at least 80%, 85%, 90% 95% or 100% identical to SEQ ID NO: 15 or SEQ ID NO:20.
Further disclosed is an isolated binding molecule, e. g., an antibody or antigen- binding fragment thereof which specifically binds to human uclein, comprising a VH amino acid sequence identical to, or identical except for one, two, three, four, five, or more amino acid substitutions to SEQ ID NO: 15 or SEQ ID NO:20..
Also disclosed is an isolated binding molecule, e.g., an dy or antigen- binding fragment thereof which specifically binds to human (x-synuclein, comprising an immunoglobulin light chain variable region (VL) amino acid sequence at least 80%, 85%, 90% 95% or 100% identical to SEQ ID NO:22 or SEQ ID NO:26.
Some embodiments disclose an isolated binding molecule, e. g., an antibody or antigen-binding fragment thereof which specifically binds to human (x-synuclein, comprising a VL amino acid sequence identical to, or cal except for one, two, three, four, five, or more amino acid tutions, to SEQ ID NO:22 or SEQ ID NO:26.
In other embodiments, an isolated antibody or n-binding fragment thereof which specifically binds to human u-synuclein comprises, consists ially of, or ts of VH and VL amino acid sequences at least 80%, 85%, 90% 95% or 100% identical to: (a) SEQ ID NO:15 and SEQ ID NO:22, respectively,(b) SEQ ID NO:15 and PCT/U82012/043701 SEQ ID NO:26, respectively,(c) SEQ ID N020 and SEQ ID NO:22 , respectively,(d) SEQ ID N020 and SEQ ID NO:26, respectively. .
Also disclosed is an isolated binding molecule, e.g., an antibody or antigen- g fragment thereof which specifically binds to human clein, comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH), where at least one, two or all three VH-CDRs of the heavy chain variable region are at least 80%, 85%, 90% or 95% identical to reference heavy chain VH-CDRl, VH- CDR2 or VH-CDR3 amino acid sequences in Fig. l, and represented by SEQ ID N016, SEQ ID NO:l7, and SEQ ID NO:18, respectively. Thus, according to this embodiment a heavy chain variable region of the invention has VH-CDRl, VH-CDR2 and VH-CDR3 polypeptide sequences related to the VH-CDRl, VH-CDR2 and VH-CDR3 amino acid ces represented by SEQ ID N016, SEQ ID NO:l7, and SEQ ID NO:18, respectively. While Fig. 1 shows VH-CDRs defined by the Kabat system, other CDR definitions, e.g., s defined by the Chothia system, are also included in the present invention, and can be easily fied by a person of ordinary skill in the art using the sequence data presented.
Also disclosed is an isolated binding molecule, e.g., an antibody or antigen- binding fragment f which specifically binds to human clein, comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH) in which the VH-CDRl, VH-CDR2 and VH-CDR3 s have ptide sequences which are cal to the VH-CDRl, VH-CDR2 and VH-CDR3 amino acid sequences represented by SEQ ID N016, SEQ ID NO:l7, and SEQ ID NO:18, respectively.
Also disclosed is an ed binding molecule, e.g., an antibody or antigen- binding fragment thereof which specifically binds to human u-synuclein, comprising, ting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH) in which the VH-CDRl, VH-CDR2 and VH-CDR3 regions have polypeptide sequences which are identical to, or identical except for one, two, three, four, five, or six amino acid substitutions in any one VH-CDR, to the VH-CDRl, VH-CDR2 or VH-CDR3 amino acid sequences represented by SEQ ID NO:l6, SEQ ID NO:l7, and SEQ ID NO:18, respectively. Also provided is an immunoglobulin heavy chain variable region (VH) in which the VH-CDRl, VH-CDR2 and 3 regions have polypeptide W0 2012/177972 PCT/U82012/043701 _ 35 _ sequences which are identical to, or identical except for five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or twenty total CDR substitutions to amino acid sequences represented by SEQ ID NO:l6, SEQ ID NO:l7, and SEQ ID NO:18, respectively. In certain embodiments the amino acid substitutions are conservative.
Also disclosed is an isolated binding molecule, e.g., an antibody or antigenbinding fragment thereof which specifically binds to human u-synuclein, comprising, consisting essentially of, or ting of an immunoglobulin light chain variable region (VL), where at least one, two, or all three of the VL-CDRs of the light chain variable region are at least 80%, 85%, 90% or 95% identical to nce light chain VL-CDRl, VL-CDR2 or VL-CDR3 amino acid sequences ented by SEQ ID NO:23, SEQ ID N024, and SEQ ID NO:25, tively. Thus, according to this embodiment a light chain variable region of the invention has VL-CDRl, VL-CDR2 and VL-CDR3 polypeptide sequences d to the polypeptides shown in Fig. l and represented by SEQ ID NO:23, SEQ ID N024, and SEQ ID NO:25, respectively. While Fig. 1 shows VL-CDRs defined by the Kabat system, other CDR definitions, e. g., VL-CDRs defined by the Chothia system, are also included in the present invention.
Also disclosed is an isolated binding molecule, e.g., an antibody or antigen- binding fragment thereof which specifically binds to human u-synuclein, comprising, consisting essentially of, or consisting of an immunoglobulin light chain variable region (VL) in which the VL-CDRl, VL-CDR2 and VL-CDR3 regions have polypeptide sequences which are cal to the VL-CDRl, VL-CDR2 and VL-CDR3 groups shown in Fig. l and ented by SEQ ID NO:23, SEQ ID N024, and SEQ ID NO:25, respectively.
Also sed is an isolated binding molecule, e.g., an antibody or antigen- binding fragment thereof which specifically binds to human u-synuclein, comprising, consisting essentially of, or consisting of an immunoglobulin light chain variable region (VL) in which the VL-CDRl, VL-CDR2 and VL-CDR3 regions have polypeptide sequences which are identical to, or cal except for one, two, three, four, five, or six amino acid substitutions in any one VL-CDR, to the VL-CDRl, VL-CDR2 or VL-CDR3 amino acid sequences represented by SEQ ID NO:23, SEQ ID N024, and SEQ ID NO:25, respectively. Also provided is an globulin light chain le region (VL) in which the VL-CDRl, VL-CDR2 and VL-CDR3 regions have polypeptide PCT/U82012/043701 sequences which are identical to, or identical except for five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen total CDR substitutions to amino acid sequences represented by SEQ ID NO:23, SEQ ID N024, and SEQ ID NO:25, tively. In certain embodiments the amino acid substitutions are conservative.
An immunoglobulin or its encoding cDNA can be further modified. Thus, in a r embodiment the method of the present invention comprises any one of the step(s) of producing a chimeric antibody, murinized antibody, single-chain antibody, Fab- fragment, bi-specific antibody, fusion antibody, labeled antibody or an analog of any one of those. Corresponding methods are known to the person skilled in the art and are bed, e.g., in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor (1988). When tives of said antibodies are obtained by the phage display technique, surface plasmon resonance as employed in the e system can be used to se the efficiency of phage antibodies which bind to the same epitope as that of any one of the antibodies described herein (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). The production of chimeric antibodies is described, for e, in international application WO89/09622. Methods for the production of humanized antibodies are described in, e.g., European application EP-Al 0 239 400 and international application WO90/0786l. A further source of antibodies to be utilized in ance with the present invention are so- called xenogeneic antibodies. The general principle for the tion of xenogeneic antibodies such as human-like antibodies in mice is described in, e.g., international applications WO91/10741, WO94/02602, WO96/34096 and WO 96/33735. As sed above, an antibody of the invention can exist in a variety of forms besides complete dies; including, for example, Fv, Fab and , as well as in single chains, such as scFvs; see e.g. international application 9344.
The antibodies of the present invention or their corresponding immunoglobulin chain(s) can be further modified using conventional ques known in the art, for example, by using amino acid deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) known in the art either alone or in combination. Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are well known to the person skilled in the art; see, e.g., Sambrook, Molecular Cloning A tory Manual, W0 2012/177972 PCT/U82012/043701 Cold Spring Harbor tory (1989) NY. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY. (1994).
Modifications of the antibody of the invention include chemical and/or enzymatic derivatizations at one or more constituent amino acids, including side chain modifications, backbone modifications, and N- and C-terminal modifications including acetylation, hydroxylation, methylation, amidation, and the attachment of carbohydrate or lipid moieties, cofactors, and the like. Likewise, the present invention encompasses the tion of ic proteins which comprise the described antibody or some fragment thereof at the amino terminus fused to heterologous molecule such as an immunostimulatory ligand at the carboxyl terminus; see, e.g., international application WOOD/30680 for corresponding technical details.
Additionally, the present invention encompasses peptides including those containing a g molecule as described above, for example containing the CDR3 region of the variable region of any one of the mentioned antibodies, in particular CDR3 of the heavy chain since it has frequently been observed that heavy chain CDR3 (HCDR3) is the region having a greater degree of variability and a predominant participation in antigen-antibody interaction. Such peptides can easily be synthesized or ed by recombinant means to produce a binding agent useful ing to the invention. Such methods are well known to those of ordinary skill in the art. Peptides can be synthesized for example, using ted peptide synthesizers which are commercially available. The peptides can also be produced by recombinant techniques by incorporating the DNA expressing the e into an expression vector and transforming cells with the expression vector to produce the peptide.
Hence, the t invention relates to any binding molecule, e.g., an antibody or g fragment thereof which is oriented s the human anti-(x-synuclein antibodies of the present invention and display the mentioned ties, z'.e. which specifically recognize u-synuclein. Such dies and binding molecules can be tested for their binding specificity and affinity by ELISA and Western Blot and immunohistochemistry as described herein, see, e.g., the Examples. Furthermore, preliminary results of uent experiments performed in accordance with the present ion revealed that the human anti-(x-synuclein antibody of the present invention, in particular antibody NI-202.22D11 recognizes clein inclusion bodies present on PCT/U82012/043701 human brain sections of patients who suffered from dementia with Lewy bodies (DLB) or son’s disease (PD). Thus, in one embodiment of the present invention, the human antibody or binding fragment, tive or variant thereof izes (x-synuclein on human DLB or PD brain sections (see, e.g., Fig. 4c).
Immortalized B cells or B memory cells can be used as a source of rearranged heavy chain and light chain loci for subsequent expression and/or genetic manipulation.
Rearranged dy genes can be reverse transcribed from appropriate mRNAs to produce cDNA. If desired, the heavy chain constant region can be exchanged for that of a different isotype or eliminated altogether. Nucleotide sequences can be engineered to remove undesired motifs (such as splice sites or restriction sties), and the codon usage can be optimized for the cell in which the antibody or fragment thereof is to be expressed. In addition, one or more mutations which alter amino acids in the variable regions can be made, e.g., to increase affinity or improve stability. The variable regions can be linked to encode single chain Fv s. Multiple Fv regions can be linked to confer g ability to more than one target or chimeric heavy and light chain combinations can be employed. Once the genetic material is available, design of analogs as described above which retain both their ability to bind the desired target is straightforward. Methods for the cloning of antibody variable regions and generation of recombinant antibodies are known to the person d in the art and are described, for example, Gilliland et al., Tissue ns 47 (1996), 1-20; Doenecke et al., ia ll (1997), 1787-1792.
Once the appropriate genetic material is obtained and, if desired, modified to encode an analog, the coding sequences, including those that encode, at a minimum, the le regions of the heavy and light chain, can be inserted into expression systems contained on vectors which can be transfected into standard recombinant host cells. A variety of such host cells can be used; for efficient processing. Typical mammalian cell lines useful for this purpose include, but are not limited to, CHO cells, HEK 293 cells, or NSO cells.
The tion of the antibody or analog is then aken by culturing the modified inant host under culture conditions appropriate for the growth of the host cells and the expression of the coding sequences. The antibodies are then recovered by isolating them from the culture. Expression systems can be designed to include signal peptides so that the resulting antibodies are secreted into the medium; however, intracellular production is also possible.
In accordance with the above, the present invention also relates to a polynucleotide ng the antibody or equivalent binding molecule of the t invention, one or more CDRs, one or more of a heavy chain or light chain variable regions or variants thereof, of an immunoglobulin chain of the anti- u-synuclein antibodies described above.
The person skilled in the art will y appreciate that the le domain of an antibody, or any portion thereof can be used for the construction of other polypeptides or antibodies of desired specificity and biological on. Thus, the present invention also es polypeptides and antibodies comprising at least one heavy chain or light chain CDR, or such CDR with 1, 2, 3, 4, or more amino acid substitutions, of antibody NI- 202.22D11, which can have substantially the same or similar binding properties as NI- 202.22D11, described in the appended examples. The person skilled in the art knows that binding affinity can be enhanced by making amino acid substitutions within the CDRs or within the hypervariable loops (Chothia and Lesk, J. Mol. Biol. 196 (1987), 901-917) which partially p with the CDRs as defined by Kabat; see, e.g., Riechmann, et al, Nature 332 (1988), 323-327. Thus, the present invention also relates to antibodies wherein one or more of the mentioned CDRs comprise one or more amino acid substitutions. In certain embodiments, an antibody of the invention comprises in one or both of its immunoglobulin chains two or all three CDRs of the variable s (original or ted) as set forth in Fig. 1.
Binding molecules, e.g., dies, or antigen-binding fragments, variants, or derivatives thereof of the invention, as known by those of ordinary skill in the art, can comprise a constant region which mediates one or more effector filnctions. For example, binding of the C1 ent of complement to an antibody constant region can activate the complement system. Activation of complement is important in the opsonization and lysis of cell ens. The activation of complement also stimulates the inflammatory response and can also be involved in autoimmune ensitivity. Further, antibodies bind to receptors on various cells via the Fc region, with a PC receptor binding site on the antibody Fc region binding to a PC receptor (FcR) on a cell. There are a number of Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors).
Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfrnent and destruction of dy-coated particles, clearance of immune xes, lysis of dy-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer and control of immunoglobulin production.
Accordingly, certain embodiments of the present invention include an antibody, or antigen-binding nt, variant, or derivative thereof, in which at least a fraction of one or more of the constant region domains has been deleted or ise altered so as to provide desired biochemical characteristics such as reduced effector functions, the ability to non-covalently dimerize, increased ability to localize at the site of u-synuclein aggregation and deposition, d serum half-life, or increased serum half-life when compared with a whole, unaltered antibody of approximately the same immunogenicity.
For example, certain antibodies for use in the diagnostic and treatment methods described herein are domain deleted antibodies which comprise a polypeptide chain similar to an immunoglobulin heavy chain, but which lack at least a portion of one or more heavy chain domains. For instance, in certain antibodies, one entire domain of the constant region of the ed antibody will be deleted, for e, all or part of the CH2 domain will be deleted. In other embodiments, certain antibodies for use in the diagnostic and treatment methods described herein have a constant region, e.g., an IgG heavy chain constant region, which is d to eliminate glycosylation, referred to elsewhere herein as aglycosylated or "agly" dies. Such 'agly" antibodies can be ed enzymatically as well as by engineering the consensus glycosylation site(s) in the constant region. While not being bound by theory, it is believed that "agly" antibodies may have an improved safety and stability profile in viva. Methods of producing sylated antibodies, having desired effector function are found for example in ational application W02005/018572, which is incorporated by reference in its entirety.
In n antibodies, or antigen-binding fragments, variants, or derivatives thereof described herein, the Fc portion can be mutated to decrease effector function using ques known in the art. For example, the on or inactivation (through point mutations or other means) of a constant region domain can reduce Fc receptor binding of the circulating modified antibody thereby increasing u-synuclein localization. In other cases, nt region modifications consistent with the instant invention moderate complement binding and thus reduce the serum half life and nonspecific association of a conjugated cytotoxin. Yet other ations of the constant region can be used to modify disulfide linkages or oligosaccharide moieties that allow for enhanced localization due to increased antigen city or antibody flexibility. The resulting physiological profile, bioavailability and other biochemical effects of the modifications, such as 0L- synuclein localization, biodistribution and serum ife, can easily be measured and quantified using well know immunological techniques without undue mentation.
In n antibodies, or antigen-binding fragments, variants, or derivatives thereof described herein, the Fc portion can be mutated or exchanged for alternative protein sequences to increase the cellular uptake of antibodies by way of example by enhancing receptor-mediated endocytosis of antibodies via Fcy receptors, LRP, or Thyl receptors or by 'SuperAntibody Technology', which is said to enable antibodies to be shuttled into living cells without harming them (Expert Opin. Biol. Ther. (2005), 237-241). For example, the generation of filSlOI‘l proteins of the antibody g region and the cognate protein ligands of cell surface receptors or bi- or multi-specific dies with a specific sequences biding to u-synuclein as well as a cell e receptor can be engineered using techniques known in the art.
In certain antibodies, or antigen-binding fragments, variants, or derivatives thereof bed herein, the Fc portion can be d or exchanged for alternative protein ces or the antibody can be chemically modified to increase its blood brain r penetration.
Modified forms of antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be made from whole precursor or parent antibodies using techniques known in the art. Exemplary techniques are sed in more detail herein. Antibodies, or antigen-binding fragments, variants, or derivatives f of the invention can be made or ctured using techniques that are known in the art. In certain embodiments, antibody molecules or fragments thereof are binantly produced," z'.e., are produced using recombinant DNA technology.
Exemplary techniques for making antibody molecules or fragments thereof are discussed in more detail elsewhere herein.
Antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention also e tives that are modified, e.g., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from specifically g to its cognate e. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e. g., by glycosylation, acetylation, pegylation, orylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical ations can be carried out by known techniques, including, but not d to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative can contain one or more non-classical amino acids.
In certain embodiments, antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention will not elicit a deleterious immune se in the animal to be treated, e. g., in a human. In certain embodiments, binding molecules, e.g., antibodies, or antigen-binding fragments thereof of the invention are derived from a patient, e. g., a human patient, and are subsequently used in the same species from which they are derived, e.g., human, alleviating or minimizing the occurrence of deleterious immune responses.
De-immunization can also be used to decrease the immunogenicity of an antibody.
As used herein, the term munization" includes alteration of an antibody to modify T cell es; see, e.g., international ations WO98/52976 and WOOD/34317. For example, VH and VL sequences from the starting antibody are analyzed and a human T cell epitope "map" from each V region showing the location of epitopes in relation to complementarity determining regions (CDRs) and other key residues within the sequence.
Individual T cell epitopes from the T cell epitope map are analyzed in order to identify alternative amino acid substitutions with a low risk of altering activity of the final antibody. A range of ative VH and VL sequences are designed comprising combinations of amino acid substitutions and these sequences are subsequently incorporated into a range of g polypeptides, e.g., u-synuclein-specific dies or immunospecific fragments thereof for use in the diagnostic and ent methods disclosed herein, which are then tested for fianction. Typically, between 12 and 24 variant antibodies are generated and tested. Complete heavy and light chain genes comprising PCT/U82012/043701 _ 43 _ modified V and human C regions are then cloned into sion vectors and the subsequent plasmids introduced into cell lines for the production of whole antibody. The antibodies are then compared in appropriate biochemical and biological assays, and the optimal variant is identified.
Monoclonal antibodies can be ed using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for e, in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988); ling et al., in: Monoclonal Antibodies and T-Cell Hybridomas Elsevier, N.Y., 563-681 (1981), said references incorporated by reference in their entireties. The term lonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, ing any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Thus, the term "monoclonal antibody" is not limited to antibodies produced through hybridoma logy. In certain embodiments, antibodies of the present invention are derived from human B cells which have been immortalized via transformation with Epstein-Barr virus, as described .
In the well known hybridoma process (Kohler et al., Nature 256 (1975), 495) the relatively short-lived, or mortal, lymphocytes from a , e.g., B cells d from a human subject as bed herein, are fused with an immortal tumor cell line (e. g.,. a myeloma cell line), thus, producing hybrid cells or "hybridomas" which are both immortal and e of producing the genetically coded antibody of the B cell. The resulting hybrids are segregated into single genetic strains by selection, dilution, and re- growth with each individual strain comprising specific genes for the ion of a single antibody. They produce antibodies, which are homogeneous against a desired antigen and, in reference to their pure genetic parentage, are termed "monoclonal".
Hybridoma cells thus prepared are seeded and grown in a suitable culture medium that can contain one or more substances that inhibit the growth or survival of the unfilsed, parental myeloma cells. Those skilled in the art will appreciate that reagents, cell lines and media for the formation, selection and growth of hybridomas are commercially available from a number of sources and standardized protocols are well established.
PCT/U82012/043701 _ 44 _ Generally, culture medium in which the oma cells are growing is assayed for production of onal antibodies against the desired antigen. The binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by in vitro assays such as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA) as described herein. After hybridoma cells are identified that produce antibodies of the desired specificity, affinity and/or activity, the clones can be subcloned by limiting dilution ures and grown by standard methods; see, e.g., Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, pp 59-103 (1986). It will fiarther be appreciated that the onal antibodies secreted by the subclones can be separated from culture medium, ascites fluid or serum by conventional purification ures such as, for example, protein-A, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity tography.
In another embodiment, lymphocytes can be ed by micromanipulation and the variable genes isolated. For example, peripheral blood clear cells can be isolated from an immunized or naturally immune mammal, e.g, a human, and cultured for about 7 days in vitro. The cultures can be screened for specific IgGs that meet the ing criteria. Cells from positive wells can be isolated. Individual Ig-producing B cells can be isolated by FACS or by identifying them in a complement-mediated hemolytic plaque assay. Ig-producing B cells can be micromanipulated into a tube and the VH and VL genes can be amplified using, e. g., . The VH and VL genes can be cloned into an antibody expression vector and transfected into cells (e. g., eukaryotic or prokaryotic cells) for expression.
Alternatively, antibody-producing cell lines can be selected and cultured using techniques well known to the skilled artisan. Such techniques are described in a y of laboratory manuals and primary publications. In this respect, techniques suitable for use in the invention as described below are described in Current Protocols in Immunology, Coligan et al., Eds., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991) which is herein incorporated by reference in its entirety, including ments.
Antibody fragments that recognize specific es can be generated by known techniques. For example, Fab and F(ab')2 fragments can be produced recombinantly or by proteolytic cleavage of immunoglobulin les, using enzymes such as papain (to W0 2012/177972 PCT/U82012/043701 _ 45 _ produce Fab fragments) or pepsin (to produce F(ab')2 fragments). F(ab')2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain. Such nts are sufficient for use, for example, in immunodiagnostic procedures involving coupling the immunospecific portions of globulins to detecting reagents such as radioisotopes.
Completely human antibodies, such as described herein, are ularly desirable for therapeutic treatment of human patients. Human antibodies of the t invention are isolated, e.g, from elderly ts who because of their age may be suspected to be at risk of developing a disorder, e.g., Parkinson’s disease, or a patient with the disorder but with an unusually stable e course. However, though it is prudent to expect that elderly healthy and symptom-free subjects, respectively, more regularly will have developed protective anti-u-synuclein antibodies than younger subjects, the latter can be used as well as source for obtaining a human antibody of the present ion. This is particularly true for younger patients who are predisposed to develop a familial form of a einopathic disease but remain symptom-free since their immune system and response functions more efficiently than that in older adults.
In one embodiment, an antibody of the invention comprises at least one heavy or light chain CDR of an antibody molecule. In another embodiment, an antibody of the invention comprises at least two CDRs from one or more antibody molecules. In r embodiment, an antibody of the invention comprises at least three CDRs from one or more antibody molecules. In r embodiment, an antibody of the invention comprises at least four CDRs from one or more antibody molecules. In r embodiment, an antibody of the invention comprises at least five CDRs from one or more antibody molecules. In another embodiment, an antibody of the invention ses at least six CDRs from one or more antibody molecules. ary antibody molecules comprising at least one CDR that can be ed in the subject dies are described herein.
Antibodies of the present invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression techniques as described herein.
In one embodiment, an antibody, or antigen-binding fragment, variant, or derivative thereof of the invention comprises a synthetic constant region wherein one or more domains are partially or entirely d ("domain-deleted antibodies"). In certain PCT/U82012/043701 _ 46 _ ments compatible modified antibodies will comprise domain deleted constructs or variants n the entire CH2 domain has been removed (ACHZ constructs). For other embodiments a short connecting peptide can be substituted for the deleted domain to provide flexibility and freedom of movement for the variable . Domain deleted constructs can be derived using a vector ng an IgG1 human constant domain, see, e.g., international applications W002/060955 and W002/096948A2. This vector is engineered to delete the CH2 domain and provide a synthetic vector expressing a domain deleted IgG1 constant region.
In certain embodiments, antibodies, or antigen-binding fragments, variants, or derivatives thereof of the present invention are minibodies. Minibodies can be made using methods described in the art, see, e.g., US patent 5,837,821 or ational application WO 94/09817.
In one embodiment, an antibody, or n-binding fragment, t, or derivative thereof of the invention comprises an immunoglobulin heavy chain having deletion or substitution of a few or even a single amino acid as long as it s association between the monomeric subunits. For example, the on of a single amino acid in selected areas of the CH2 domain can be enough to ntially reduce Fc binding and thereby increase u-synuclein localization. Similarly, one or more constant region domains that control the effector function (e. g. complement binding) can be deleted. Such partial ons of the constant regions can improve selected characteristics of the antibody (serum half-life) while leaving other desirable functions associated with the subject nt region domain intact. Moreover, as alluded to above, the constant s of the disclosed antibodies can be synthetic through the mutation or substitution of one or more amino acids that enhances the profile of the resulting construct. In this respect it can be possible to disrupt the activity provided by a conserved binding site (e.g.
Fc binding) while substantially maintaining the configuration and immunogenic profile of the modified antibody. Yet other embodiments comprise the addition of one or more amino acids to the constant region to enhance ble characteristics such as effector function or provide for more cytotoxin or carbohydrate attachment. In such embodiments it can be desirable to insert or replicate specific sequences derived from selected constant region domains.
The present invention also provides antibodies that comprise, consist essentially of, or consist of, variants (including derivatives) of antibody molecules (e. g., the VH regions and/or VL regions) described herein, which antibodies or fragments f immunospecifically bind to u-synuclein. Standard techniques known to those of skill in the art can be used to introduce ons in the nucleotide sequence encoding an antibody, including, but not limited to, site-directed mutagenesis and diated mutagenesis which result in amino acid substitutions. Variants (including derivatives) can encode less than 50 amino acid tutions, less than 40 amino acid substitutions, less than 30 amino acid tutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, 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 , VH-CDRl, VH-CDRZ, VH-CDR3, VL region, VL-CDRl, VL- CDR2, or VL-CDR3. 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 charge. Families of amino acid es having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., , arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, e, phenylalanine, methionine, phan), 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 sequence, such as by saturation mutagenesis, and the resultant s can be screened for biological activity to identify mutants that retain activity (e.g., the ability to bind u-synuclein).
For example, it is possible to introduce mutations only in framework regions or only in CDR regions of an antibody molecule. Introduced mutations can be silent or neutral missense mutations, e.g., have no, or little, effect on an antibody’s y to bind antigen, indeed some such ons do not alter the amino acid sequence ever.
These types of mutations can be useful to optimize codon usage, or improve a hybridoma’s antibody production. Codon-optimized coding regions encoding antibodies WO 77972 PCT/U82012/043701 _ 48 _ of the t invention are disclosed elsewhere herein. Alternatively, non-neutral missense mutations can alter an antibody’s ability to bind antigen. The location of most silent and neutral missense mutations is likely to be in the framework regions, while the location of most non-neutral missense mutations is likely to be in CDR, though this is not an absolute requirement. 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 specificity). Following mutagenesis, the encoded protein can routinely be expressed and the fianctional and/or biological activity of the d n, (e. g., ability to immunospecifically bind at least one epitope of u-synuclein) can be determined using ques described herein or by routinely modifying techniques known in the art. 111. Polynucleotides Encoding Antibodies A polynucleotide encoding an antibody, or antigen-binding fragment, variant, or derivative thereof can be composed of any polyribonucleotide or polydeoxribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. For example, a polynucleotide encoding an antibody, or n-binding fragment, t, or derivative thereof can be composed of single- and double-stranded DNA, DNA that is a mixture of - and -stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded s, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, a polynucleotide encoding an antibody, or antigen-binding fragment, variant, or derivative thereof can be ed of triple- stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide encoding an antibody, or antigen-binding fragment, variant, or derivative thereof can also contain one or more modified bases or DNA or RNA backbones modified for ity or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" es chemically, enzymatically, or metabolically modified forms.
An isolated polynucleotide encoding a non-natural variant of a polypeptide derived from an immunoglobulin (e.g., an immunoglobulin heavy chain portion or light chain portion) can be d by introducing one or more nucleotide substitutions, additions or ons into the nucleotide sequence of the immunoglobulin such that one PCT/U82012/043701 _ 49 _ or more amino acid substitutions, additions or deletions are uced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions can be made at one or more non-essential amino acid residues.
As is well known, RNA can be isolated from the al B cells, hybridoma cells or from other transformed cells by standard techniques, such as guanidinium isothiocyanate extraction and precipitation followed by centrifilgation or chromatography.
Where desirable, mRNA can be isolated from total RNA by rd techniques such as chromatography on oligo dT cellulose. Suitable techniques are familiar in the art. In one embodiment, cDNAs that encode the light and the heavy chains of the antibody can be made, either simultaneously or separately, using reverse riptase and DNA polymerase in accordance with well known methods. PCR can be initiated by consensus constant region primers or by more specific primers based on the published heavy and light chain DNA and amino acid sequences. As discussed above, PCR also can be used to e DNA clones encoding the antibody light and heavy chains. In this case the libraries can be screened by consensus primers or larger homologous probes, such as human constant region .
DNA, typically d DNA, can be ed from the cells using techniques known in the art, restriction mapped and sequenced in accordance with standard, well known techniques set forth in detail, e.g., in the foregoing references relating to recombinant DNA techniques. Of course, the DNA can be synthetic according to the present invention at any point during the isolation process or subsequent analysis.
One embodiment provides an isolated polynucleotide sing, ting ially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain variable region (VH) amino acid sequence at least 80%, 85%, 90% 95% or 100% identical to SEQ ID NO:lS or SEQ ID NO:20.
Another embodiment es an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding a VH amino acid sequence identical to, or identical except for one, two, three, four, five, or more amino acid substitutions to SEQ ID NO:15 or SEQ ID NO:20.
Another ment provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain W0 2012/177972 2012/043701 variable region (VH), where at least one, two or all three of the CDRs of the heavy chain variable region are at least 80%, 85%, 90% or 95% identical to reference heavy chain VH-CDRl, VH-CDR2 or VH-CDR3 amino acid sequences represented by SEQ ID N016, SEQ ID NO:17, and SEQ ID NO:18, respectively. Thus, this embodiment provides an ed polynucleotide encoding a heavy chain variable region of the invention which has VH-CDRl, VH-CDR2 and VH-CDR3 amino acid sequences related to those represented by SEQ ID N016, SEQ ID NO:17, and SEQ ID NO:18, respectively, as shown in Fig. 1.
In another ment, the present invention provides an isolated polynucleotide comprising, consisting ially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain variable region (VH) in which the VH-CDRl, VH-CDRZ, and VH-CDR3 regions have polypeptide sequences which are identical to the VH-CDRl, VH-CDRZ, and VH-CDR3 groups represented by SEQ ID N016, SEQ ID NO:17, and SEQ ID NO:18, respectively, as shown in Fig. 1.
A fiarther embodiment provides an ed binding molecule e. g., an dy or antigen-binding nt comprising the VH d by the polynucleotide which specifically or preferentially binds to human (x-synuclein.
Another embodiment provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin light chain variable region (VL) amino acid sequence at least 80%, 85%, 90% 95% or 100% identical toSEQ ID NO:22 or SEQ ID NO:26.
A fiarther embodiment provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid ng a VL amino acid sequence identical to, or identical except for one, two, three, four, five, or more amino acid substitutions to SEQ ID NO:22 or SEQ ID NO:26.
Another embodiment provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a c acid encoding an immunoglobulin light chain variable region (VL), where at least one, two, or all three of the VL-CDRs of the light chain variable region are at least 80%, 85%, 90% or 95% identical to reference light chain VL-CDRl, 2 or VL-CDR3 amino acid sequences represented by SEQ ID N023, SEQ ID N024, and SEQ ID NO:25, respectively. Thus, this embodiment provides an isolated polynucleotide ng a light chain variable region of the invention which has VL-CDRl, VL-CDR2 and VL-CDR3 amino acid sequences d to those represented by SEQ ID N023, SEQ ID N024, and SEQ ID NO:25, respectively, as shown in Fig. 1.
In another embodiment, the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin light chain variable region (VL) in which the VL-CDRl , VL-CDRZ, and VL-CDR3 regions have polypeptide sequences which are identical to the VH-CDRl, VH- CDR2, and 3 groups represented by SEQ ID N016, SEQ ID NO:17, and SEQ ID NO:18, respectively, as shown in Fig. 1.
A fiarther ment provides an isolated binding molecule e. g., an antibody or antigen-binding fragment comprising the VL encoded by the polynucleotide which specifically or preferentially binds to human (x-synuclein.
As known in the art, "sequence identity" between two polypeptides or two polynucleotides is determined by comparing the amino acid or nucleic acid sequence of one ptide or polynucleotide to the sequence of a second ptide or cleotide. When discussed herein, whether any particular polypeptide is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to another polypeptide can be determined using methods and computer programs/software known in the art such as, but not limited to, the BESTFIT program (Wisconsin Sequence is Package, Version 8 for Unix, Genetics Computer Group, sity Research Park, 575 Science Drive, Madison, WI 53711). BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in d Mathematics 2 (1981), 482-489, to find the best segment of homology between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present ion, the parameters are set, of course, such that the percentage of identity is calculated over the full 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 d.
In one embodiment of the present invention, the cleotide comprises, consists essentially of, or consists of a nucleic acid having a polynucleotide sequence of the VH set forth in SEQ ID NO:19 or SEQ ID NO:21, or the VL set forth in SEQ ID NO:27 or SEQ ID NO:28. In this respect, the person skilled in the art will readily appreciate that the cleotides encoding at least the variable domain of the light W0 2012/177972 PCT/U82012/043701 and/or heavy chain can encode the le domain of both immunoglobulin chains or only one.
The present ion also includes fragments of the polynucleotides of the ion, as described elsewhere. Additionally polynucleotides which encode fusion polynucleotides, Fab fragments, and other tives, as described herein, are also plated by the invention.
The polynucleotides can be produced or manufactured by any method known in the art. For example, if the nucleotide ce of the antibody is known, a polynucleotide encoding the dy can be assembled from chemically synthesized oligonucleotides, e.g., as described in Kutmeier et al., BioTechniques 17 (1994), 242, which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
Alternatively, a polynucleotide ng an antibody, or antigen-binding fragment, variant, or derivative thereof can be generated from nucleic acid from a le . If a clone containing a nucleic acid encoding a particular antibody is not ble, but the sequence of the antibody le is known, a nucleic acid encoding the antibody can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, such as polyA+ RNA, isolated from, any tissue or cells expressing the a-synuclein-specific antibody, such as hybridoma cells selected to express an antibody) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.
Once the nucleotide sequence and ponding amino acid sequence of the dy, or antigen-binding fragment, variant, or derivative thereof is determined, its nucleotide sequence can be manipulated using methods well known in the art for the manipulation of tide sequences, e. g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. (1990) and Ausubel et al., eds., Current Protocols in Molecular W0 2012/177972 PCT/U82012/043701 _ 53 _ y, John Wiley & Sons, NY (1998), which are both incorporated by reference herein in their entireties), to generate antibodies having a different amino acid ce, for example to create amino acid substitutions, deletions, and/or insertions.
IV. Expression of Antibody Polypeptides ing manipulation of the isolated genetic material to provide antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention, the polynucleotides encoding the antibodies are typically inserted in an expression vector for introduction into host cells that can be used to produce the desired ty of antibody.
Recombinant expression of an antibody, or fragment, derivative or analog thereof, e.g., a heavy or light chain of an dy which binds to a target molecule is described herein.
Once a polynucleotide ng an antibody molecule or a heavy or light chain of an antibody, or n thereof (e.g., containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the tion of the antibody molecule can be ed by recombinant DNA technology using techniques well known in the art.
Thus, methods for ing a protein by expressing a polynucleotide containing an antibody encoding nucleotide ce are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing dy coding sequences and appropriate transcriptional and ational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in viva genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors can include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., international applications WO 07 and WO 89/01036; and US patent no. 5,122,464) and the variable domain of the dy can be cloned into such a vector for expression of the entire heavy or light chain.
The term "vector" or "expression vector" is used herein to mean vectors used in accordance with the present invention as a vehicle for introducing into and expressing a desired gene in a host cell. As known to those skilled in the art, such vectors can easily be selected from the group consisting of plasmids, phages, Viruses and iruses. In general, vectors compatible with the instant invention will comprise a selection marker, W0 2012/177972 PCT/U82012/043701 appropriate restriction sites to facilitate cloning of the desired gene and the ability to enter and/or replicate in eukaryotic or prokaryotic cells. For the purposes of this invention, numerous expression vector systems can be employed. For example, one class of vector utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, ia virus, baculovirus, iruses (RSV, MMTV or MOMLV) or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites. Additionally, cells which have integrated the DNA into their chromosomes can be selected by introducing one or more markers which allow selection of transfected host cells. The marker can provide for prototrophy to an ophic host, biocide resistance (e. g., antibiotics) or resistance to heavy metals such as copper. The selectable marker gene can either be directly linked to the DNA sequences to be expressed, or introduced into the same cell by nsformation. Additional ts can also be added for optimal sis of mRNA. These elements can e signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals.
In certain embodiments the cloned variable region genes are inserted into an expression vector along with the heavy and light chain constant region genes (e.g., human) as discussed above. In one embodiment, this is effected using a proprietary expression vector of Biogen IDEC, Inc., ed to as NEOSPLA, disclosed in US patent no. 6,159,730. This vector contains the cytomegalovirus promoter/enhancer, the mouse beta globin major promoter, the SV40 origin of replication, the bovine grth hormone polyadenylation sequence, neomycin phosphotransferase exon 1 and exon 2, the dihydrofolate reductase gene and leader sequence. This vector has been found to result in very high level expression of antibodies upon incorporation of variable and constant region genes, transfection in CHO cells, followed by selection in G418 containing medium and methotrexate amplification. Of course, any expression vector which is capable of eliciting expression in eukaryotic cells can be used in the present invention.
Examples of suitable vectors include, but are not limited to plasmids pcDNA3, Zeo, pCR3.l, pEFl/His, pIND/GS, MVZ, pSV4O/Ze02, pTRACER- HCMV, 5-His, pVAXl, and pZeoSV2 (available from Invitrogen, San Diego, CA), and plasmid pCI (available from Promega, Madison, WI). In general, screening large numbers of ormed cells for those which s suitably high levels if _ 55 _ globulin heavy and light chains is routine experimentation which can be carried out, for example, by robotic systems. Vector systems are also taught in US patent nos. ,736,137 and 5,658,570, each of which is incorporated by reference in its entirety herein.
This system provides for high expression levels, e. g., > 30 pg/cell/day. Other exemplary vector systems are disclosed e.g., in US patent no. 6,413,777.
In other embodiments the antibodies, or antigen-binding fragments, variants, or derivatives thereof of the ion can be expressed using polycistronic constructs such as those disclosed in US patent application publication no. 2003-0157641 A1 and incorporated herein in its entirety. In these expression s, multiple gene products of interest such as heavy and light chains of antibodies can be produced from a single polycistronic construct. These systems advantageously use an internal ribosome entry site (IRES) to provide relatively high levels of antibodies. Compatible IRES sequences are disclosed in US patent no. 6,193,980 which is also orated herein. Those skilled in the art will iate that such expression systems can be used to effectively produce the full range of antibodies disclosed in the instant ation.
More generally, once the vector or DNA sequence encoding a monomeric subunit of the antibody has been prepared, the expression vector can be introduced into an appropriate host cell. uction of the plasmid into the host cell can be accomplished by various techniques well known to those of skill in the art. These include, but are not limited to, transfection including lipotransfection using, e.g., Fugene or lipofectamine, protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact Virus. Typically, d introduction into the host is Via standard calcium phosphate co-precipitation method. The host cells harboring the expression construct are grown under conditions appropriate to the production of the light chains and heavy chains, and d for heavy and/or light chain n synthesis.
Exemplary assay techniques e enzyme-linked immunosorbent assay ), radioimmunoassay (RIA), or fluorescence-activated cell sorter analysis (FACS), immunohistochemistry and the like.
The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody for use in the s described . Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the ion, or a heavy or light chain thereof, W0 2012/177972 2012/043701 operably linked to a heterologous promoter. For the expression of double-chained antibodies, s encoding both the heavy and light chains can be inserted into a host cell for expression of the entire immunoglobulin molecule, as detailed below.
The host cell can be nsfected with two expression vectors of the invention, the first vector encoding a heavy chain derived ptide and the second vector encoding a light chain d polypeptide. The two vectors can contain identical selectable markers which enable equal expression of heavy and light chain ptides.
Alternatively, a single vector can be used which encodes both heavy and light chain polypeptides. In such situations, the light chain is advantageously placed before the heavy chain to avoid an excess of toxic free heavy chain; see Proudfoot, Nature 322 (1986), 52; Kohler, Proc. Natl. Acad. Sci. USA 77 (1980), 2197. The coding sequences for the heavy and light chains can comprise cDNA or c DNA.
As used herein, "host cells" refers to cells which harbor vectors constructed using recombinant DNA techniques and encoding at least one heterologous gene. In descriptions of processes for isolation of antibodies from recombinant hosts, the terms "cell" and "cell culture" are used interchangeably to denote the source of antibody unless it is clearly specified otherwise. In other words, recovery of polypeptide from the "cells" can mean either from spun down whole cells, or from the cell culture containing both the medium and the suspended cells.
A variety of xpression vector systems can be utilized to express antibody molecules for use in the methods described . Such host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells which can, when transformed or transfected with the riate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. 0011', B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, ) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems ed with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems ed with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression s (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e. g., COS, CHO, NSO, BLK, 293, 3T3 cells) harboring recombinant expression constructs containing ers derived from the genome of mammalian cells (e.g., othionein promoter) or from ian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Bacterial cells such as Escherichia coli, or eukaryotic cells, ally for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese Hamster Ovary (CHO) cells, in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective sion system for antibodies; see, e. g., Foecking et al., Gene 45 (1986), 101; Cockett et al., Bio/Technology 8 (1990), 2.
The host cell line used for protein expression is often of ian origin; those skilled in the art are credited with ability to determine particular host cell lines which are best suited for the desired gene product to be expressed therein. Exemplary host cell lines e, but are not limited to, CH0 (Chinese Hamster Ovary), DG44 and DUXBll (Chinese Hamster Ovary lines, DHFR minus), HELA (human al carcinoma), CV1 (monkey kidney line), COS (a derivative of CV1 with SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK, W138, R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line), SP2/O (mouse myeloma), P3x63- Ag3.653 (mouse myeloma), BFA-lclBPT (bovine endothelial cells), RAJI (human lymphocyte) and 293 (human kidney). Host cell lines are typically available from commercial services, the an Tissue Culture Collection or from published literature.
In addition, a host cell strain can be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e. g., ylation) and processing (e. g., cleavage) of protein products can be ant for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational sing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used.
For long-term, high-yield production of recombinant ns, stable expression is used. For example, cell lines which stably express the dy molecule can be engineered. Rather than using expression vectors which contain viral s of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g, promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the n DNA, engineered cells are allowed to grow for, e.g, 1-2 days in an enriched media, and then are ed to a selective media. The selectable marker in the recombinant plasmid confers ance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines which stably express the antibody molecule.
A number of selection s can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11 (1977), 223), nthine- guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48 (1992), 202), and adenine phosphoribosyltransferase (Lowy et al., Cell 22 (1980), 817) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, etabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers ance to methotrexate r et al., Natl. Acad. Sci. USA 77 (1980), 357; O'Hare et al., Proc. Natl. Acad. Sci. USA 78 (1981), 1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78 (1981), 2072); neo, which confers resistance to the aminoglycoside G-418 Goldspiel et al., Clinical Pharmacy 12 , 488-505; Wu and Wu, Biotherapy 3 (1991), 87-95; Tolstoshev, Ann. Rev. col. Toxicol. 32 (1993), 573-596; Mulligan, Science 260 (1993), 926- 932; and Morgan and Anderson, Ann. Rev. Biochem. 62 (1993), 191-217; TIB TECH 11 (1993), 155-215; and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30 (1984), 147. Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY ; Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and W0 2012/177972 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated by reference herein in their entireties.
The expression levels of an antibody le can be sed by vector amplification, for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Academic Press, New York, Vol. 3. (1987). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the dy will also increase; see Crouse et al., Mol. Cell. Biol. 3 , 257.
In vitro production allows scale-up to give large amounts of the desired polypeptides. Techniques for mammalian cell cultivation under tissue e conditions are known in the art and include homogeneous suspension culture, e.g. in an airlift reactor or in a continuous stirrer reactor, or lized or entrapped cell culture, e. g. in hollow fibers, microcapsules, on agarose eads or ceramic cartridges. If necessary and/or d, the solutions of polypeptides can be purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose or immuno-affinity chromatography, e. g., after preferential biosynthesis of a synthetic hinge region polypeptide or prior to or subsequent to the HIC chromatography step described herein.
Genes encoding antibodies, or n-binding fragments, variants, or derivatives thereof of the invention can also be expressed in mmalian cells such as bacteria or insect or yeast or plant cells. Bacteria which readily take up nucleic acids include members of the enterobacteriaceae, such as strains of Escherichia coli or Salmonella; aceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, and Haemophilus influenzae. It will filrther be appreciated that, when expressed in bacteria, the heterologous ptides typically become part of inclusion bodies. The heterologous polypeptides must be isolated, purified and then led into onal molecules.
Where tetravalent forms of antibodies are desired, the subunits will then self-assemble into tetravalent antibodies; see, e.g., international application W002/096948.
In bacterial systems, a number of expression vectors can be advantageously selected depending upon the use intended for the antibody molecule being sed. For example, when a large quantity of such a protein is to be produced, for the generation of ceutical itions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified can be used.
Such s include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2 (1983), 1791), in which the antibody coding ce can be ligated individually into the vector in frame with the lacZ coding region so that a filSlOI‘l protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13 (1985), 3101-3109; Van Heeke & Schuster, J. Biol. Chem. 24 (1989), 5503-5509); and the like. pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S- transferase (GST). In general, such fusion ns are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix of glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene t can be released from the GST moiety.
In addition to prokaryotes, eukaryotic microbes can also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among otic microorganisms although a number of other strains are commonly available, e.g., Pichia pastoris. For expression in Saccharomyces, the d YRp7, for example, hcomb et al., Nature 282 (1979), 39; Kingsman et al., Gene 7 (1979), 141; Tschemper et al., Gene 10 (1980), 157) is commonly used. This plasmid already contains the TRPl gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-l (Jones, Genetics 85 , 12).
The presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by grth in the absence of tryptophan.
In an insect system, Autographa calz’form’ca nuclear polyhedrosis virus ) is typically used as a vector to s foreign genes. The virus grows in Spodoptera frugz'perda cells. The antibody coding ce can be cloned individually into non- essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
PCT/U82012/043701 _ 61 _ Once an antibody molecule of the invention has been recombinantly expressed, the whole antibodies, their dimers, dual light and heavy chains, or other immunoglobulin forms of the present invention, can be purified according to standard procedures of the art, including for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, e. g. ammonium sulfate precipitation, or by any other standard technique for the purification of proteins; see, e.g., Scopes, "Protein Purification", Springer Verlag, NY. . Alternatively, a method for increasing the affinity of antibodies of the invention is disclosed in US patent publication 2002-0123057 Al.
V. Fusion Proteins and Conjugates In n embodiments, the antibody polypeptide comprises an amino acid sequence or one or more moieties not normally associated with an antibody. ary modifications are described in more detail below. For example, in some embodiments a -chain fv antibody nt of the invention can comprise a flexible linker sequence, or can be d to add a functional moiety (e.g., PEG, a drug, a toxin, or a label such as a cent, radioactive, enzyme, nuclear magnetic, heavy metal and the like) In certain embodiments, an antibody polypeptide of the invention comprises, ts essentially of, or consists of a fusion protein. Fusion proteins are chimeric molecules which comprise, for example, an immunoglobulin clein-binding domain with at least one target binding site, and at least one heterologous portion, z'.e., a portion with which it is not naturally linked in nature. The amino acid sequences can normally exist in separate proteins that are brought together in the fusion polypeptide or they can normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide. Fusion proteins can be created, for example, by chemical synthesis, or by ng and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.
The term "heterologous" as applied to a polynucleotide or a polypeptide, means that the cleotide or polypeptide is derived from a ct entity from that of the rest of the entity to which it is being compared. For ce, as used herein, a "heterologous polypeptide" to be fused to an antibody, or an antigen-binding fragment, WO 77972 PCT/U82012/043701 _ 62 _ variant, or analog thereof is d from a non-immunoglobulin polypeptide of the same species, or an immunoglobulin or non-immunoglobulin polypeptide of a different species.
As sed in more detail elsewhere herein, antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can r be recombinantly fused to a logous polypeptide at the N- or C-terminus or ally conjugated (including nt and non-covalent conjugations) to polypeptides or other compositions. For example, antibodies can be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins; see, e. g., international applications 8495; WO9l/l4438; 2624; US patent no. 5,314,995; and European patent application EP 0 396 387.
Antibodies, or antigen-binding fragments, variants, or derivatives f of the invention can be composed of amino acids joined to each other by peptide bonds or modified e bonds, z'.e., peptide isosteres, and can contain amino acids other than the gene-encoded amino acids. Antibodies can be modified by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous ch literature. Modifications can occur anywhere in the antibody, including the peptide backbone, the amino acid side- chains and the amino or carboxyl termini, or on moieties such as carbohydrates. It will be appreciated that the same type of modification can be t in the same or varying degrees at several sites in a given antibody. Also, a given antibody can contain many types of modifications. Antibodies can be branched, for example, as a result of ubiquitination, and they can be cyclic, with or without ing. Cyclic, branched, and branched cyclic antibodies can result from posttranslation natural processes or can be made by synthetic methods. Modifications include acetylation, acylation, ADP- lation, amidation, covalent attachment of flaVin, covalent attachment of a heme moiety, covalent attachment of a tide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, WO 77972 PCT/U82012/043701 _ 63 _ myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated on of amino acids to proteins such as arginylation, and tination; see, e.g, ns - Structure And Molecular Properties, T. E. Creighton, W. H. Freeman and Company, New York 2nd Ed., (1993); Posttranslational Covalent Modification 0f Proteins, B. C.
Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth.
Enzymol. 182 (1990), 626-646; Rattan et al., Ann. NY Acad. Sci. 663 (1992), 48-62).
The present invention also provides for 11 proteins comprising an antibody, or antigen-binding fragment, variant, or derivative thereof, and a heterologous polypeptide.
In one embodiment, a filSlOI‘l protein of the ion comprises, consists essentially of, or consists of, a polypeptide having the amino acid sequence of any one or more of the VH s of an antibody of the ion or the amino acid ce of any one or more of the VL regions of an antibody of the invention or fragments or ts f, and a heterologous polypeptide sequence. In another ment, a fusion protein for use in the diagnostic and treatment methods disclosed herein comprises, consists essentially of, or consists of a polypeptide having the amino acid sequence of any one, two, three of the VH-CDRs of an antibody, or fragments, variants, or derivatives thereof, or the amino acid sequence of any one, two, three of the VL-CDRs of an antibody, or fragments, variants, or tives thereof, and a heterologous polypeptide sequence. In one embodiment, the fusion n comprises a polypeptide having the amino acid sequence of a VH-CDR3 of an antibody of the present invention, or fragment, derivative, or variant thereof, and a heterologous polypeptide sequence, which fusion protein specifically binds to a- synuclein. In another embodiment, a filSlOI‘l protein comprises a polypeptide having the amino acid sequence of at least one VH region of an antibody of the invention and the amino acid sequence of at least one VL region of an antibody of the invention or fragments, derivatives or variants thereof, and a heterologous ptide sequence. In some embodiments, the VH and VL regions of the fusion n correspond to a single source antibody (or scFv or Fab fragment) which specifically binds a-synuclein. In yet another embodiment, a fusion protein for use in the diagnostic and treatment methods disclosed herein comprises a polypeptide having the amino acid sequence of any one, two, three or more of the VH CDRs of an antibody and the amino acid sequence of any one, two, three or more of the VL CDRs of an antibody, or fragments or variants thereof, PCT/U82012/043701 _ 64 _ and a heterologous polypeptide sequence. In certain embodiments, two, three, four, five, six, or more of the VH-CDR(s) or VL-CDR(s) correspond to single source antibody (or scFv or Fab nt) of the invention. Nucleic acid molecules encoding these fusion proteins are also encompassed by the invention.
Exemplary filSlOIl ns reported in the literature include filSlOIlS of the T cell receptor (Gascoigne et al., Proc. Natl. Acad. Sci. USA 84 , 2936-2940; CD4 (Capon et al., Nature 337 (1989), 525-531; Traunecker et al., Nature 339 (1989), 68-70; Zettmeissl et al., DNA Cell Biol. USA 9 (1990), 347-353; and Bym et al., Nature 344 , 667-670); L-selectin g receptor) (Watson et al., J. Cell. Biol. 110 (1990), 2221-2229; and Watson et al., Nature 349 (1991), 164-167); CD44 (Aruffo et al., Cell 61 (1990), 1303-1313); CD28 and B7 (Linsley et al., J. Exp. Med. 173 (1991),721-730); CTLA-4 (Lisley et al., J. Exp. Med. 174 (1991), 9); CD22 (Stamenkovic et al., Cell 66 (1991), 1133-1144); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88 (1991), 10535-10539; Lesslauer et al., Eur. J. Immunol. 27 (1991), 886; and Peppel et al., J. Exp. Med. 174 (1991), 1483-1489 (1991); and IgE receptor a (Ridgway and Gorman, J. Cell. Biol. 115 (1991), Abstract No. 1448).
As discussed elsewhere herein, antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be fused to logous polypeptides to increase the in viva half life of the polypeptides or for use in immunoassays using methods known in the art. For e, in one embodiment, PEG can be conjugated to the dies of the invention to increase their half-life in viva; see, e. g., Leong et al., Cytokine 16 , 106-119; Adv. in Drug Deliv. Rev. 54 (2002), 531; or Weir et al., m. Soc. Transactions 30 , 512.
Moreover, antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be fused to marker sequences, such as a peptide to facilitate their purification or detection. In certain embodiments, the marker amino acid sequence is a hexa-histidine peptide (HIS), such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif, , among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86 (1989), 821-824, for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37 (1984), 767) and the "flag" tag.
Fusion proteins can be prepared using s that are well known in the art; see for example US patent nos. 5,116,964 and 5,225,538. The e site at which the fusion is made can be selected empirically to optimize the secretion or binding characteristics of the fiJsion protein. DNA encoding the fusion protein is then transfected into a host cell for expression.
Antibodies of the present invention can be used in non-conjugated form or can be conjugated to at least one of a y of molecules, e. g., to improve the therapeutic ties of the le, to facilitate target detection, or for imaging or y of the patient. Antibodies, or antigen-binding fragments, ts, or derivatives thereof of the invention can be d or conjugated either before or after purification, when purification is performed. In particular, antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be conjugated to therapeutic agents, prodrugs, peptides, ns, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.
Conjugates that are toxins including tional antibodies have been widely described in the art. The toxins can be coupled to the antibodies by conventional coupling techniques or immunotoxins containing protein toxin portions can be produced as fusion proteins. The antibodies of the present invention can be used in a corresponding way to obtain such toxins. Illustrative of such immunotoxins are those described by Byers, Seminars Cell. Biol. 2 (1991), 59-70 and by Fanger, Immunol. Today 12 (1991), 51-54.
Those skilled in the art will appreciate that conjugates can also be assembled using a variety of techniques depending on the selected agent to be conjugated. For example, conjugates with biotin are prepared 6. g. by reacting an (x-synuclein binding polypeptide with an activated ester of biotin such as the biotin N-hydroxysuccinimide ester. Similarly, conjugates with a fluorescent marker can be prepared in the presence of a coupling agent, 6.g. those listed herein, or by reaction with an isothiocyanate, such as fluorescein-isothiocyanate. Conjugates of the antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention are prepared in an ous manner.
PCT/U82012/043701 _ 66 _ The present invention r encompasses antibodies, or antigen-binding nts, variants, or derivatives thereof of the invention conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, demonstrate presence of a neurological disease, to indicate the risk of getting a neurological disease, to monitor the development or progression of a neurological e, z'.e. synucleinopathic disease as part of a clinical testing procedure to, e.g., ine the efficacy of a given treatment and/or prevention regimen. Detection can be facilitated by coupling the antibody, or antigen-binding fragment, variant, or derivative f to a detectable substance. Examples of detectable substances include s enzymes, etic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission aphies, and nonradioactive paramagnetic metal ions; see, e.g., US patent no. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, ne phosphatase, B-galactosidase, or acetylcholinesterase; examples of le prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; es of bioluminescent als include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 125I, 131I, 111In or 99Tc.
An antibody, or antigen-binding fragment, variant, or tive thereof also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the uminescent-tagged antibody is then ined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent ng compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
One of the ways in which an antibody, or n-binding fragment, variant, or derivative thereof can be ably labeled is by linking the same to an enzyme and using the linked product in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked Immunosorbent Assay (ELISA)" Microbiological Associates Quarterly Publication, Walkersville, Md., Diagnostic Horizons 2 (1978), 1-7); Voller et al., J. Clin.
W0 2012/177972 PCT/U82012/043701 _ 67 _ Pathol. 31 (1978), 507-520; Butler, Meth. Enzymol. 73 (1981), 482-523; Maggio, E. (ed.), Enzyme Immunoassay, CRC Press, Boca Raton, Fla., (1980); Ishikawa, E. et al., (eds.), Enzyme Immunoassay, Kgaku Shoin, Tokyo (1981). The enzyme, which is bound to the dy will react with an appropriate substrate, such as a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by Visual means. Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-S-steroid isomerase, yeast alcohol ogenase, alpha- glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline atase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucosephosphate dehydrogenase, glucoamylase and acetylcholinesterase. Additionally, the detection can be accomplished by colorimetric methods which employ a chromogenic ate for the enzyme. Detection can also be accomplished by Visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
Detection can also be accomplished using any of a variety of other immunoassays.
For example, by radioactively labeling the antibody, or antigen-binding fragment, variant, or derivative thereof, it is possible to detect the antibody h the use of a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmanoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, (March, , which is incorporated by reference herein). The radioactive isotope can be detected by means including, but not limited to, a gamma counter, a scintillation counter, or autoradiography.
An antibody, or antigen-binding fragment, variant, or derivative thereof can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals can be ed to the dy using such metal ing groups as diethylenetriaminepentacetic acid (DTPA) or nediaminetetraacetic acid (EDTA).
Techniques for conjugating various moieties to an antibody, or antigen-binding fragment, variant, or derivative f are well known, see, e.g., Amon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer y", in Monoclonal Antibodies And Cancer Therapy, ld et al. (eds.), pp. 243-56 (Alan R.
PCT/U82012/043701 _ 68 _ Liss, Inc. (1985); Hellstrom et al., "Antibodies For Drug ry", in Controlled Drug ry (2nd Ed.), Robinson et al. (eds.), Marcel Dekker, Inc., pp. 623-53 (1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds), pp. 6 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Imrnunol. Rev. 62 (1982), 119-158.
As mentioned, in certain embodiments, a moiety that enhances the stability or efficacy of a binding molecule, e.g., a binding polypeptide, e. g., an antibody or immunospecif1c nt thereof can be conjugated. For example, in one embodiment, PEG can be conjugated to the binding molecules of the invention to increase their half- life in vivo. Leong et al., Cytokine 16 (2001), 106; Adv. in Drug Deliv. Rev. 54 , 531; or Weir et al., Biochem. Soc. ctions 30 (2002), 512.
VI. Compositions and Methods ofUse The present invention relates to compositions comprising the aforementioned 0L- ein binding molecule, e.g, antibody or antigen-binding fragment thereof of the present invention or derivative or t thereof, or the cleotide, vector or cell of the invention. The composition of the present invention can further comprise a pharmaceutically acceptable carrier. Furthermore, the pharmaceutical composition of the present invention can comprise further agents such as interleukins or interferons depending on the intended use of the pharmaceutical composition. For example, for use in the treatment of Parkinson’s disease the additional agent can be selected from the group consisting of small organic molecules, x-synuclein antibodies, and combinations thereof. Hence, in one ment the present invention relates to the use of the 0L- synuclein binding molecule, e.g, dy or antigen-binding fragment thereof of the present ion or of a binding molecule having substantially the same binding specificities of any one thereof, the cleotide, the vector or the cell of the present invention for the ation of a pharmaceutical or diagnostic composition for prophylactic and therapeutic treatment of a synucleinopathic e, monitoring the progression of a synucleinopathic disease or a response to a synucleinopathic disease PCT/U82012/043701 _ 69 _ treatment in a subject or for determining a subject's risk for developing a synucleinopathic disease.
Hence, in one embodiment the present invention s to a method of treating a neurological disorder characterized by abnormal accumulation and/or deposition of 0L- synuclein in the brain and the central nervous system, respectively, which method comprises administering to a subject in need thereof a therapeutically effective amount of an anti-u-synuclein binding molecule, antibody, cleotide, vector or cell of the instant invention. In certain embodiments NI-202.22Dll or a fragment, variant or derivative thereof is delivered. The term "neurological disorder" includes but is not limited to synucleinopathic diseases such as Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple systems atrophy (MSA). Unless stated otherwise, the terms neurodegenerative, neurological or neuropsychiatric are used interchangeably A particular advantage of the therapeutic approach of the present invention lies in the fact that the antibodies of the present invention are derived from B cells or B memory cells from elderly subjects with no signs of Parkinsonism and thus are, with a certain probability, capable of preventing a clinically manifest synucleinopathic disease, or of diminishing the risk of the occurrence of the ally manifest disease, or of delaying the onset or progression of the clinically manifest disease. Typically, the antibodies of the t invention also have already successfully gone through c maturation, z'.e. the optimization with respect to selectivity and effectiveness in the high affinity binding to the target u-synuclein molecule by means of somatic ion of the variable regions of the dy.
The knowledge that such cells in viva, e. g. in a human, have not been activated by means of related or other logical ns or cell structures in the sense of an autoimmunological or allergic reaction is also of great medical importance since this es a considerably increased chance of successfully living through the clinical test . So to speak, efficiency, acceptability and tolerability have already been demonstrated before the preclinical and al development of the prophylactic or therapeutic antibody in at least one human subject. It can thus be expected that the human anti-(x-synuclein dies of the present ion, both its target structure-specific W0 2012/177972 PCT/U82012/043701 _ 70 _ efficiency as therapeutic agent and its decreased probability of side effects significantly increase its clinical probability of success.
The present invention also provides a pharmaceutical and diagnostic, respectively, pack or kit comprising one or more containers filled with one or more of the above described ingredients, e.g. anti-u-synuclein dy, binding fragment, derivative or t thereof, polynucleotide, vector or cell of the present invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects al by the agency of manufacture, use or sale for human administration. In addition or alternatively the kit comprises reagents and/or instructions for use in appropriate diagnostic assays. The composition, 6.g. kit of the t invention is of course particularly le for the risk assessment, diagnosis, prevention and treatment of a disorder which is accompanied with the ce of u-synuclein, and in particular applicable for the treatment of Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple systems atrophy (MSA).
The pharmaceutical itions of the t ion can be formulated according to s well known in the art; see for example Remington: The Science and Practice of Pharmacy (2000) by the University of Sciences in Philadelphia, ISBN 0 306472. es of suitable pharmaceutical rs are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose.
Administration of the suitable itions can be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, intranasal, topical or intradermal administration or spinal or brain delivery. Aerosol formulations such as nasal spray formulations include purified aqueous or other solutions of the active agent with preservative agents and isotonic agents. Such ations can be adjusted to a pH and isotonic state compatible with the nasal mucous nes. Formulations for rectal or vaginal administration can be presented as a suppository with a suitable carrier.
Furthermore, whereas the present invention includes the now standard (though fortunately infrequent) procedure of drilling a small hole in the skull to administer a drug W0 2012/177972 PCT/U82012/043701 _ 71 _ of the t invention, in a one aspect, the binding molecule, especially antibody or antibody based drug of the present invention can cross the blood-brain barrier, which allows for intravenous or oral administration.
The dosage regimen will be determined by the attending ian and clinical factors. 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 administration, l health, and other drugs being administered concurrently. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non- aqueous solvents are propylene glycol, hylene , vegetable oils such as olive oil, and able organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous es e fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. vatives and other additives can also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
Furthermore, the pharmaceutical ition of the invention can comprise fiarther agents such as dopamine or psychopharmacologic drugs, depending on the intended use of the pharmaceutical composition.
Furthermore, a pharmaceutical composition can be formulated as a vaccine, for example, if the pharmaceutical ition of the invention comprises an anti-0L- synuclein antibody or g nt, derivative or variant thereof for passive immunization. As mentioned in the background section, oligomeric species of 0L- synuclein have been reported extracellularly in plasma and CSF (El-Agnaf et al., FASEB J. 20 (2006), 419—425) and passive immunization studies in mouse models of son’s disease show that ellular mouse monoclonal antibodies against u-synuclein can reduce accumulation of intracellular u-synuclein aggregates (Masliah et al., Neuron, 46 (2005), 857—868). Accordingly it is prudent to expect that the human anti-u-synuclein antibodies and equivalent u-synuclein binding molecules of the present invention are particularly useful as a vaccine for the tion or amelioration of synucleinopathic diseases such as PD, DLB and MSA.
In one embodiment, it is ial to use recombinant Fab (rFab) and single chain fragments (scFvs) of the antibody of the present invention, which might more readily penetrate a cell ne. For example, Robert et al., Protein Eng. Des. Sel. (2008) Oct 16; 81741-0134, published online ahead, be the use of ic recombinant Fab (rFab) and single chain fragments (scFvs) of monoclonal antibody WO-2 which recognizes an epitope in the N-terminal region of AB. The engineered fragments were able to (i) prevent amyloid fibrillization, (ii) disaggregate preformed ABl-42 fibrils and (iii) t ABl-42 oligomer-mediated neurotoxicity in vitro as efficiently as the whole IgG molecule. The perceived ages of using small Fab and scFv engineered antibody formats which lack the effector fianction include more efficient passage across the blood-brain barrier and minimizing the risk of triggering inflammatory side reactions.
Furthermore, besides scFv and single-domain antibodies retain the binding city of full-length antibodies, they can be expressed as single genes and intracellularly in mammalian cells as intrabodies, with the potential for alteration of the folding, interactions, modifications, or subcellular localization of their targets; see for review, e.g., Miller and Messer, Molecular Therapy 12 (2005), 394—401.
In a different approach Muller et al., Expert Opin. Biol. Ther. (2005), 237-241, describe a technology platform, so-called 'SuperAntibody Technology', which is said to enable antibodies to be shuttled into living cells without harming them. Such cell- penetrating antibodies open new diagnostic and therapeutic windows. The term 'TransMabs' has been coined for these antibodies.
A r embodiment es co-administration or sequential administration of other neuroprotective agents useful for ng a synucleinopathic disease. In one embodiment, the additional agent is comprised in the pharmaceutical ition of the t invention. es of neuroprotective agents which can be used to treat a subject include, but are not limited to, an acetylcholinesterase inhibitor, a glutamatergic receptor antagonist, kinase inhibitors, HDAC inhibitors, anti-inflammatory agents, divalproex sodium, or any combination thereof es of other rotective agents that can be used concomitant with pharmaceutical composition of the present invention are described in the art; see, e.g. international application WO2007/Oll907. In one embodiment, the additional agent is dopamine or a dopamine receptor agonist.
W0 2012/177972 PCT/U82012/043701 _ 73 _ In a filrther embodiment of the present invention the u-synuclein binding molecules, in particular antibodies of the present invention can also be co-administered or administered before or after transplantation y with neural transplants or stem cell y useful for treating a synucleinopathic disease. Such ches with transplants of embryonic mesencephalic neurons have been performed in patients with Parkinson’s disease with the aim of replacing the neurons that are lost in the disease and reinstating dopaminergic neurotransmission in the striatum. After 11-16 years post transplantation, the d neurons were found to contain Lewy bodies and Lewy neurites. This spread of u-synuclein pathology from the host to the grated tissues can be prevented by co- administration of u-synuclein binding molecules, in particular antibodies of the present ion.
A therapeutically effective dose or amount refers to that amount of the active ingredient sufficient to ameliorate the symptoms or condition. Therapeutic efficacy and toxicity of such compounds can be ined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between eutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LDso/EDSO. In n embodiments, the therapeutic agent in the composition is present in an amount sufficient to restore or preserve normal behavior and/or cognitive ties in case of PD, DLB or other synucleinopathic diseases.
From the foregoing, it is evident that the present invention encompasses any use of an clein binding molecule comprising at least one CDR of NI-202.22Dll or fragments, variants, or derivatives thereof, in particular for diagnosing and/or treatment of a synucleinopathic disease as mentioned above. The g molecule can be an antibody of the present invention or an immunoglobulin chain thereof. In addition, the present invention relates to anti-idiotypic antibodies of any one of the mentioned antibodies described herein. These are antibodies or other binding molecules which bind to the unique antigenic peptide sequence located on an antibody's le region near the antigen-binding site and are useful, e.g., for the ion of anti-(x-synuclein antibodies in sample of a t.
In another embodiment the present invention relates to a diagnostic composition comprising any one of the above described clein binding molecules, antibodies, W0 2012/177972 PCT/U82012/043701 _ 74 _ antigen-binding fragments, polynucleotides, s or cells of the invention and optionally suitable means for detection such as reagents conventionally used in immuno or nucleic acid based diagnostic methods. The antibodies of the invention are, for example, suited for use in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. Examples of assays which can utilize the antibody of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA), the sandwich (immunometric , flow cytometry and the Western blot assay. The antigens and antibodies of the invention can be bound to many different carriers and used to isolate cells specif1cally bound thereto. Examples of well known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, n, nylon, amyloses, natural and d celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble or insoluble for the purposes of the invention. There are many different labels and methods of labeling known to those of ordinary skill in the art. es of the types of labels which can be used in the t invention include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds, and bioluminescent compounds; see also the embodiments discussed hereinabove.
By a filrther embodiment, the u-synuclein binding molecules, in particular dies of the present invention are used in a method for the diagnosis of a disorder in an individual by obtaining a body fluid sample from the tested individual which can be a blood sample, a lymph sample or any other body fluid sample and ting the body fluid sample with an antibody of the instant invention under conditions enabling the formation of antibody-antigen complexes. The level of such xes 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 n bound by the antibodies of the invention can also be used. Thus, the present invention relates to an in vitro immunoassay comprising the binding molecule, e.g., antibody or antigen-binding fragment thereof of the invention.
In this context, the present invention also s to means specifically designed for this purpose. For e, an antibody-based array can be used, which is for example loaded with antibodies or equivalent antigen-binding molecules of the present invention which specifically ize u-synuclein. Design of microarray immunoassays is summarized in Kusnezow et al., Mol. Cell Proteomics 5 (2006), 696. Accordingly, the present invention also relates to microarrays loaded with u-synuclein binding molecules fied in accordance with the present invention.
In one embodiment, the t invention relates to a method of diagnosing a synucleinopathic disease in a subject, the method comprising: (a) assessing the level, localization, conformation or a combination thereof of u-synuclein in a subject to be diagnosed with the antibody or fragment thereof of any one of the invention and (b) comparing the level, localization, conformation or combination thereof of u-synuclein in the subject to one or more reference rds derived from one or more l samples, wherein a difference or similarity between the level, localization, conformation or ation thereof of u-synuclein in the subject and the nce standard indicates whether the subject has a synucleinopathic disease.
The subject to be diagnosed can be asymptomatic or nical for the disease.
The reference rd can be from a patient with a synucleinopathic disease, for example PD, DLB or MSA, where a similarity n the level, localization, conformation or combination thereof of u-synuclein in the subject to be diagnosed and the reference standard indicates that the subject to be diagnosed has a synucleinopathic disease.
Alternatively, or in addition a reference standard is derived from a subject does not have a einopathic disease. In n embodiments, the subject to be diagnosed and the reference standard(s) are age-matched. The analysis can be done in vivo, or via a sample isolated from the subject to be diagnosed, e. g., any body fluid suspected to contain 0L- synuclein, for example a blood, CSF, or urine sample The level, localization, and/or conformation of u-synuclein can be assessed by any le method known in the art comprising, e.g., analyzing u-synuclein by one or more techniques chosen from Western blot, immunoprecipitation, -linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescent activated cell sorting (FACS), two-dimensional gel electrophoresis, 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 W0 77972 2012/043701 _ 76 _ chromatography (HPLC), fast n liquid chromatography (FPLC), multidimensional liquid chromatography (LC) followed by tandem mass spectrometry (MS/MS), and laser densitometry. In vivo imaging of (x-synuclein can ses positron emission tomography (PET), single photon emission tomography (SPECT), near infrared (NIR) optical imaging or magnetic nce imaging (MRI).
Methods of diagnosing a synucleinopathic disease such as PD, DLB, or MSA, monitoring a synucleinopathic disease progression, and monitoring a synucleinopathic disease treatment using antibodies and related means which can be adapted in accordance with the t invention are also described in international application /011907. Similarly, antibody based detection methods for (x-synuclein are described in international applications WO99/50300, W02005/047860, /021255 and W02008/103472, the disclosure content of all being incorporated herein by reference. Those methods can be applied as described but with an (x-synuclein specific antibody, binding fragment, derivative or t of the present invention.
These and other embodiments are sed and encompassed by the description and examples of the present invention. Further ture concerning any one of the materials, methods, uses and compounds to be employed in accordance with the present invention can be retrieved from public libraries and databases, using for example electronic devices. For example the public database "Medline" can be utilized, which is hosted by the National Center for Biotechnology Information and/or the National y of ne at the National Institutes of Health. Further databases and web addresses, such as those of the European Bioinformatics ute (EBI), which is part of the European Molecular Biology Laboratory (EMBL) are known to the person skilled in the art and can also be obtained using intemet search engines. An ew of patent information in biotechnology and a survey of relevant sources of patent information useful for pective searching and for current awareness is given in Berks, TIBTECH 12 (1994), 352-364.
The above disclosure generally describes the present invention. Unless otherwise stated, a term as used herein is given the definition as provided in the Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, 1997, revised 2000 and reprinted 2003, ISBN 0 19 850673 2. Several documents are cited throughout the text of this specification. The contents of all cited references (including literature references, 2012/043701 _ 77 _ issued s, published patent applications as cited throughout this application and manufacturer's specifications, instructions, etc) are hereby expressly incorporated by reference; however, there is no ion that any document cited is indeed prior art as to the present invention.
A more complete understanding can be obtained by nce to the ing specific examples which are provided herein for purposes of illustration only and are not intended to limit the scope of the invention.
EXAMPLES The examples which follow fiarther illustrate the invention, but should not be construed to limit the scope of the invention in any way. The ing experiments in Examples 1 and 2 are illustrated and described with respect to antibody NI-202.3G12, NI- F4, and NI-202.3D8 as cloned, z'.e. containing primer induced mutations at the very N—termini of the framework 1 Ig-variable regions and not being adjusted to the germ line (GL) sequences of human variable heavy and light chains; see Figure 1. However, the other antibodies of the NI-202 series, in particular those with the adjusted GL sequences are structurally similar and thus can be expected to provide comparable results. These antibodies were expressed as human IgG1 molecules. The experiments in examples 3 and 4 are illustrated and described with respect to dy NI-202.12F4 with primer d mutations at the N—termini of the Ig-variable regions being adjusted to the germ line (GL) sequences of human variable heavy and light chains; see Figure 1. This antibody was sed as a chimeric le where the adjusted human variable domains were fused to mouse IgG2a constant regions to allow for chronic dosing studies in transgenic mouse models without to induce a mouse anti-human immune response.
Material and methods ed descriptions of conventional methods, such as those employed herein can be found in the cited literature. Unless indicated otherwise below, identification of (X- synuclein-speciflc B cells and molecular g of u-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 application W0 2012/177972 PCT/U82012/043701 _ 78 _ published as W02008/081008 and of l86 published as W02010/069603, the disclosure content of which are incorporated herein by reference in their entireties.
Purification of antigen Recombinant His-u-synuclein was obtained by recombinant expression in Escherichia coli and subsequent purification using heat induced precipitation, Nickel y-, anion exchange- and size exclusion-chromatography.
For example, a DNA uct comprising the cDNA encoding u-synuclein under the control of the T7 promotor was used to transform an appropriate Escherichia coli strain such as BL21(DE3) and expression of 200 ml cell culture was induced by the addition of lmM isopropyl iogalactopyranoside (IPTG). Cells were ted after 4 hrs induction at 37°C and then resuspended in 20 ml 50mM Tris, 150 mM NaCl pH 8, followed by sonification. After boiling for 15 min, the heat resistant l7000g supernatant was collected. Similar, heat-resistant l7000g supernatant from mock Escherichia coli was collected. After heat resistant l7000g supernatant (20 ml) from Escherichia coli expressing His-tagged u-synuclein was adjusted to 50 mM Tris, 300 mM NaCl, 20 mM Imidazole, pH 8, it was loaded onto a HisTrap HP lml (GE Life e) column and HIS-(x-synuclein was eluted with an 30-500mM ole gradient. Fractions ning HIS-u-synuclein were pooled and then diluted 1:10 with 50 mM Tris pH 8. Diluted pooled fractions were applied to a HiTrap Q HP lml (GE Life Science) column and bound proteins were eluted in a 30-1000 mM NaCl gradient. Finally, eluates containing HIS-(x-synuclein were further purified using high performance gel ion (Superdex 200 10/300 GL). This purification procedure yields HIS-u-synuclein with a purity grade of around 99% as estimated by SDS-PAGE and Coomassie staining. Concentration of purified n has been determined using a BCA assay (Pierce). u-symuclein antibody ing 96 well half area Microplates (Corning) were coated with purified HIS-0t- synuclein or d-synuclein (rPeptide) at a standard concentration of 2ug/ml in coating buffer (PBS pH 9.6) ght at 4°C. Plates were washed in PBS-T pH 7.6 and non- specific binding sites were blocked for 1 hr at RT with PBS-T containing 2% BSA (Sigma, Buchs, Switzerland). B cell conditioned medium was preabsorbed for 1 hr at RT with 10% Heat-resistant E. coli proteins in 1% BSA. This preabsorption step had been developed after several previous attempts of ELISA screening were unsuccessful in identifying human (x-synuclein c antibodies. Thus, fortunately it turned out that orption of the ELISA plate with heat-resistant E. coli proteins excludes screening for false positive hits such as sticky antibodies and antibodies directed against E. coli protein inations probably present in purified recombinant (x-synuclein samples.
Preabsorbed medium was then transferred from memory B cell culture plates to ELISA plates and incubated for 2 hrs at RT. ELISA plates were washed in PBS-T and then incubated with horse radish peroxidase conjugated donkey anti-human IgG (Fcy fragment specific) polyclonal antibodies. After washing with PBS-T, binding of human antibodies was determined by measurement of HRP activity in a standard colorimetric assay.
Molecular cloning of u-symuclein antibodies Samples containing memory B cells were obtained from eers >60 years of age. All eers had in common to lack any sign of Parkinsonism. Living B cells of selected memory B cell cultures are ted and mRNA is prepared. Immunoglobulin heavy and light chain sequences are then obtained using Ig-framework 1 specific primers for all human variable heavy and light chain families as 5’ primers in combination with primers specific for all human I segments (heavy and kappa light chain) and C segments (lambda light chain) as 3’primers (Marks et (1]., Mol. Biol. 222 (1991), 581-597; de Haard et al., J. Biol. Chem. 26 (1999), 18218-18230).
Identification of the antibody clone with the desired specificity is performed by re- screening on ELISA upon recombinant expression of te dies. inant expression of complete human IgGl dies or chimeric IgG2a antibodies is achieved upon insertion of the variable heavy and light chain sequences "in the correct reading frame" into expression vectors that complement the variable region ce with a sequence encoding a leader peptide at the 5’-end and at the 3’-end with a sequence encoding the appropriate constant domain(s). To that end the primers contained restriction sites designed to tate cloning of the variable heavy and light chain sequences into antibody expression vectors. Heavy chain immunoglobulin are expressed by inserting the immunoglobulin heavy chain RT-PCR product in frame into a heavy chain expression vector bearing a signal peptide and the nt domains of human immunoglobulin gamma 1 or mouse immunoglobulin gamma 2a. Kappa light chain immunoglobulin is sed by inserting the kappa light chain RT-PCR-product of NI-202.3D8 in frame into a light chain expression vector providing a signal peptide and the constant domain of human kappa light chain immunoglobulin. NI-202.12F4 and NI-202.3G12 lambda light chain immunoglobulins are expressed by inserting the lambda light chain RT-PCR- product in frame into a lambda light chain expression vector providing a signal peptide and the nt domain of human or mouse lambda light chain immunoglobulin.
Functional inant monoclonal antibodies were obtained upon co- ection into HEK293 or 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 human monoclonal antibody was subsequently purified from the conditioned medium using a rd Protein A column purification.
Antibodies Pan synuclein antibody Syn211 (Sigma) was used according to manufacturer’s protocol. Recombinant human u-synuclein antibodies N1202.22Gll and N1202.12F4 are antibodies of this invention. They were expressed in HEK293 or CHO cells and then conditioned media was directly used in subsequent applications unless otherwise stated.
Direct ELISA Antigens were coated at ted concentration in PBS pH 9.6 onto 96 well half area microplates (Corning) overnight at 4°C. Plates were washed in PBS-T pH 7.6 and non-specific binding sites were blocked for 1 hr at RT with PBS-T ning 2% BSA (Sigma). Probes (Primary antibodies) were then transferred to wells and incubated for 2 hrs at RT. After washing in PBS-T pH 7.6, wells were incubated with horse radish peroxidase (HRP)-conjugated polyclonal anti-human (for recombinant human antibodies), anti-rabbit (for pan synuclein antibody) or anti-mouse (for LB509 or Syn2l l) secondary antibodies for 1 hr at RT. After rigorous washing in PBS-T, binding of probes was ined by measurement of HRP ty in a standard colorimetric assay using ,5'-tetramethylbiphenyl-4,4'-diamine (Sigma) as chromogenic substrate. _ 81 _ Peptide scan for epitope mapping The entire sequence of human u-synuclein was synthesized as pping peptides, with lengths of 15 amino acids (aa) and an overlap of ll aa, coupled via a flexible linker to cellulose membrane (JPT, Berlin, Germany). A membrane that comprises a total of 33 peptides was rinsed in methanol and then blocked with Rotiblock (Roth, Karlsruhe, y). The membrane was ted with indicated antibodies diluted in blocking solution and then with horse radish peroxidase (HRP)-labeled secondary dy for 1 hr. Between incubations the membrane was washed 3x with PBS-T for 5 min. The membrane was then developed using ECL plus Western Blotting Detection Reagents (GE Healthcare).
In-solution ELISA NI-202.12F4 (2 ug/ml) diluted in sodium bicarbonate buffer (pH 9.6) was coated at 4°C overnight onto ELISA plates. Then the plate was blocked with 2% BSA PBS-T and subsequently washed with PBS-T. Indicated biotinylated u-synuclein peptides were added and after 2 hrs incubation the plates were washed with PBS-T. After incubation with HRP labeled streptavidin for 1 hr, binding was determined by measurement of HRP activity in a standard colorimetric assay.
Example 1: Human derived u-synuclein antibody NI-202.21Dll is selective for human 0L- synuclein u-,B- and y-synuclein are highly homologous proteins that are predominantly expressed in the nervous system, skeletal muscle and heart. clein is ly linked to a broad spectrum of CNS diseases whereas B-synuclein can be a neuroprotective n. Thus the ion provides therapeutic antibodies against ogical 0L- synuclein variants which do not cross react with B- and y-synuclein. In order to t potential therapeutic use of NI-202.21Dl l, the antibody was tested for binding to (1-, B- and y-synuclein in a direct ELISA. Recombinant u-,B and y-synuclein was coated onto ELISA plates at equal concentration and then either incubated with recombinant NI- 202.21Dll or a control pan synuclein antibody. The pan-synuclein dy detects all three synuclein proteins but NI-202.21Dll ys selective binding for u-synuclein (Fig 2a).
WO 77972 PCT/U82012/043701 _ 82 _ Human and mouse u-synuclein are highly conserved proteins. To probe if recombinant NI-202.21Dll preferentially binds human vs murine (x-synuclein, recombinant His-tagged human or murine (x-synuclein were coated onto ELISA plates at equal tration and then tested for NI-202.21Dll and NI-202.12F4 g (Fig 2b).
NI-202.21Dll detects only human (x-synuclein s NI-202.12F4 detects both human and murine (x-synuclein in this direct ELISA (see PCT Publication No.
Al). Together these findings demonstrate that NI-202.21Dll is highly selective for human u-synuclein.
Example 2: NI-202.21Dll shows preferential binding to human a-synuclein at high coating concentrations pointing to a conformational epitope The half maximal effective concentration (ECSO) ting the potency of NI- 202.21Dll was determined for low and high coating concentrations of recombinant 0t- synuclein using a direct (x-synuclein ELISA. High affinity binding of recombinant NI- 202.21Dll with an ECSO of ~200 pM was observed for high coating concentrations of 0L- synuclein protein (20 ug/ml). At lower concentrations of (x-synuclein, a sharp decrease in affinity was observed (Fig 3). These characteristics are in strong contrast to commercially available dy synle that is also detecting an epitope in the C-terminal domain of 0t- synuclein. This finding suggests that NI-202.21Dll prefers an epitope that is formed or exposed under high density conditions such as found in high molecular weight species of u-synuclein. e 3: Recombinant NI-202.22Dll binds to ogical (x-synuclein species in the brain.
Binding of .21Dll to human (x-synuclein was filrther characterized by immunohistochemical ng of brain sections from uclein transgenic mice and from a patient with a neuropathologically confirmed synucleinopathy (Dementia with Lewy ). .21Dll shows prominent staining of Lewy Body and Lewy Neurite like inclusions on Proteinase K treated paraffin sections from brain tissue of transgenic mice overexpressing human u-synuclein A53T (Fig 4a). No N1202-21Dll staining was detected in brain sections from wild-type mice supporting that NI-202.21Dll is specific for human (x-synuclein (Fig 4b). NI-202.21Dll also detected pathological (x-synuclein in human brain tissue of a patient with Dementia with Lewy Body (Fig 4c). These results PCT/U82012/043701 _ 83 _ show that human-derived antibody NI-202.21D11 detects pathological u-synuclein in brain.
Example 4: Mapping the epitope of human derived clein-specific antibody NI- D11 to an epitope within C-terminal domain of human clein. u-synuclein is a 140 amino acids (aa) long natively unfolded protein that is ed of three domains. These are the N-terminal amphipathic repeat region (aa 1- 60), the center region (aa 61-95) and the acidic C-terminal region (aa 96-140). (A) In order to get an initial understanding for the .21D11 binding domain, recombinant u-synuclein truncations were tested for NI-202.21D11 binding in a direct ELISA.
Recombinant clein truncations from residues 1-60, 1-95, 61-140 and 96-140 were coated onto ELISA plates and then incubated with recombinant NI-202.21D11. Binding of NI-202.21D11 was only observed to u-synuclein truncations 61-140 and 96-140 demonstrating that NI-202.21D11 binds to the C-terminal acidic domain of u-synuclein (Fig. 5a).
In order to understand the recognition sequence of NI-202.21D11 in more detail, NI-202.21D11 was tested for binding to overlapping linear 15-mer peptides that cover the entire human u-synuclein amino acid sequence. Adjacent peptides share an overlap of 11 residues and peptides are C-terminally spotted to a cellulose support membrane. NI- 202.21D11 bound to three pping peptides namely es 109-123 (B08), 113-127 (B09) and 117-131 (B10) of human u-synuclein (Fig. 5b). This result suggests the minimal ition sequence within the C-terminus of u-synuclein required for NI- 202.21D11 binding is PVDPDNE (117-123). Notably, NI-202.21D11 bound peptide B10 slightly less than peptides B08 and B09. Thus es 113-117 within a-synuclein may influence on NI-202.21D11 g.
Almost no binding of NI-202.21D11 to mouse u-synuclein was observed in a direct ELISA (Fig. 2B). Sequence alignment of the determined epitope sequence of NI- 202.21D11 (PVDPEE) to the corresponding murine sequence E) suggest that D121 and N122 are key amino acids for selectivity of NI-202.21D11 for human vs. murine a-synuclein. In order to confirm the key role of D121/N 122, recombinant d human a-synuclein D121G/N122S was produced and tested for NI202.21D11 binding in a direct ELISA. As shown in Figure 5c NI-202.21D11 showed almost no binding to human u-synuclein D121G/N122S compared to wt human u-synuclein. A control pan- PCT/U82012/043701 _ 84 _ synuclein antibody was used as normalization l for equal coating of synuclein proteins.
These results show that NI-202.21D11 is a human-derived uclein antibody detecting a C-terminal epitope (residues 117-123) within human (x-synuclein and that amino acids D12 1/N 122 contribute to human vs. murine (x-synuclein the selectivity.
Example 5: NI-202.12F4 detects epitope within (x-synuclein 4-15 and K10 in (x-synuclein is key amino acid for (x-synuclein selectivity ofNI-202. 12F4 In order to understand the recognition sequence (epitope) of NI-202.12F4 in more , overlapping linear 15-mer peptides that cover the entire human (x-synuclein amino acid sequence were tested for NI-202.12F4 binding by immunoblotting. Adjacent peptides share an overlap of 11 es and peptides were C-terminally spotted to a cellulose support ne. NI-202.12F4 only bound to the very N-terminal peptide (A01) g that epitope is within residues 1-15 (Fig. 6a). Since NI-202.12F4 does not bind to peptide (A02) residues 5-20, the epitope starts between residues 1 and 5. To determine the exact start e of the epitope, synthetic (x-synuclein peptides were tested for .12F4 binding in an in-solutz'on binding ELISA. First in order to validate in- solution binding ELISA, synthetic peptides (x-synuclein 1-30 and 5-30 were tested for NI- 202.12F4 binding. NI-202.12F4 bound (x-synuclein 1-30 but not 5-30 validating the assay by confirming the epitope starts between residue 1 and 5 (Fig. 6b). Next, (x-synuclein 4-30 was tested for NI-202.12F4 binding. As show in Figure 6b NI-202.12F4 bound to 0L- synuclein 4-30. These results show that NI-202.12F4 epitope starts at residue 4. .12F4 ively bound to (x-synuclein but not B- and y-synuclein.
Sequence alignment of the NI-202.12F4 epitope containing sequence (u-synuclein 4-15) with the ponding B-synuclein sequence showed that these sequences only differed in one amino acid. Lysine at position 10 in (x-synuclein is replaced by methionine in B- synuclein. Thus 12F4 should bind to clein M10K but not (x-synuclein K10M. For experimental confirmation, recombinant wt and K10M (x-synuclein, and wt and M10K B-synuclein were tested for NI-202.12F4 binding in a direct ELISA. As predicted N1202.12F4 only bound to wt (x-synuclein and B-synuclein M10K but not to wt B-synuclein and (x-synuclein K10M (Fig 6c). A pan-synuclein antibody bound to all four recombinant proteins equally well demonstrating equal g onto ELISA plates.
W0 2012/177972 PCT/U82012/043701 _ 85 _ All er these experiments show that the epitope for N1202.12F4 is localized within residues 4-15 of u-synuclein. The epitope starts at residue 4 and ends between residue 11-15. Lysine at position 10 in (x-synuclein accounts for the specificity of NI- 202.12F4 for u- versus B-synuclein.

Claims (54)

What we claim is:
1. An isolated antibody or n-binding fragment thereof that specifically binds to human α-synuclein (SEQ ID NO:1), said antibody or n-binding nt comprising: a VH CDR1 region comprising the amino acid ce of SEQ ID NO:16 or a ce that differs from SEQ ID NO:16 by one amino acid; a VH CDR2 region comprising the amino acid sequence of SEQ ID NO:17 or a sequence that differs from SEQ ID NO:17 by one amino acid; a VH CDR3 region comprising the amino acid sequence of SEQ ID NO:18 or a sequence that differs from SEQ ID NO:18 by one amino acid; a VL CDR1 region comprising the amino acid sequence of SEQ ID NO:23 or a sequence that differs from SEQ ID NO:23 by one amino acid; a VL CDR2 region comprising the amino acid sequence of SEQ ID NO:24 or a sequence that differs from SEQ ID NO:24 by one amino acid; and a VL CDR3 region comprising the amino acid sequence of SEQ ID NO:25 or a sequence that differs from SEQ ID NO:25 by one amino acid.
2. The antibody or antigen-binding nt of claim 1, comprising a VH and a VL that are respectively at least 95% identical to SEQ ID NO:15 and SEQ ID NO:22; SEQ ID NO:15 and SEQ ID NO:26; SEQ ID NO:20 and SEQ ID NO:22; or SEQ ID NO:20 and SEQ ID NO:26.
3. The antibody or antigen-binding fragment of claim 1 or claim 2, wherein the VH ses a polypeptide sequence at least 95% identical to SEQ ID NO:15 or SEQ ID NO:20.
4. The antibody or antigen-binding fragment of any one of claims 1 to 3, wherein the VH comprises SEQ ID NO:15 or SEQ ID NO:20.
5. The antibody or antigen-binding fragment of claim 1 or claim 2, wherein the VL comprises a polypeptide sequence at least 95% identical to SEQ ID NO:22 or SEQ ID NO:26.
6. The antibody or antigen-binding fragment of claim 1 or claim 2, wherein the VL comprises SEQ ID NO:22 or SEQ ID NO:26.
7. The antibody or antigen-binding fragment of claim 1 or claim 2, wherein the VH and VL respectively comprise: SEQ ID NO:15 and SEQ ID NO:22; SEQ ID NO:15 and SEQ ID NO:26; SEQ ID NO:20 and SEQ ID NO:22; or SEQ ID NO:20 and SEQ ID NO:26.
8. The antibody or antigen-binding fragment of claim 1, wherein: the CDR1, CDR2 and CDR3 regions of the VH comprise amino acid sequences SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, respectively; and the CDR1, CDR2 and CDR3 regions of the VL se amino acid sequences SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25, respectively.
9. An isolated dy or n-binding fragment thereof that specifically binds to human α-synuclein (SEQ ID NO:1) comprising: a VH CDR1 region comprising the amino acid sequence of SEQ ID NO:16; a VH CDR2 region comprising the amino acid sequence of SEQ ID NO:17; a VH CDR3 region comprising the amino acid sequence of SEQ ID NO:18; a VL CDR1 region comprising the amino acid sequence of SEQ ID NO:23; a VL CDR2 region comprising the amino acid sequence of SEQ ID NO:24; and a VL CDR3 region comprising the amino acid sequence of SEQ ID NO:25.
10. The antibody or antigen-binding fragment thereof of claim 9, comprising: SEQ ID NO:15 and SEQ ID NO:22; SEQ ID NO:15 and SEQ ID NO:26; SEQ ID NO:20 and SEQ ID NO:22; or SEQ ID NO:20 and SEQ ID NO:26.
11. The antibody or antigen-binding fragment of any one of claims 1 to 10, further comprising a signal peptide fused to the VH.
12. The antibody or antigen-binding nt of any one of claims 1 to 11, further comprising a signal peptide fused to the VL.
13. The antibody or antigen-binding fragment of any one of claims 1 to 12, further comprising a CH1 domain fused to the VH.
14. The antibody or antigen-binding fragment of any one of claims 1 to 13, further comprising a CH2 domain fused to the VH.
15. The antibody or antigen-binding nt of any one of claims 1 to 14, further comprising a CH3 domain fused to the VH.
16. The antibody or antigen-binding fragment of any one of claims 1 to 15, further comprising a hinge region fused to the VH.
17. The antibody or antigen-binding fragment of any one of claims 1 to 16, further comprising a CK domain fused to the VL.
18. The antibody or antigen-binding fragment of any one of claims 1 to 16, further comprising a CL domain fused to the VL.
19. The antibody or antigen-binding fragment of any one of claims 1, 8, 9 and 11 to 18, wherein said antibody or antigen-binding fragment is ic.
20. The antibody or antigen-binding fragment of any one of claims 1 to 18, wherein said antibody or antigen-binding nt is fully human.
21. The antibody or antigen-binding nt of any one of claims 1 to 20, wherein said antibody or antigen-binding fragment is a Fab fragment, a Fab' fragment or a F(ab)2 fragment.
22. The antibody or n-binding fragment of any one of claims 1 to 20, wherein said antibody or antigen-binding fragment is a single chain antibody.
23. The antibody or antigen-binding fragment of any one of claims 1 to 20, wherein said antibody or antigen-binding fragment is a single-chain Fv fragment.
24. The antibody or n-binding fragment of any one of claims 1 to 23, further comprising a heterologous polypeptide fused thereto.
25. The antibody or antigen-binding fragment of any one of claims 1 to 24 conjugated to an agent selected from the group consisting of a eutic agent, a g, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical agent and PEG.
26. A composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 25 and a carrier.
27. An isolated polynucleotide comprising a nucleic acid encoding the antibody or antigenbinding fragment thereof of any one of claims 1 to 25.
28. A composition comprising the polynucleotide of claim 27 and a carrier.
29. A vector comprising the polynucleotide of claim 27.
30. The vector of claim 29, wherein said polynucleotide is ly associated with a promoter.
31. The vector of claim 30, wherein the polynucleotide includes a polynucleotide encoding a VH and a polynucleotide encoding a VL and wherein the polynucleotide encoding the VH and the polynucleotide encoding the VL are co-transcribed from a single promoter operably associated ith, but are separately translated.
32. The vector of claim 31, r comprising an IRES sequence disposed between the polynucleotide encoding the VH and the cleotide encoding the VL.
33. The vector of claim 31, wherein the polynucleotide encoding the VH and the polynucleotide encoding the VL are tely transcribed, each being operably ated with a separate promoter.
34. The vector of claim 33, wherein each said separate promoter is ent.
35. An isolated host cell comprising the polynucleotide of claim 27 or the vector of any one of claims 29 to 34.
36. A method of producing an anti-human α-synuclein antibody or antigen-binding fragment thereof, said method comprising culturing the host cell of claim 35 and recovering the antibody or antigen-binding fragment thereof.
37. An anti-human clein antibody or antigen-binding fragment thereof produced by the method of claim 36.
38. Use of the antibody of antigen-binding fragment of any one of claims 1 to 25 and 37, the composition of claim 26 or claim 28, the polynucleotide of claim 27, the vector of any one of claims 29 to 34 or the host cell of claim 35 in the manufacture of a medicament for treating or preventing a synucleinopathic disease in a subject.
39. Use of the antibody or antigen-binding fragment of any one of claims 1 to 25 and 37 in the manufacture of a medicament for diagnosing whether a subject has a synucleinopathic disease, wherein said sing comprises: (a) assessing a level, localization, conformation or a combination thereof of lein in the t to be diagnosed with the medicament; and (b) comparing the level, localization, conformation or combination thereof of said αsynuclein in the subject to one or more reference standards derived from one or more control samples, wherein a difference or similarity between the level, localization, conformation or combination thereof of said α-synuclein in the subject and the one or more reference standards indicates whether the subject has a einopathic disease.
40. The use of claim 38 or claim 39, wherein the synucleinopathic disease is Parkinson's disease (PD), dementia with Lewy bodies (DLB), multiple systems y (MSA) or a combination thereof.
41. The use of any one of claims 38 to 40, n the subject is a mammal.
42. The use of claim 41, wherein the mammal is a human.
43. The use of claim 42, wherein the level, localization, conformation or combination f of α-synuclein in the subject is measured by in vivo imaging.
44. The use of claim 43, wherein said in vivo imaging comprises positron emission tomography (PET), single photon emission tomography (SPECT), near infrared (NIR) optical imaging or magnetic resonance imaging (MRI).
45. An isolated antibody or antigen-binding fragment thereof as defined in any one of claims 1 to 10 and as substantially described herein with reference to the section entitled “DETAILED DESCRIPTION OF THE INVENTION”.
46. A composition comprising the dy or antigen-binding nt of claim 45 and a
47. An isolated polynucleotide comprising a nucleic acid encoding the antibody or antigenbinding fragment of claim 45.
48. A ition comprising the polynucleotide of claim 47 and a carrier.
49. A vector comprising the polynucleotide of claim 47.
50. An isolated host cell comprising the polynucleotide of claim 47 or the vector of claim
51. A method of producing an anti-human α-synuclein antibody or antigen-binding fragment, said method comprising culturing the host cell of claim 50 and recovering the antibody or antigen-binding fragment thereof.
52. An anti-human α-synuclein antibody or antigen-binding fragment thereof produced by the method of claim 51.
53. Use of the antibody of n-binding fragment of claim 45 or claim 52, the composition of claim 46 or claim 48, the polynucleotide of claim 47, the vector of claim 49 or the host cell of claim 50 in the manufacture of a medicament for treating or preventing a synucleinopathic disease in a subject.
54. Use of the antibody or antigen-binding fragment of claim 45 or claim 52 in the manufacture of a medicament for diagnosing whether a subject has a synucleinopathic disease, wherein said sing comprises: (a) assessing a level, localization, conformation or a ation f of αsynuclein in the subject to be diagnosed with the medicament; and (b) comparing the level, localization, conformation or combination thereof of said lein in the subject to one or more nce standards derived from one or more control wherein a difference or similarity between the level, localization, mation or combination thereof of said α-synuclein in the subject and the one or more reference standards indicates whether the subject has a synucleinopathic disease.
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