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WO2025158162A1 - Anticorps anti-alpha-synucléine - Google Patents

Anticorps anti-alpha-synucléine

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Publication number
WO2025158162A1
WO2025158162A1 PCT/GB2025/050134 GB2025050134W WO2025158162A1 WO 2025158162 A1 WO2025158162 A1 WO 2025158162A1 GB 2025050134 W GB2025050134 W GB 2025050134W WO 2025158162 A1 WO2025158162 A1 WO 2025158162A1
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WO
WIPO (PCT)
Prior art keywords
antibody
antigen
binding fragment
alpha
synuclein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/GB2025/050134
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English (en)
Inventor
Francesco APRILE
Dimitra GIALAMA
Devkee VADUKUL
Rebecca THRUSH
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ip2ipo Innovations Ltd
Original Assignee
Imperial College Innovations Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Imperial College Innovations Ltd filed Critical Imperial College Innovations Ltd
Publication of WO2025158162A1 publication Critical patent/WO2025158162A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • 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
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • Alpha- Antibody The invention relates to alpha-synuclein antibodies, and particularly, although not exclusively, to antibodies which target the P1 motif of alpha-synuclein.
  • the invention extends to compositions comprising the antibodies, including pharmaceutical compositions and kits.
  • the invention also extends to methods of making and using the antibodies, for example in therapy and diagnosis of neurodegenerative disorders, such as Parkinson’s disease.
  • Antibodies are characterised by high binding affinity and specificity for their targets, making them ideal probes for biomedical research and appealing clinical molecules 1,2 .
  • antibody-discovery technology has evolved rapidly and there is now a plethora of in vitro selection approaches to generate such molecules, including cell and cell-free display 3,4 .
  • ⁇ -syn The amyloid aggregation of the protein ⁇ -synuclein ( ⁇ -syn) is associated with pathologies referred to as synucleinopathies, which include Parkinson’s disease (PD) and Multiple system atrophy (MSA) 11 .
  • ⁇ -Syn is 140-residues long and abundantly expressed in dopaminergic neurons 11 . Under physiological conditions, its role involves the regulation of presynaptic terminal size and activity by controlling the distribution of neurotransmitter vesicles 12 .
  • N- terminal domain (residues 1–60), important for the interaction with other biomolecules 13–15 , the non-amyloid- ⁇ component (NAC) region (residues 61–95), which has high amyloid propensity 16 , and a C-terminal tail (residues 96–140), whose truncation promotes aggregation 17 .
  • NAC non-amyloid- ⁇ component
  • C-terminal tail (residues 96–140), whose truncation promotes aggregation 17 .
  • the 7-residue-long motif P1 (residues 36-42) has been identified as a master regulator of ⁇ -syn aggregation 18 , and deletion of this motif has been shown to prevent ⁇ -syn aggregation at physiological pH in vitro.
  • the inventors carried out ribosome display on a single-domain antibody (sdAb) raised against a peptide encompassing the region P1 of ⁇ -synuclein, and then performed a multi-parametric screening, in which antibody candidates were ranked based on their ability to specifically bind to the aforementioned peptide and inhibit the aggregation of full-length ⁇ -syn ( Figure 1).
  • sdAb single-domain antibody
  • Figure 1 the inventors identified an antibody that can inhibit the toxic ⁇ -synuclein amyloid aggregation by binding to the P1 motif in a specific manner.
  • an antibody or antigen-binding fragment thereof that specifically binds to the P1 motif of alpha- synuclein, or a variant or fragment thereof.
  • the inventors have developed an antibody that is capable of inhibiting the toxic clustering of the protein, alpha-synuclein, through binding to its P1 motif.
  • the inventors demonstrated that alpha-synuclein aggregation is strongly inhibited by the antibody according to the invention, one embodiment of which is referred to herein “ ⁇ SP1”.
  • the inventors surprisingly discovered that the antibody is effective at very low concentrations in vitro, because 1 molecule of antibody per 100 molecules of alpha-synuclein is sufficient for providing a strong inhibition.
  • the inventors have demonstrated that the antibody according to the invention binds to the P1 motif of alpha-synuclein aggregates. For example, as discussed in Example 6, binding of ⁇ SP1 increases synuclein aggregates, suggesting that this antibody is specific for aggregated forms of ⁇ -synuclein (see Figure 5A).
  • the antibody or antigen-binding fragment thereof specifically binds to the P1 motif of aggregated alpha-synuclein.
  • the antibody or antigen-binding fragment thereof specifically binds to the P1 motif of alpha-synuclein aggregates. It will be appreciated that some of these aggregates are those which are formed during the early stages of aggregation. In some embodiments, the antibody or antigen-binding fragment thereof is capable of inhibiting the aggregation of alpha-synuclein. In an embodiment, the antibody or antigen-binding fragment thereof is capable of inhibiting the aggregation of alpha- synuclein into toxic amyloid fibrils.
  • the amino acid sequence of alpha-synuclein may be represented by Gene ID: 6622 (Genomic Accession (Homo sapiens synuclein alpha (SNCA), RefSeqGene on chromosome 4): NG_011851), which is provided herein as SEQ ID No: 1, as follows: MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAV VTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA [SEQ ID No: 1]
  • the antibody or antigen-binding fragment thereof may therefore bind specifically to a region between amino acid positions 36 and 42 of SEQ ID No: 1 (shown in bold above), which corresponds to the P1 motif of alpha-synuclein.
  • SEQ ID No: 1 is the sequence of the wild type protein, though the antibody or antigen-binding fragment thereof can bind to variants or fragments of SEQ ID NO: 1.
  • the amino acid sequence of the P1 motif of alpha-synuclein may be provided herein as SEQ ID No: 2, as follows: GVLYVGS [SEQ ID No: 2]
  • the or antigen-binding fragment thereof binds to the P1 motif comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 2, or a variant or fragment thereof.
  • the invention extends to both whole antibodies (i.e.
  • the antibody or antigen-binding fragment thereof may be monovalent, divalent or polyvalent.
  • Monovalent antibodies are dimers (HL) comprising a heavy (H) chain associated by a disulphide bridge with a light chain (L).
  • Divalent antibodies are tetramer (H2L2) comprising two dimers associated by at least one disulphide bridge.
  • Polyvalent antibodies may also be produced, for example by linking multiple dimers.
  • the basic structure of an antibody molecule consists of two identical light chains and two identical heavy chains which associate non-covalently and can be linked by disulphide bonds.
  • Each heavy and light chain contains an amino-terminal variable region of about 110 amino acids, and constant sequences in the remainder of the chain.
  • the variable region includes several hypervariable regions, or Complementarity Determining Regions (CDRs), that form the antigen-binding site of the antibody molecule and determine its specificity for the antigen, i.e. the P1 motif, or variant or fragment thereof (e.g. an epitope).
  • CDRs Complementarity Determining Regions
  • On either side of the CDRs of the heavy and light chains is a framework region, a relatively conserved sequence of amino acids that anchors and orients the CDRs.
  • Antibody fragments may include a bi-specific antibody (BsAb) or a chimeric antigen receptor (CAR).
  • Each heavy chain typically comprises a heavy chain variable region (VH) and a heavy chain constant region.
  • the heavy chain constant region typically comprises three domains, CH1, CH2, and CH3.
  • Each light chain typically comprises a light chain variable region (VL) and a light chain constant region.
  • the light chain constant region typically comprises one domain, abbreviated CL.
  • Each heavy chain and light chain generally comprise three CDRs and four frameworks (FRs), arranged in the following order (from N-terminus to C- terminus): FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4.
  • the CDRs are involved in antigen-binding and confer antigen specificity and binding affinity to the antibody.
  • IgA immunoglobulin A
  • IgD immunoglobulin D
  • IgE immunoglobulin G
  • IgM immunoglobulin M
  • IgG immunoglobulin G
  • IgM immunoglobulin M
  • the IgG antibody class may be preferred.
  • the light chain from any vertebrate species can be assigned to one of two types, called kappa and lambda, based on the sequence of the constant domain.
  • the constant region consists of one of five heavy chain sequences ( ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ ) and one of two light chain sequences ( ⁇ or ⁇ ).
  • the heavy chain constant region sequences determine the isotype of the antibody and the effector functions of the molecule.
  • the antibody or antigen-binding fragment thereof is isolated or purified.
  • the antibody or antigen-binding fragment thereof comprises a polyclonal antibody, or an antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof may be generated in a rabbit, mouse or rat.
  • the antibody or antigen-binding fragment thereof is obtained by immunising a host animal with a P1 protein, or a variant or fragment thereof, and then collecting the antibody or antigen-binding fragment thereof.
  • the host animal may be a rabbit.
  • the antibody or antigen-binding fragment thereof comprises a monoclonal antibody or an antigen-binding fragment thereof.
  • the antibody or fragment thereof of may be mammalian.
  • the antibody of the invention is a human antibody.
  • human antibody can mean an antibody, such as a monoclonal antibody, which comprises substantially the same heavy and light chain CDR amino acid sequences as found in a particular human antibody exhibiting immunospecificity for the P1 motif of alpha- synuclein, or a variant or fragment
  • An amino acid sequence which is substantially the same as a heavy or light chain CDR, exhibits a considerable amount of sequence identity when compared to a reference sequence. Such identity is definitively known or recognisable as representing the amino acid sequence of the particular human antibody.
  • Substantially the same heavy and light chain CDR amino acid sequence can have, for example, minor modifications or conservative substitutions of amino acids.
  • human antibody or fragment thereof maintains its function of selectively binding to the P1 motif of alpha-synuclein, or a variant or fragment thereof.
  • the antibody or antigen-binding fragment thereof may be produced using a cell-free discovery method, for example ribosome display.
  • the term “human monoclonal antibody” can include a monoclonal antibody with substantially or entirely human CDR amino acid sequences produced, for example by recombinant methods, such as production by a phage library, by lymphocytes or by hybridoma cells.
  • monoclonal antibody refers to an antibody from a population of substantially homogeneous antibodies, derived from the same B cell.
  • a population of substantially homogeneous antibodies comprises antibodies that are substantially similar and that bind the same epitope(s), except for variants that may normally arise during production of the monoclonal antibody. Such variants are generally present in only minor amounts.
  • a monoclonal antibody is typically obtained by a process that includes the selection of a single antibody from a plurality of antibodies. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones.
  • the selected antibody can be further altered, for example, to improve affinity for the target (by so-called “affinity maturation”), to humanize the antibody, to improve its production in cell culture, and/or to reduce its immunogenicity in a subject.
  • affinity maturation can mean an antibody from a non-human species (e.g. mouse or rabbit) whose protein sequences have been modified to increase their similarity to antibodies produced naturally in humans.
  • the antibody may be a recombinant antibody.
  • recombinant human antibody can include a human antibody produced using recombinant DNA technology.
  • the term “antigen-binding fragment” can mean a region of the antibody having specific binding affinity for its target antigen, for example, the P1 motif of alpha- synuclein, or a variant or fragment thereof.
  • the fragment is an epitope.
  • the epitope may be linear or conformational.
  • the antigen-binding region may be a hypervariable CDR or a functional portion thereof.
  • the term “functional portion” of a CDR can mean a sequence within the CDR which shows specific affinity for the target antigen.
  • the functional portion of a CDR may comprise a ligand which specifically binds to the P1 motif of alpha-synuclein, or a fragment thereof.
  • CDR can mean a hypervariable region in the heavy and light variable chains. There may be one, two, three or more CDRs in each of the heavy and light chains of the antibody. Normally, there are at least three CDRs on each chain which, when configured together, form the antigen-binding site, i.e. the three- dimensional combining site with which the antigen binds or specifically reacts. It has however been postulated that there may be four CDRs in the heavy chains of some antibodies.
  • the definition of CDR also includes overlapping or subsets of amino acid residues when compared against each other. The exact residue numbers which encompass a particular CDR or a functional portion thereof will vary depending on the sequence and size of the CDR.
  • amino acid sequence boundaries of a CDR can be determined by using any of a number of known numbering schemes, including those described by Kabat et al., supra (“Kabat” numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927- 948 (“Chothia” numbering scheme); MacCallum et al., 1996, J. Mol. Biol.262:732- 745 (“Contact” numbering scheme); Lefranc et al., Dev. Comp.
  • the term “functional fragment” of an antibody can mean a portion of the antibody which retains a functional activity.
  • a functional activity can be, for example antigen binding activity or specificity.
  • a functional activity can also be, for example, an effector function provided by an region.
  • the term “functional fragment” is also intended to include, for example, fragments produced by protease digestion or reduction of a human monoclonal antibody and by recombinant DNA methods known to those skilled in the art.
  • Human monoclonal antibody functional fragments include, for example individual heavy or light chains and fragments thereof, such as VL, VH and Fd; monovalent fragments, such as Fv, Fab, and Fab'; bivalent fragments such as F(ab')2; single chain Fv (scFv); and Fc fragments.
  • the Fc fragment of the antibody may be disabled by introducing amino acid substitutions into the Fc region, which silence or reduce the effector function of the antibody.
  • VL fragment can mean a fragment of the light chain of a human monoclonal antibody which includes all or part of the light chain variable region, including the CDRs.
  • a VL fragment can further include light chain constant region sequences.
  • VH fragment can mean a fragment of the heavy chain of a human monoclonal antibody which includes all or part of the heavy chain variable region, including the CDRs.
  • Fd fragment can mean the heavy chain variable region coupled to the first heavy chain constant region, i.e. VH and CH-1. The “Fd fragment” does not include the light chain, or the second and third constant regions of the heavy chain.
  • Fv fragment can mean a monovalent antigen-binding fragment of a human monoclonal antibody, including all or part of the variable regions of the heavy and light chains, and absent of the constant regions of the heavy and light chains.
  • the variable regions of the heavy and light chains include, for example, the CDRs.
  • an Fv fragment includes all or part of the amino terminal variable region of about 110 amino acids of both the heavy and light chains.
  • Fab fragment can mean a monovalent antigen-binding fragment of a human monoclonal antibody that is larger than an Fv fragment.
  • a Fab fragment includes the variable regions, and all or part of the first constant domain of the heavy and light chains.
  • a Fab fragment additionally includes, for example, amino acid residues from about 110 to about 220 of the heavy and light chains.
  • Fab' fragment can mean a monovalent antigen-binding fragment of a human monoclonal antibody that is larger than a Fab fragment.
  • a Fab' fragment includes all of the light chain, all of the variable region of the heavy chain, and all or part of the first and second constant domains of the heavy chain.
  • a Fab' fragment can additionally include some or all of amino acid residues 220 to 330 of the heavy chain.
  • the term “F(ab')2 fragment” can mean a bivalent antigen-binding fragment of a human monoclonal antibody.
  • An F(ab')2 fragment includes, for example, all or part of the variable regions of two heavy chains-and two light chains, and can further include all or part of the first constant domains of two heavy chains and two light chains.
  • single chain Fv can mean a fusion of the variable regions of the heavy (VH) and light chains (VL) connected with a short linker peptide.
  • BsAb bispecific antibody
  • BsAb can mean a bispecific antibody comprising two scFv linked to each other by a shorter linked peptide.
  • the antigen-binding fragment thereof is a single domain antibody (sdAb) (also referred to as a nanobody).
  • sdAb is an antibody fragment consisting of a single monomeric variable antibody domain (referred to as a VHH).
  • the antigen-binding fragment thereof is a single-chain antibody, an intrabody, a peptide (e.g. a bicyclic peptide), or any other type of fragment or protein scaffold.
  • a peptide e.g. a bicyclic peptide
  • the exact boundaries of a fragment of an antibody are not important, so long as the fragment maintains a functional activity.
  • a functional fragment of the antibody may comprise or consist of a fragment with substantially the same heavy and light chain variable regions as the human antibody.
  • the antibody binding fragment thereof is immunospecific for an epitope within the P1 motif of alpha-synuclein.
  • the antigen-binding fragment thereof may comprise or consist of any of the fragments selected from a group consisting of VH, VL, Fd, Fv, Fab, Fab', scFv, F (ab')2 and Fc fragment.
  • the antigen-binding fragment thereof may comprise or consist of any one of the antigen binding region sequences of the VL, any one of the antigen binding region sequences of the VH, or a combination of VL and VH antigen binding regions of a human antibody.
  • VH and VL antigen binding region sequences may be determined by those skilled in the art depending on the desired affinity and specificity and the intended use of the antigen-binding fragment.
  • Functional fragments or antigen-binding fragments of antibodies may be readily produced and isolated using methods well known to those skilled in the art. Such methods include, for example, proteolytic methods, recombinant methods and chemical synthesis. Proteolytic methods for the isolation of functional fragments comprise using human antibodies as a starting material. Enzymes suitable for proteolysis of human immunoglobulins may include, for example, papain, and pepsin. The appropriate enzyme may be readily chosen by one skilled in the art, depending on, for example, whether monovalent or bivalent fragments are required.
  • papain cleavage results in two monovalent Fab' fragments that bind antigen and an Fc fragment.
  • Pepsin cleavage results in a bivalent F (ab') fragment.
  • An F (ab')2 fragment of the invention may be further reduced using, for example, DTT or 2-mercaptoethanol to produce two monovalent Fab' fragments.
  • Functional or antigen-binding fragments of antibodies produced by proteolysis may be purified by affinity and column chromatographic procedures. For example, undigested antibodies and Fc fragments may be removed by binding to protein A. Additionally, functional fragments may be purified by virtue of their charge and size, using, for example, ion exchange and gel filtration chromatography. Such methods are well known to those skilled in the art.
  • the antibody or antigen-binding fragment thereof may be produced by recombinant methodology.
  • Such regions may include, for example, all or part of the variable region of the heavy and light chains.
  • such regions include the antigen binding regions of the heavy and light chains, such as the antigen binding sites, in particular the CDRs.
  • the polynucleotide encoding the antibody or antigen-binding fragment thereof according to the invention may be produced using methods known to those skilled in the art.
  • the polynucleotide encoding the antibody or antigen-binding fragment thereof may be directly synthesized by methods of oligonucleotide synthesis known in the art.
  • the term “immunospecificity” can mean the binding region of the antibody or antigen-binding fragment thereof is capable of immunoreacting with the P1 motif of alpha-synuclein, or a variant or fragment thereof, by specifically binding therewith.
  • the antibody or antigen-binding fragment thereof can selectively interact with an antigen (the P1 motif of alpha-synuclein) with an affinity constant of approximately 10 -5 to 10 -13 M -1 , typically 10 -6 to 10 -9 M -1 , or more typically 10 -10 to 10 -12 M -1 .
  • the term “immunoreact” can mean the binding region is capable of eliciting an immune response upon binding with the P1 motif of alpha-synuclein, or an epitope thereof.
  • epitope can mean any region of an antigen with the ability to elicit, and combine with, a binding region of the antibody or antigen-binding fragment thereof.
  • the epitope may be linear. This can mean that the antibody interacts with a plurality of continuous amino acids of the antigen, and so the epitope can consist of these defined amino acids. Alternatively, the epitope may be conformational, i.e. non-linear or discontinuous. This can mean that the antibody interacts with multiple, distinct segments from the primary amino acid sequence of the antigen.
  • the inventors surprisingly discovered that the antibody or antigen- binding fragment thereof according to the invention binds better to alpha-synuclein aggregates rather than alpha-synuclein monomers.
  • the antibody or antigen-binding fragment thereof binds to a conformational epitope.
  • the antibody referred to herein as ⁇ SP1 is able to specifically bind to the P1 motif of aggregated ⁇ -synuclein aggregates and inhibit toxic amyloid aggregation of alpha-synuclein.
  • the antibody or antigen-binding fragment thereof is referred to herein as ⁇ SP1.
  • the antibody or antigen-binding fragment thereof may comprise a CDR1 domain of SEQ ID No: 3, which is provided herein, as follows: RHQWME [SEQ ID No: 3]
  • the antibody or antigen-binding fragment thereof comprises a CDR1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 3, or a variant or fragment thereof.
  • the antibody or antigen-binding fragment thereof may comprise a CDR2 domain of SEQ ID No: 4, which is provided herein, as follows: SSFGQWTK [SEQ ID No: 4]
  • typically the antibody or antigen-binding fragment thereof comprises a CDR2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 4, or a variant or fragment thereof.
  • the antibody or antigen-binding fragment thereof may comprise a CDR3 domain of SEQ ID No: 5, which is provided herein, as follows: VVEVGYEYY [SEQ ID No: 5]
  • the antibody or antigen-binding fragment thereof comprises a CDR3 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 5, or a variant or fragment thereof.
  • the antibody binding fragment thereof comprises at least one, at least two, or at least three CDRs.
  • the antibody or antigen-binding fragment thereof comprises at least CDR3.
  • the antibody or antigen-binding fragment thereof comprises a CDR1 domain comprising or consisting of SEQ ID No: 3, a CDR2 domain comprising or consisting of SEQ ID No: 4 and/or a CDR3 domain comprising or consisting of SEQ ID No: 5. In some embodiments, however, the antibody or antigen-binding fragment thereof comprises a CDR1 domain comprising or consisting of SEQ ID No: 3, a CDR2 domain comprising or consisting of SEQ ID No: 4 and a CDR3 domain comprising or consisting of SEQ ID No: 5.
  • the antibody or antigen-binding fragment thereof may comprise a variable domain (VHH) sequence as set out in SEQ ID No: 19, which is provided herein, as follows: QVQLVESGGGLVQAGGSLRLSCAASGFPVRHQWMEWYRQAPGKEREWVAAISSFGQWTKYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCVVEVGYEYYGQGTQVTVS [SEQ ID No: 19]
  • the antibody or antigen-binding fragment thereof comprises a variable domain (VHH) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 19, or a variant or fragment thereof.
  • variable domain (VHH) region is referred to herein as SEQ ID No: 20, as follows: CAGGTTCAGCTGGTTGAGAGCGGTGGTGGCCTGGTCCAAGCTGGCGGTTCGCTGCGTCTGAGCTGCGCCG CAAGCGGTTTCCCGGTGAGGCATCAGTGGATGGAATGGTATCGTCAGGCCCCGGGCAAAGAACGTGAGTG GGTCGCGGCGATTTCTAGCTTCGGTCAATGGACGAAATACGCAGATTCTGTTAAGGGCCGCTTTACCATC AGCCGCGACAACGCGAAGAATACGGTCTATTTGCAGATGAATAGCCTGAAACCGGAAGATACCGCGGTTT ACTACTGTGTTGTGGAAGTGGGTTACGAATACTACGGCCAAGGTACCCAAGTGACTGTGAGC [SEQ ID No: 20]
  • the antibody or antigen-binding fragment thereof comprises a variable domain (VHH) region encoded by a nucleic acid sequence as substantially set
  • the antibody or binding fragment thereof comprises a PelB sequence.
  • the PelB sequence is disposed N-terminal to the CDR domain, typically the CDR1 domain.
  • the amino acid sequence of PelB is provided herein as SEQ ID No: 6, as follows: MSKYLLPTAAAGLLLLAAQPAMA [SEQ ID No: 6]
  • the antibody or antigen-binding fragment thereof comprises a Myc tag.
  • the Myc sequence is disposed C-terminal to the CDR domain, for example the CDR3 domain.
  • the amino acid sequence of the Myc tag is provided herein as SEQ ID No: 7, as follows: EQKLISEEDL [SEQ ID No: 7]
  • the antibody or antigen-binding fragment thereof comprises a His tag.
  • the His tag sequence is disposed C-terminal to the CDR domain, such as the CDR3 domain, and more typically the Myc tag.
  • the amino acid sequence of the His tag is provided herein as SEQ ID No: 8, as follows: HHHHHH [SEQ ID No: 8]
  • the antibody or antigen-binding fragment thereof may comprise a variable domain (VHH) sequence as set out in SEQ ID No: 9, which is provided herein, as follows: MSKYLLPTAAAGLLLLAAQPAMAGSSSQVQLVESGGGLVQAGGSLRLSCAASGFPVRHQWMEWYRQAPGK EREWVAAISSFGQWTKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVVEVGYEYYGQGTQVTV SAGRAGEQKLISEEDLNSAVDHHHHHH [SEQ ID No: 9]
  • the antibody or antigen-binding fragment thereof comprises a variable domain (VHH) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 9, or a variant or fragment thereof.
  • VHH variable domain
  • SEQ ID No: 10 One embodiment of a nucleotide encoding the variable domain (VHH) region is referred to herein as SEQ ID No: 10, as follows: ATGAGTAAATATCTGCTGCCGACCGCAGCAGCGGGTCTGCTGCTGCTGGCAGCCCAGCCTGCAATGGCCG GCTCTTCAAGTCAGGTTCAGCTGGTTGAGAGCGGTGGTGGCCTGGTCCAAGCTGGCGGTTCGCTGCGTCT GAGCTGCGCCGCAAGCGGTTTCCCGGTGAGGCATCAGTGGATGGAATGGTATCGTCAGGCCCCGGGCAAA GAACGTGAGTGGGTCGCGGCGATTTCTAGCTTCGGTCAATGGACGAAATACGCAGATTCTGTTAAGGGCC GCTTTACCATCAGCCGCGACAACGCGAAGAATACGGTCTATTTGCAGATGAATAGCCTGAAACCGGAAGA TACCGCGGTTTACTACTGTGTTGTGGAAGTGGGTTACGAATACTACGGCCAAGGTACCCAAGTG
  • the ability of the antibody or antigen-binding fragment thereof according to the first aspect of the invention to inhibit alpha-synuclein aggregation means that it has significant utility as a therapeutic agent in its own right, and may be used in the treatment, amelioration or prevention of a condition caused by alpha-synuclein aggregation, such as Parkinson’s disease.
  • an antibody or antigen-binding fragment thereof according to the first aspect for use in therapy.
  • an antibody or antigen-binding fragment thereof according to the first aspect for use in treating, preventing or ameliorating a condition associated with or caused by alpha-synuclein aggregation.
  • a method of treating, preventing or ameliorating a condition associated with or caused by alpha- synuclein aggregation in a subject comprising administering, or having administered, to a patient in need of such treatment, a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to the first aspect.
  • the condition associated with or caused by alpha-synuclein aggregation may be referred to as a synucleinopathy.
  • the condition associated with or caused by alpha-synuclein aggregation is a neurodegenerative disorder.
  • associated with or caused by alpha-synuclein aggregation is selected from the group consisting of: Parkinson’s disease, dementia with Lewy bodies (DLB), multiple system atrophy (MSA), Alzheimer’s disease, and pure autonomic failure (PAF).
  • agents antibodies or antigen-binding fragments thereof according to the invention (referred to herein as “agents”) may be used in a monotherapy (e.g. the use of an antibody alone), for treating, ameliorating or preventing a condition associated with or caused by alpha-synuclein aggregation.
  • agents according to the invention may be used as an adjunct to, or in combination with, known therapies for treating, ameliorating, or preventing a condition associated with or caused by alpha-synuclein aggregation, such as levodopa and other dopaminergic drugs, inhibitors of dopamine metabolism, and dopamine receptor agonists (which focus on controlling or delaying the symptoms).
  • Agents according to the invention may also be used as an adjunct to, or in combination with putative disease-modifying treatments, including drugs to reduce the levels of alpha-synuclein, promote the restoration of the mitochondrial function, and reduce cellular oxidative stress, microglia activation and neuroinflammation.
  • the agents according to the invention may be combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used.
  • the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension or any other suitable form that may be administered to a person or animal in need of treatment.
  • vehicle of medicaments according to the invention should be one which is well-tolerated by the subject to whom it is given.
  • Medicaments comprising agents of the invention may be used in a number of ways.
  • compositions comprising agents and medicaments of the invention may be administered by inhalation (e.g. intranasally).
  • Compositions may also be formulated for topical use. For instance, creams or ointments may be applied to the skin.
  • the agent may be delivered by gene therapy, for example through a AAV vector.
  • Agents and medicaments according to invention may also be incorporated within a slow- or delayed-release device. Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months. The device may be located at least adjacent the treatment site.
  • agents and medicaments according to the invention may be administered to a subject by injection into the blood stream or directly into a site requiring treatment. Injections may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion), or intradermal (bolus or infusion). It will be appreciated that the amount of the antibody or antigen-binding fragment thereof (i.e. agent) that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of the agent, and whether it is being used as a monotherapy or in a combined therapy.
  • the frequency of administration will also be influenced by the half-life of the agent within the subject being treated.
  • Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular agent in use, the strength of the pharmaceutical composition, the mode of administration, and the advancement of the neurodegenerative disease. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.
  • a daily dose of between 0.01 ⁇ g/kg of body weight and 100mg/kg of body weight of agent according to the invention may be used for treating, ameliorating, or preventing a neurodegenerative disease, depending upon which agent.
  • the daily dose of agent is between 1 ⁇ g/kg of body weight and 100mg/kg of body weight, more typically between 10 ⁇ g/kg and 10mg/kg body weight, and most typically between approximately 100 ⁇ g/kg and 10mg/kg body weight.
  • the agent may be administered before, during or after onset of a neurodegenerative disorder. Daily doses may be given as a single administration (e.g. a single daily injection). Alternatively, the agent may require administration twice or more times during a day. As agents may be administered as two (or more depending upon the severity of the neurodegenerative disorder being treated) daily doses of between 0.07 ⁇ g and 700 mg (i.e. assuming a body weight of 70 kg).
  • a patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3- or 4-hourly intervals thereafter.
  • a slow release device may be used to provide optimal doses of agents according to the invention to a patient without the need to administer repeated doses.
  • Known procedures such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials, etc.), may be used to form specific formulations of the agents according to the invention and precise therapeutic regimes (such as daily doses of the agents and the frequency of administration).
  • a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to the first aspect, and optionally a pharmaceutically acceptable vehicle.
  • the invention also provides in a sixth aspect, a process for making the pharmaceutical composition according to the fifth aspect, the process comprising combining a therapeutically effective amount of an antibody or antigen-binding fragment thereof as defined in the first aspect, with a pharmaceutically acceptable vehicle.
  • a “subject” may be a vertebrate, mammal, or domestic animal.
  • medicaments according to the invention may be used to treat any mammal, for example livestock (e.g. a horse), pets, or may be used in other veterinary applications.
  • the subject is a human being.
  • a “therapeutically effective amount” of the antibody or antigen-binding fragment thereof is any amount which, when administered to a subject, is the amount of agent that is needed to treat the neurodegenerative disorder, or produce the desired effect.
  • the therapeutically effective amount of antibody or antigen-binding fragment thereof used may be from about 0.1 ng/kg to about 100 mg/kg, and typically from about 1 ng/kg to about 10 mg/kg. It is considered that the amount of antibody or antigen-binding fragment is an amount from about 10 ng/kg to about 10 mg/kg, and most typically from about 50 ng/kg to about 5 mg/kg.
  • a “pharmaceutically acceptable vehicle” as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.
  • the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet.
  • a solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet-disintegrating agents.
  • the vehicle may also be an encapsulating material.
  • the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention.
  • the active agent In tablets, the active agent may be mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets typically contain up to 99% of the active agents.
  • Suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.
  • the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like.
  • the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution.
  • Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions.
  • the active agent according to the invention may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats.
  • a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats.
  • the liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators.
  • suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, typically sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g.
  • liquid vehicle for parenteral administration, can also be an oily ester such as and isopropyl myristate.
  • Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration.
  • the liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.
  • Liquid pharmaceutical compositions which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and particularly subcutaneous injection.
  • the agent may be prepared as a sterile solid composition that may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.
  • the agents and compositions of the invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like.
  • the agents used according to the invention can also be administered orally either in liquid or solid composition form.
  • compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions.
  • forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.
  • the invention also extends to methods for producing the antibody, or antigen- binding fragment of the first aspect, and the antibody, or antigen-binding fragment so produced.
  • an antibody or antigen-binding fragment thereof obtained by a method comprising:- (i) immunising a host organism with the P1 motif of alpha-synuclein, or a fragment or variant thereof; and (ii) collecting an antibody or antigen-binding fragment thereof from the host.
  • the host may be a mammal, and may be a human, rabbit, mouse, or chicken. In some embodiments, the host is a rabbit.
  • the method may comprise host with at least 50 ⁇ g, 75 ⁇ g, or 100 ⁇ g of the immunogen. In some embodiments, the method comprises subsequently immunising the host with a first boost of at least 25 ⁇ g, 35 ⁇ g or 50 ⁇ g of immunogen.
  • the first boost is administered at least 20, 30 or 40 days after the first immunisation.
  • the method comprises subsequently immunising the host with a second boost of at least 25 ⁇ g, 35 ⁇ g or 50 ⁇ g of immunogen.
  • the method comprises bleeding the host animal, and then typically collecting the antibody or antigen-binding fragment thereof from the blood, most typically blood serum.
  • the method comprises bleeding the host animal at least 30, 40 or 50 days after the first immunisation.
  • the blood serum is passed through a gravity column with covalently bound peptide-support. Following washing, the antibody or antigen- binding fragment thereof is typically eluted in buffer, which is typically acidic buffer, and the solution may then be neutralized.
  • the method may further comprise dialysis against a suitable buffer (e.g. PBS) and, optionally, lyophilisation.
  • a suitable buffer e.g. PBS
  • an antibody or antigen-binding fragment thereof obtained by a method comprising selecting an antibody or antigen-binding fragment thereof that specifically binds to the P1 motif of alpha- synuclein, using directed evolution or a computational approach.
  • a polynucleotide sequence encoding the antibody or antigen binding fragment thereof as defined in the first aspect.
  • an expression cassette comprising a polynucleotide sequence according to the eighth aspect.
  • the polynucleotide sequence encoding the antibody or antigen binding fragment thereof of the invention is typically harboured in a recombinant vector, for example a recombinant vector for delivery into a host cell of interest to enable production of the antibody or antigen binding fragment thereof.
  • a recombinant vector comprising the expression cassette according to the ninth aspect.
  • the vector encoding the antibody or antigen binding fragment thereof of the first aspect may for example be a plasmid, cosmid or phage and/or be a viral vector.
  • Such recombinant vectors are highly useful in the delivery systems of the invention for transforming cells with the nucleotide sequences.
  • the nucleotide sequences may typically be a DNA sequence, and it is this DNA sequence which encodes the antibody or antigen binding fragment thereof sequence forming the antibody or antigen binding fragment thereof of the first aspect.
  • Recombinant vectors encoding the antibody or antigen binding fragment thereof of the first aspect may also include other functional elements. For example, they may further comprise a variety of other functional elements including a suitable promoter for initiating transgene expression upon introduction of the vector in a host cell.
  • the vector is typically capable of autonomously replicating in the nucleus of the host cell. In this case, elements which induce or regulate DNA replication may be required in the recombinant vector.
  • the recombinant vector may be designed such that it integrates into the genome of a host cell.
  • Suitable promoters may include the SV40 promoter, CMV, EF1a, PGK, viral long terminal repeats, as well as inducible promoters, such as the Tetracycline inducible system, as examples.
  • the cassette or vector may also comprise a terminator, such as the Beta globin, SV40 polyadenylation sequences or synthetic polyadenylation sequences.
  • the recombinant vector may also comprise a promoter or regulator or enhancer to control expression of the nucleic acid as required.
  • the vector may also comprise DNA coding for a gene that may be used as a selectable marker in the cloning process, i.e. to enable selection of cells that have been transfected or transformed, and to enable the selection of cells harbouring vectors incorporating heterologous DNA.
  • a selectable marker for example, ampicillin, neomycin, puromycin or chloramphenicol resistance is envisaged.
  • the selectable marker gene may be in a different vector to be used simultaneously with the vector containing the transgene.
  • the cassette or vector may also comprise DNA involved with regulating expression of the nucleotide sequence, or for targeting the expressed polypeptide to a certain part of the host cell.
  • Purified vector may be inserted directly into a host cell by suitable means, e.g. direct endocytotic uptake.
  • the vector may be introduced directly into a host cell (e.g. a eukaryotic or prokaryotic cell) by transfection, infection, electroporation, microinjection, cell fusion, protoplast fusion, calcium phosphate, cationic lipid-based lipofection, polymer or dendrimer-based methods or ballistic bombardment.
  • a host cell e.g. a eukaryotic or prokaryotic cell
  • vectors of the invention may be introduced directly into a host cell using a particle gun.
  • the delivery system may provide the polynucleotide to the host cell without it being incorporated in a vector.
  • the nucleic acid molecule may be incorporated within a liposome or virus particle.
  • a “naked” polynucleotide may be inserted into a host cell by a suitable means e.g. direct endocytotic uptake.
  • a host cell comprising the polynucleotide sequence according to the eighth aspect, the expression cassette according to the ninth aspect, or the vector according to the tenth aspect.
  • the host cell may be a eukaryotic or prokaryotic host cell. In some embodiments, the host cell is a eukaryotic host cell.
  • the host cell is a mammalian host cell such as NS0 murine myeloma cells, PER.C6® human cells, Human embryonic kidney 293 cells or Chinese hamster ovary (CHO) cells. Most In some embodiments, the host cell is a CHO cell.
  • a method of preparing the antibody or antigen binding fragment according to the first aspect comprising: a) introducing, into a host cell, the vector of the tenth aspect; and b) culturing the host cell under conditions to result in the production of the antibody or antigen binding fragment according to the first aspect.
  • the host cell of step a) may be a eukaryotic or prokaryotic host cell.
  • the host cell is a eukaryotic host cell. More
  • the host cell is a mammalian host cell such as NS0 murine myeloma cells, PER.C6® human cells, Human embryonic kidney 293 cells or Chinese hamster ovary (CHO) cells. Most In some embodiments, the host cell is a CHO cell.
  • the method may further comprise (c) centrifuging and/or filtering the cell culture media to obtain a cell culture supernatant comprising the antibody or antigen binding fragment thereof.
  • the method may further comprise (d) separating and purifying the antibody or antigen binding fragment thereof from the cell culture supernatant.
  • purification is performed by at least one chromatographic step. Suitable chromatographic steps include affinity chromatography and/or ion exchange chromatography.
  • affinity chromatography is protein A chromatography.
  • Ion exchange chromatography may be anionic exchange chromatography and/or cationic exchange chromatography.
  • step (d) comprises separating and purifying the antibody or antigen binding fragment thereof from the cell culture supernatant by: i) protein A chromatography; ii) anionic exchange chromatography; and/or iii) cationic exchange chromatography.
  • the method may further comprise (e) filtering the purified antibody or antigen binding fragment thereof resulting from step (d).
  • step (e) comprises virus filtration.
  • virus filtration typically the purified antibody or antigen binding fragment thereof resulting from step (d) is filtered using a virus filtration membrane. Suitable membranes would be known to those skilled in the art.
  • alpha-synuclein aggregation is increased in a number of conditions, including neurodegenerative disorders, such as Parkinson’s disease and multiple system atrophy (MSA).
  • the antibody or antigen- binding fragment thereof may be used as a robust diagnostic tool by detecting the presence, and determining the concentration of, alpha-synuclein aggregates.
  • the antibody or antigen-binding fragment thereof according to the first aspect for use in diagnosis or prognosis.
  • the antibody or antigen-binding fragment thereof according to the first aspect for use in diagnosing or prognosing a condition associated caused by alpha-synuclein aggregation.
  • a method of diagnosing or prognosing a condition associated with or caused by alpha-synuclein aggregation in a subject comprising detecting alpha-synuclein aggregates in a biological sample obtained from the subject with the antibody or antigen-binding fragment thereof according to the first aspect.
  • the condition associated with or caused by alpha-synuclein aggregation may be referred to as a synucleinopathy.
  • the condition associated with or caused by alpha-synuclein aggregation is a neurodegenerative disorder.
  • the condition associated with or caused by alpha-synuclein aggregation is selected from the group consisting of: Parkinson’s disease, dementia with Lewy bodies (DLB), multiple system atrophy (MSA), Alzheimer’s disease, and pure autonomic failure (PAF).
  • the method may be an in vitro or ex vivo method. In some embodiments, the method is an in vitro method.
  • the use or method may comprise determining the level of alpha-synuclein aggregates in a subject, typically wherein an increase in the concentration of alpha- synuclein aggregates in the biological sample when compared to a reference concentration from a healthy control population is indicative of a condition caused by alpha-synuclein aggregation or a poor prognosis.
  • a 1 fold increase of alpha-synuclein aggregates when compared to the reference from a healthy control population is indicative of a condition caused by alpha-synuclein aggregation or a poor prognosis.
  • a 2 fold, 3 fold, 4 fold or 5 fold increase of alpha-synuclein aggregates when compared to the reference from a healthy control population is indicative of a condition caused by alpha-synuclein aggregation or a poor prognosis.
  • a 10 fold, 50 fold or 100 fold increase of alpha-synuclein aggregates when compared to the reference from a healthy control population is indicative of a condition caused by alpha-synuclein aggregation or a poor prognosis.
  • kits for diagnosing a subject suffering from a condition associated with or caused by alpha- synuclein aggregation, or for providing a prognosis of the subject’s condition comprising an antibody or antigen-binding fragment thereof according to the first aspect for detecting alpha-synuclein aggregates in a sample from a test subject.
  • the kit may further comprise instructions for use and/or a receptacle for obtaining a biological sample from a subject.
  • the condition caused by alpha-synuclein aggregation is as defined above.
  • Prognosis may relate to determining the therapeutic outcome in a subject that has been diagnosed with a condition caused by alpha-synuclein aggregation.
  • Prognosis may relate to predicting the rate of progression or improvement and/or the duration of a condition caused by alpha-synuclein aggregation in a subject, the probability of survival, and/or the efficacy of various treatment regimes.
  • a poor prognosis may be indicative of progression of a condition caused by alpha- synuclein aggregation, low probability of survival and reduced efficacy of a treatment regime.
  • the sample comprises a biological sample.
  • the sample may be any material that is obtainable from a subject from which protein is obtainable.
  • the biological sample may be tissue or a biological fluid.
  • the biological sample may be any material that is obtainable from the subject from which blood plasma, endothelial cells, megakaryocytes, and platelets are obtainable.
  • the sample may be blood, plasma, serum, spinal fluid, urine, sweat, saliva, tears, breast aspirate, breast milk, prostate fluid, seminal fluid, vaginal fluid, stool, cervical scraping, cytes, amniotic fluid, intraocular fluid, mucous, moisture in breath, animal tissue, cell lysates, tumour tissue, hair, skin, buccal scrapings, lymph, interstitial fluid, nails, bone marrow, cartilage, prions, bone powder, ear wax, lymph, granuloma, cancer biopsy or combinations thereof.
  • the sample may be a liquid aspirate.
  • the sample may be bronchial alveolar lavage (BAL), ascites, pleural lavage, or pericardial lavage.
  • the sample may comprise blood, urine, tissue etc.
  • the biological sample comprises a blood sample.
  • the blood may be venous or arterial blood. Blood samples may be assayed immediately. Alternatively, the blood sample may be stored at low temperatures, for example in a fridge or even frozen before the method is conducted. Alternatively, the blood sample may be stored at room temperature, for example between 18 to 22 degrees Celsius, before the method is conducted.
  • the blood sample may comprise comprises blood serum.
  • the blood sample may comprise blood plasma. In some embodiments, however, the detection is carried out on whole blood and most typically the blood sample is peripheral blood. The blood may be further processed before the use of the fifteenth aspect is performed.
  • an anticoagulant such as citrate (such as sodium citrate), hirudin, heparin, PPACK, or sodium fluoride may be added.
  • the sample collection container may contain an anticoagulant in order to prevent the blood sample from clotting.
  • the sample may comprise blood plasma, endothelial cells, megakaryocytes, and/or platelets. It will be appreciated that the invention extends to any nucleic acid or peptide or variant, derivative or analogue thereof, which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including variants or fragments thereof.
  • substantially the amino acid/nucleotide/peptide sequence can be a sequence that has at least 40% sequence identity with the amino acid/nucleotide/peptide sequences of any one of the sequences referred to herein, for example 40% identity with the sequence identified as SEQ ID Nos: 1-20 and so on.
  • Amino acid/polynucleotide/polypeptide sequences with a sequence identity which is greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged.
  • the amino acid/polynucleotide/polypeptide sequence has at least 85% identity with any of the sequences referred to, more at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity and, most preferably at least 99% identity with any of the sequences referred to herein.
  • the skilled technician will appreciate how to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences. In order to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences, an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value.
  • the percentage identity for two sequences may take different values depending on:- (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional form and constants. Having made the alignment, there are many different ways of calculating percentage identity between the two sequences.
  • the method used to align the sequences for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison
  • the parameters used by the alignment method for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.
  • percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance. Hence, it will be appreciated that the accurate alignment of protein or DNA sequences is a complex process.
  • calculation of percentage identities between two amino acid/polynucleotide/polypeptide sequences may then be calculated from such an alignment as (N/T)*100, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps and either including or excluding overhangs. In some embodiments, overhangs are included in the calculation.
  • Alternative methods for identifying similar sequences will be known to those skilled in the art.
  • a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to DNA sequences or their complements under stringent conditions.
  • the inventors mean the nucleotide hybridises to filter-bound DNA or RNA in 3x sodium chloride/sodium citrate (SSC) at approximately 45oC followed by at least one wash in 0.2x SSC/0.1% SDS at approximately 20-65oC.
  • a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from the sequences shown in, for example, in those of SEQ ID Nos: 1 to 20 that are amino acid sequences. Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof.
  • Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent (synonymous) change.
  • Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change.
  • small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine.
  • Non-polar, hydrophobic amino acids include phenylalanine, tryptophan and polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine.
  • the positively charged (basic) amino acids include lysine, arginine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids.
  • A) Ribosome Display Panning Cycle three cycles of ribosome display panning against a synthetic peptide encompassing the P1 motif of alpha-synuclein were performed.
  • FIG. 1 shows the selection of the antibody according to the invention referred to as “ ⁇ SP1”, by ELISA and ThT assay.
  • ⁇ SP1 the antibody according to the invention referred to as “ ⁇ SP1”, by ELISA and ThT assay.
  • Data are represented as the ratio of the half time (t50) of ⁇ -syn aggregation in the presence of sdAbs and of a control without sdAb, i.e., aggregation score.
  • C Plot of the binding and aggregation score where ⁇ SP1 (clone A6) is highlighted. The black arrows in A, B, and C indicate the values for ⁇ SP1.
  • D Schematic representation of the naive library showing the framework regions (FWRs) and CDRs of the antibodies, as well as the consensus CDR sequences and the CDR sequences of ⁇ SP1.
  • FT flow through
  • W1 wash 1
  • W2 wash 2
  • E1 elution 1
  • E2 elution 2.
  • B Characterisation of the different steps of size exclusion chromatography (SEC). Representative chromatogram (left) and Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE, right) are shown.
  • C Far-UV circular dichroism (CD) spectrum of antibody ⁇ SP1 (10 ⁇ M) at pH 7.4, 25°C.
  • Figure 5 shows the inhibition of the aggregation of ⁇ -syn by ⁇ SP1.
  • A) ThT assay of ⁇ -syn alone (squares) and in the presence of different concentrations of ⁇ SP1 (from light to dark grey, 1:100, 1:50, 1:20, 1:10 [ ⁇ SP1]: [ ⁇ -syn]). N 3, individual replicates are shown.
  • Figure 6 shows the inhibition of ⁇ -syn aggregation by ⁇ SP1 under conditions which promote the formation of the oligomers of ⁇ -syn. This process is called secondary nucleation.
  • A) ThT assay of WT ⁇ -syn under secondary nucleation conditions at varying concentrations of ⁇ SP1 (1:500, 1:250, 1:100, 1:50, 1:20, and in black 0:1, [ ⁇ SP1]: [ ⁇ -syn]), N 3, individual replicates are shown.
  • FIG. 7 illustrates the specificity of ⁇ SP1 for the oligomers of ⁇ -syn over other protein species.
  • B) ThT assay of 1 ⁇ M A ⁇ 42 in the absence (filled symbols) or presence (empty symbols) of 1:10 [ ⁇ SP1]: [A ⁇ 42], N 3.
  • the error bars represent the standard error of the mean (SEM) of the biological replicates. Each biological replicate is the average of 4–10 technical replicates.
  • B Caspase 3/7 activation in SH-SHY5Y cells exposed to ⁇ -syn aggregates formed in the absence or presence of ⁇ SP1. Cells were treated with ⁇ -syn aggregated in the absence or presence of ⁇ SP1 (molar ratio of 1:20, [ ⁇ SP1]: [ ⁇ -syn]) for 72 h and caspase 3/7 activation was measured as a readout of apoptosis. Luminescence values of the different conditions were normalized over those of the cells incubated with only ⁇ - syn. The averages of 3 biological replicates are shown.
  • the error bars represent SEM of the biological replicates. Each biological replicate is the average of 4–10 technical replicates.
  • Statistical analysis shown in (A) and (B) was carried out by one-way ANOVA with Tukey’s multiple comparison test where P > 0.05 (ns), 0.05 ⁇ P > 0.01 (*), 0.01 ⁇ P > 0.001 (**), 0.001 ⁇ P > 0.0001 (***), and P ⁇ 0.0001 (****). Examples The inventors have generated an antibody that can inhibit ⁇ -syn aggregation via binding to aggregates. The antibody binds specifically to the P1 motif of ⁇ -syn aggregates, and so inhibits the toxic clustering of the protein ⁇ -syn into amyloid fibrils, through binding to its P1 motif.
  • E. coli strain XL1-Blue (Agilent) was used.
  • Synthetic target peptides (P1 and S1) were purchased by Genescript. The peptides were N-terminally biotinylated and C-terminally amidated.
  • Plasmid Constructions DNA primers utilised for cloning of recombinant DNA are listed in Table 1. All enzymes for cloning of recombinant DNA were purchased from New England Biolabs. All plasmids used are listed in Table 2.
  • the mutation TAC136TAT was previously introduced into the pT7-7 ⁇ -syn WT plasmid (a gift from Hilal Lashuel, Addgene plasmid #36046). The resulting plasmid will be called pT7-7 ⁇ -syn WT. Table 1.
  • Plasmids used in this study Plasmid Encodes Source pRDV5 plasmid containing Loop sybody Sybody generation toolbox a loop Sybody (Addgene #1000000160). pT7-7 Wild-type ⁇ -syn Addgene # 36046 pSB_init N/A Sybody generation toolbox (Addgene #1000000160). A6pSB_init Antibody ⁇ SP1 This work Ribosome display against P1 The first round of ribosome display was performed as previously described 20 , and the second and third round of ribosome display were performed as previously described 7 with some modifications.
  • the PUREfrex 2.1 kit and the DS supplement translation mix were used (GeneFrontier Corporation, Kashiwa, Japan).
  • the kit components were prepared to a total volume of 9.3 ⁇ l and incubated at 37 °C for 5 minutes.
  • the concave RNA library was added to the translation mix (0.7 ⁇ l corresponding to 1.6 ⁇ 10 12 mRNA strands) and incubated at 37 ⁇ C for 30 minutes.
  • Dynabeads To coat the Dynabeads they were incubated with 100 ⁇ l WTB-BSA containing 500 nM biotinylated P1 for 20 minutes. After 3 washes of 500 ⁇ l WTB-BSA complexes were added to the beads and incubated for 20 minutes. After 2 washes of the beads with 500 ⁇ l WTB the beads were placed in a fresh tube. Another wash with 500 ⁇ l WTB followed and RNA elution took place by adding 100 ⁇ l 50 mM Tris/acetate pH 7.4, supplemented with 150 mM NaCl, 50 mM EDTA pH 8 and 100 ⁇ g/ml yeast RNA and incubating for 10 minutes at room temperature.
  • the RNeasy micro kit (Qiagen) was used to purify the RNA, which was eluted in 14 ⁇ l RNase-free water.
  • the eluted RNA was mixed with 2 ⁇ l of RT_Primer at 100 ⁇ M and 4 ⁇ l of 10 mM dNTPs and heated to 65 ⁇ C for 5 minutes. Then, the sample was cooled on ice and a RT reaction of 40 ⁇ l was prepared according to the manufacturer’s instructions (Affinity Script, Agilent). The reaction was incubated for 1 hour at 37 ⁇ C and at 95 ⁇ C for 5 minutes.
  • a PCR purification kit (Macherey Nagel) was used to purify the cDNA, which was eluted in 30 ⁇ l elution buffer. PCR amplification followed using 29 ⁇ l of the purified cDNA, Q5 High-Fidelity DNA Polymerase (NEB) and primers Med_FX_for and Med_FX_rev for the concave and loop library. Gel purification of the PCR product followed, and the product was used as template for assembly PCR using megaprimers to append the flanking regions for the in vitro transcription step. The megaprimers were prepared as previously described.
  • the assembled PCR product was gel purified and translated to RNA using a 10 ⁇ l reaction of the T7 RiboMAX Large Scale RNA Production System (Promega). RNA purification followed using the RNeasy kit (Qiagen) and the purified RNA was used as input material of the next round of ribosome display. The second round was performed according to the first round using Dynabeads MyOne Streptavidin C1. For the third round, Dynabeads MyOne Streptavidin T1 were used, and after cDNA amplification the PCR product was subsequently cloned into the pSB_init vector by FX cloning for further analysis as previously described 20 .
  • single antibody clones were expressed in BL21(DE3) cells in 1 ml terrific broth supplemented with 25 mg/ml chloramphenicol in a 96 deep well plate of 2 ml volume.
  • periplasmic extraction buffer 20% (w/v) sucrose, 50 mM Tris, 25 mM EDTA, pH 8.0, 0.5 ⁇ g/ml lysozyme.
  • the lysate was diluted with 900 ⁇ l Tris-buffered saline (TBS) supplemented with 1 mM MgCl2 and centrifuged to pellet cell debris.
  • the supernatant was used as the periplasmic cell extract for subsequent ELISA and aggregation assay steps.
  • Maxi-Sorp immunoplates (Nunc) were used after overnight coating with 100 ⁇ l/well of 5 ⁇ g/ml Protein A in TBS. After washing 3 times with 250 ⁇ l TBS, the plate was blocked with 250 ⁇ l TBS-BSA (TBS supplemented with 0,5% BSA).
  • the absorbance of 650 nm was measured using a CLARIOstar Plus microplate reader.
  • Wild-type ⁇ -Syn Expression & Purification The pT7-7 ⁇ -syn WT plasmid was transformed into BL21(DE3) (NEB) and wild-type or mutant ⁇ -syn expression was induced with 1 mM IPTG at OD600 of 0.7 in LB supplemented with ampicillin (100 ⁇ g/ml) at 37°C for 4 hours. The cells were pelleted and resuspended in buffer A (20 mM Tris-HCl, 1 mM EDTA, pH 8.0) with a protease inhibitor tablet (EDTA-free, Roche).
  • ⁇ -Syn was then purified as previously described 25 . Briefly, the crude extract was incubated at 80°C for 30 min to induce the misfolding and precipitation of all proteins except ⁇ -syn. The sample was then centrifuged at 35,000 x g for 30 min supernatant was incubated in the presence of 10 mg/mL of streptomycin sulphate for 20 min at 4°C and centrifuged at 35,000 x g to precipitate and remove the nucleic acids. The proteins were finally isolated by precipitation using 360 mg/mL ammonium sulfate.
  • Antibody expression and purification Antibody ⁇ SP1 was expressed in BL21(DE3) cells (NEB), which were grown in lysogeny broth containing 25 ⁇ g/ml chloramphenicol to an OD 600 of 0.7 at 37 ⁇ C. Protein expression was induced with 0.02% (w/v) L-arabinose at 22 ⁇ C for 18 hours. Cells were lysed in TBS (20 HCl pH 7.5, 150 mM NaCl) supplemented with 30 mM imidazole and 1 tablet of protease inhibitors (Roche) per liter of culture using a sonicator.
  • Ni-NTA slurry (Qiagen) was used for each purification.
  • the beads were washed twice with 25 ml TBS containing 30 mM imidazole pH 7.5 and the his- tagged antibody was eluted twice with 10 ml TBS containing 300 mM imidazole pH 7.5.
  • the Ni-NTA purified antibodies were dialyzed against TBS to remove imidazole.
  • Antibodies were concentrated using centrifugal filters with a 3 kDa cut-off (Amicon Ultra-4) and separated by SEC using Superdex 200 Increase 10/300 GL (GE Healthcare, Glattbrugg, Switzerland) in PBS pH 7.4. Superdex 75 Increase pg column (GE Healthcare). For protein concentration determination the absorbance at 280 nm was measured and theoretical extinction coefficients were calculated using ExPASy ProtParam. Western blot analysis Protein samples were analysed on NuPAGE Bis-Tris protein gels (Invitrogen). For Western blotting, proteins were transferred to nitrocellulose (NC) membranes (Invitrogen) for 7 minutes at 20 V on an iblot2 gel transfer device (Invitrogen).
  • NC nitrocellulose
  • Membranes were blocked with 5% nonfat dried milk in Tris buffered saline containing 0.1% Tween-20 (TBST) for 1 hour at room temperature. After washing with TBST three times, the membrane was incubated with a rabbit monoclonal anti- ⁇ -syn antibody (Abcam, ab138501) at 1:1000 dilution in TBST overnight. After washing with TBST three times, the membrane was incubated with a goat anti- rabbit secondary antibody Alexa Fluor 555 conjugated (Invitrogen) at 1:2000 dilution in TBST for 1 hour at room temperature.
  • TBST Tris buffered saline containing 0.1% Tween-20
  • a buffer spectrum was subtracted from each time point.
  • a ⁇ 42 purification A ⁇ 42 was purified as previously described.
  • fusion protein the A ⁇ 42 peptide conjugated to a spider silk domain (called fusion protein) for solubility purposes was expressed in BL21 E. coli cells. Cultures were grown in Lennox broth supplemented with 50 ⁇ g/ml kanamycin at 37°C with shaking at 200 rpm until an OD of 0.8 and protein expression was induced with 1mM IPTG at 20°C overnight with shaking at 200 rpm. Cells were collected by centrifugation and the pellet was resuspended in 20 mM Tris–HCl and 8 M urea, pH 8. Following sonication (15s on, 45s off pulses with 20% amplitude), centrifugation was used to remove cellular debris.
  • the filtered supernatant was loaded onto two HisTrap 5 mL columns (Cytiva, Little Chalfont, UK) in tandem that had been pre-equilibrated with binding buffer (20 mM Tris–HCl and 8 M urea, pH 8 supplemented with 15 mM imidazole). Following several column volume washes with binding buffer, the fusion protein was eluted from the column with 5 column volumes of elution buffer (20 mM Tris–HCl and 8 M urea, pH 8 supplemented with 300 mM imidazole). Eluted fusion protein was dialyzed overnight against 20 mM Tris–HCl, pH 8 to remove imidazole.
  • TEV protease was then added to the fusion protein in a 1:15 molar ratio (TEV protease: fusion protein was added to cleave the spider silk domain) at 4°C overnight.7M guanidine-HCL was then added to the sample and incubated on ice for at least 2 h before size exclusion chromatography using a Superdex 75 Increase pg 10/600 column (Cytiva) in 20mM phosphate buffer supplemented with 200 ⁇ M EDTA, pH 8.
  • Monomer concentration (in ⁇ M) was determined from the size exclusion chromatogram using the following calculation: ⁇ 280 ⁇ ⁇ where A280 is the absorbance at elution peak of A ⁇ 42, 0.2 is the path length (cm) of the ATKA Pure (Cytiva, UK) and 1490 M–1 cm–1 is the molecular coefficient of A ⁇ 42. ThT ⁇ -syn and A ⁇ 42 aggregation assays using periplasmic extracts or pure protein at neutral pH.
  • ThT ThT ⁇ -syn aggregation assay using periplasmic extracts
  • ⁇ - syn 30 ⁇ M protein monomer solutions in PBS pH 7.4 were aggregated in the presence of approximately 75 % v/v periplasmic extract, 12 ⁇ M Thioflavin T (ThT) and 0.02% NaN3.150 ⁇ L of each sample (1 replicate only) were loaded into a 96 well full-area plate (non-binding, clear bottomed) and incubated at 37°C for approximately 40 hours in a CLARIOstar Plus microplate reader (BMG Labtech, Germany).
  • Cells were collected by centrifugation, resuspended in buffer A (20 mM Tris-HCl, 1 mM EDTA, pH 8.0) with a protease inhibitor tablet (EDTA-free, Roche) and sonicated for 5 minutes with 15 second pulses and 45 second breaks. Cellular debris was cleared by centrifugation and the supernatant was boiled at 80°C for 20 minutes. This was then centrifuged at max speed ( ⁇ 18,000 rpm) for 20 minutes and the supernatant was collected. Streptomycin was gradually added to reach a final concentration of 10mg/ml to precipitate DNA and incubated at 4C for 20 minutes.
  • buffer A (20 mM Tris-HCl, 1 mM EDTA, pH 8.0
  • EDTA-free protease inhibitor tablet
  • Streptomycin was gradually added to reach a final concentration of 10mg/ml to precipitate DNA and incubated at 4C for 20 minutes.
  • Enzyme-linked immunosorbent assay to assess ⁇ SP1 binding to ⁇ -syn aggregates 20 ⁇ M ⁇ -syn was aggregated as described above and 20 ⁇ l aliquots were taken at T0, 5 h, 10 h, 24 h and 48 h. These samples were immobilised in triplicates onto a 96 well Maxisorp ELISA plate (Nunc) and incubated at 4°C. The plate was then washed with TBS (20 mM Tris, pH 7.4, and 100 mM NaCl) three times and blocked with 5% bovine serum albumin (BSA) in TBS overnight.
  • TBS bovine serum albumin
  • the plate was then washed six time with TBS and incubated with 30 ⁇ l 2 ⁇ M ⁇ SP1 per well at room temperature for 1 hour with constant shaking.
  • the plate was then washed six times with TBS and incubated with 30 ⁇ L solutions of rabbit polyclonal 6x His tag horseradish peroxidase (HRP) conjugated (Abcam) at a 1:4000 dilution in 5% BSA-TBS for 1 h at room temperature with constant shaking.
  • the plate was finally washed three times with TBS, twice with TBS supplemented with 0.02% Tween20, and three times again with TBS.
  • the amount of bound ⁇ SP1 was quantified using the 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific) as per manufacturer’s instructions and the absorbance at 450nm was read using the CLARIOstar plate reader (BMG Labtech).
  • ⁇ -syn preformed fibril seeds 100 - 200 ⁇ M WT ⁇ -syn (PBS, pH 7.4) was aggregated in an Eppendorf tube at 37 °C with shaking (400 - 500 rpm) for approximately 4 d in the presence 0.02% NaN3.
  • the fibrils were diluted to ⁇ 100 ⁇ M and their concentration remeasured. Following further dilution to 5 ⁇ M in PBS, pH 4.8 the fibrils were sonicated on ice at 20 % 5 s pulse, 5 s rest for 3 cycles using a probe sonicator.
  • a probe sonicator [Flagmeier, P.; Meisl, G.; Vendruscolo, M.; Knowles, T. P.; Dobson, C. M.; Buell, A. K.; Galvagnion, C., Mutations associated with familial Parkinson's disease alter the initiation and amplification steps of alpha-synuclein aggregation.
  • Fluorescence intensity measurements were taken using spiral averaging (3 mm diameter) every 420 s using excitation 440 nm, dichroic 460 nm and emission 480 nm filters, 4 gains and 50 flashes per well. All data is background corrected.
  • FRET-based titrations To obtain a Kd value of binding for ⁇ SP1 to ⁇ -syn monomers, oligomers and fibrils, an A140C ⁇ -syn variant was created and purified to allow for an Alexa Flour 488 C5-maleimide (Thermo Fisher Scientific) conjugation. Labelling of the protein was achieved following the manufacturer’s instructions.
  • Alexa fluor 488-labelled oligomers were obtained by lyophilising a solution of 1:10 labelled:unlabelled ⁇ -syn monomers overnight. The lyophilised protein was then resuspended in the same volume of PBS as before lypholization to maintain the 1:10 labelled:unlabelled molar ratio (80:800 ⁇ M, respectively), and incubated at 37°C for 20-24 h. Following ultracentrifugation as described above, monomers were removed by four centrifugations with a 100 kDa molecular cut off filter.488-labelled fibrils were obtained by seeding 100 ⁇ M 488-monomers with 10 ⁇ M PFFs at pH 7.4 under quiescent conditions for 48 h.
  • Labelled fibrils were collected by centrifugation at max speed ( ⁇ 17,000 g) for 30 min and washed with PBS to remove any soluble species.
  • labelled oligomers and concentration was determined as described above, accounting for the fluorophore absorbance at 494 nm and a correction factor of 0.11, as per manufacturer’s instructions.
  • ⁇ SP1 was labelled with an NTA Alexa Fluor 647 kit (Thermo Fisher Scientific) as per manufacturer’s instructions.
  • a range of labelled ⁇ -syn monomer and fibril concentrations were added to labelled 0.5 ⁇ M ⁇ SP1 in triplicate and fluorescence intensity was measured using an excitation and emission wavelength of 493 nm and 672 nm, respectively.
  • MTS cell viability assay SH-SY5Y neuroblastoma cells were cultured in RPMI medium supplemented with 10% (v/v) fetal bovine serum under the 5% CO2 condition. Cells were not used past passage 19. For MTS assays, 10,000 cells per well were plated in 96-well plates 24 h prior to being incubated with protein samples.
  • P1 is poorly accessible in the mature fibrils as shown by cryogenic electron microscopy (CryoEM) 19 .
  • cryogenic electron microscopy (CryoEM) 19 .
  • antibodies for P1 may show preferential binding for aggregation intermediates rather than mature fibrils, which are considered less toxic.
  • the inventors screened a large synthetic sdAb library of concave architecture 7 against this target. Three rounds of ribosome display were coupled to an enzyme linked immunosorbent assay (ELISA) and screening of protein aggregation inhibition. Specifically, the sdAbs were expressed in the Escherichia coli (E.
  • Example 2 Ribosome display and screening A library of synthetic sdAbs based on an anti-GFP antibody of concave architecture was used 7,20 .
  • the synthetic sdAbs have a 6-residue complementarity-determining region 3 (CDR3).
  • the library contains 15 randomised amino acids, and its theoretical diversity is 8.3 x 10 17 .
  • the inventors used purified translation components from E. coli from the commercially available PURE system 21 .
  • Ribosome display was subsequently coupled with a binding screening method as previously described for synthetic sdAb discovery against membrane proteins 7,20 .
  • This method involves FX cloning 22 of single antibodies into the expression vector pSB_init, which generates a fusion of the PelB leader sequence and the antibody.
  • the antibody is targeted to the E. coli periplasm, where disulphide bonds can readily be the vector appends C-terminal Myc- and His-tags to the antibody for detection by ELISA and, subsequently, immobilised metal affinity chromatography (IMAC) purification.
  • IMAC immobilised metal affinity chromatography
  • Example 3 Binding of antibodies to P1 motif
  • the inventors performed ELISA against peptide P1 using the periplasmic extracts of E. coli that contained each antibody candidate ( Figures 1, 2, and 3).
  • an additional ELISA was performed against the scrambled peptide S1 containing the P1 residues in a different order (YSGGLVV; SEQ ID No: 11).
  • the ELISA data provide important information indicative of the specificity and the binding affinity of the antibodies for peptide P1.
  • the screening method is based on the property of interest, which is the inhibition of ⁇ - syn aggregation. Therefore, as a preliminary step to assess the effect of the antibodies on the aggregation of ⁇ -syn, the inventors performed ThT aggregation assays, the results of which are shown in Figure 2B and 3. To bypass the purification step and acquire preliminary data for as many antibodies as possible, recombinant wild-type ⁇ -syn was aggregated in the presence of the E. coli periplasmic extracts containing the antibodies. Given the highly specific binding, A6 was taken forward for additional characterisation. Hereafter, this antibody will be referred to as ⁇ SP1.
  • Example 4 Structural characterisation of ⁇ SP1 ⁇ SP1 was overexpressed in the E. coli periplasm and purified using IMAC followed by size exclusion chromatography (SEC) ( Figure 4A,B). The final purification yield was 0.033 mg/L culture. Circular dichroism (CD) spectroscopy was performed to assess the structural integrity of the purified protein. The data show that the ⁇ SP1 CD spectrum is compatible with a ⁇ -sheet fold ( Figure 4C) characteristic of immunoglobulin domains 23 .
  • SEC size exclusion chromatography
  • Example 5 – ⁇ SP1 inhibition of ⁇ -syn aggregation To determine whether ⁇ SP1 influences the in vitro aggregation of ⁇ -syn, ThT fluorescence assays were performed on ⁇ -syn solutions containing varying ⁇ SP1 concentrations. The results show that ⁇ -syn aggregation is strongly inhibited by ⁇ SP1 and that this inhibition is the concentration of ⁇ SP1 ( Figure 5A). The inhibitory effect by ⁇ SP1 was also confirmed by negative staining and transmission electron microscopy (Figure 5B).
  • the electron micrographs show there are fibrillar aggregates at the end of aggregation for ⁇ -synuclein alone, but not when ⁇ -synuclein is aggregated in the presence of the antibody ⁇ SP1 at a 1:100 molar ratio.
  • the soluble protein fraction at the endpoint of aggregation was isolated by centrifugation and the amount of soluble ⁇ -syn was quantified by western blotting and densitometry.
  • Figure 5C the amount of soluble ⁇ -syn increases at increasing concentrations of ⁇ SP1, in agreement with the ThT results.
  • ⁇ SP1 showed a clear inhibitory effect on ⁇ -syn secondary nucleation at sub-stoichiometric concentrations, i.e., molar ratios of ⁇ SP1 to ⁇ -syn as low as 1:50 (Figure 6A). CD spectroscopy was used to confirm that ⁇ SP1 was stable under these buffer conditions. ( Figure 6B). Homogeneity of seed size was ensured by brief sonication and confirmed by Dynamic Light Scattering (DLS) ( Figure 6C-D).
  • DLS Dynamic Light Scattering
  • Example 6 Specificity of ⁇ SP1 antibody for ⁇ -syn aggregates
  • ⁇ SP1 preferentially interacts with ⁇ -syn aggregates
  • the binding of ⁇ SP1 for different aggregated species of ⁇ -syn was tested. To do so, samples were collected at different incubation times during the in vitro aggregation of ⁇ -syn. Then, they were analysed by ELISA using ⁇ SP1 as primary antibody (Figure 7A). The results show that ⁇ SP1 reactivity is higher for ⁇ - syn samples collected at late times of aggregation, proving that the antibody displays a preferential binding for ⁇ -syn aggregates.
  • the wells of the ELISA plate were coated with the same amount of concentration of protein and increasing amounts of ⁇ SP1 as a primary antibody in an indirect ELISA set-up.
  • the results show that the ELISA signal was significantly reduced when the wells were coated with ⁇ -syn ⁇ P1. This observation is in agreement with ⁇ SP1 binding to P1.
  • the ThT assays were performed against another target known to form amyloids (Figure 8B), the 42-residue long amyloid- ⁇ peptide (A ⁇ 42) 24 . These assays were performed with a molar ratio of ⁇ SP1 to A ⁇ 42 equal to 1:10, which was the maximum molar ratio tested for ⁇ -syn.
  • Example 7 – ⁇ SP1 protects cells against ⁇ syn-induced toxicity ⁇ -Syn aggregates are reported to cause cellular toxicity. Therefore, the inventors assessed the ability of ⁇ SP1 to prevent this mechanism in SH-SY5Y neuroblastoma cells. To do so, they carried out cell viability (MTS) and apoptosis (caspase 3/7 activation) assays on cells incubated with ⁇ -syn aggregated in the absence or presence of ⁇ SP1 (1:20 molar ratio [ ⁇ SP1][ ⁇ -syn].
  • MTS cell viability
  • apoptosis caspase 3/7 activation
  • the inventors have surprisingly identified an antibody that is able to bind specifically to the P1 motif of alpha-synuclein aggregates.
  • this antibody is capable of inhibiting the toxic clustering of the protein alpha-synuclein into amyloid fibrils, through binding to its P1 motif.
  • ⁇ SP1 the antibody according to the invention
  • ⁇ SP1 strongly inhibits the aggregation of alpha-synuclein into toxic fibrils via preferential interaction with alpha-synuclein aggregates.
  • this antibody is effective at very low concentrations in vitro (1 molecule of antibody per 100 molecules of alpha-synuclein is sufficient to provide a strong inhibition).
  • the antibody according to the invention can be used to treat several conditions associated with the toxic amyloid aggregation of alpha-synuclein, including Parkinson’s disease (PD) and Multiple system atrophy (MSA).
  • PD Parkinson’s disease
  • MSA Multiple system atrophy
  • Non-amyloid component sequence is essential and ⁇ -sheet formation is prerequisite to aggregation.
  • Doherty, C. P. A. et al. A short motif in the N-terminal region of ⁇ -synuclein is critical for both aggregation and function.
  • the antibody or antigen-binding fragment thereof according to any preceding claim wherein the antibody or antigen-binding fragment thereof binds specifically to a region between amino acid positions 36 and 42 of alpha-synuclein, as substantially set out in SEQ ID No: 1. 5.
  • the antibody or antigen-binding fragment thereof according to any preceding claim wherein the antibody or antigen-binding fragment thereof binds to the P1 motif comprising or consisting of an amino acid sequence as substantially set out in SEQ ID No: 2, or a variant or fragment thereof.
  • the antibody or antigen-binding fragment thereof according to any preceding claim, wherein the antibody or antigen-binding fragment thereof is a single domain antibody (sdAb). 7.
  • the antibody or antigen-binding fragment thereof comprises: (i) a CDR1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 3, or a variant or fragment thereof; (ii) a CDR2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 4, or a variant or fragment thereof; and/or (iii) a CDR3 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 5, or a variant or fragment thereof,

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Abstract

L'invention concerne des anticorps anti-alpha-synucléine et, en particulier, bien que non exclusivement, des anticorps ciblant le motif P1 de l'alpha-synucléine. L'invention concerne des compositions comprenant les anticorps, y compris des compositions pharmaceutiques et des kits. L'invention concerne également des procédés de production et d'utilisation des anticorps, par exemple en thérapie et en diagnostic de troubles neurodégénératifs, tels que la maladie de Parkinson.
PCT/GB2025/050134 2024-01-25 2025-01-24 Anticorps anti-alpha-synucléine Pending WO2025158162A1 (fr)

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