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WO2014188269A1 - Agregats de superoxyde dismutase - Google Patents

Agregats de superoxyde dismutase Download PDF

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Publication number
WO2014188269A1
WO2014188269A1 PCT/IB2014/001008 IB2014001008W WO2014188269A1 WO 2014188269 A1 WO2014188269 A1 WO 2014188269A1 IB 2014001008 W IB2014001008 W IB 2014001008W WO 2014188269 A1 WO2014188269 A1 WO 2014188269A1
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amino acid
acid sequence
equivalent
binding specificity
sequence seq
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Stefan L. Marklund
Peter M. ANDERSEN
Thomas BRÄNNSTRÖM
Mikael Oliveberg
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A61K38/446—Superoxide dismutase (1.15)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/23—Immunoglobulins specific features characterized by taxonomic origin from birds
    • 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
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/90283—Oxidoreductases (1.) acting on superoxide radicals as acceptor (1.15)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders

Definitions

  • the present invention relates to aggregates of human superoxide dismutase (hSODl), and their use in diagnosis and in methods for the identification and development of new therapeutical agents for use in treatment for amyotrophic lateral sclerosis (ALS)
  • hSODl human superoxide dismutase
  • ALS is a fatal neurodegenerative syndrome characterized by progressive loss of motor neurons in the cortex, brainstem and spinal cord.
  • the clinical hallmark is focal onset dysfunction of motor neurons resulting in paresis and wasting of skeletal muscles, which then spreads to become generalized, including invasion of other areas of CNS.
  • the median age of onset is 58 years, the median survival time is only 26 months and the lifetime risk for developing ALS is about 1 :400.
  • no effective treatment is available.
  • ALS Compared to the other diseases ALS offers some advantages for research: (i) A common cause is mutations in SOD1 (Rosen et al., 1993; Andersen et al., 2003) and currently >170 of such ALS-associated mutations have been found, which aid mechanistic studies; (ii) there are multiple, reliable mutant SOD 1 -expressing transgenic models, e.g. the G93A, D90A, G85R, G127instggg forms of human SOD1 (hSODl), (Gurney et al., 1994; Jonsson et al.
  • SOD1 is a globular enzyme that is amenable to atomic-resolution structural analysis (Nordlund et al., 2009; Generalum et al., 2009) and has well characterised folding and unfolding pathways (Nordlund and Oliverberg, 2006; Leinartaite et al., 2010).
  • SODl is a ubiquitously expressed antioxidant enzyme, which is primarily is located to the cytosol, but is also found in the nucleus and the inter-membrane space of mitochondria.
  • SODl is composed of two equal 153 amino acids long non-covalently bound subunits. Each subunit contains a Cu ion which confers the enzymatic activity and a Zn ion which is important for the stability. There are 4 cysteine residues (C6, C57, CI 1 1 and C146) and two of them (C57-C146) form an intrasubunit disulphide bond. Structural disulphide bonds are rare in the strongly reducing cytosol, and the bond could be an Achilles heel of the protein.
  • the present inventors have, using an assay based on eight anti-hSODl peptide antibodies, identified two varieties of aggregates of hSODl with distinct molecular structures: type A and type B.
  • type A and type B aggregates of hSODl are associated with different characters and severities of disease progression.
  • the type A and type B aggregates of hSODl according to the invention are different from the hSODl aggregates that can be induced to form in vitro.
  • one aspect of the present invention provides methods for identifying and characterizing aggregates of aggregate forming polypeptides, said method comprising analysing the reactivity of said aggregate forming polypeptide with one or more of a panel of antibodies directed to short peptide sequences derived from the amino acid sequence of said aggregate forming polypeptide.
  • an aspect of the present invention provides a method for typing an aggregate (including a fibril) of a polypeptide in a test sample, typically wherein the polypeptide is capable of forming at least two structurally distinct types of aggregates, said method comprising - (i) determining the reactivity of the aggregate in the test sample with one or more, such as 2, 3, 4, 5, 6, 7, 8 or more, different antibody preparations in a panel of antibody preparations,
  • each antibody preparation in the panel has binding specificity to a peptide sequence derived from the amino acid sequence of the polypeptide, such that different antibody preparations in the panel have binding specificity to different peptides, and
  • step (i) wherein at least one of the antibody preparations of the panel, for which reactivity to the aggregate is determined in step (i), displays differential reactivity to at least two structurally distinct types of the aggregate;
  • step (ii) attributing a type to the aggregate in the test sample, based on the determined level(s) of reactivity with the one or more antibody preparations in the panel as determined in step (i).
  • the method of the invention has been used to identify and isolate structurally distinct types of aggregates of a polypeptide, then (as discussed further below) it will be possible to generate antibodies, preferably monoclonal antibodies, which bind specifically to a specific conformational epitope for each aggregate type, preferably wherein the generated antibodies (i) lack of reactivity with soluble native or denatured polypeptide, (ii) lack of reactivity with peptide sequences derived from the amino acid sequence of the polypeptide; and/or (iii) lack of reactivity with at least one alternative structurally distinct type of aggregates of a polypeptide.
  • antibodies preferably monoclonal antibodies, which bind specifically to a specific conformational epitope for each aggregate type, preferably wherein the generated antibodies (i) lack of reactivity with soluble native or denatured polypeptide, (ii) lack of reactivity with peptide sequences derived from the amino acid sequence of the polypeptide; and/or (iii) lack of reactivity
  • Such antibodies may be used in the above-mentioned method of typing in addition to, or in place of, any one or more (or even all) of the one or more antibody preparation in the panel which have binding specificity to a peptide sequence derived from the amino acid sequence of the polypeptide.
  • an aggregate is a structurally ordered assembly of protein polypeptides, where the repetitive units are structurally different from the native state, either in terms of sequence connectivity (domain swapped-based aggregates), in terms of unit-unit interface (aggregates based on native-like units), or in terms of tertiary structure (amyloid-type of aggregates).
  • the aggregates can also be mixtures of these basic structural architectures.
  • the aggregates can be uniformly repetitive, i.e. with structurally identical, monomeric subunits (first-order subunits), or being composed of more complex repetitive units containing several protein polypeptides with identical or diverse structures following the basic architectures outlined above (high- order subunits).
  • the aggregate may comprise a fibril-like spine of stacked ⁇ sheet structures, optionally with domain-swapped material based on inter- molecular native contacts (a simplified cartoon in Figure 9C), wherein the sequence regions outside the ordered aggregate core, which will have lost their native contacts, form disordered fringes.
  • inter- molecular native contacts a simplified cartoon in Figure 9C
  • the term "aggregates" is used herein preferably to exclude dimers, and optionally (although not necessarily) to further exclude low-level oligomers of the polypeptide.
  • Low-level oligomers are intended to include oligomers containing less than, for example, about 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 (such as 3- 20, 3-15 or 3-10) polypeptide molecules.
  • aggregates are non-soluble or substantially more resistant to solubilisation than monomers and dimers, and optionally also low-level oligomers.
  • the aggregate may be visible by light microscopy when viewed at a magnification with the range of 12.5 to 1000 times, whereas non-aggregated polypeptide would typically not be visible within this range of magnification.
  • aggregates may possess a dimension of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500 nm or more, in one or more dimensions, whereas non-aggregated polypeptide would typically not possess such dimensions.
  • an aggregate present in a tissue homogenate e.g. in APBS (see below) supplemented by 1.8 mJVI EDTA, ImM DTT and 1% (w/v) of the detergent NP40
  • a non-aggregated polypeptide typically would not form a pellet under such conditions.
  • the methods may be practiced on oligomers, such as low-level oligomers as described above, and, in that embodiment, the term aggregate is intended to include oligomers, such as oligomers containing at least 5, 10, 15, 20, 25, 30, 40, 50 60 or more polypeptide molecules. In one embodiment, the aggregate may be a fibril.
  • the term "typing" is intended to include the meaning of discriminating between structurally distinct forms of aggregates of the same polypeptide.
  • the term "typing" can refer to discriminating between structurally distinct forms of aggregates of the same polypeptide wherein the structurally distinct forms each comprise a fibril-like spine of stacked ⁇ sheet structures, optionally with domain-swapped material based on inter-molecular native contacts (a simplified cartoon in Figure 9C), wherein the sequence regions outside the ordered aggregate core, which will have lost their native contacts, form disordered fringes.
  • Aggregates for typing according to the present invention may be obtained from biological sources or created in vitro.
  • the aggregates are obtained from biological sources, such as from a human or animal ALS patient (optionally wherein the ALS is sporadic or familial), a human or animal diagnosed as possessing a mutated SOD1 gene, or a transgenic animal carrying a wild-type or mutated hSODl gene.
  • biological sources such as from a human or animal ALS patient (optionally wherein the ALS is sporadic or familial), a human or animal diagnosed as possessing a mutated SOD1 gene, or a transgenic animal carrying a wild-type or mutated hSODl gene.
  • Suitable tissue sources from which aggregates may be obtained from such humans or animals include the spinal cord (such as the cervical spinal cord, the thoracic spinal cord, and/ or the lumbar spinal cord), the brain (such as the hippocampus, the ventral cingulate gyrus, the frontal cortex, the middle and/or superior temporal gyrus, the striatum, and/or the mesencephalon), the liver, the kidney, the blood, plasma, serum, cerebrospinal fluid, or isolated cells, such as leukocytes or lymphocytes.
  • the human or animal from which the aggregate sample is taken may be living, or deceased, at the time of obtaining the sample.
  • the human may be aged up to 20 years, or at least 20, 30 40, 50, 60, 70, 75, 80, 85, 90, 95 or 100 years of age at the time of taking of the sample (or at the time of death, in the case of a sample from a deceased human).
  • the animal may be aged up to 20 % of the mean lifespan of that species or strain of animal, or at least 5, 10, 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95 or 100 % of the mean lifespan at the time of taking of the sample (or at the time of death, in the case of a sample from a deceased human).
  • the aggregate sample is preferably obtained from its source, and maintained for the purposes of typing, under conditions that do not substantially contribute to a change in the conformation of the aggregate present therein.
  • the sample should not be exposed to extremes of pH (generally, operating within the range of pH 5.5 - 9 will be acceptable, and preferably pH values below 5.5, 5.0. 4.5, or 4.0 should be avoided; likewise pH values above 9.0, 9.5, 10.0, or 10.5 should preferably be avoided);; conditions that contribute to the proteolysis of the protein in the aggregate; and/or denaturing agents (such as trichloroacetic acid or the like) at levels that would contribute to a change in the conformation of the aggregate present therein.
  • extremes of pH generally, operating within the range of pH 5.5 - 9 will be acceptable, and preferably pH values below 5.5, 5.0. 4.5, or 4.0 should be avoided; likewise pH values above 9.0, 9.5, 10.0, or 10.5 should preferably be avoided
  • denaturing agents such as trich
  • the test sample Prior to typing, the test sample may, or may not, be subjected to purification step to render it substantially free of non-aggregated forms of the polypeptide, including monomelic forms and dimeric forms, denatured forms and/or misfolded forms of the polypeptide, and optionally low-level oligomeric forms.
  • purification step to render it substantially free of non-aggregated forms of the polypeptide, including monomelic forms and dimeric forms, denatured forms and/or misfolded forms of the polypeptide, and optionally low-level oligomeric forms.
  • Such pre-typing purification is, however, not necessary for the practice of the present invention. Nevertheless, such forms can typically be separated from aggregated forms of the polypeptide by techniques well known in the art, such as centrifugation, as discussed in the examples.
  • an antibody preparation having binding specificity for peptides consisting of the sequence of SEQ ID NO: 1 or 2 will bind to at least monomelic forms, misfolded dimeric forms, and/or denatured forms of hSODl, but not to type A or type B aggregates, and so can be used to assess whether samples of type A or type B aggregates are free of such species.
  • the methods of the present invention can be used for identifying epitopes and peptide sequences of the aggregate forming polypeptide, which are specifically exposed in pathological aggregates of said aggregate forming polypeptide, said epitopes being useful as vaccine candidates and as target for identifying therapeutical antibodies and other types of binding molecules, including small molecules, which potentially can be used in the treatment of the related diseases.
  • the present invention employs one or more different antibody preparations in a panel of antibody preparations, wherein each antibody preparation in the panel has binding specificity to a peptide sequence derived from the amino acid sequence of the polypeptide, such that different antibody preparations in the panel have binding specificity to different peptides.
  • the, or each, peptide sequence derived from the amino acid sequence of the polypeptide contains no more than 25, 20, 15, 10 or fewer contiguous amino acids from the amino acid sequence of the polypeptide.
  • peptides contain less then the full polypeptide, such as the full naturally occurring protein (e.g. is smaller than hSODl in embodiments intended to type aggregates of hSODl).
  • The, or each, peptide sequence may optionally comprise one or more additional amino acids at the N-terminus or C-terminus, typically in which the additional amino acids do not match the amino acids found at a corresponding position in the sequence of the polypeptide.
  • peptides may be less than 40, 35, 30, 25, 20, 19, 18, 17, 16, or 15 in length, and optionally contain no more than 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 contiguous amino acids from the amino acid sequence of the polypeptide.
  • Particularly preferred peptides may be 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length.
  • the peptide consists of no more than 25, 20, 15, 10 or fewer amino acids, which are contiguous amino acids from the amino acid sequence of the polypeptide.
  • binding specificity includes the meaning that there is no cross-reactivity between the peptide sequences targeted by the different antibody preparations in the panel.
  • the different antibody preparations in the panel each bind to different peptide sequences derived from the amino acid sequence of the polypeptide.
  • an antibody preparation can be said to bind specifically to a peptide (a 'target' peptide) sequence if a binding reaction can be seen in a dot blot assay (such as described in the Examples) between the antibody preparation and the target peptide that is at least 3, 4, 5, 6, 7, 8, 9, 10 or more times higher than - the background reaction;
  • any other peptide of 10-15 amino acids in length derived from the amino acid sequence of the polypeptide of interest without overlapping with the target peptide.
  • This may, for example, be adjudged with reference to an internal control, such as the level of binding reactivity to another antibody preparation in the panel.
  • an internal control such as the level of binding reactivity to another antibody preparation in the panel.
  • antibody preparations with binding specificity to peptides consisting of the sequence of SEQ ID NOs: 5, 6 and 7, respectively display differential reactivity to at least two structurally distinct types of hSODl aggregates.
  • the antibodies present in an antibody preparation within the panel can be polyclonal or monoclonal.
  • Polyclonal antibody preparations may be preferred in some instances, since this may render the panels more resilient to the existence of unanticipated point mutations in the polypeptides under assessment, and continue to permit the assessment of structural variations in the aggregates of such mutants.
  • monoclonal antibody preparations may be preferred in other instances, in particular since this may permit further selectivity between polypeptide mutants as well as assessment of structural variations in the aggregates.
  • the antibody preparations are peptide specific polyclonal sera, optionally generated in an animal model selected from rabbits, chicken, mice, goat, sheep, donkey, camelids, or other animals which are available for antibody generation.
  • the peptide specific polyclonal sera may, for example, be generated by immunization of the animals with a peptide having a specific amino acid sequence, preferably a peptide having an amino acid sequence selected from the amino acid sequences SEQ ID NO: 1-15, the obtained polyclonal sera purified by affinity chromatography using the same peptide as used as immunogen, resulting in a peptide specific polyclonal sera.
  • the peptides may be linked, directly or indirectly, to a carrier.
  • a carrier may be used, including well-known carriers such as keyhole-limpet haemocynanin (KLH) or an albumin, such as human serum albumin (HSA) or bovine serum albumin (BSA).
  • KLH keyhole-limpet haemocynanin
  • HSA human serum albumin
  • BSA bovine serum albumin
  • a carrier function should be present in any immunogenic formulation in order to stimulate, or enhance stimulation of, the immune system. It is thought that the best carriers embody (or, together with the antigen, create) a T-cell epitope.
  • the peptides may be associated, for example by cross-linking, with a separate carrier, such as serum albumins, myoglobins, bacterial toxoids and keyhole limpet haemocyanin.
  • a separate carrier such as serum albumins, myoglobins, bacterial toxoids and keyhole limpet haemocyanin.
  • More recently developed carriers which induce T-cell help in the immune response include the hepatitis-B core antigen (also called the nucleocapsid protein), presumed T-cell epitopes such as Thr-Ala-Ser-Gly-Val-Ala-Glu- Thr-Thr-Asn-Cys, beta-galactosidase and the 163-171 peptide of interleukin-1.
  • the latter compound may variously be regarded as a carrier or as an adjuvant or as both.
  • sequence of the polypeptide form which the peptide is derived may be a cysteine-containing sequence, or the peptide may be engineered to comprise, consist essentially of, or consist of, the peptide sequence from the polypeptide of interest plus an additional cysteine (which most preferably will be a terminal cysteine).
  • cross-linking agents include those listed as such in the Sigma and Pierce catalogues, for example glutaraldehyde, carbodiimide and succinimidyl 4-(N- maleimidomethyl)cyclohexane-l-carboxyIate, the latter agent exploiting the -SH group on the C-terminal cysteine residue (if present).
  • each antibody preparation in the panel has binding specificity to a peptide sequence derived from the amino acid sequence of the polypeptide.
  • the peptides should be short, typically less than 30, 25, 20, 19, 18, 17, 16, or 15 in length. Particularly preferred peptides may be 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length.
  • peptides When such short antibodies are used to raise antibodies, such as in a method as described above for obtaining polyclonal sera, the peptides will typically have no fixed conformation at the point of immunization (and/or if there is a conformation it is most likely different from the conformation of the corresponding segment in the native protein). Thus, peptides may, in one embodiment, be characterized as "short” if they can be used to raise antibodies that react with a non-native (such as denatured) conformation of the full polypeptide and not with the full polypeptide in native conformation.
  • a non-native such as denatured
  • the panel contains multiple different preparations of antibodies, wherein the different antibody preparations bind specifically to different peptides derived from the full polypeptide, such that the whole panel is capable of binding to locations on the full polypeptide that, in sum, are greater than 50%, 60%, 70%, 80%, 85%, 90%, 95% or more, such as substantially 100%, of the full polypeptide.
  • the panel in addition to the at least one antibody preparation of the panel which displays differential reactivity to at least two structurally distinct types of the aggregate, further comprises one or more antibody preparations that distinguish the polypeptide when in aggregated form from the polypeptide when in monomeric, dimeric, denatured and/or misfolded form.
  • the panel may comprise one or more antibody preparations that have binding specificity for the polypeptide when in monomeric, dimeric, denatured and/or misfolded form, but not in aggregated form.
  • the absence of reactivity of such antibody preparations with the aggregate in the sample can be taken into account in confirming that the aggregate in the sample is, indeed, in aggregated form and/or that the sample is clear of non-aggregated polypeptide.
  • the present invention takes advantage of this knowledge to use an epitope-mapping type assay to type different aggregate structures, since it appears that antibody preparations with binding specificity to peptide sequences that are found in the aggregate core have no reactivity with the aggregate, because of the ordered structure and perhaps also because of steric hindrance, whereas antibody preparations with binding specificity to short peptides derived from sequences found in the disordered fringes in the aggregate fibrils should be reactive with the aggregate.
  • short peptides can raise antibodies that react with a non-native conformation of the full polypeptide, and not with the full polypeptide in native conformation, and so these antibodies can bind to sequence present in the disordered fringes of the aggregated polypeptide, but typically not to the same sequence in the native polypeptide.
  • the method of the invention has been used to identify and isolate structurally distinct types of aggregates of a polypeptide, then (as discussed further below) it will be possible to generate antibodies, preferably monoclonal antibodies, which bind specifically to a specific conformational epitope for each aggregate type, preferably wherein the generated antibodies (i) lack of reactivity with soluble native or denatured polypeptide, (ii) lack of reactivity with peptide sequences derived from the amino acid sequence of the polypeptide; and/or (iii) lack of reactivity with at least one alternative structurally distinct type of aggregates of a polypeptide.
  • antibodies preferably monoclonal antibodies, which bind specifically to a specific conformational epitope for each aggregate type, preferably wherein the generated antibodies (i) lack of reactivity with soluble native or denatured polypeptide, (ii) lack of reactivity with peptide sequences derived from the amino acid sequence of the polypeptide; and/or (iii) lack of reactivity
  • Such antibodies may be used in the above-mentioned method of typing in addition to, or in place of, any one or more (or even all) of the one or more antibody preparation in the panel which have binding specificity to a peptide sequence derived from the amino acid sequence of the polypeptide.
  • the aggregate forming polypeptide can, in a preferred embodiment, be superoxide dismutase (SOD), such as a human superoxide dismutase (hSOD), preferably human superoxide dismutase 1 (hSODl).
  • hSODl may be wild-type, or may be a mutant hSODl, such as a mutant SOD1 that is associated with ALS, such as the D90A (i.e.
  • mutant hSODl proteins associated with ALS and/or altered aggregate formation are known in the art, and may be useful in the practice of the present invention, including any one or more of the following mutations: A4S, A4T, A4V, C6F, C6G, C6S, V7E, L8Q, L8V, G10R, G10V, G12R, V14G, V14M, G16A, G16S, N19S, F20C, E21K, E21G, Q22L, Q22R, G27delGP, V29A, G37R, G37V, L38R, L38V, G41D, G41S, H43R, F45C, H46D, H46R, V47A, V47F, H48R, H48Q, E49K, T54R, S59I, G61R, N65S, P66R, L67P
  • One particularly preferred embodiment provides methods for identifying and characterizing aggregates of hSODl with distinct molecular structures, said method comprising analysing the reactivity of a hSODl aggregate with one or more of a panel of antibodies directed to short peptide sequences derived from the amino acid sequence of hSODl, and/or comprising analysing the reactivity of a hSODl aggregate with one or more antibodies as defined by claims 26 or 27, below.
  • the particularly preferred embodiment provides a method for typing an hSODl aggregate (including a fibril) in a test sample, said method comprising -
  • each antibody preparation in the panel has binding specificity to a peptide sequence derived from the amino acid sequence of hSODl, such that different antibody preparations in the panel have binding specificity to different peptides, and
  • step (i) wherein at least one of the antibody preparations of the panel, for which reactivity to the aggregate is determined in step (i), displays differential reactivity to at least two structurally distinct types of hSODl aggregate;
  • step (ii) attributing a type to the hSODl aggregate in the test sample, based on the determined level(s) of reactivity with the one or more antibody preparations in the panel as determined in step (i).
  • step (i) may comprise determining the reactivity of the hSODl aggregate in the test sample with one or more antibodies as defined by claims 26 or 27, below, and step (ii) may comprise attributing a type to the hSODl aggregate in the test sample, based on the determined level(s) of reactivity with the one or more antibodies as determined in step (i).
  • this alternative may also be used in combination with the method described in the preceding paragraph.
  • the present application may also be practiced on other aggregate forming polypeptides which may, for example, include Alpha-Synuclein/Parkinson's disease, Beta- amyloid/Alzheimer's disease, TAU/Alzheimer's disease, IAPP (Amylin)/Diabetes mellitus type 2, Serum Amyloid A/Rheumatoid arthritis, PrP/Transmissible spongiform encephalopathy, Transthyretin/Familial amyloid polyneuropathy, Huntingtin/Huntington's disease.
  • Alpha-Synuclein/Parkinson's disease Beta- amyloid/Alzheimer's disease
  • TAU/Alzheimer's disease IAPP (Amylin)/Diabetes mellitus type 2
  • Serum Amyloid A/Rheumatoid arthritis PrP/Transmissible spongiform encephalopathy
  • hSODl human superoxide dismutase 1
  • each antibody preparation in the panel has binding specificity to a peptide sequence derived from the amino acid sequence of hSODl, such that different antibody preparations in the panel have binding specificity to different peptides, and
  • step (i) wherein at least one of the antibody preparations of the panel, for which reactivity to the hSODl aggregate is determined in step (i), displays differential reactivity to at least two structurally distinct types of aggregate of the hSODl D90A mutant; and (ii) attributing a type to the aggregate in the test sample, based on the determined level(s) of reactivity with the one or more antibody preparations of the panel as determined in step
  • step (i) may comprise determining the reactivity of the hSODl aggregate in the test sample with one or more antibodies as defined by claims 26 or 27, below, and step (ii) may comprise attributing a type to the hSODl aggregate in the test sample, based on the determined level(s) of reactivity with the one or more antibodies as determined in step (i).
  • step (i) may comprise determining the reactivity of the hSODl aggregate in the test sample with one or more antibodies as defined by claims 26 or 27, below, and step (ii) may comprise attributing a type to the hSODl aggregate in the test sample, based on the determined level(s) of reactivity with the one or more antibodies as determined in step (i).
  • this alternative may also be used in combination with the method described in the preceding paragraph.
  • hSODl polypeptide in the hSODl aggregate may be wild-type, or may be a mutant hSODl, such as a mutant SOD1 that is associated with ALS, including any one or more of the following mutations: A4S, A4T, A4V, C6F, C6G, C6S, V7E, L8Q, L8V, G10R, G10V, G12R, V14G, V14M, G16A, G16S, N19S, F20C, E21K, E21G, Q22L, Q22R, G27delGP, V29A, G37R, G37V, L38R, L38V, G41D, G41S, H43R, F45C, H46D, H46R, V47A, V47F, H48R, H48Q, E49 , T54R, S59I, G61R, N65S, P66R, L67P, L67R, G72C, G
  • the foregoing methods of the present invention can also be used for identifying epitopes and peptide sequences of aggregates of hSODl, which are specifically exposed in aggregates of hSODl , particularly pathological aggregates of hSODl , said epitopes and peptide sequences being useful as vaccine candidates and as target for identifying therapeutical antibodies and other types of binding molecules, including small molecules, which potentially can be used in the treatment of ALS.
  • the panel of antibody preparations comprises or consists of one or more antibody preparations selected from
  • the whole panel of antibody preparations for use in the method of the present invention has binding specificity peptides that correspond to locations on the full hSODl polypeptide that, in sum, is greater than 50%, 60%, 70%, 80%, 85%, 90%, 95% or more, such as substantially 100%, of the full hSODl polypeptide
  • the antibody panel may comprise or consist of antibodies as defined by claims 26 or 27, below. The skilled person will appreciate that this alternative may also be used in combination with the method described in the preceding paragraph.
  • the panel of antibody preparations comprises, or consists of, two or more (such as 3, 4, 5, 6, 7, 8 or more) different antibody preparations, and wherein - at least one antibody preparation in the panel serves as a control and has binding specificity to a peptide consisting of the amino acid sequence SEQ ID NO: 4 or an equivalent thereof, and
  • one or more other antibody preparations in the panel display differential reactivity (relative to the control) to at least two structurally distinct types of hSODl aggregate, and the one or more other antibody preparations in the panel comprise, consist essentially of, or consist of -
  • the binding specificity of preparation(s) with differential reactivity in the panel further comprises an antibody preparation which has binding specificity to a peptide consisting of the amino acid sequence SEQ ID NO: 8 or an equivalent thereof.
  • the method further comprises determining the reactivity of the aggregate in the test sample with an antibody preparation which has binding specificity to a peptide consisting of the amino acid sequence SEQ ID NO: 3 or an equivalent thereof.
  • the method further comprises determining the reactivity of the aggregate in the test sample with -
  • the hSODl aggregate may be identified as Type A, if the measured level of reactivity of the aggregate with the control preparation which has binding specificity to a peptide consisting of the amino acid sequence SEQ ID NO:4 or an equivalent thereof is greater than the level of reactivity of the aggregate with the or each of the antibody preparations in the panel having differential reactivity and which have binding specificity to peptides consisting of the amino acid sequences of each of SEQ ID NOs: 5, 6 and/or 7 (and optionally, also SEQ ID NO: 8), or an equivalents thereof, respectively.
  • the hSODl aggregate may be identified as Type A, if the measured level of reactivity of the aggregate is highest for X, lower than X for Y, and lower than Y and optionally not detectable for Z, wherein - X is the measured level of reactivity of the aggregate to the control antibody preparation which has binding specificity to a peptide consisting of the amino acid sequence SEQ ED NO:4 or an equivalent thereof,
  • Y is the measured levels of reactivity of the aggregate to antibody preparations which have binding specificity to peptides consisting of the amino acid sequences of each of SEQ ID NOs: 3, 5 and 8, or equivalents thereof, respectively, and
  • Z is the measured levels of reactivity of the aggregate to antibody preparations which have binding specificity to peptides consisting of the amino acid sequences of each of SEQ
  • the hSODl aggregate may be identified as Type B, if the measured level of reactivity of the aggregate with the control preparation is less than the level of reactivity of the aggregate with the or each of the antibody preparations in the panel having differential reactivity and which have binding specificity to peptides consisting of the amino acid sequences of each of SEQ ID NOs: 5, 6 and/or 7 (and optionally, also SEQ ID NO: 8), or equivalents thereof, respectively.
  • the hSODl aggregate may be identified as Type B, if the measured level of reactivity of the aggregate is highest for X, lower than X for Y, and lower than Y and optionally not detectable for Z, wherein - X is the measured levels of reactivity of the aggregate to antibody preparations which have binding specificity to peptides consisting of the amino acid sequences of each of SEQ ID NOs: 5, 6 and 7, or equivalents thereof, respectively,
  • Y is the measured level of reactivity of the aggregate to the control antibody preparation which has binding specificity to a peptide consisting of the amino acid sequence SEQ ID NO:4 or an equivalent thereof and/or the measured level of reactivity of the aggregate to the antibody preparation which has binding specificity to a peptide consisting of the amino acid sequence SEQ ID NO: 8 or an equivalent thereof, and
  • Z is the measured level of reactivity of the aggregate to antibody preparations which have binding specificity to peptides consisting of the amino acid sequences of each of SEQ ID NOs: 1 and 2, or equivalents thereof, respectively.
  • the panel of antibody preparations for use in the methods of the invention comprises an antibody preparation which has binding specificity to a peptide consisting of the amino acid sequence SEQ ID NO:4 or an equivalent thereof; and one, two, or all three of -
  • an antibody preparation which has binding specificity to a peptide consisting of the amino acid sequence SEQ ID NO: 8 or an equivalent thereof.
  • the panel of antibody preparations for use in the methods of the invention comprises eight different antibody preparations which, respectively, have binding specificity to a peptide consisting of the amino acid sequence of each of SEQ ID NO: l, 2, 3, 4, 5, 6, 7 and 8, or equivalents thereof.
  • an "equivalent" of a defined SEQ ID NO is intended to refer to a sequence that comprises, consists essentially of, or consists of, a portion of the primary amino acid sequence of hSODl, wherein the portion is typically 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more amino acids in length, and wherein the portion includes 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more amino acids of the defined SEQ ID NO.
  • peptides comprising the sequences of any of SEQ ID Nos: 1 , 2, 3, 4,5 ,6, 7, 8, 9, 10, 1 1, 12, 13, 14 and/or 15, and the "equivalents" thereof, may optionally exclude peptides which comprise, or consist, any of the sequences DLGKGGNEESTKTGNAGS (optionally, with N-terminal truncation of D, DL, DLG or DLGK, an/or C-terminal truncation of S, GS, AGS or NAGS); NPLSRKHGGPKDEE (optionally with N-terminal truncation of N, NP or NPL and/or C- terminal truncation of E, EE or DE); IKGLTEGLHGF; HCIIGRTLVVH (optionally with N-terminal truncation of H or HC and/or C-terminal truncation of H or VH; GLHGFHVH; RLA
  • the "equivalent" of a defined SEQ ED NO does not overlap with any other defined SEQ ID NO in these claims by more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids, and preferably wherein the equivalent does not include any overlap with any of the other defined SEQ ID NO in these claims.
  • SEQ ID NO: 3 overlaps with SEQ ID NOs 9 and 10, but not with SEQ ID NO: 2, and so a greater level of overlap may be tolerated between equivalents of SEQ ID NO 3 and either or both of SEQ ID NOs 9 and 10, than with SEQ ID NO:2.
  • SEQ ID NO: 3 overlaps with SEQ ID NOs 9 and 10 but not with SEQ ID NO: 2
  • a greater level of overlap may be tolerated between equivalents of SEQ ID NO 3 and either or both of SEQ ID NOs 9 and 10, than with SEQ ID NO:2.
  • SEQ ID NO:2 overlaps with SEQ ID NOs 9 and 10
  • an antibody preparation which has binding specificity to the "equivalent" of a defined SEQ ID NO may also possesses binding specificity to a peptide consisting of the defined SEQ ID NO.
  • a peptide comprising the "equivalent" of a defined SEQ ID NO may be capable of competitively inhibiting the binding of a peptide consisting of the defined SEQ ID NO to an antibody preparation raised against the defined SEQ ID NO.
  • an antibody preparation which binds specifically to a defined SEQ ID NO may bind specifically to a peptide consisting of that defined SEQ ID NO in preference to (such as, with at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 40 or 50 times greater reactivity, relative to background binding in a dot blot assay as described in the present examples, than to) a peptide which consists of any of the sequences DLGKGGNEEST TGNAGS (optionally, with N-terminal truncation of D, DL, DLG or DLGK, an/or C-terminal truncation of S, GS, AGS or NAGS); NPLS RKHGGPKDEE (optionally with N-terminal truncation of N, NP or NPL and/or C- terminal truncation of E, EE or DE); IKGLTEGLHGF; HCIIGRTLVVH (optionally with N-terminal trun
  • isolated can include the meaning that the aggregate is substantially free of structurally different forms of aggregates of the same polypeptide.
  • substantially free may include the meaning of no more than 50%, 40%, 30%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% or less (by weight) of a preparation comprising the isolated aggregate possesses a structurally different form of aggregate from the predominating aggregate form in the preparation.
  • a preparation comprising, consisting essentially or, or consisting of, an isolated aggregate of hSODl, wherein the isolated aggregate is characterized by:
  • this isolated aggregate is characterized by:
  • the aggregates are of type A.
  • Type A aggregates are characterized by:
  • Type A aggregates may optionally be further characterized by:
  • a preparation comprising, consisting essentially or, or consisting of, an isolated aggregate of hSODl, wherein the isolated aggregate is characterized by:
  • this isolated aggregate is characterized by:
  • Type B aggregates may be characterized by:
  • a specific aggregate may be said to be reacting with an antibody, or antibody preparation, if a substantial reaction can be seen between the aggregate and antibody / antibody preparation in a dot blot assay (such as the assay as described in the Examples), wherein a substantial reaction can be a reaction that is least 3, 4, 5, 6, 7, 8, 9, 10 or more times higher than -
  • a specific aggregate is said to not be reacting with an antibody if no substantial reaction can be seen in a dot blot assay as described in the Examples.
  • Type A aggregates of hSODl form in the spinal cord and brain and can be isolated from transgenic mice expressing the G93A, G85R and wild- type hSODl .
  • Type A and Type B aggregates form in the spinal cord and brain of and can be isolated from transgenic mice expressing the D90A form of hSODl .
  • Type B aggregates of hSODl are more neurotoxic than type A aggregates, and give rise to accelerated neurodegeneration.
  • methods of the present invention for typing aggregates of polypeptides may further comprise the step of attributing a diagnosis, prognosis or disease-related prediction to the subject.
  • the hSODl aggregates according to the invention can be isolated from spinal cord or brain of transgenic mice expressing wild-type hSODl or mutant forms of hSODl .
  • the hSODl aggregates according to the invention are different from previously reported hSODl aggregates formed in vitro.
  • hSODl aggregates according to the invention can be generated and isolated in vitro by methods comprising seeding solutions of hSODl with hSODl aggregates according to the invention, allowing hSODl aggregates to be formed and isolating the formed aggregates.
  • the present invention also provides a method of producing Type A hSODl aggregate, the method comprising seeding a solution of hSODl with a Type A hSODl aggregate (optionally, wherein the Type A hSODl aggregate has been identified as such by a method of the present invention), and allowing further hSOD 1 aggregates to be formed in the solution.
  • the method may comprise the further step of typing the thus-formed hSODl aggregates by a typing method of the present invention. Additionally, or alternatively, the method may further comprise the step of isolating the thus-formed aggregate, and optionally formulating the aggregate in a composition with carriers or diluents.
  • the present invention also provides a method of producing Type B hSODl aggregate, the method comprising seeding a solution of hSODl with a Type B hSODl aggregate (optionally, wherein the Type A hSODl aggregate has been identified as such by a method of the present invention), and allowing further hSODl aggregates to be formed in the solution.
  • the method may comprise the further step of typing the thus-formed hSODl aggregates by a typing method of the present invention. Additionally, or alternatively, the method may further comprise the step of isolating the thus-formed aggregate, and optionally formulating the aggregate in a composition with carriers or diluents.
  • Suitable carriers or diluents according to the present application must be “acceptable” in the sense of being compatible with the other components in the compositions of the invention and not deleterious to the recipients thereof.
  • the carriers may be water or saline which will be sterile and pyrogen free.
  • the hSODl aggregates according to the invention can be used to generate aggregate specific antibodies. Accordingly, another aspect of the present invention provides methods for generation of antibodies specific for pathological aggregates of hSODl , said methods comprising the use of isolated hSODl aggregates according to the present invention, such as in the immunization of animals and collection of polyclonal sera, or in panning antibody libraries for specifically binding antibodies.
  • Another aspect of the present invention provides methods for the generation of monoclonal antibodies using isolated type A or type B hSODl aggregates of the present invention.
  • the type A and type B aggregates should each contain specific conformational epitopes that are not bound by the anti-peptide antibodies discussed above. Such specific conformational epitopes may, for example, be present in ordered parts of the aggregates.
  • the isolated type A or type B hSODl aggregates used for the generation of such monoclonal antibodies may be generated by seeding solution of hSODl in vitro with type A or type B, as described above. Such aggregates that are generated by in vitro seeding may be preferred for this purpose, since their level of purity may be higher than type A or type B aggregates that have been purified from biological sources.
  • such monoclonal antibodies are further screened to identify those demonstrating a lack of reactivity with any one or more, such as all eight, of the peptides consisting of the sequences defined by SE ID NOs: 1, 2, 3, 4 ,5, 6, 7, and 8.
  • Monoclonal antibodies fitting such binding criteria may be a preferred form to use therapeutically, since they would not react with mature and immature soluble forms of SOD1 in the tissues.
  • Another aspect of the present invention provides antibodies directed to the hSODl aggregates according to the present invention.
  • Such antibodies may be useful in the treatment, monitoring and diagnosis of ALS.
  • the antibodies may have binding specificity to a peptide that comprises, consists essentially of, or consists of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, or an equivalent of any one thereof and/or monoclonal antibodies obtained using isolated type A or type B hSODl aggregates of the present invention as discussed above (and/or as defined in claims 26 or 27 below).
  • antibodies having binding specificity to a peptide that comprises, consists essentially of, or consists of any of SEQ ID NOs: 3, 4, 5, 8, 10, 11, 12, 14, or an equivalent of any one thereof, and/or monoclonal antibodies obtained using isolated type A or type B hSODl aggregates of the present invention as discussed above (and/or as defined in claims 26 or 27 below), are of greatest interest for providing direct therapeutic effects for ALS.
  • the present invention provides a pharmaceutically acceptable composition, comprising a peptide corresponding to amino acid sequences specifically exposed on pathological aggregates of hSODl, and preferably selected from peptides comprising, consisting essentially of, or consisting of
  • the formulation further comprises one or more additional components, such as components selected from the group consisting of pharmaceutically acceptable carriers, diluents, adjuvants, delivery agents or the like.
  • compositions for use in medicine Also provided is the foregoing pharmaceutically acceptable composition for use in medicine. Also provided is the foregoing pharmaceutically acceptable for use in the treatment and/or prophylaxis of ALS, including the delay of onset, reduction in the speed of progression and/or reduction in the symptoms of ALS.
  • the present invention provides a pharmaceutically acceptable composition, the composition comprising, consisting essentially of, or consisting of, one or more antibody preparations, wherein the or each antibody preparation has binding specificity to a peptide corresponding to amino acid sequences specifically exposed on pathological aggregates of hSODl, such as a peptide that comprises, consists essentially of, or consists of - a) the amino acid sequence SEQ ID NO: 3 or an equivalent thereof,
  • monoclonal antibodies obtained using isolated type A or type B hSODl aggregates of the present invention as discussed above, optionally, wherein the peptide is linked, directly or indirectly, to another entity, such as a carrier; and
  • composition further comprises one or more additional components, such as components selected from the group consisting of pharmaceutically acceptable carriers, diluents, delivery agents or the like.
  • the antibodies in the antibody preparations may be polyclonal or monoclonal.
  • the antibodies are preferably monoclonal antibodies.
  • the antibodies may be human or non-human.
  • the antibodies will be human (e.g. human polyclonal or human monoclonal) or will be humanised.
  • Antibodies for use in the present invention includes antibody fragments.
  • Antibody fragments will retain the binding specificity of the parent antibody.
  • the antibody fragments can be generated by standard molecular biology techniques or by cleavage of purified antibodies using enzymes (e.g. pepsin or papain) that generates these fragments.
  • enzymes e.g. pepsin or papain
  • Such antibody fragments according to the invention are exemplified, but not limited to, single chain antibodies, Fv, scFv, Fab, F(ab')2, Fab', Fd, dAb, CDR, or scFv-Fc fragments or nanobodies, and diabodies, or any fragment that may have been stabilized by e.g. PEGylation.
  • Fab, Fv, ScFv and dAb antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the said fragments.
  • Whole antibodies, and F(ab')2 fragments are "bivalent". By “bivalent” we mean that the said antibodies and F(ab')2 fragments have two antigen combining sites. In contrast, Fab, Fv, ScFv and dAb fragments are monovalent, having only one antigen combining sites.
  • antibodies for use in the present invention are antibodies preparation which binds specifically to a defined SEQ ID NO an/or to isolated type A or type B hSODl aggregates in preference to (such as, with at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 40 or 50 times greater reactivity, relative to background binding in a dot blot assay as described in the present examples, than to) a peptide which consists of any of the sequences DLGKGGNEESTKTGNAGS (optionally, with N-terminal truncation of D, DL, DLG or DLGK, an/or C-terminal truncation of S, GS, AGS or NAGS); NPLSRKHGGPKDEE (optionally with N-terminal truncation of N, NP or NPL and/or C-terminal truncation of E, EE or DE); IKGLTEGLHGF; HCIIGRTLVVH (optionally with N-terminal truncation of H or HC and
  • compositions may be given by any suitable means of administration, typically parenterally.
  • the composition is preferably given by injection, but can in practice be administered by any suitable means that allows the peptide to provoke an immune response in the subject to which it is administered.
  • the pharmaceutical compositions of the invention can be administered to a subject parenterally, for example, intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intramuscularly or subcutaneously, or they may be administered by infusion techniques. They may be best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.
  • the preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • the daily dosage level of the peptides or antibodies will usually be from 1 to 1000 mg per adult (i.e. from about 0.015 to 15 mg/kg), administered in single or divided doses.
  • the physician in any event will determine the actual dosage which will be most suitable for any individual patient and it will vary with the age, weight and response of the particular patient.
  • the above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited and such are within the scope of this invention.
  • a pharmaceutical compositions of the invention is administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal.
  • a subject for the purposes of the present invention can be any human or animal, such as a mouse or a dog.
  • the subject may have been diagnosed with ALS, or at risk of ALS (such as having a family history of ALS).
  • the subject may be homozygous or hemizygous for a mutant SODl gene, such as a mutant hSODl, or be homozygous for wildtype SODl .
  • the subject is an animal, it may be a transgenic animal that carries a wild-type and/or mutant hSODl gene and thus, for example, an animal that is the subject of the present invention may be a animal model of ALS.
  • the present invention also includes a method of assessing the therapeutic potential of a test treatment for ALS, the method comprising using the foregoing methods of the present invention to assess the ability of test therapy to modulate the type, amount, rate of formation or destruction, and/or tissue distribution of hSODl aggregate.
  • a method may comprise assessing the type, amount, rate of formation or destruction, and/or tissue distribution of hSODl aggregate in cohorts of test subjects, wherein at least one cohort receives the test therapy, and another cohort receives a placebo or alternative therapy.
  • Yet another aspect of the present invention provides antibodies or antibody fragments directed to epitopes specifically exposed on pathological aggregates of hSODl, such as on a type A aggregate or a type B aggregate as defined herein, for passive immunization for the treatment and prophylaxis of ALS, preferably the antibodies and antibody fragments are selected from antibodies and antibody fragments specific for an aggregate of hSODl according to the invention, more preferably the antibodies and antibody fragments are selected from
  • the antibodies and/or antibody fragments may be polyclonal or monoclonal.
  • the antibodies and/or antibody fragments are preferably monoclonal antibodies.
  • the antibodies and/or antibody fragments may be human or non-human.
  • the antibodies and/or antibody fragments are intended for administration to humans, then they will be human (e.g. human polyclonal or human monoclonal) or will be humanised.
  • Antibody fragments will retain the binding specificity of the parent antibody.
  • the antibody fragments can be generated by standard molecular biology techniques or by cleavage of purified antibodies using enzymes (e.g. pepsin or papain) that generates these fragments.
  • Such antibody fragments according to the invention are exemplified, but not limited to, single chain antibodies, Fv, scFv, Fab, F(ab')2, Fab', Fd, dAb, CDR, or scFv-Fc fragments or nanobodies, and diabodies, or any fragment that may have been stabilized by e.g. PEGylation.
  • antibody fragments rather than whole antibodies
  • the smaller size of the fragments may lead to improved pharmacological properties, such as better penetration of solid tissue.
  • Effector functions of whole antibodies, such as complement binding, are removed.
  • Fab, Fv, ScFv and dAb antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the said fragments.
  • antibodies and/or antibody fragments for use in the present invention binds specifically to a defined SEQ ID NO an/or to isolated type A or type B hSODl aggregates in preference to (such as, with at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 40 or 50 times greater reactivity, relative to background binding in a dot blot assay as described in the present examples, than to) a peptide which consists of any of the sequences DLGKGGNEESTKTGNAGS (optionally, with N-terminal truncation of D, DL, DLG or DLGK, an/or C-terminal truncation of S, GS, AGS or NAGS); NPLSRKHGGPKDEE (optionally with N-terminal truncation of N, NP or NPL and/or C-terminal truncation of E, EE or DE); IKGLTEGLHGF; HCIIGRTLVVH (optionally with N-terminal truncation of H or
  • Another aspect of the present invention provides methods for the identification and characterization of substances or compounds that bind to, interfere with the formation of, and/or promotes the degradation of pathological aggregates of hSODl according to the present invention.
  • Substance or compounds that bind to, interfere with the formation of, and/or promote the degradation of aggregates of hSODl are potentially useful for the treatment and/or prevention of ALS.
  • the present invention also provides a method for the identification of a substance or compound that binds to, interfere with the formation of, and/or promotes the degradation of pathological aggregates of hSODl, said method comprising one or more of the steps of:
  • test substance or compound b) contacting said test substance or compound with an aggregate of hSODl as defined above, c) determining if the test substance or compound binds to the aggregate of hSODl, inhibits the formation of aggregates of hSODl and/or promotes the degradation of aggregates of hSODl,
  • the method may further comprise the step of determining if the test substance or compound alters a neurotoxic and/or neurodegenerating effect of the aggregates of hSODl.
  • the method may further comprising the step of formulating the substance or compound in a pharmaceutically acceptable composition, optionally wherein the composition further comprises one or more additional components, such as components selected from the group consisting of pharmaceutically acceptable carriers, diluents, delivery agents or the like.
  • the present invention also provides a method for the treatment and/or prophylaxis of ALS, including the delay of onset, reduction in the speed of progression and/or reduction in the symptoms of ALS, in a subject, the method comprising the administration of a pharmaceutically acceptable composition to the subject, wherein the pharmaceutical composition comprises the substance or compound that has been as potentially suitable for the treatment of ALS.
  • Candidate compounds which may be tested in the methods according to the invention include simple organic molecules, commonly known as "small molecules", for example those having a molecular weight of less than 2000 Daltons. The methods may also be used to screen compound libraries such as peptide libraries, including synthetic peptide libraries and peptide phage libraries. Once an inhibitor or stimulator of aggregate activity is identified then medicinal chemistry techniques can be applied to further refine its properties, for example to enhance efficacy and/or reduce side effects.
  • Candidate compounds which may be tested in the methods according to the invention further include antibodies, antibody fragments, such as a Fab fragment, a (Fab)2 fragment, a single chain Fab fragment, a single chain Fv fragment, or a single chain Fv dimer.
  • the compound to be tested can be selected from engineered non-natural receptor derivatives, such as derivatives of the anticalin, Affibody, FNfnlO, neocarzinostatin, ankyrin repeat protein, PDH finger, CDR3 grafted green fluorescent protein, and E. coli periplasmic binding protein scaffolds.
  • engineered non-natural receptor derivatives such as derivatives of the anticalin, Affibody, FNfnlO, neocarzinostatin, ankyrin repeat protein, PDH finger, CDR3 grafted green fluorescent protein, and E. coli periplasmic binding protein scaffolds.
  • the antibodies are selected from:
  • antibodies to be used are selected from
  • the antibodies to be used are antibodies directed to the amino acid sequences SEQ ID NO: 4.
  • Another aspect of the present invention provides methods for identifying and/or quantifying the amount of hSODl aggregates and/or hSODl fibrils present in a biological sample obtained from a subject.
  • the biological sample can be blood, plasma, serum, cerebrospinal fluid, or isolated cells, preferably leukocytes or lymphocytes. Such methods are potentially useful for diagnosis and monitoring of ALS:
  • the method may comprise the use of antibodies directed to peptide sequences derived from the amino acid sequence of hSODl .
  • the present invention also provides a method for identifying and/or quantifying the amount of hSODl aggregates (including hSODl fibrils) present in a biological sample obtained from a subject, said method comprising the use of a panel comprising, consisting essentially of, or consisting of, one or more (such as 2, 3, 4, 5, 6, 7, 8 or more) antibody preparations selected from
  • an antibody preparation which has binding specificity to a peptide consisting of the amino acid sequence SEQ ID NO:8, or an equivalent thereof.
  • an antibody preparation which has binding specificity to a peptide consisting of the amino acid sequence SEQ ID NO: 14, or an equivalent thereof, and/or o) an antibody preparation which has binding specificity to a peptide consisting of the amino acid sequence SEQ ID NO: 15, or an equivalent thereof.
  • antibodies to be used are selected from
  • antibodies to be used are antibodies directed to the amino acid sequences SEQ ID NO: 4 or an equivalent thereof.
  • the method may comprise the use of one or more antibodies as defined by claims 26 or 27, below.
  • the skilled person will appreciate that this alternative may also be used in combination with the method described in the preceding paragraphs.
  • the antibodies can be labeled with a fluorescent label.
  • two different antibodies directed to the same amino acid sequence are used.
  • the two antibodies are each labeled with a different fluorescent label, the fluorescent labels being able to undergo fluorescent resonance energy transfer (FRET).
  • FRET fluorescent resonance energy transfer
  • fluorescent resonance energy transfer FRET
  • the method for identifying and/or quantifying the amount of hSODl aggregates (including hSODl fibrils) present in a biological sample obtained from a subject may comprising the use of a first and a second antibody preparation,
  • first antibody preparation has binding specificity to an amino acid sequence which is the same as, or equivalent to, an amino acid sequence to which the second antibody preparation has binding specificity, and wherein
  • the antibodies in the first preparation are labeled with a first fluorescent label
  • the antibodies in the second preparation are labeled with a second fluorescent label
  • the first and second fluorescent labels are different and, together, are able to undergo fluorescent resonance energy transfer (FRET).
  • FRET fluorescent resonance energy transfer
  • the first antibody preparation has binding specificity to an amino acid sequence which is equivalent to an amino acid sequence to which the second antibody preparation has binding specificity, if the first antibody preparation can competitively inhibit the binding of the second antibody preparation to a peptide consisting of the amino acid sequence to which it binds.
  • FRET is not the only way to detect oligomers and aggregates using antibodies reacting with the same epitope or equivalent epitopes.
  • the method may, for example, employ the same antibody labeled with two different markers. The point would be that each monomelic disordered SOD1 molecule would react with only one antibody.
  • the figure shows the two non-covalently associated hSODl subunits in different colors.
  • the segments corresponding to the peptides used for immunization are black. Note that several of the peptide segments are well exposed on the surface. Still none of the anti- peptide antibodies reacts with native hSODl, and all with misfolded hSODl (cf. Figure 2A).
  • a 200 x g supernatant of a spinal cord homogenate from a terminally ill G93 A mouse was captured in filter dots in 10 wells, as described under Methods. Human SOD1 captured in the dots was dissolved by boiling in sample buffer. The 200 x g supernatant was also added to 6 replicate tubes and centrifuged at 25,000 x g for 30 min. The supernatants and washed pellets were collected. In the immunoblot, the volumes applied resulted in the pellets being 4 times more diluted and the 25,000 x g superaatants 10 times more diluted than the filter dot samples. A replicate immunoblot was run with the murine SOD 1 -specific antibody to assay for the presence of aggregated murine SOD1 in the homogenate. Figure 4. Development of the assay: analysis of hSODl captured in cellulose acetate filters.
  • Non-reduced immunoblots of hSODl captured on filters from homogenates of spinal cord and brain from a terminally ill G93A mouse.
  • the homogenates were sonicated in buffer containing either 1% NP-40 or 0.1% SDS.
  • the filter dots were punched out and extracted in sample buffer.
  • SDS-PAGE one gel was subjected to in-gel reduction, to ensure that there was equal representation of reduced and oxidized hSODl and hSODl disulfide-coupled to other proteins 21 .
  • the major proportion of trapped hSODl was subunits lacking the disulfide bond and a species with intermediate mobility perhaps representing subunits with non-native disulfide bond.
  • Type A and type B aggregates in non-symptomatic and terminal D90A mice.
  • Spinal cord homogenates from non-symptomatic and terminal D90A mice were analyzed for type
  • a (O, ⁇ ) and type B ( ⁇ , A) aggregates using the 57-72 and 1 1 1-127 antibodies, respectively.
  • Open symbols represent non-symptomatic and full symbols terminal mice, x, +, the results with the 57 - 72 and 111 - 127 antibodies, respectively, for these mice were below the mean + 2 SD values for non-transgenic controls (0.0026 and 0.0048, respectively, cf. Table 2).
  • Figure 7 Relation between lifespan and disease duration in transgenic mice expressing different mutant hSODls.
  • mice were examined twice a weak. Criteria for onset were reduced ability to extend hind legs or tremor when held by the tails, or gait disturbances, or foreleg weakness (in particular G85R mice). The latter was evaluated by letting the mouse grip the wire grid of the cage, while being slightly pulled from behind. Gait was evaluated by watching the mouse walk on a smooth surface. In the duration study, the mice were deemed terminally ill and sacrificed when they no longer could reach the food in the cages. The investigation of the mouse strains was conducted during 2004, 2005 and 2006 when the lifespans, in particular for G93A mice, were shorter than is currently observed.
  • Antibodies raised against short peptides in hSODl do not react with native hSODl, only with disordered forms of the protein: background to the principles behind the epitope-mapping assay.
  • Peptides corresponding to amino acids 3-20, 24-39, 43-57, 57-72, 80-96, 100-1 15, and 131-153 in hSODl were coupled to keyhole limpet hemocyanin and used to raise antibodies in rabbits.
  • the antibodies were purified with Protein A-Sepharose (GE Healthcare, Uppsala, Sweden) followed by Sulfolink gel with the respective peptides coupled (Pierce, Rockford, IL).
  • the antibodies were then coupled to Protein A-Sepharose and the resulting beads incubated in pH 7 PBS containing 5 ⁇ g/ml hSODl that was either native (Figure 9A) or had been denatured by exposure to guanidinium chloride and a chelator followed by dialysis ( Figure 9B). Following washes, bound hSODl was analyzed by western immunoblots. The native hSODl solutions were incubated twice with the antibody beads, with the intention to capture any traces of denatured hSODl in the first, to make the second more representative for the reaction of the peptide antibodies with native hSODl .
  • Short peptides such as those used here, have generally no ordered structure, and if so not necessarily the same structure as in native hSODl .
  • the resulting antibodies will not bind to any specific structure in the rigid native hSODl molecule, but only to flexible segments that can adapt to the antigen-binding sites.
  • protein aggregates are typically composed of a fibril-like spine of stacked ⁇ sheet structures, which is sometimes decorated with domain-swapped material based on inter-molecular native contacts (a simplified cartoon in Figure 9C). The sequence regions outside this ordered aggregate core, which will have lost their native contacts, form disordered fringes.
  • mice hemizygous G93AGur (Gl), homozygous line 134 D90A mice (Jonsson et al., 2006b), and hemizygous G85R (Bruijin et al., 1997) and homozygous wild-type hSODl (wt-hSODl) transgenic mice (Graffmo et al., 2012).
  • mice The lines that carried mutant hSODls were backcrossed 10-30 generations in C57B1/6 mice, whereas the homozygous wt-hSODl mice were in CBA background.
  • C57B1/6 and SOD1 knockout mice (Reaume et al., 1996) were used. The mice were considered terminally ill if they could not right themselves within 5 seconds after being put on their side.
  • the use and maintenance of the mice and the experimental protocol described in this article were approved by the Ethics Committee for Animal Research at Umea University.
  • Antibodies to peptides corresponding to amino acids 4-20, 24-39, 43-57, 57-72, 80-96, 100-115, and 131-153 in hSODl were coupled to keyhole limpet hemocyanin and raised in rabbits as previously described (Jonsson et al., 2004).
  • the antibodies were purified with Protein A-Sepharose (GE Healthcare, Uppsala, Sweden) followed by Sulfolink gel with the respective peptides coupled (Pierce, Rockford, IL).
  • Antibodies to amino acids 1 1 1-132 and 123-132 in G127X mutant hSODl were similarly prepared.
  • Amino acids 128-132 represent a neosequence in the mutant, which is why the 111-132 antibody only can react with epitopes among amino acids 1 1 1-127 in full-length hSODl. It is therefore designated so in assays involving full-length hSODls.
  • the 123-132 antibody shows no reactivity with full-length hSODl (Jonsson et al., 2004).
  • For analysis of murine SOD1 a specific antibody raised against a peptide corresponding to amino acids 24-36 in the sequence was used.
  • mice were killed by intraperitoneal injection of pentobarbital.
  • the dissected tissues were homogenized with an Ultraturrax apparatus (IKA, Staufen, Germany) for 30 s and sonication for 1 min in 25 volumes of ice-cold APBS (137 mM NaCl, 2.7 mM KC1, 4.3 mM Na2HP04, 1.4 mM KH2P04, pH 7.0) supplemented with 1.8 mM EDTA, 1 mM dithithreitol (DTT), and the antiproteolytic cocktail Complete® without EDTA (Roche Diagnostics, Basel, Switzerland).
  • Ultraturrax apparatus IKA, Staufen, Germany
  • tissue homogenates were added to 20 volumes of the APBS with DTT and EDTA containing 1% of the detergent NP40, sonicated for 30 s and then centrifuged at 200 x g for 10 min.
  • the supernatants were stepwise diluted 1+1 in the APBS, and 100 ⁇ captured on 0.22 ⁇ cellulose acetate filters in a 96-well dot-blot apparatus (Whatman GmbH, Dassel, Germany). The wells were then washed with 3 x 300 ⁇ of the APBS without NP40.
  • the filters were cut and incubated overnight at 4°C with the anti-hSODl peptide antibodies at 0.01 ⁇ g/ml (the 4-20 and 100-1 15 antibodies at 0.02 ⁇ g/ml) dissolved in the blocking buffer. After washing, the blots were thereafter developed with HRP -substituted goat anti -rabbit Ig antibodies (Dako 1/42000) and ECL Advance (GE Healthcare), recorded in a Chemidoc apparatus and evaluated with Quantity One software (BioRad).
  • one homogenate of a spinal cord from a terminal G93A mouse was designated as a standard (set to 1) and kept in multiple aliquots at -80 °C. Dilutions series (1 + 1) of this standard were run in 1 or 2 lanes on all filters and were stained with the 57-72 antibody. All blots of all homogenates with all antibodies were quantified against this standard. To facilitate comparison of staining patterns, in some cases the staining intensities of the 8 antibodies of individual homogenates were normalized against the staining of the homogenate with the 57 - 72 antibody (taken as 100%). Fragilities of types A and B aggregates.
  • Human SOD1 was induced to aggregate in vitro under 8 different conditions where the first 6 aggregations were carried out with 100 ⁇ (subunits) wild-type hSODl, were monitored by eye and generally required 3 - 5 days under shaking to develop.
  • Human SOD1 was rendered in apo form by incubation in 3 M guanidinium chloride and EDTA. The apo hSODl was then dialyzed and incubated at 37°C in 100 mM Na phosphate, pH 7.0, and 20 mM dithiothreitol (DTT) to reduce the C57-C146 disulfide bond.
  • DTT dithiothreitol
  • the aggregates were collected by centrifugation at 20,000 x g for 15 min, washed 5- times with PBS and kept as pellets at -80 °C. The pellets were then suspended by sonication for 2 x 15 s in 1+25 homogenates of brain from SOD1 knockout mice, made in a similar way to the tissue homogenates in the epitope-mapping assay. The resulting aggregate suspensions were then analyzed by the epitope-mapping assay similar to the tissue extracts. The suspensions were diluted to varying extents with PBS, 1.8 mM EDTA, 1 mM DTT and Complete to produce intensities in the assay that were similar to those from spinal cord homogenates. Electron microscopy
  • Carbon-coated 200 mesh copper grids were applied on top of 20 ⁇ droplets of aggregate preparations 7 and 8 and blotted for 5 min.
  • the grid was stained with 1% (w/v) uranyl acetate and allowed to dry in air. Images of 18,000-fold magnification were recorded in a Tecnai G2 Spirit BioTWIN microscope with a tungsten filament operating at 80 kV.
  • the western immunoblots were generally carried out as previously described (Jonsson et al., 2004) using an antibody raised in chicken against a peptide corresponding to amino acids 57-72 of the hSODl sequence.
  • the chemiluminescence of the blots was recorded in a ChemiDoc apparatus and analyzed with Quantity One software (BioRad).
  • IAM iodoacetamide
  • 60 mM iodoacetamide (IAM) (and no DTT) was added to the homogenization buffer to block free sulfhydryl groups.
  • the SDS-PAGE separations were then carried out without reductant but with 100 mM IAM in the sample buffer (non-reduced immunoblot).
  • Reduced hSODl in such blots has an immunoreactivity that is 20-fold higher than that of disulfide-oxidized subunits (Jonsson et al., 2006a; Zetterstrom et al. 2007b). This is advantageous for demonstration of small proportions of reduced subunits.
  • the SDS-PAGE gels were in some cases soaked in 2% mercaptoethanol for 10 min prior to electroblotting to the membrane (in-gel reduction).
  • the assay employed 8 polyclonal rabbit anti-hSODl peptide antibody preparations, raised to a panel of 8 peptides that, between them, cover over 90% of the hSODl sequence.
  • the peptide antibodies are all specific for misfolded/unfolded/non-native/disordered (in the following text the term misfolded will be used) hSODl species; there is no reaction with natively folded hSODl (Forsberg et al., PLos ONE 2010). This lack of reactivity is not simply explained by exposure or concealment of the peptides in the native hSODl structure, since several of the sequences are well exposed on the surface.
  • Short peptides such as those used here as antigens have generally no ordered structure, and if so, not necessarily the same as in native hSODl .
  • the resulting antibodies will not bind to any specific structure in the rigid native hSODl molecule, but only to flexible segments that can adapt to the antigen-binding sites.
  • the hSODl aggregates were captured on 0.2 ⁇ cellulose acetate filters in a 96-well dot-blot apparatus. The filters were then stained similar to western immunoblots using the 8 anti-hSODl peptide antibodies (Figure 2B). Antibody concentrations estimated to bind equally well to soluble misfolded hSODl species present in spinal cord extracts were used. Around 3% of the soluble hSODl in G93A spinal cords is misfolded (Zetterstrom et al., 2007). When equal amounts of peptide antibodies immobilized on Sepharose were incubated with a G93A spinal cord extract, they were found to capture similar amounts of such hSODl from an extract (Figure 2A). The 4-20 and 100-1 15 antibodies bound about half as much as the others and they were used in double concentration in the dot-blot assay. There is a close to linear relationship between staining intensity and antibody concentration in the dot-blot assay (not shown).
  • protein aggregates are typically composed of a fibril-like spine of stacked ⁇ sheet structures, which is sometimes decorated with domain-swapped material based on inter-molecular native contacts (Eisenberg and Jucker, 2012).
  • the sequence regions outside this ordered aggregate core form disordered fringes.
  • this aggregate core has no reactivity to the antibodies, because of the ordered structure and perhaps also because of steric hindrance. In contrast the disordered fringes should be reactive.
  • Two structural varieties of hSODl aggregates are formed in transgenic ALS model mice
  • Figure 2B shows epitope-mapping assay patterns for the spinal cord and brain of a terminally ill G93A mouse.
  • the patterns were distinctly different from the patterns of soluble misfolded G93A hSODl from the spinal cord extract of 100-day-old G93A mouse, as captured by the same antibodies (cf. Figure 2A).
  • the results show that aggregates formed in vivo are not amorphous, but have well-defined and clearly distinguishable structures.
  • the upper data set represents absolute results compared with the results for the G93A standard with the 57-72 antibody (set to 1).
  • the lower set shows means for the groups with the results for the individual mice with the different antibodies calculated relative to the 57-72 antibody (set to 100%).
  • the lowest row presents blank reactions given by 4 C57B1/6 and 4 SOD1 knockout mice (only absolute results). There were no systematic differences between the 2 groups and these non-transgenic controls are therefore presented together. All data are given as mean ⁇ SD.
  • type A and B patterns staining intensities with the 57-72 and 1 11-127 antibodies, as well as ratios between the staining intensities, respectively, cf. Figure 5A
  • Type A and type B aggregates appear to coexist in D90A mice, and the longer the lifespan the more the type A aggregates predominate.
  • non-symptomatic mice within the same age-span were examined ( Figure 5B, Figure 5C, open symbols). These mice contained mainly type A aggregates, but apparently less than mice that had become terminally ill around the same age. Because the type B aggregates also appeared to show some reactivity with the 57-72 antibody ( Figure 5A), the type A aggregation in the non- symptomatic mice appeared to fit the trajectory of type A aggregation seen in the terminally ill mice.
  • type B aggregation can occur in D90A mice, and when it is initiated, seemingly for stochastic reasons, there is rapid spread of the aggregation and neurodegeneration. If type B aggregation is not initiated, the mice have a long lifespan and eventually die bearing only type A aggregates.
  • the type A aggregate level in the oldest D90A mouse was within the range seen in terminally ill G93A mice ( Figure 2D, Figure 5B). The relationships between epitope-mapping patterns and lifespans of individual G93A, G85R, and wt-hSODl mice were also explored, but no age-related differences were found.
  • mice with short lifespans are primarily killed by rapidly evolving type B aggregation, their disease durations would be expected to be short. Conversely, mice with long lifespans should first be affected by slowly evolving type A aggregation, and then after various times also type B aggregation, resulting in longer durations. To explore this notion the lifespans of mice of the different transgenic models were plotted against the disease durations (Figure 7). A distinct positive correlation was found in the D90A mice, but less so in the other examined models.
  • hSODl primarily aggregates in the spinal cord, when the level of the protein is similar to that in the brain (Graffmo et al., 2013; Jonsson et al., 2006).
  • One explanation could be that unfolded hSODl monomers that lack the stabilizing C57-C146 disulfide bond are present at higher levels in the spinal cord than in brain and other tissues in the transgenic mice (Zetterstrom et al., 2007; Zetterstrom et al., 2013), and that such species are the primary precursors for aggregation.
  • the aggregates in transgenic mice are mostly composed of disulfide-reduced monomers (Karch et al., 2009; Bergemalm et al., 2010), and loss of the disulfide bond promotes in vitro aggregation of hSODl (Chattopadhyay et al., 2008; Furukawa et al., 2008). Moreover, agitation-induced fibrillation of recombinant hSODl in vitro has been found to progress by fragmentation-limited kinetics from globally unfolded monomers (Lang et al., 2012).
  • D90A mice show large variations in lifespan, and type A and type B aggregates coexisted in most of them (Figure 5B, Figure 5D). The longer the lifespan, the more type A aggregates predominate. Mice within the age range of terminal disease, but which still lacked symptoms, contained mainly type A aggregates. The findings suggest that type A aggregation is a default for the 4 hSODl variants examined in this study. The time-course of type A aggregation did not appear to differ much between individual D90A mice, but became detectable much later than in G93A mice ( Figure 2D, Figure 5D).
  • D90A mice naturally draw attention to further analogies with the prion protein diseases, where the disease manifestations are variable and linked to different strains of prions (Colby & Prusiner 201 1).
  • the D90A mutation causes ALS with a phenotype that deviates from ALS in general: there are commonly both sensory symptoms and bladder control disturbances (Andersen et al., 1996).
  • D90A mice also show bladder control problems, which seldom appear in other hSODl transgenic model mice (Jonsson et al., 2006). Perhaps these phenotype deviations are related to the occurrence of type B aggregates.
  • aggregates can be demonstrated in the G93A model long before any appearance of injury (Figure 2D) (Kanning et al., 2010). Yet, the identities of the major harmful molecular species formed in the aggregations are still open to conjecture. There is likely a continuum of aggregate species from small fibril fragments to large aggregates. The latter are gradually sequestered into inclusion bodies, and in that form most likely rendered innocuous (Chen et al., 201 1). Oligomers, with a structure different from A or B fibrils containing a few hSODl monomers, might conceivably correlate with the content of aggregates. However, no such hSODl species have with certainty been identified so far.
  • fibril fragments which would more directly correlate with the aggregate load. They are known to be more cytotoxic than larger aggregates (Xue et al., 2010), and the smaller size would facilitate spread both within cells including neurites, and in the interstitial space of the CNS.
  • the mechanisms by which fibrils are fragmented in the cells are not well understood, but could involve thermal and mechanical impacts at a molecular length scale, the action of various classes of chaperones (sun et al., 2008; Winkler et al., 2012), and the impact of pulse waves.
  • Disulfide cross-linked protein represents a significant fraction of ALS- associated Cu, Zn-superoxide dismutase aggregates in spinal cords of model mice. Proc. Natl. Acad. Sci. USA 103, 7148-7153 (2006).
  • ALSOD the Amyotrophic Lateral Sclerosis Online Database. AmyotrophXateral Scler. 9, 249-250 (2008).

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Abstract

La présente invention porte sur un procédé de typage d'agrégat (y compris une fibrille) d'un polypeptide dans un échantillon d'essai, tel qu'une superoxyde dismutase humaine (hSODI), le polypeptide étant apte à former au moins deux types structurellement distincts d'agrégats, ledit procédé comprenant (i) déterminer la réactivité de l'agrégat dans l'échantillon d'essai avec une ou plusieurs préparations d'anticorps différentes dans un panel de préparations d'anticorps, chaque préparation d'anticorps dans le panel ayant une spécificité de liaison à une séquence peptidique dérivée de la séquence d'acides aminés du polypeptide, de telle sorte que différentes préparations d'anticorps dans le panel ont une spécificité de liaison à différents peptides, et au moins une des préparations d'anticorps du panel, pour laquelle une réactivité vis-à-vis de l'agrégat est déterminée à l'étape (i), affichant une réactivité différentielle vis-à-vis d'au moins deux types structurellement distincts de l'agrégat ; et (ii) attribuer un type à l'agrégat dans l'échantillon d'essai, sur la base du ou des niveaux déterminés de réactivité avec la ou les préparations d'anticorps dans le panel telle que déterminée dans l'étape (i).
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