WO2014191493A1 - Single domain antibodies against sod1 and their use in medicine - Google Patents
Single domain antibodies against sod1 and their use in medicine Download PDFInfo
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- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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- 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®
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- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- C07K2317/77—Internalization into the cell
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- C07K2319/42—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a HA(hemagglutinin)-tag
Definitions
- the present application relates to the field of single domain antibodies (also called nanobodies), more particularly single domain antibodies against SODl protein isoforms. It also relates to the use of these nanobodies in medicine. Accordingly, methods to treat a disease using these nanobodies are provided herein.
- the single domain antibodies are particularly envisaged for treatment of ALS.
- ALS is an adult-onset neurodegenerative disease that affects the upper and lower motoneurons (MNs) in the cerebral cortex, brain stem and spinal cord. It results in progressive MN loss, muscle paralysis and atrophy leading to death within few years. Most ALS patients are thought to have a sporadic form of the disease, but in about 10% ALS is inherited or familial (FALS). In about 20% of patients with FALS the disease is caused by mutations in the gene encoding Cu/Zn-superoxide dismutase (SODl) (Rosen et al., 1993). Other causes are mutations in TARP, FUS/TLS, VCP and C90RF72.
- SODl Cu/Zn-superoxide dismutase
- wild type SODl has been suggested to play a role in the pathogenesis of sporadic ALS 8 . It has been proposed that in sporadic ALS wild-type SODl undergoes secondary modifications (e.g. through oxidation or demetalation), misfolds and is toxic to motor neurons in a very similar manner to what is seen with mutant SODl 9 . Using a conformation-specific antibody that recognizes the misfolded species selectively, pathogenic SODl has been found in motor neurons of at least a part of sporadic ALS patients 1 . Most interestingly, the toxic effect of astrocytes from sporadic ALS patients (thus not harboring SOD1 mutations) on motor neurons is dependent on the presence of SOD1 2 .
- reducing the levels of the pathogenic SOD1 is an interesting strategy to treat patients with mutant SODl-associated familial ALS, as well as patients with sporadic ALS.
- anti-sense oligonucleotide, siRNA-based and immunological approaches have been developed. For the latter, both active and passive immunisation is under investigation.
- approaches using siRNA and conventional antibodies come with significant problems (e.g. half-life, distribution in the CNS, uptake by neurons...), and none of these approaches has thus far led to a therapy for ALS.
- the therapies should also be efficient in clearing mutant SOD1, and rescue the toxic gain of functions associated with these mutant SOD1 isoforms, or indeed with wild-type SOD1 isoforms that also show toxic gain of function.
- ALS amyotrophic lateral sclerosis
- VHH domain can be cloned and the product, called nanobody or single domain antibody, shows high target specificity and affinity, and low inherent toxicity 15"17 .
- nanobodies are highly soluble, extremely stable, have refolding capacity, can be administered by means other than injection, and are easy to manufacture 18 .
- Nanobodies have a potential use as therapeutics in many fields 3 ' 4 . Nanobodies have, like conventional forms of antibodies, applications as diagnostic markers 5,6 . Furthermore, nanobodies can be used as structural probes of protein misfolding and fibril formation 7 . Nanobodies that are capable of influencing the aggregation pathway of a disease-related protein, either by inhibiting the formation of particular intermediates or by neutralizing their neurotoxic effects, could also serve as potential therapeutics. Nanobodies have additional properties that make them particularly appealing as therapeutic and diagnostic agents, including low immunogenicity due to high sequence similarities with human VH family III 8 . Particularly important for their potential use in neurodegenerative disorders, they have been shown to be able in some cases at least to cross the blood-brain barrier efficiently 9 .
- Nanobodies against a-synuclein 10 n , ⁇ -amyloid 12 13 and hungtintin 14 have been already raised, and it has been reported to inhibit a-synuclein, ⁇ -amyloid and huntingtin aggregation in in vitro models for Parkinson's, Alzheimer's and Huntington's disease.
- ALS is an adult-onset fatal neurodegenerative disease that is familial in about 10 % of patients. In about 20% of patients with familial ALS (FALS) the disease is caused by gain-of-toxic-function mutations in the gene encoding Cu/Zn-superoxide dismutase (SODl). To reduce the expression of mutant SODl in the motor neurons, we have raised anti-SODl heavy chain antibodies in dromedaries and alpacas, and cloned their N-terminal antigen binding VHH region, coding for anti-SODl nanobodies.
- SODl Cu/Zn-superoxide dismutase
- SODl nanobodies have high affinity for SODl in vitro and dose-dependently block its fibril formation, their expression reduces mutant SODl levels and rescues mutant SODl-induced axonopathy in zebrafish and inhibits SODl aggregates formation in in vitro models for ALS.
- SODl nanobody also rescues mutant SODl-induced axonopathy in zebrafish when added in the tank water.
- SODl is human SODl.
- the single domain antibodies bind to mutant SODl, i.e. they recognize an epitope that is present in a mutated form of the SODl protein.
- the mutant SODl is characterized by a mutation of amino acids at position 4, 93 and/or 113, particularly by an A4V, G93A, and/or G113W mutation.
- the single domain antibodies bind both wildtype and mutant SODl (i.e.
- the single domain antibody is an inhibitory single domain antibody against SODl.
- the nanobody interferes with the superoxide dismutase function of SODl.
- the inhibitory single domain antibody inhibits the toxic gain of function of mutant SODl protein.
- the single domain antibody interferes with (inhibits, prevents, reverses or slows) the formation of SODl aggregates; and/or the single domain antibody can counter the phenotypic changes caused by expression of the mutant SODl protein (e.g. axonopathy).
- the single domain antibody has a sequence selected from the group of SEQ ID NOs: 1-14. According to alternative embodiments, the single domain antibody shares the sequence of the complementary determining regions of these sequences.
- the single domain antibody may be provided as such or may be fused to further moieties. According to particular embodiments, the single domain antibody is fused to a tag. According to further particular embodiments, the tag to which the single domain antibody is fused is a His-tag, HA-tag, and/or Myc- tag.
- SODl normally is a soluble cytoplasmic protein, although a detrimental or neurotoxic role has been ascribed both to extracellular secreted SODl and cytoplasmic mutant SODl.
- the nanobody is able to enter cells, particularly neuronal cells. This may be an inherent property of the nanobody, or may be achieved by further fusion to moieties or tags that allow cellular uptake.
- the single domain antibodies are not provided as such, but are provided as nucleic acid molecules, i.e. nucleic acid molecules encoding single domain antibodies against SODl as herein described. Also provided are vectors comprising such nucleic acids or nucleic acid molecules. According to yet further embodiments, host cells are provided comprising such nucleic acids or such vectors.
- the single domain antibodies are provided herein for use in medicine. That is to say, the single domain antibodies against SODl are provided for use as a medicament.
- the nucleic acid molecules encoding the single domain antibodies or for the vectors containing such nucleic acids.
- the single domain antibodies are provided for use in treatment of amyotrophic lateral sclerosis (ALS).
- ALS amyotrophic lateral sclerosis
- the single domain antibody may be provided as protein, or may be administered as a nucleic acid molecule encoding a single domain antibody against SODl, or as a vector comprising such nucleic acid molecule. If the single domain antibody is administered as protein, it is particularly envisaged that it is administered intracerebroventricularly, such as e.g. through injection or pump.
- the single domain antibody is provided as a nucleic acid or a vector, it is particularly envisaged that the single domain antibody is administered through gene therapy.
- the methods further comprise a step of monitoring the progression of ALS (or ALS symptoms) in the subject.
- kits comprising a single domain antibody against SODl and a pharmaceutically acceptable excipient.
- the single domain antibody may be provided as protein, as a nucleic acid encoding a single domain antibody against SODl, or as a vector comprising such nucleic acid.
- FIG. 1 SODl nanobodies have high affinity for SODl in vitro and block its fibril formation.
- FIG. 2 SODl nanobodies reduce SODl aggregation in HeLa cells.
- A Expression of HA-tag Nb and SOD1-CFP in HeLa cells.
- B Nb expressed in mammalian cells retain binding capacity for SODl as shown after co-purifying together SODl Nb and SODl.
- C Analysis of the nanobodies in transiently transfected HeLa cells by Blue Native Polyacrylamide gel electrophoresis (BN-PAGE).
- Figure 3 SODl aggregates formation and HA-tagged nanobody expression after transient transfection in NSC-34 cells.
- mutant SODl aggregates could be observed when cells were transfected with (B) eGFP-SODl G93A and (C) eGFP-SODl A4V but not with (A) eGFP-SODl WT .
- Control and SODl nanobodies could be detected by IHQ (D) and Western blot (E) after transfection through the HA-tag.
- Figure 4 SODl nanobody reduces the number of cells with mSODl aggregates after transfection.
- SODl nanobody After SODl nanobody and mSODl co-transfection in NSC-34 cells, SODl nanobody reduces the number of cells with S0D1 G93A (A) and SODl A4V (B) aggregates. This reduction is dose dependent (C). The appearance of the aggregates could vary between the different mutations without any apparent correlation, and the SODl nanobody did not affect either this appearance.
- FIG. 5 SODl nanobody expressed in NSC34 cells recognizes SODl protein and does not decrease hSODl levels. After SODl and nanobody co-transfection, SODl nanobody (A) but not control nanobody (B) can recognize and bind to endogenous, WT and mutant SODl (both G93A and A4V). However, SODl nanobody does not reduce hSODl levels after co-transfection (C).
- Injected SODl nanobody in zebrafish can also be detected through the HA-tag.
- B Expression levels of SODl decrease with the co-injection of SODl nanobody and SODl A4V , but not when co-injected with control nanobody.
- C Shortening of motor neuron axons is partially rescued with the co-injection of SODl A4V with SODl nanobody but not with the control nanobody.
- FIG. 7 The SODl nanobody (protein) colocalizes with mutant SODl aggregates.
- the SODl nanobody can be detected through its His-tag by Western blot and when it is added to the NSC-34 culture medium (B) can be found associated with the cells. Colocalization of the mutant SODl and the SODl nanobody (but not the control nanobody) was found by fluorescence (C) and confocal microscopy (D).
- the SODl nanobody can enter the cell through its His-tag and reduce the number of cells with mSODl aggregates. After cell fractionation, SODl nanobody can be found in the cytosol (A) and after immunoprecipitation it has found to bind SODl (B). 48 hours after administration in the cell medium, the SODl nanobody inhibits aggregate formation in a dose-dependent manner (C). The inhibition is blocked when the His-tag is removed from the nanobody (D).
- FIG 9 The SODl nanobody reduces SOD activity. When the SODl nanobody is added to the cell medium, the SOD activity is decreased. Read-out are OD values, inversely correlated with SOD activity (using OxiSelectTM Superoxide Dismutase Activity Assay of Cell Biolabs).
- Figure 10 Effect of the SODl nanobody treatment in injected zebrafish. The SODl nanobody can increase the axonal length and rescue the phenotype induced by mutant SODl in injected zebrafish when added to the tank water.
- FIG. 11 SODl nanobody can be detected in the mouse CNS after injection and binds SODl.
- the SODl nanobody Map of the mouse brain and scheme of the ICV injection. After one single injection, the SODl nanobody can be found in different areas of the brain (B) and spinal cord (C). (D) The SODl nanobody is clear 12 hours after injection and weakly detected after 24h. The SOD1 nanobody, through its His- tag, can be immunoprecipitated with SOD1 in injected hSODl G93A mice (E).
- Figure 12 The survival and disease onset are increased in treated hSODl G93A mice. Survival (A) and disease onset (D) of hSODl G93A mice treated with the SOD1 nanobody are delayed when the treatment is administrated starting at P60, at P90 (B) and P120 (C), 3 times/week. With daily injections starting at P90 the survival is also significantly increased.
- FIG. 13 The SOD1 nanobody rescues MN death. The number of small MN, but not of big MN was increased at P145 in mice treated with SOD1 nanobody from P60 (A). The number of MNJ did not vary (B).
- FIG. 14 The SOD1 nanobody recognizes neurotoxic forms of SOD1.
- the SOD1 nanobody In injected hSODl G93A mice, the SOD1 nanobody, through its His-tag, can be immunoprecipitated with the antibody anti P2X 4 , that selectively recognizes special conformers of mutant SOD1 that are neurotoxic.
- FIG. 15 Amino acid sequences of 14 different Nanobodies specific for human SOD1 (superoxide dismutase) [Cu-Zn]. All Nanobodies originate from VHH germline sequences. The complementarity determining regions (CDRs) are shown in bold. The top 7 Nanobodies were isolated from an immune library from a dromedary. The bottom 7 Nanobodies were isolated from an immune library from an alpaca. The above 14 different Nanobodies represent 4 different groups shown in 4 different colours. Nanobodies belonging to the same group (with the same colour) are very similar and their amino acid sequences suggest that they are from clonally-related B-cells resulting from somatic hypermutation or from the same B-cell but diversified due to PCR error during library construction. The gaps were introduced in order to align sequences.
- a “single-domain antibody” also referred to as “nanobody” herein, is an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. With a molecular weight of only 12-15 kDa, single-domain antibodies are much smaller than common antibodies (150-160 kDa) which are composed of two heavy protein chains and two light chains, and even smaller than Fab fragments ( ⁇ 50 kDa, one light chain and half a heavy chain) and single-chain variable fragments ( ⁇ 25 kDa, two variable domains, one from a light and one from a heavy chain).
- SODl refers to the gene superoxide dismutase 1 and its encoded protein (Gene ID: 6647 for the human gene).
- the enzyme SODl binds copper and zinc ions and is one of three superoxide dismutases responsible for destroying free superoxide radicals in the body. Mutations in this gene have been linked to familial amyotrophic lateral sclerosis, and several pieces of evidence also show that wild-type SODl, under conditions of cellular stress, is implicated in a significant fraction of sporadic ALS cases. Over 150 mutations of SODl have been linked to ALS (cf.
- mutant SODl in particularly refers to SODl containing one or more mutations that are linked to ALS.
- Selected examples include those listed in the OMIM database under entry 147450, i.e.
- inhibitory refers to the fact that the nanobody can inhibit the function and/or activity of its target protein.
- wild-type SODl this means that the superoxide dismutase activity is inhibited.
- mutant SODl that has a gain of function, typically it is meant that the (toxic) new function is inhibited, although this may also mean that the enzymatic activity is inhibited, or inhibited as well.
- inhibition may result in decrease of aggregation of mutant SODl.
- inhibition or decrease in toxic function may also be evaluated as an increase of another parameter, e.g.
- the inhibition may be evaluated by an increase in axonal length or an extended life span.
- “Inhibitory” can mean full inhibition (no enzymatic activity and/or toxic effect is observable) or may mean partial inhibition. For instance, inhibition can mean 10% inhibition, 20% inhibition, 25% inhibition, 30% inhibition, 40% inhibition or more. Particularly, inhibition will be at least 50%, e.g. 50% inhibition, 60% inhibition, 70% inhibition, 75% inhibition, 80% inhibition, 90% inhibition, 95% inhibition or more. % inhibition typically will be evaluated against a suitable control (e.g. treatment with an irrelevant nanobody, or a wild-type subject versus a diseased subject), as will be readily chosen by the skilled person.
- a suitable control e.g. treatment with an irrelevant nanobody, or a wild-type subject versus a diseased subject
- ALS or "amyotrophic lateral sclerosis” as used herein, sometimes also known as Lou Gehrig's disease, is a neurodegenerative disorder characterized by the death of motor neurons in the brain, brainstem, and spinal cord, resulting in fatal paralysis. It is a genetically heterogeneous disorder, described under entry 105400 in the OMIM database. A particular subset of ALS is ALS with SODl involvement, either through mutated SODl (OMIM entry 147450) or in cases where wild-type SODl is involved (typically in conditions of cellular stress).
- a "subject” as used herein refers to an animal that can develop ALS wherein SODl is involved (e.g. through misfolding). Typically, the animal is a mammal. Most particularly, the subject is a human. It is an object of the invention to provide single domain antibodies (or nanobodies) against SODl. According to particular embodiments, SODl is human SODl. According to alternative, but not exclusive, embodiments, the single domain antibodies bind to mutant SODl, i.e. they recognize an epitope that is present in a mutated form of the SODl protein.
- the mutant SODl is characterized by a mutation of amino acids at position 4, 93 and/or 113, particularly by an A4V, G93A, and/or G113W mutation.
- the single domain antibodies bind both wildtype and mutant SODl (i.e. they recognize an epitope present in the wildtype protein and at least one (but possibly more) mutated isoform).
- the single domain antibody is an inhibitory single domain antibody against SODl.
- the nanobody interferes with the superoxide dismutase function of SODl.
- the inhibitory single domain antibody inhibits the toxic gain of function of mutant SODl protein.
- the single domain antibody interferes with (inhibits, prevents, reverses or slows) the formation of SODl aggregates; and/or the single domain antibody can counter the phenotypic changes caused by expression of the mutant SODl protein (e.g. axonopathy).
- the single domain antibody has a sequence selected from the group of SEQ ID NOs: 1-14.
- the single domain antibody shares the sequence of the complementary determining regions (CDRs) of these sequences, fitted in a suitable framework region.
- CDRs complementary determining regions
- the 3 CDR sequences correspond to GGDTRPYITYWMG (SEQ ID NO: 15), TIYTGGSGTYYSDSVEG (SEQ ID NO: 16) and GNGALPPGRRLSPQNMDT (SEQ ID NO: 17) respectively.
- the CDR sequences correspond to ETLFSLYAMG (SEQ ID NO: 18) or ESLFSLYAMG (SEQ ID NO: 19), TISGGGEGTGNYADPVKG (SEQ ID NO: 20) and YGTNLAP (SEQ ID NO: 21) respectively.
- the CDR sequences correspond to GLPYRTVFMG (SEQ ID NO: 22) or GLPYRVVFMG (SEQ ID NO: 23), VINADGVSTYYADSVKG (SEQ ID NO: 24), and NHFFDYSRDPLATAEYNY (SEQ ID NO: 25) respectively.
- sequences of the CDRs are GYTFSSYCMG (SEQ ID NO: 26), TIISDGSTYYADSVKG (SEQ ID NO: 27) and RSGGVCSGRASRYNY (SEQ ID NO: 28) respectively.
- the single domain antibody may be provided as such or may be fused to further moieties. According to particular embodiments, the single domain antibody is fused to a tag. According to further particular embodiments, the tag to which the single domain antibody is fused is a His-tag, HA-tag, and/or Myc- tag.
- SODl normally is a soluble cytoplasmic protein, although a detrimental or neurotoxic role has been ascribed both to extracellular secreted SODl and cytoplasmic mutant SODl.
- the nanobody is able to enter cells, particularly neuronal cells. This may be an inherent property of the nanobody, or may be achieved by further fusion to moieties or tags that allow cellular uptake.
- the single domain antibodies are not provided as such, but are provided as nucleic acid molecules, i.e. nucleic acid molecules encoding single domain antibodies against SODl as herein described. Also provided are vectors comprising such nucleic acids or nucleic acid molecules. According to yet further embodiments, host cells are provided comprising such nucleic acids or such vectors.
- the single domain antibodies are provided herein for use in medicine. That is to say, the single domain antibodies against SODl are provided for use as a medicament.
- the single domain antibodies against SODl are provided for use as a medicament.
- the nucleic acid molecules encoding the single domain antibodies, or for the vectors containing such nucleic acids are provided for use in treatment of amyotrophic lateral sclerosis (ALS).
- ALS amyotrophic lateral sclerosis
- the single domain antibody may be provided as protein, or may be administered as a nucleic acid molecule encoding a single domain antibody against SODl, or as a vector comprising such nucleic acid molecule. If the single domain antibody is administered as protein, it is particularly envisaged that it is administered intracerebroventricularly, such as e.g. through injection or pump.
- the single domain antibody is provided as a nucleic acid or a vector, it is particularly envisaged that the single domain antibody is administered through gene therapy.
- the methods further comprise a step of monitoring the progression of ALS (or ALS symptoms) in the subject.
- kits comprising a single domain antibody against SODl and a pharmaceutically acceptable excipient.
- the single domain antibody may be provided as protein, as a nucleic acid encoding a single domain antibody against SODl, or as a vector comprising such nucleic acid.
- Nanobodies were generated as described before .
- an alpaca and a dromedary were injected subcutaneously on days 0, 7, 14, 21, 28, 35 with about 250 ⁇ g of human superoxide dismutase [Cu-Zn] (SOD1) per injection.
- SOD1 human superoxide dismutase
- antibodies of different IgG subclasses were obtained by successive affinity chromatography on protein A and protein G columns.
- Total plasma and three purified IgG subclasses (IgGl, lgG2 and lgG3) from both alpaca and dromedary were tested by ELISA to assess the immune response to SOD1.
- VHH libraries (one from the alpaca and one from the dromedary immunized with SOD1) were constructed using conventional methods 16 17 and screened for the presence of SODl-specific nanobodies. To this end, total NA from peripheral blood lymphocytes was used as template for first strand cDNA synthesis with oligo(dT) primer. Using this cDNA, the VHH encoding sequences were amplified by PCR, digested with Pstl and Notl, and cloned into the Pstl and Notl sites of the phagemid vector pHEN4.
- a VHH library of about 10 s independent transformants was obtained. About 74% of these transformants harboured the vector with the right insert size.
- a VHH library of about 2.5 ⁇ 10 s independent transformants was obtained. About 84% of the transformants from the dromedary library harboured the vector with the right insert size.
- Each library was subject to four consecutive rounds of panning, performed on solid-phase coated antigen (concentration: 100 ⁇ g/ml, 10 ⁇ g/well).
- the enrichment for antigen-specific phages after each round of panning was assessed by comparing the number of phages eluted from antigen-coated wells with the number of phages eluted from only-blocked wells.
- the enrichment was also evaluated by polyclonal phage ELISA. Based on these assays, the library obtained from alpaca was enriched for antigen-specific phages only after 4 th round of panning. In contrast, the library from dromedary was enriched for antigen-specific phages after 2 nd , 3 rd and 4 th rounds, with best enrichment factors after 2 nd and 3 rd rounds.
- Table 1 Overview of the obtained SODl nanobodies after the immunization and their characteristics.
- the heat of binding of selected nanobodies to SODl was measured using the Omega isothermal titration calorimeter (Microcal).
- Samples containing SODl in TBS were titrated with selected nanobodies in TBS at in isothermal chamber kept at the constant temperature of 25 ⁇ .
- Samples were filtered through 0.2 mM syringe and degassed before measurements.
- Aliquots (10 ⁇ ) of nanobodies were added consequently each 10 minutes (28 aliquotes in total) to allow for the chamber to equilibrate.
- the resulting change in the heat required to equilibrate the chamber to the constant temperature was recorded and processed using the single-site binding equation in the Origin 7.0 software (Microcal).
- the proteins for TEM studies were expressed in E. coli and purified as described above. Samples containing either SODl (0.2 mM) alone or SODl with equimolar concentrations of selected nanobodies were imaged after incubation for 4 weeks with shaking in 50 mM Tris-HCI (pH 8) at 25°C. Where indicated, DTT and EDTA were added to concentrations of 40 and 10 mM respectively to facilitate fibrillar aggregation of SODl. Aliquots (5 ⁇ ) of the incubated protein preparations were adsorbed to carbon-coated FormVar film on 400-mesh copper grids (Piano GmbH, Germany) for 1 min.
- the grids were blotted, washed twice in 50 ⁇ droplets of Milli-Q water, and stained with 1% (wt/vol) uranylacetate (Sigma). Samples were studied with a JEOL JEM-2100 microscope at 200 kV. Images were processed using iTEM software. DNA constructs and manipulation
- Coding DNA of SODl was amplified from human brain cDNA library (Invitrogen) and cloned into BamHI/Xhol sites of pCDNA4a (Invitrogen) to produce MycHis 6 tagged SODl construct for mammalian expression 19 .
- SOD1-CFP construct was prepared by subcloning of SODl from pCDNA4 into BamHI/Xhol sites of pCDNA3-CFP (Addgene #13030).
- a pcDNA plasmid containing full-length cDNA of wild type or mutant (A4V) SODl behind a T7 promoter was linearized with Asp718l and m NA was transcribed in vitro using a m MESSAGE mMACHINE T3 Kit (Ambion, Huntingdon, UK) followed by purification with a MEGAclearTMKit (Ambion, Huntingdon, UK).
- the nanobody mRNAs were similarly produced using a m MESSAGE mMACHINE T7 Kit (Ambion, Huntingdon, UK).
- the SODl nanobody protein was produced by the Protein Service Facility of VIB (Gent, Belgium). A His tag was added to better detect it, followed by a mCaspase-3 recognition site to remove the fusion partner.
- the protein sequence of the nanobody without initial methionine and the tags is: QVQLQESGGGSVQAGGSLRLACVASGGDTRPYITYWMGWYRQAPGKEREGVATIYTGGSGTYYSDSVEGRFTISQD KAQRTVYLQMNDLKPEDTAMYYCAAGNGALPPGRRLSPQNMDTWGPGTQVTVSS (SEQ ID NO: 1)
- HeLa Human carcinoma
- ATCC American Type Culture Collection
- Dulbecco's Modified Eagle Medium supplemented with 10 mM Hepes buffer. All media were supplemented with 10% FBS, 100 units/ml penicillin and 100 ⁇ g/ml streptomycin/2 mM L-glutamine. All tissue culture media and supplements were obtained from Difco.
- Cells were transfected using Fugene (Roche) according to the manufacturer's protocol. In a typical transfection, 2x10 s cells were transfected with 1 ⁇ g of DNA dissolved in 100 ⁇ of DMEM.
- the mouse motor neuron-like hybrid cell line NSC-34 (hybrid cell line produced by fusion of motor neuron enriched, embryonic mouse spinal cord cells with mouse neuroblastoma 20 was purchased from CELLutions Biosystems (Toronto, Canada). Cells were subcultured in 6-well plates (2x 10 s cells per well) and transiently transfected with plasmids (10 ⁇ g DNA per well) using a 1:1 ratio of Lipofectamine 2000 (Invitrogen, Gent, Belgium) to DNA. Expression vectors for human SODl WT -EGFP, S0D1 G93A -EGFP, and SODl A4V -EGFP were used for transfection.
- Embryos were co-injected with mRNA encoding the anti-SODl nanobody or a Control Nanobody raised against another protein ( ⁇ -lactamase) at concentrations ranging from 0.25- 25ng ⁇ l. Embryos were then stored in Danieau water (50 mM NaCI, 0.7 mM KCI, 0,4 mM MgS0 4 .7H 2 0 0,6 mM Ca(N0 3 )2.4H 2 0, 0,5 mM HEPES) at 27.5-28.5 °C. Analysis of motor neuron outgrowth
- morphologically normal zebrafish embryos were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) and immunostained using mouse anti-synaptic vesicle 2 (1/200; Developmental Studies Hybridoma Bank) and secondary Alexa Fluor 555 anti-mouse antibody (1/500; Molecular Probes) in order to visualize motor neurons.
- Observers blind to injection and treatment conditions measured the axonal length of the first five ventral motor axons after the yolk sac in each embryo using Lucia software (PSI, version 4.9) and the average of these five lengths was calculated for each embryo.
- Protein lysates were prepared from zebrafish embryos following removal of the protein-rich yolk sac by triturating within a thin tipped glass pipette. The embryos were then lysed in T-PE buffer and homogenized using a manual dounce.
- Cell medium was removed from the cell culture and cells were washed with PBS and harvested. Cells lysates were obtained by adding T-PER buffer and homogenizing by pipetting.
- Protein concentrations were determined using the micro-BCA protein assay reaction kit 207 (Pierce, Rockford, IL). Samples were separated according to size through denaturing 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). An equal amount of protein from each sample was heated at 100°C for 5 minutes with an equivalent volume of sample buffer (containing 8% SDS and 2% mercaptoethanol) and loaded onto polyacrylamide gels. The proteins were electrotransferred to a PVDF membrane in Tris-glycine-methanol buffer. The membrane was blocked for 1 hour at room temperature (RT) in a blocking solution mixture of 5% nonfat dry milk, 0.1% Tween 20, and TBS.
- RT room temperature
- the membrane was then incubated for 2 hours at RT with primary antibody (1/1000 anti SOD1, Sheep pAB, Calbiochem-Merck Chemicals, 1/1000 anti-SODl, rabbit pAb, Stressgen, 1/1000 anti HA goat pAB, Abeam, Cambridge, UK, 1/5000 anti ⁇ -actin mAB, Sigma-Aldrich, 1/1000 anti-Hsp70, mouse pAb, Stressgen).
- the membrane was rinsed once with TBS-Tween 20 for 15 minutes, washed twice with blocking solution for 5 minutes, and then incubated for 1 hour at RT in peroxidase-labeled secondary antibodies from Santa Cruz Biotechnology (1/5000, Santa Cruz, CA).
- the blot was washed once for 15 minutes, once for 10 minutes, and 3 times for 5 minutes, and then processed for analysis using a Supersignal chemiluminescent detection kit (Pierce), as described by the manufacturer and a Bio ad GelDoc System.
- a Supersignal chemiluminescent detection kit Pieris, as described by the manufacturer and a Bio ad GelDoc System.
- mice were anesthetized using 10% pentobarbital sodium in PBS. After transcardiac perfusion with ice- cold PBS followed by fixation with 4% paraformaldehyde, spinal cords were dissected and postfixed for 2 hours in 4% paraformaldehyde. Spinal cord sections (16 ⁇ ) were cut on a cryostat (Leica, Wetzlar, Germany), mounted in gelatinized slides. Samples were incubated in PBS with 0.1% Triton X-100 (PBST) and blocked in PBST with 10% normal donkey serum (Sigma, St Louis, MO) for 1 hour.
- PBST Triton X-100
- mice anti-NeuN mouse anti-NeuN (Millipore, Billerica, MA)
- rabbit anti P2X 4 Alomone Labs, Jerusalem, Irsael
- Antibodies were incubated overnight at 4°C. After 3 washing steps, the samples were incubated with the corresponding secondary antibodies (Alexa Fluor 555 or Alexa Fluor 488; Invitrogen). After 3 washing steps, sections were mounted using Vectashield (Vector Laboratories, Burlingame, CA) (with 4', 6-diamidino-2- phenylindole) and analyzed under a fluorescence microscope (DMIRB; Leica).
- mice were littermate and gender matched.
- P90 and P120 we started the intracerebroventricular administration of the nanobody (three times a week) and the evaluation of the injected mice.
- the hanging wire test we gave each mouse three trials of 60 s, two times a week.
- mice were anesthetized by isoflurane inhalation. Following anesthesia, the fur was shaved from the top of the skull, and the mouse scalp was disinfected with ethanol. The animal was then positioned on the stereotaxic apparatus, the head was fixed using nonrupture ear bars and a 2-cm midsagittal skin incision was made on the scalp in order to visualize the skull landmarker bregma (formed by the cross of the coronal and sagittal sutures).
- the cannula was firmly cemented to the skull with dental cement. Then we sutured the skin with a non-absorbable, sterile, surgical silk suture. Finally, we kept the animal warm on a temperature-controlled heating pad ( ⁇ 37°C) until its full recovery.
- cryostat sections (20 ⁇ ) with hematoxylin and eosin (H&E), modified Gomori trichrome and nicotinamide adenosine dinucleotide (NADH)-tetrazolium reductase.
- H&E hematoxylin and eosin
- NADH nicotinamide adenosine dinucleotide
- NSC-34 ceil were cultured, transfected with EGFP-S0D1 and treated with nanobody as described before. After 48h of culture, dismutase activity was determined (Cell Biolabs, San Diego, CA) as described by the manufacturer. Statistics
- SODl nanobodies (nucleic acids) have high affinity for SODl in vitro
- SODl nanobodies(nucleic acids) block fibril formation by SODl
- TEM Transmission Electron Microscopy
- SODl nanobodies reduce SODl aggregation in HeLa cells
- the nanobodies were expressed as HA-tagged or HA-Myc-His 6 tagged constructs in HeLa cells ( Figure 2A).
- pull-down of HA-Myc-His 6 tagged nanobodies was performed to ensure that the nanobodies expressed in mammalian cells retain binding capacity for SODl.
- HeLa cells were transiently co-transfected with Nbl, Nb2, Nb3 and Nb4 and SOD1-CFP, lysed and nanobodies were purified on Ni- agarose as described in Materials and Methods.
- SOD1-CFP was co-purifying together with Nbl, Nb2 and Nb3, indicating that they bind SODl efficiently when expressed in HeLa cells ( Figure 2A).
- Example 3 The SODl nanobody (nucleic acids) clears SODl and rescues the axonopathy in zebrafish in vivo.
- the SODl nanobody clears SODl and rescues the axonopathy in zebrafish in vivo
- SODl nanobody rescued the axonal phenotype induced by mutant SODl
- This axopathy was rescued when fish were co-injected with the SODl nanobody (2.5- 25ng/ul, p ⁇ 0.0031), but not the control nanobody ( Figure 6C).
- the SODl nanobody (protein) can enter the cell, reduce the number of transfected NSC-34 cells with SODl aggregates and rescue the axonopathy in zebrafish in vivo.
- the SODl nanobody can enter the cell and reduce the number of cells with SODl aggregates
- the SOD1 nanobody has a mCaspase-3 sequence to remove the fusion tag from the nanobody
- the cells were treated with SOD1 nanobody pre incubated with Caspase-3, with the His fraction or with the nanobody fraction, the number of cells with aggregates did not decrease (Figure 8D), showing that the entrance of the nanobody to the cell was due to the His tag.
- the SOD1 nanobody reduces SOD activity in transfected NSC-34 cells
- the assay principle is that Superoxide anions (02-) are generated by a Xanthine/Xanthine Oxidase (XOD) system, and then detected with a Chromagen Solution, provided by the kit. However, in the presence of SOD, these superoxide anion concentrations are reduced, yielding less colorimetric signal. Thus, more colorimetric signal means less SOD activity.
- XOD Xanthine/Xanthine Oxidase
- zebrafish is a good model to investigate ALS pathogenesis and possible treatments.
- SOD1 nanobody protein
- 30hpf we fixed the zebrafish and then we measured axonal length.
- SOD1 nanobody could rescue the axonal length in injected zebrafish when added directly to the tank water (Figure 10).
- Figure 10 The SOD1 nanobody (protein) prevents motorneuron death, delays disease onset and prolongs lifespan in a mouse model for ALS.
- the nanobody was also detected 6, 12 and 24 hours after injection in both brain and spinal cord (Figure 11D).
- CMAP compound muscle axonal potential
- ALS is a neurodegenerative disease where around 90% of patients have no familial history and are considered to have the sporadic form. ALS is familial in 10% of patients, and in about 20% of FALS patients the disease is caused by mutations in the gene encoding SODl (see Bento-Abreu 26 and obberecht and Philips 27 for review). The elimination of mutant SODl, the primary cause of motor neuron toxicity, is an obvious therapeutic strategy. This has been achieved previously by the viral delivery of RNAi against SODl 28 29 , by intracerebroventricular administration of antisense oligonucleotides 30 and by crossbreeding mutant SODl mice with mice that express a shRNA against mutant SODl 31 . Hence, gene silencing holds great promise as a therapy for ALS 32 (and in fact for many neurodegenerative diseases). The first clinical studies investigating the feasibility of these approaches in humans are currently underway.
- Nanobodies are a novel form of antibodies developed from the discovery that antibodies within camelids can function without light chains and can bind antigens through a single N-terminal (VHH) domain. Harnessing this fact, dromedary and alpaca were immunized with human SODl and the VHH of the resulting camelid antibodies was cloned to produce an anti SODl nanobody. In this study we have identified four different isotypes of nanobodies that were selected after consecutive rounds of phage display. Based on preliminary selection, we have chosen two nanobodies (one from alpaca and one from dromedary) for further characterization in vitro. The one that had higher affinity was selected for further characterization in vivo.
- our anti-SODl nanobody is not specific for mutant SODl, here we demonstrate the effect of the anti-SODl nanobody at disrupting formation of high molecular weight species by mutant SODl in vitro, that nanobodies can be expressed in mammalian cells, reducing mutant SODl aggregation in different cell lines, that they can rescue related axonopathy induced by mutant SODl in zebrafish and also its beneficial effect in delaying onset and extending lifespan in a mouse model for ALS.
- SODl and chaperone levels remain the same, meaning that the SODl expression is not altered and the effect is mostly due to the binding of SODl. We could not see interactions with other elements involved in protein elimination pathways. In our in vitro experiments, once the aggregates are formed, the presence of the SOD nanobody cannot decrease the number of cells with aggregates, meaning that it could only reduce toxicity when binds oligomeric forms of SODl.
- Zebrafish is a model of ALS where a toxic gain of function of a mutated SODl protein results in a neuronal phenotype 22 .
- the embryonic nature of this model for a neurodegenerative disease has several major advantages. Treatment of embryos with small compound libraries is more feasible when compared with ALS rodent models and is likely to have more potential than the in vitro models currently used for chemical screening. Another advantage is that drug testing can be performed within 2 days.
- SODl nanobody could decrease levels of human SODl protein following mRNA injection in zebrafish.
- the fact that in zebrafish embryos the proteosome system is upregulated might explain why mSODl protein levels decreases in the zebrafish model.
- the beneficial effect of the SODl nanobody, injected or added to the water, can be observed in the rescue of the axonal length induced by the injection of mutant SODl.
- nanobodies are not able to cross the cell membrane.
- Our experiments showed that the SODl nanobody is able to enter the cell when it is added to the cell medium. We could demonstrate that this characteristic is due to the His tag attached to the nanobody, because when we removed it, we did not find inhibition of aggregate formation. Although we did not investigate the mechanism further, we hypothesize that the high positive charge of the molecule could favour the interaction with the cell membrane and its internalization.
- SODl nanobody was used to treat hSODl G93A mice, a well-established model to investigate ALS.
- the injected SODl nanobody was able to bind SODl, although we cannot distinguish between cytoplasmic or extracellular SODl 34 or between cell types.
- SODl nanobody significantly inhibits MN death, increases survival and delays disease onset in hSODl G93A mice.
- SODl nanobody binds mutant SODl, which is associated with ALS 35 , including specific neurotoxic conformers 25 .
- ALS 35 including specific neurotoxic conformers 25 .
- the binding of mutant SODl and the blocking of its toxic effect appears to be cause of the neuroprotection in these transgenic animals.
- the SODl nanobody is not selective for mutant human SODl but can also bind WT human SODl and endogenous SODl. However, this feature does not imply any apparent toxicity or side effects in our experiments, as it was described before 38 39 .
- Nanobodies have a wide range of advantages and applications compared with conventional antibodies. Besides the size and the stability, they are easy to produce and also to modify in order to change their properties.
- the intracerebroventricular infusion of the Fab fragment of a monoclonal antibody against misfolded forms of SODl 40 , the immunization with mutant SODl 41 and the isolation of single chain fragments of variable regions (scFvs) of antibodies directed against SODl and its expression as intrabodies 42 have been published already and they imply a therapeutic approach, but our promising findings could have a direct application in ALS therapeutics, not only in SODl-linked familial cases, where they could have an obvious role, but also in sporadic cases 1,43,44 , where it has been demonstrated the implication of WT SODl in the pathogenesis of ALS. Obviously some modifications would be needed in order to make them suitable for patients, but SODl nanobodies could be a novel and real therapy for ALS patients.
- V(L) human light chain variable domain
- Van Hoecke, A et al. EPHA4 is a disease modifier of amyotrophic lateral sclerosis in animal models and in humans. Nature medicine 18, 1418-1422 (2012).
- RNAi selectively silences mutant SODl and achieves significant therapeutic benefit in vivo. Neurobiology of disease 23, 578-586 (2006).
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Abstract
Single domain antibodies against SOD1and their use in medicine Abstract The present application relates to the field of single domain antibodies (also called nanobodies), more particularly single domain antibodies against SOD1 protein isoforms. It also relates to the use of these nanobodies in medicine. Accordingly, methods to treat a disease using these nanobodies are provided herein. The single domain antibodies are particularly envisaged for treatment of ALS.
Description
SINGLE DOMAIN ANTIBODIES AGAINST SODl AND THEIR USE IN MEDICINE
Field of the invention
The present application relates to the field of single domain antibodies (also called nanobodies), more particularly single domain antibodies against SODl protein isoforms. It also relates to the use of these nanobodies in medicine. Accordingly, methods to treat a disease using these nanobodies are provided herein. The single domain antibodies are particularly envisaged for treatment of ALS.
Background
There currently is no effective treatment for neurodegenerative diseases such as Alzheimer's disease, Parkinson's and Huntington's disease, and amyotrophic lateral sclerosis (ALS). In all of these disorders, proteins in which mutations induce a toxic gain of function are thought to play a causal or pathogenic role. This newly acquired toxic function often is either multifactorial or incompletely understood. Reducing the expression of the mutant protein is an obvious therapeutic strategy of which the success is independent of the understanding of the pathogenic action of the protein.
ALS is an adult-onset neurodegenerative disease that affects the upper and lower motoneurons (MNs) in the cerebral cortex, brain stem and spinal cord. It results in progressive MN loss, muscle paralysis and atrophy leading to death within few years. Most ALS patients are thought to have a sporadic form of the disease, but in about 10% ALS is inherited or familial (FALS). In about 20% of patients with FALS the disease is caused by mutations in the gene encoding Cu/Zn-superoxide dismutase (SODl) (Rosen et al., 1993). Other causes are mutations in TARP, FUS/TLS, VCP and C90RF72. Overexpression of mutated human SODl in transgenic animals, both mice (Gurney et al., 1994) and rats (Nagai et al., 2001), results in the development of a lethal motor neuron disease. More than 150 distinct SODl point mutations have been described including cases in which enzymatic activity is increased, decreased or non-altered (cf. the ALS Online genetics Database at http://alsod.iop.kcl.ac.uk/). Therefore, a toxic gain of function is generally accepted to underlie neuronal toxicity of mutant SODl. Mutant SODl has been found to be misfolded4"5, and it is hypothesized that this toxicity is related to the formation of high- molecular-weight complexes and in a final stage, the formation of aggregates6"7, a hallmark of many neurodegenerative diseases (Zhang and Zhu, 2006, Ross and Poirier, 2004).
Furthermore, wild type SODl has been suggested to play a role in the pathogenesis of sporadic ALS8. It has been proposed that in sporadic ALS wild-type SODl undergoes secondary modifications (e.g. through oxidation or demetalation), misfolds and is toxic to motor neurons in a very similar manner to what is seen with mutant SODl9. Using a conformation-specific antibody that recognizes the misfolded species selectively, pathogenic SODl has been found in motor neurons of at least a part of sporadic
ALS patients1. Most interestingly, the toxic effect of astrocytes from sporadic ALS patients (thus not harboring SOD1 mutations) on motor neurons is dependent on the presence of SOD12.
Therefore, reducing the levels of the pathogenic SOD1 is an interesting strategy to treat patients with mutant SODl-associated familial ALS, as well as patients with sporadic ALS. To achieve this, anti-sense oligonucleotide, siRNA-based and immunological approaches have been developed. For the latter, both active and passive immunisation is under investigation. However, approaches using siRNA and conventional antibodies come with significant problems (e.g. half-life, distribution in the CNS, uptake by neurons...), and none of these approaches has thus far led to a therapy for ALS.
Accordingly, there is a pressing need for new therapies for ALS, particularly therapies that address the above-mentioned problems. Of course, the therapies should also be efficient in clearing mutant SOD1, and rescue the toxic gain of functions associated with these mutant SOD1 isoforms, or indeed with wild-type SOD1 isoforms that also show toxic gain of function.
Summary
Many neurodegenerative diseases such as Alzheimer's and Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis (ALS) are caused by a mutant protein which, through the presence of this mutation, gains a function that is toxic for the cell. Reducing the levels of the pathogenic mutant protein would be one strategy to treat patients suffering from these disorders. To avoid all problems inherent with the use of conventional antibodies, we have explored whether nanobodies can be used in the treatment of neurodegenerative disease, and in particular of ALS.
In 1993, Hamers-Castermans et al.14 described that camelids and some sharks produce functional antibodies devoid of light chains of which the single variable N-terminal domain was fully capable of antigen binding. This so called VHH domain can be cloned and the product, called nanobody or single domain antibody, shows high target specificity and affinity, and low inherent toxicity15"17. Furthermore, nanobodies are highly soluble, extremely stable, have refolding capacity, can be administered by means other than injection, and are easy to manufacture18.
Nanobodies have a potential use as therapeutics in many fields 3'4. Nanobodies have, like conventional forms of antibodies, applications as diagnostic markers5,6. Furthermore, nanobodies can be used as structural probes of protein misfolding and fibril formation7. Nanobodies that are capable of influencing the aggregation pathway of a disease-related protein, either by inhibiting the formation of particular intermediates or by neutralizing their neurotoxic effects, could also serve as potential therapeutics.
Nanobodies have additional properties that make them particularly appealing as therapeutic and diagnostic agents, including low immunogenicity due to high sequence similarities with human VH family III8. Particularly important for their potential use in neurodegenerative disorders, they have been shown to be able in some cases at least to cross the blood-brain barrier efficiently9. Nanobodies against a-synuclein10 n, β-amyloid12 13 and hungtintin14 have been already raised, and it has been reported to inhibit a-synuclein, β-amyloid and huntingtin aggregation in in vitro models for Parkinson's, Alzheimer's and Huntington's disease.
ALS is an adult-onset fatal neurodegenerative disease that is familial in about 10 % of patients. In about 20% of patients with familial ALS (FALS) the disease is caused by gain-of-toxic-function mutations in the gene encoding Cu/Zn-superoxide dismutase (SODl). To reduce the expression of mutant SODl in the motor neurons, we have raised anti-SODl heavy chain antibodies in dromedaries and alpacas, and cloned their N-terminal antigen binding VHH region, coding for anti-SODl nanobodies.
The therapeutic potential of such nanobodies was investigated in ALS, by studying their effect on mutant SODl-induced toxicity in vitro and in vivo and testing it in different ALS models. Different approaches were used: transfection or injection of constructs that express a SODl nanobody and also the administration of SODl nanobody itself. It could be shown that SODl nanobodies have high affinity for SODl in vitro and dose-dependently block its fibril formation, their expression reduces mutant SODl levels and rescues mutant SODl-induced axonopathy in zebrafish and inhibits SODl aggregates formation in in vitro models for ALS. In vivo, SODl nanobody also rescues mutant SODl-induced axonopathy in zebrafish when added in the tank water. Moreover, administering a nanobody to symptomatic hSODlG93A mice, a murine model for ALS, extends their lifespan in a dose-dependent way. These data demonstrate the potential use of SODl nanobodies as a novel therapeutic strategy for ALS. Moreover, they might be broadly applicable for neurodegenerative disease in general caused by toxicity of a mutant protein.
Thus, it is an object of the invention to provide single domain antibodies (or nanobodies) against SODl. According to particular embodiments, SODl is human SODl. According to alternative, but not exclusive, embodiments, the single domain antibodies bind to mutant SODl, i.e. they recognize an epitope that is present in a mutated form of the SODl protein. According to further particular embodiments, the mutant SODl is characterized by a mutation of amino acids at position 4, 93 and/or 113, particularly by an A4V, G93A, and/or G113W mutation. According to yet further embodiments, the single domain antibodies bind both wildtype and mutant SODl (i.e. they recognize an epitope present in the wildtype protein and at least one (but possibly more) mutated isoform).
According to particular embodiments, the single domain antibody is an inhibitory single domain antibody against SODl. Typically, this means that the nanobody interferes with the superoxide dismutase function of SODl. However, according to particular embodiments, the inhibitory single domain antibody inhibits the toxic gain of function of mutant SODl protein. Most particularly, the single domain antibody interferes with (inhibits, prevents, reverses or slows) the formation of SODl aggregates; and/or the single domain antibody can counter the phenotypic changes caused by expression of the mutant SODl protein (e.g. axonopathy).
According to particular embodiments, the single domain antibody has a sequence selected from the group of SEQ ID NOs: 1-14. According to alternative embodiments, the single domain antibody shares the sequence of the complementary determining regions of these sequences.
The single domain antibody may be provided as such or may be fused to further moieties. According to particular embodiments, the single domain antibody is fused to a tag. According to further particular embodiments, the tag to which the single domain antibody is fused is a His-tag, HA-tag, and/or Myc- tag.
SODl normally is a soluble cytoplasmic protein, although a detrimental or neurotoxic role has been ascribed both to extracellular secreted SODl and cytoplasmic mutant SODl. In order to be able to inhibit intracellular forms of SODl, according to particular embodiments, the nanobody is able to enter cells, particularly neuronal cells. This may be an inherent property of the nanobody, or may be achieved by further fusion to moieties or tags that allow cellular uptake.
According to particular embodiments, the single domain antibodies are not provided as such, but are provided as nucleic acid molecules, i.e. nucleic acid molecules encoding single domain antibodies against SODl as herein described. Also provided are vectors comprising such nucleic acids or nucleic acid molecules. According to yet further embodiments, host cells are provided comprising such nucleic acids or such vectors.
According to a further aspect, the single domain antibodies are provided herein for use in medicine. That is to say, the single domain antibodies against SODl are provided for use as a medicament. The same goes for the nucleic acid molecules encoding the single domain antibodies, or for the vectors containing such nucleic acids. According to particular embodiments, the single domain antibodies (or nucleic acids encoding them, or vectors comprising such nucleic acids) are provided for use in treatment of amyotrophic lateral sclerosis (ALS).
This is equivalent as saying that methods are provided for treating ALS, or of improving symptoms of ALS in a subject in need thereof, comprising administering a single domain antibody against SODl to
said subject. Here also, the single domain antibody may be provided as protein, or may be administered as a nucleic acid molecule encoding a single domain antibody against SODl, or as a vector comprising such nucleic acid molecule. If the single domain antibody is administered as protein, it is particularly envisaged that it is administered intracerebroventricularly, such as e.g. through injection or pump.
In case the single domain antibody is provided as a nucleic acid or a vector, it is particularly envisaged that the single domain antibody is administered through gene therapy.
According to particular embodiments, the methods further comprise a step of monitoring the progression of ALS (or ALS symptoms) in the subject.
According to further embodiments, kits are provided comprising a single domain antibody against SODl and a pharmaceutically acceptable excipient. According to particular embodiments, the single domain antibody may be provided as protein, as a nucleic acid encoding a single domain antibody against SODl, or as a vector comprising such nucleic acid. Brief description of the Figures
Figure 1: SODl nanobodies have high affinity for SODl in vitro and block its fibril formation. (A)
Western Dot Blot showing the binding of nanobodies to immobilized recombinant SODl. (B) Characterization of the binding properties of the nanobodies. (C) The SODl Nbl and Nb2 block fibril formation in vitro. (D) TEM pictures showing that Nbl prevent aggregate formation in samples containing both Nbl and SODl.
Figure 2: SODl nanobodies reduce SODl aggregation in HeLa cells. (A) Expression of HA-tag Nb and SOD1-CFP in HeLa cells. (B) Nb expressed in mammalian cells retain binding capacity for SODl as shown after co-purifying together SODl Nb and SODl. (C) Analysis of the nanobodies in transiently transfected HeLa cells by Blue Native Polyacrylamide gel electrophoresis (BN-PAGE). Figure 3: SODl aggregates formation and HA-tagged nanobody expression after transient transfection in NSC-34 cells. 24 hours after transfection, mutant SODl aggregates could be observed when cells were transfected with (B) eGFP-SODlG93A and (C) eGFP-SODlA4V but not with (A) eGFP-SODlWT. Control and SODl nanobodies could be detected by IHQ (D) and Western blot (E) after transfection through the HA-tag. Figure 4: SODl nanobody reduces the number of cells with mSODl aggregates after transfection.
After SODl nanobody and mSODl co-transfection in NSC-34 cells, SODl nanobody reduces the number of cells with S0D1G93A (A) and SODlA4V (B) aggregates. This reduction is dose dependent (C). The
appearance of the aggregates could vary between the different mutations without any apparent correlation, and the SODl nanobody did not affect either this appearance.
Figure 5: SODl nanobody expressed in NSC34 cells recognizes SODl protein and does not decrease hSODl levels. After SODl and nanobody co-transfection, SODl nanobody (A) but not control nanobody (B) can recognize and bind to endogenous, WT and mutant SODl (both G93A and A4V). However, SODl nanobody does not reduce hSODl levels after co-transfection (C).
Figure 6: SODl nanobody reduces SODlA4V protein level and abolishes its toxicity in zebrafish. (A)
Injected SODl nanobody in zebrafish can also be detected through the HA-tag. (B) Expression levels of SODl decrease with the co-injection of SODl nanobody and SODlA4V, but not when co-injected with control nanobody. (C) Shortening of motor neuron axons is partially rescued with the co-injection of SODlA4V with SODl nanobody but not with the control nanobody.
Figure 7: The SODl nanobody (protein) colocalizes with mutant SODl aggregates. (A) The SODl nanobody can be detected through its His-tag by Western blot and when it is added to the NSC-34 culture medium (B) can be found associated with the cells. Colocalization of the mutant SODl and the SODl nanobody (but not the control nanobody) was found by fluorescence (C) and confocal microscopy (D).
Figure 8: The SODl nanobody can enter the cell through its His-tag and reduce the number of cells with mSODl aggregates. After cell fractionation, SODl nanobody can be found in the cytosol (A) and after immunoprecipitation it has found to bind SODl (B). 48 hours after administration in the cell medium, the SODl nanobody inhibits aggregate formation in a dose-dependent manner (C). The inhibition is blocked when the His-tag is removed from the nanobody (D).
Figure 9: The SODl nanobody reduces SOD activity. When the SODl nanobody is added to the cell medium, the SOD activity is decreased. Read-out are OD values, inversely correlated with SOD activity (using OxiSelect™ Superoxide Dismutase Activity Assay of Cell Biolabs). Figure 10: Effect of the SODl nanobody treatment in injected zebrafish. The SODl nanobody can increase the axonal length and rescue the phenotype induced by mutant SODl in injected zebrafish when added to the tank water.
Figure 11: SODl nanobody can be detected in the mouse CNS after injection and binds SODl. (A)
Map of the mouse brain and scheme of the ICV injection. After one single injection, the SODl nanobody can be found in different areas of the brain (B) and spinal cord (C). (D) The SODl nanobody
is clear 12 hours after injection and weakly detected after 24h. The SOD1 nanobody, through its His- tag, can be immunoprecipitated with SOD1 in injected hSODlG93A mice (E).
Figure 12: The survival and disease onset are increased in treated hSODlG93A mice. Survival (A) and disease onset (D) of hSODlG93A mice treated with the SOD1 nanobody are delayed when the treatment is administrated starting at P60, at P90 (B) and P120 (C), 3 times/week. With daily injections starting at P90 the survival is also significantly increased.
Figure 13: The SOD1 nanobody rescues MN death. The number of small MN, but not of big MN was increased at P145 in mice treated with SOD1 nanobody from P60 (A). The number of MNJ did not vary (B).
Figure 14: The SOD1 nanobody recognizes neurotoxic forms of SOD1. In injected hSODlG93A mice, the SOD1 nanobody, through its His-tag, can be immunoprecipitated with the antibody anti P2X4, that selectively recognizes special conformers of mutant SOD1 that are neurotoxic.
Figure 15: Amino acid sequences of 14 different Nanobodies specific for human SOD1 (superoxide dismutase) [Cu-Zn]. All Nanobodies originate from VHH germline sequences. The complementarity determining regions (CDRs) are shown in bold. The top 7 Nanobodies were isolated from an immune library from a dromedary. The bottom 7 Nanobodies were isolated from an immune library from an alpaca. The above 14 different Nanobodies represent 4 different groups shown in 4 different colours. Nanobodies belonging to the same group (with the same colour) are very similar and their amino acid sequences suggest that they are from clonally-related B-cells resulting from somatic hypermutation or from the same B-cell but diversified due to PCR error during library construction. The gaps were introduced in order to align sequences.
Detailed description
Definitions
The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated.
Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
The following terms or definitions are provided solely to aid in the understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999), for definitions and terms of the art. The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.
A "single-domain antibody" (sdAb), also referred to as "nanobody" herein, is an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. With a molecular weight of only 12-15 kDa, single-domain antibodies are much smaller than common antibodies (150-160 kDa) which are composed of two heavy protein chains and two light chains, and even smaller than Fab fragments (~50 kDa, one light chain and half a heavy chain) and single-chain variable fragments (~25 kDa, two variable domains, one from a light and one from a heavy chain). The term "SODl" as used herein refers to the gene superoxide dismutase 1 and its encoded protein (Gene ID: 6647 for the human gene). The enzyme SODl binds copper and zinc ions and is one of three superoxide dismutases responsible for destroying free superoxide radicals in the body. Mutations in this gene have been linked to familial amyotrophic lateral sclerosis, and several pieces of evidence also show that wild-type SODl, under conditions of cellular stress, is implicated in a significant fraction of sporadic ALS cases. Over 150 mutations of SODl have been linked to ALS (cf. the ALS Online genetics Database at http://alsod.iop.kcl.ac.uk/; see also Olubunmi Abel, John F Powell, Peter M. Andersen, Ammar Al-Chalabi "ALSoD: A user-friendly online bioinformatics tool for amyotrophic lateral sclerosis genetics." Hum Mutat. 2012); "mutant SODl" in particularly refers to SODl containing one or more mutations that are linked to ALS. Selected examples (listed as 1 letter amino acid abbreviations, with numbering referring to the human protein) include those listed in the OMIM database under entry 147450, i.e. SODl A4V, G93A, G113W, H46 , G37R, L38V, G41D, H43R, G85R, G93C, G93A, E100G,
L106V, I113T, A4T, D90A, I104F, L144S, A145T; IVS4AS, T-G, -10; C6F, T151I, E21K, S134N, L84V, G16S, L126X; IVS4AS, A-G, -11; G72S, G12 , F45C, H80A, D96N; 6-BP DEL, GGACCA; IVS4AS, C-G, -304.
The term "inhibitory" as used in the phrase "inhibitory single domain antibody" or "inhibitory nanobody" herein, refers to the fact that the nanobody can inhibit the function and/or activity of its target protein. In case of wild-type SODl, this means that the superoxide dismutase activity is inhibited. In case of mutant SODl that has a gain of function, typically it is meant that the (toxic) new function is inhibited, although this may also mean that the enzymatic activity is inhibited, or inhibited as well. For instance, inhibition may result in decrease of aggregation of mutant SODl. Importantly, inhibition or decrease in toxic function may also be evaluated as an increase of another parameter, e.g. the inhibition may be evaluated by an increase in axonal length or an extended life span. "Inhibitory" can mean full inhibition (no enzymatic activity and/or toxic effect is observable) or may mean partial inhibition. For instance, inhibition can mean 10% inhibition, 20% inhibition, 25% inhibition, 30% inhibition, 40% inhibition or more. Particularly, inhibition will be at least 50%, e.g. 50% inhibition, 60% inhibition, 70% inhibition, 75% inhibition, 80% inhibition, 90% inhibition, 95% inhibition or more. % inhibition typically will be evaluated against a suitable control (e.g. treatment with an irrelevant nanobody, or a wild-type subject versus a diseased subject), as will be readily chosen by the skilled person. The term "ALS" or "amyotrophic lateral sclerosis" as used herein, sometimes also known as Lou Gehrig's disease, is a neurodegenerative disorder characterized by the death of motor neurons in the brain, brainstem, and spinal cord, resulting in fatal paralysis. It is a genetically heterogeneous disorder, described under entry 105400 in the OMIM database. A particular subset of ALS is ALS with SODl involvement, either through mutated SODl (OMIM entry 147450) or in cases where wild-type SODl is involved (typically in conditions of cellular stress).
A "subject" as used herein refers to an animal that can develop ALS wherein SODl is involved (e.g. through misfolding). Typically, the animal is a mammal. Most particularly, the subject is a human. It is an object of the invention to provide single domain antibodies (or nanobodies) against SODl. According to particular embodiments, SODl is human SODl. According to alternative, but not exclusive, embodiments, the single domain antibodies bind to mutant SODl, i.e. they recognize an epitope that is present in a mutated form of the SODl protein. According to further particular embodiments, the mutant SODl is characterized by a mutation of amino acids at position 4, 93 and/or 113, particularly by an A4V, G93A, and/or G113W mutation. According to yet further embodiments,
the single domain antibodies bind both wildtype and mutant SODl (i.e. they recognize an epitope present in the wildtype protein and at least one (but possibly more) mutated isoform).
According to particular embodiments, the single domain antibody is an inhibitory single domain antibody against SODl. Typically, this means that the nanobody interferes with the superoxide dismutase function of SODl. However, according to particular embodiments, the inhibitory single domain antibody inhibits the toxic gain of function of mutant SODl protein. Most particularly, the single domain antibody interferes with (inhibits, prevents, reverses or slows) the formation of SODl aggregates; and/or the single domain antibody can counter the phenotypic changes caused by expression of the mutant SODl protein (e.g. axonopathy).
According to particular embodiments, the single domain antibody has a sequence selected from the group of SEQ ID NOs: 1-14. According to alternative embodiments, the single domain antibody shares the sequence of the complementary determining regions (CDRs) of these sequences, fitted in a suitable framework region. For Nb2 (SEQ ID NO: 1) and related nanobodies, the 3 CDR sequences correspond to GGDTRPYITYWMG (SEQ ID NO: 15), TIYTGGSGTYYSDSVEG (SEQ ID NO: 16) and GNGALPPGRRLSPQNMDT (SEQ ID NO: 17) respectively. For Nbl and related nanobodies, the CDR sequences correspond to ETLFSLYAMG (SEQ ID NO: 18) or ESLFSLYAMG (SEQ ID NO: 19), TISGGGEGTGNYADPVKG (SEQ ID NO: 20) and YGTNLAP (SEQ ID NO: 21) respectively. For Nb3 and related nanobodies, the CDR sequences correspond to GLPYRTVFMG (SEQ ID NO: 22) or GLPYRVVFMG (SEQ ID NO: 23), VINADGVSTYYADSVKG (SEQ ID NO: 24), and NHFFDYSRDPLATAEYNY (SEQ ID NO: 25) respectively. For Nb4, the sequences of the CDRs are GYTFSSYCMG (SEQ ID NO: 26), TIISDGSTYYADSVKG (SEQ ID NO: 27) and RSGGVCSGRASRYNY (SEQ ID NO: 28) respectively.
The single domain antibody may be provided as such or may be fused to further moieties. According to particular embodiments, the single domain antibody is fused to a tag. According to further particular embodiments, the tag to which the single domain antibody is fused is a His-tag, HA-tag, and/or Myc- tag.
SODl normally is a soluble cytoplasmic protein, although a detrimental or neurotoxic role has been ascribed both to extracellular secreted SODl and cytoplasmic mutant SODl. In order to be able to inhibit intracellular forms of SODl, according to particular embodiments, the nanobody is able to enter cells, particularly neuronal cells. This may be an inherent property of the nanobody, or may be achieved by further fusion to moieties or tags that allow cellular uptake.
According to particular embodiments, the single domain antibodies are not provided as such, but are provided as nucleic acid molecules, i.e. nucleic acid molecules encoding single domain antibodies against SODl as herein described. Also provided are vectors comprising such nucleic acids or nucleic
acid molecules. According to yet further embodiments, host cells are provided comprising such nucleic acids or such vectors.
According to a further aspect, the single domain antibodies are provided herein for use in medicine. That is to say, the single domain antibodies against SODl are provided for use as a medicament. The same goes for the nucleic acid molecules encoding the single domain antibodies, or for the vectors containing such nucleic acids. According to particular embodiments, the single domain antibodies (or nucleic acids encoding them, or vectors comprising such nucleic acids) are provided for use in treatment of amyotrophic lateral sclerosis (ALS). This is equivalent as saying that methods are provided for treating ALS, or of improving symptoms of ALS in a subject in need thereof, comprising administering a single domain antibody against SODl to said subject. Here also, the single domain antibody may be provided as protein, or may be administered as a nucleic acid molecule encoding a single domain antibody against SODl, or as a vector comprising such nucleic acid molecule. If the single domain antibody is administered as protein, it is particularly envisaged that it is administered intracerebroventricularly, such as e.g. through injection or pump.
In case the single domain antibody is provided as a nucleic acid or a vector, it is particularly envisaged that the single domain antibody is administered through gene therapy.
According to particular embodiments, the methods further comprise a step of monitoring the progression of ALS (or ALS symptoms) in the subject.
According to further embodiments, kits are provided comprising a single domain antibody against SODl and a pharmaceutically acceptable excipient. According to particular embodiments, the single domain antibody may be provided as protein, as a nucleic acid encoding a single domain antibody against SODl, or as a vector comprising such nucleic acid.
It is to be understood that although particular embodiments, specific configurations as well as materials and/or molecules, have been discussed herein for cells and methods according to the present invention, various changes or modifications in form and detail may be made without departing from the scope and spirit of this invention. The following examples are provided to better illustrate particular embodiments, and they should not be considered limiting the application. The application is limited only by the claims.
Examples
MATERIALS AN D METHODS Generation of SODl na no bodies
Nanobodies were generated as described before . In brief, an alpaca and a dromedary were injected subcutaneously on days 0, 7, 14, 21, 28, 35 with about 250 μg of human superoxide dismutase [Cu-Zn] (SOD1) per injection. After these six rounds of immunization, antibodies of different IgG subclasses were obtained by successive affinity chromatography on protein A and protein G columns. Total plasma and three purified IgG subclasses (IgGl, lgG2 and lgG3) from both alpaca and dromedary were tested by ELISA to assess the immune response to SOD1. In the dromedary, there was immune response in all IgG subclasses with best response in IgGl. The immune response raised in alpaca was very low. Two VHH libraries (one from the alpaca and one from the dromedary immunized with SOD1) were constructed using conventional methods16 17 and screened for the presence of SODl-specific nanobodies. To this end, total NA from peripheral blood lymphocytes was used as template for first strand cDNA synthesis with oligo(dT) primer. Using this cDNA, the VHH encoding sequences were amplified by PCR, digested with Pstl and Notl, and cloned into the Pstl and Notl sites of the phagemid vector pHEN4. From the alpaca, a VHH library of about 10s independent transformants was obtained. About 74% of these transformants harboured the vector with the right insert size. In a similar way, from the dromedary, a VHH library of about 2.5 χ 10s independent transformants was obtained. About 84% of the transformants from the dromedary library harboured the vector with the right insert size. Each library was subject to four consecutive rounds of panning, performed on solid-phase coated antigen (concentration: 100 μg/ml, 10 μg/well). The enrichment for antigen-specific phages after each round of panning was assessed by comparing the number of phages eluted from antigen-coated wells with the number of phages eluted from only-blocked wells. The enrichment was also evaluated by polyclonal phage ELISA. Based on these assays, the library obtained from alpaca was enriched for antigen-specific phages only after 4th round of panning. In contrast, the library from dromedary was enriched for antigen-specific phages after 2nd, 3rd and 4th rounds, with best enrichment factors after 2nd and 3rd rounds.
From the alpaca library, 189 individual colonies identified after the 4th round of panning were randomly selected and analyzed by ELISA for the presence of SODl-specific nanobodies in their periplasmic extracts. Out of 189 colonies, 129 scored positive in this assay. Sequencing of 46 of these positive colonies identified 7 different nanobodies. All these 7 nanobodies belong to the same group.
From dromedary library, 142 individual colonies (47 from the 2nd and 95 from the 4th round of panning) were randomly selected and analyzed by ELISA for their specificity for SOD1. Out of these 142 colonies, 64 colonies (42 from 2nd round and 22 from 4th round) scored positive in this assay. Sequencing of 36 positive colonies identified 7 different nanobodies representing 3 different groups. These vary only by single-point mutations away from the variable regions that are involved in antigen binding. These mutations are likely derived from PCR errors during construction of libraries. From these two libraries,
one nanobody (Nbl) was selected from alpaca library, and three nanobodies (Nb2, Nb3, Nb4) were selected from dromedary library, each representing an individual group (Table 1). An alignment of all nanobodies is provided in figure 15.
Table 1: Overview of the obtained SODl nanobodies after the immunization and their characteristics.
These 4 constructs were expressed in E. coli by subcloning into BamHI/Xhol sites of or pET30a (Novagen), to obtain His6-tagged peptides. These were purified using conventional Ni-affinity purification protocol (Qiagen). Briefly, proteins were overexpressed in C41 (DE3) cells overnight at 2!?C in TB medium after induction with 1 mM IPTG. Cells were lysed by high-pressure cell cracker in lysis buffer (TBS containing 15 mM imidazole), and supernatant was cleared by centrifugation at 12,000 rpm for 20 minutes. Supernatant was incubated with Ni-agarose for 30 minutes, followed by washes with 200 volumes of lysis buffer, and eluted in TBS containing 250 mM imidazole. In a second step, nanobodies were purified by size-exclusion chromatography on Superdex S-75 columns in TBS buffer and concentrated using Centricon units (Millipore).
Isothermal titration calorimetrics
The heat of binding of selected nanobodies to SODl was measured using the Omega isothermal titration calorimeter (Microcal). Samples containing SODl in TBS were titrated with selected nanobodies in TBS at in isothermal chamber kept at the constant temperature of 25^. Samples were filtered through 0.2 mM syringe and degassed before measurements. Aliquots (10 μί) of nanobodies were added consequently each 10 minutes (28 aliquotes in total) to allow for the chamber to equilibrate. The resulting change in the heat required to equilibrate the chamber to the constant
temperature was recorded and processed using the single-site binding equation in the Origin 7.0 software (Microcal).
Transmission Electron Microscopy
The proteins for TEM studies were expressed in E. coli and purified as described above. Samples containing either SODl (0.2 mM) alone or SODl with equimolar concentrations of selected nanobodies were imaged after incubation for 4 weeks with shaking in 50 mM Tris-HCI (pH 8) at 25°C. Where indicated, DTT and EDTA were added to concentrations of 40 and 10 mM respectively to facilitate fibrillar aggregation of SODl. Aliquots (5 μί) of the incubated protein preparations were adsorbed to carbon-coated FormVar film on 400-mesh copper grids (Piano GmbH, Germany) for 1 min. The grids were blotted, washed twice in 50 μί droplets of Milli-Q water, and stained with 1% (wt/vol) uranylacetate (Sigma). Samples were studied with a JEOL JEM-2100 microscope at 200 kV. Images were processed using iTEM software. DNA constructs and manipulation
Coding DNA of SODl was amplified from human brain cDNA library (Invitrogen) and cloned into BamHI/Xhol sites of pCDNA4a (Invitrogen) to produce MycHis6 tagged SODl construct for mammalian expression 19. SOD1-CFP construct was prepared by subcloning of SODl from pCDNA4 into BamHI/Xhol sites of pCDNA3-CFP (Addgene #13030). For zebrafish injections, a pcDNA plasmid containing full-length cDNA of wild type or mutant (A4V) SODl behind a T7 promoter was linearized with Asp718l and m NA was transcribed in vitro using a m MESSAGE mMACHINE T3 Kit (Ambion, Huntingdon, UK) followed by purification with a MEGAclear™Kit (Ambion, Huntingdon, UK). The nanobody mRNAs were similarly produced using a m MESSAGE mMACHINE T7 Kit (Ambion, Huntingdon, UK). Expression vectors for human SODlWT enhanced green fluorescent protein (EGFP), S0D1G93A-EGFP, and SODlA4V-EGFP, that were used for NSC-34 transfection, were kindly donated by Prof. Esquerda from Universitat de Lleida, Spain.
Nanobody ( protein) production
The SODl nanobody protein was produced by the Protein Service Facility of VIB (Gent, Belgium). A His tag was added to better detect it, followed by a mCaspase-3 recognition site to remove the fusion partner. The protein sequence of the nanobody without initial methionine and the tags is:
QVQLQESGGGSVQAGGSLRLACVASGGDTRPYITYWMGWYRQAPGKEREGVATIYTGGSGTYYSDSVEGRFTISQD KAQRTVYLQMNDLKPEDTAMYYCAAGNGALPPGRRLSPQNMDTWGPGTQVTVSS (SEQ ID NO: 1)
To remove the His-tag, Caspase-3 (RD Systems) was added to the nanobody and it was incubated overnight at 37°C. Using His Select Niquel Magnetic Agarose Beads (Sigma) we could separate the His- tag from the nanobody.
Cell Culture
Human carcinoma (HeLa) cell lines were obtained from the American Type Culture Collection (ATCC) and cultured according to standard mammalian tissue culture protocols in Dulbecco's Modified Eagle Medium supplemented with 10 mM Hepes buffer. All media were supplemented with 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin/2 mM L-glutamine. All tissue culture media and supplements were obtained from Difco. Cells were transfected using Fugene (Roche) according to the manufacturer's protocol. In a typical transfection, 2x10s cells were transfected with 1 μg of DNA dissolved in 100 μΙ of DMEM.
The mouse motor neuron-like hybrid cell line NSC-34 (hybrid cell line produced by fusion of motor neuron enriched, embryonic mouse spinal cord cells with mouse neuroblastoma 20 was purchased from CELLutions Biosystems (Toronto, Canada). Cells were subcultured in 6-well plates (2x 10s cells per well) and transiently transfected with plasmids (10 μg DNA per well) using a 1:1 ratio of Lipofectamine 2000 (Invitrogen, Gent, Belgium) to DNA. Expression vectors for human SODlWT-EGFP, S0D1G93A-EGFP, and SODlA4V-EGFP were used for transfection. Cells were fixed at 24 to 72 hours with 4% paraformaldehyde and permeabilized with PBS 0.1% Triton X-100, blocked in normal goat serum and incubated overnight at 4 °C in goat anti-HA or rabbit anti His antibody (1/500). Immunoreactivity was visualized after incubation with Cy5-conjugated anti-goat secondary antibody 1/500 in PBS from Invitrogen (Carlsbad, CA) under a Fluorescence microscope (DMIRB; Leica) or Confocal microscopy (Zeiss 200M microscope, Munich, Germany).
Zebrafish maintenance and embryo injection
All experiments were approved and performed in accordance with the guidelines of the Ethical Committee for Animal Experimentation, K.U. Leuven. Adult zebrafish (Danio rerio, AB strain) and embryos were maintained under standard laboratory conditions. Zebrafish embryo microinjections were made using a FemtoJet injection setup (Eppendorf, Hamburg, Germany). Each injection was made in the 1-4 cell stage of the zebrafish embryo and involved delivery of 2.14nl of 1 μg/μl of SOD1 mRNA, accomplished by an injection pressure of less than 4.5 psi, which produced a droplet diameter of 160 μιτι on a micrometer. Embryos were co-injected with mRNA encoding the anti-SODl nanobody or a
Control Nanobody raised against another protein (β-lactamase) at concentrations ranging from 0.25- 25ng^l. Embryos were then stored in Danieau water (50 mM NaCI, 0.7 mM KCI, 0,4 mM MgS04.7H20 0,6 mM Ca(N03)2.4H20, 0,5 mM HEPES) at 27.5-28.5 °C. Analysis of motor neuron outgrowth
At 30 hours post-fertilization (hpf) morphologically normal zebrafish embryos were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) and immunostained using mouse anti-synaptic vesicle 2 (1/200; Developmental Studies Hybridoma Bank) and secondary Alexa Fluor 555 anti-mouse antibody (1/500; Molecular Probes) in order to visualize motor neurons. Observers blind to injection and treatment conditions measured the axonal length of the first five ventral motor axons after the yolk sac in each embryo using Lucia software (PSI, version 4.9) and the average of these five lengths was calculated for each embryo.
Electrophoresis and western blotting
Protein lysates were prepared from zebrafish embryos following removal of the protein-rich yolk sac by triturating within a thin tipped glass pipette. The embryos were then lysed in T-PE buffer and homogenized using a manual dounce.
Cell medium was removed from the cell culture and cells were washed with PBS and harvested. Cells lysates were obtained by adding T-PER buffer and homogenizing by pipetting.
Protein concentrations were determined using the micro-BCA protein assay reaction kit 207 (Pierce, Rockford, IL). Samples were separated according to size through denaturing 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). An equal amount of protein from each sample was heated at 100°C for 5 minutes with an equivalent volume of sample buffer (containing 8% SDS and 2% mercaptoethanol) and loaded onto polyacrylamide gels. The proteins were electrotransferred to a PVDF membrane in Tris-glycine-methanol buffer. The membrane was blocked for 1 hour at room temperature (RT) in a blocking solution mixture of 5% nonfat dry milk, 0.1% Tween 20, and TBS. The membrane was then incubated for 2 hours at RT with primary antibody (1/1000 anti SOD1, Sheep pAB, Calbiochem-Merck Chemicals, 1/1000 anti-SODl, rabbit pAb, Stressgen, 1/1000 anti HA goat pAB, Abeam, Cambridge, UK, 1/5000 anti β-actin mAB, Sigma-Aldrich, 1/1000 anti-Hsp70, mouse pAb, Stressgen). The membrane was rinsed once with TBS-Tween 20 for 15 minutes, washed twice with blocking solution for 5 minutes, and then incubated for 1 hour at RT in peroxidase-labeled secondary antibodies from Santa Cruz Biotechnology (1/5000, Santa Cruz, CA). The blot was washed once for 15 minutes, once for 10 minutes, and 3 times for 5 minutes, and then processed for analysis
using a Supersignal chemiluminescent detection kit (Pierce), as described by the manufacturer and a Bio ad GelDoc System.
Immunocitochemistry
Mice were anesthetized using 10% pentobarbital sodium in PBS. After transcardiac perfusion with ice- cold PBS followed by fixation with 4% paraformaldehyde, spinal cords were dissected and postfixed for 2 hours in 4% paraformaldehyde. Spinal cord sections (16 μιτι) were cut on a cryostat (Leica, Wetzlar, Germany), mounted in gelatinized slides. Samples were incubated in PBS with 0.1% Triton X-100 (PBST) and blocked in PBST with 10% normal donkey serum (Sigma, St Louis, MO) for 1 hour. The following primary antibodies were used: mouse anti-NeuN (Millipore, Billerica, MA), and rabbit anti P2X4 (Alomone Labs, Jerusalem, Irsael). Antibodies were incubated overnight at 4°C. After 3 washing steps, the samples were incubated with the corresponding secondary antibodies (Alexa Fluor 555 or Alexa Fluor 488; Invitrogen). After 3 washing steps, sections were mounted using Vectashield (Vector Laboratories, Burlingame, CA) (with 4', 6-diamidino-2- phenylindole) and analyzed under a fluorescence microscope (DMIRB; Leica).
Immunoprecipitation
Samples were centrifuged in a microcentrifuge for 10 minat 12,000 rpm and at a temperature of 4°C and then placed on ice. The supernatant was aspirated and placed in a fresh tube, then kept on ice and incubated with the antibody (1/1000 anti-SODl, Calbiochem) overnight at 4°C, under agitation. The protein A Sepharose beads (Amersham Pharmacia Biotech AB) were used to immunoprecipitate. The lysate-beads mixture was incubated at 4°C under rotary agitation for 4 h. After that, the samples were centrifuged, the supernatant was collected and the beads were washed in lysis buffer three times. Finally, the last supernatant was removed, 25 μΙ of 2x loading buffer were added to the beads, and both the first collected supernatant and the samples were boiled at 95-100°C for 5 min for ulterior SDS PAGE and western blot.
Treatment and evaluation of mice
We maintained adult mice overexpressing human S0D1G93A (C57BL/6 background) in accordance with the Guide of Care and Use of Experimental Animals of the Ethical Committee of KU Leuven. The Ethical
Committee of KU Leuven approved all animal experiments. The experiments were littermate and gender matched. At post natal day 60 (P60), P90 and P120 we started the intracerebroventricular administration of the nanobody (three times a week) and the evaluation of the injected mice. We weigh the mice and we used the hanging wire test and the rotarod treadmill (Ugo Basile, Comerio,
Italy) to evaluate the motor performance. For the hanging wire test, we gave each mouse three trials of 60 s, two times a week. For rotarod treadmill mice had to walk at 15 r.p.m over the course of 180 s. We gave each mouse three trials of 180 s, two times a week. We killed mice when they could no longer roll over within 20 s after being placed on their backs, and we considered this time point as the time of death.
Intracerebroventricular cannulation in mice
Before surgery, animals were anesthetized by isoflurane inhalation. Following anesthesia, the fur was shaved from the top of the skull, and the mouse scalp was disinfected with ethanol. The animal was then positioned on the stereotaxic apparatus, the head was fixed using nonrupture ear bars and a 2-cm midsagittal skin incision was made on the scalp in order to visualize the skull landmarker bregma (formed by the cross of the coronal and sagittal sutures). A microdrill was used to perform a small hole (1 mm of diameter) on the left side of the skull according to the previously defined stereotaxic intracerebroventricular coordinates: Anteroposterior = -0.1 mm; Mediolateral - +1.0 mm (left side), from bregma. Stereotaxic coordinates were determined from the mouse brain atlas (George Paxinos and Keith B.J. Franklin, Academic Press, 2005). Localization of the final point of injection was previously confirmed by injection of 1 microliter of colorant (comassie blue) in a small subgroup of animals. A stainless steel guide cannula with a tubing below the pedestal of 3mm (Bilaney, Dusseldorf, Germany), was inserted into the hole made previously. The cannula was firmly cemented to the skull with dental cement. Then we sutured the skin with a non-absorbable, sterile, surgical silk suture. Finally, we kept the animal warm on a temperature-controlled heating pad (~37°C) until its full recovery.
Motoneurons and neuromuscular junctions counting
We snap-froze gastrocnemius muscle in isopentane, cooled by immersion in liquid nitrogen. We stained cryostat sections (20 μιτι) with hematoxylin and eosin (H&E), modified Gomori trichrome and nicotinamide adenosine dinucleotide (NADH)-tetrazolium reductase. To visualize neuromuscular junctions, we immunostained longitudinal cryostat sections (40 μηι) with NF-200 (1:200, Sigma, N4142), and Alexa-488-conjugated a-bungarotoxin (1:500, Invitrogen, B13422). To perform cresyl violet (Sigma) on spinal cord, we first fixed (4% paraformaldehyde), dehydrated (30% sucrose) and snap-froze spinal cord in Tissue-Tec (Sakura) and then made cryostat sections of 20um thickness. We calculated the area of normal-appearing neurons in the ventral horn of the lumbar spinal cord on every tenth slide for a total of ten slides per animal using Axiovision 4 software (Zeiss) and determined the number of neurons in different size groups. We considered neurons in the ventral horn of the lumbar spinal cord, with a cell body area >250 μηι2, motor neurons.
SODl activity
NSC-34 ceil were cultured, transfected with EGFP-S0D1 and treated with nanobody as described before. After 48h of culture, dismutase activity was determined (Cell Biolabs, San Diego, CA) as described by the manufacturer. Statistics
Data are shown as mean ± SEM. Student-t test was used to calculate significance. When more than 2 groups were compared, a one-way analysis of variance with Tukey least significant difference post hoc test was used. Example 1. Generation and characterization of single domain antibodies against SODl
SODl nanobodies (nucleic acids) have high affinity for SODl in vitro
The binding of nanobodies to immobilized recombinant SODl was verified by Western Dot Blot (Figure 1A). Strong binding was detected with Nbl and Nb2, but not the Nb3 and Nb4, suggesting that the recombinant Nb3 was not fully active. To further characterize the binding properties of the nanobodies, Isothermal Titration Calorimetry (ITC) was performed to determine affinities (Kd), enthalpies (ΔΗ) and entropies (AS) of binding18. Solution containing SODl was titrated with nanobodies in isothermic chamber at 25°C, and the heat required to dissipate the energy of binding was recorded for each titration (Figure IB, lower panel). These data were fitted into one-site binding model to calculate affinity of binding Kd, ΔΗ and AS (Figure IB, upper panel). Both Nbl and Nb2 were found to bind SODl with near-micromolar Kd: ~1 μΜ and ~0.2 μΜ for Nb2 and Nbl respectively (Table 1 and Figure IB). The high enthalpy of interaction (-15 kCal per mole) indicated efficient binding for both nanobodies. Calculation of Kd and thermodynamic parameters of binding for Nb3 was not possible due to its low affinity and limitations on the amount of the ligand.
SODl nanobodies(nucleic acids) block fibril formation by SODl
To determine if nanobodies can affect SODl aggregation in vitro, formation of fibrils by SODl in the presence of nanobodies was characterized by Transmission Electron Microscopy (TEM). It has been previously shown that SODl forms amyloid fibrils in conditions where binding of metal ions and assembly of SODl dimer is compromised by addition of Guanidinium Chloride or disulfide reducing agents and EDTA21. Thus, TEM was used to probe formation of amyloid fibrils by SODl destabilized by addition of DTT and EDTA. Both SODl WT (not shown) and A4V mutant very efficiently formed visible precipitates under these conditions that consisted entirely of amyloid fibrils as seen by TEM (Figure 1C). The appearance of SODl fibrils was similar to that of previously published studies, and we did not observe any amorphous aggregates under these conditions. However, in the presence of equimolar
concentrations of Nb2, formation of amyloid fibrils was completely suppressed, resulting in the formation of small or amorphous aggregates (Figure IC). This result suggests that binding of Nb2 interferes with formation of cross-beta structure by destabilized SODl, probably through steric or conformational hindrance. Nbl was less effective in suppression of amyloid formation by SODl, as aggregates were mainly amorphous, but also contained some amyloid fibrils (Figure IC). This result might reflect difference in Kd of Nbl and Nb2 for SODl as well as a difference in binding sites on SODl. To determine if Nbl can block SODl fibrils formation under less stringent condition, we also incubated it with the non-destabilized SODl in 50 mM Tris, pH 8.0 for 1 month with shacking at 25°C. In these conditions, SODl can form both amorphous and amyloid fibrils, but fibril formation is very inefficient as most of the protein stays soluble in the solution. Indeed, we observed formation of some SODl fibrils by TEM under these conditions, although no visible precipitate was formed (Figure ID). By contrast, no aggregates could be seen by TEM in samples containing both Nbl and SODl. Hence, we conclude that both Nbl and Nb2 can suppress formation of fibrils by SODl in vitro.
SODl nanobodies (nucleic acids) reduce SODl aggregation in HeLa cells
To determine the effect of the nanobodies on aggregation of SODl in cell culture, the nanobodies were expressed as HA-tagged or HA-Myc-His6 tagged constructs in HeLa cells (Figure 2A). Initially, pull-down of HA-Myc-His6 tagged nanobodies (Nbl, Nb2, Nb3 and Nb4) was performed to ensure that the nanobodies expressed in mammalian cells retain binding capacity for SODl. HeLa cells were transiently co-transfected with Nbl, Nb2, Nb3 and Nb4 and SOD1-CFP, lysed and nanobodies were purified on Ni- agarose as described in Materials and Methods. SOD1-CFP was co-purifying together with Nbl, Nb2 and Nb3, indicating that they bind SODl efficiently when expressed in HeLa cells (Figure 2A).
The oligomeric state of SODlA4V co-expressed with the nanobodies in transiently transfected HeLa cells was analyzed by Blue Native Polyacrylamide gel electrophoresis (BN-PAGE) following lysis under non- denaturing condition in non-ionic detergent MPE buffer in order to preserve intracellular aggregates. While most A4V migrated as monomers and dimers in a bottom part of BN-PAGE, some of it formed a smear that extended to the upper limit of fractionation on the gel (several thousands kDa), indicating formation of high molecular weight (HMW) oligomeric species of A4V (Figure 2C). By contrast, in lysates of cells co-transfected with A4V and Nbl or Nb2, A4V was migrating entirely as low molecular weight oligomers (Figure 2C).
This data demonstrates that Nbl and Nb2 inhibit aggregation of SODlA4V, with the Nb2 being the most effective. We therefore further investigated the effect of this nanobody in mutant SODl-associated disease models.
Example 2. SODl nanobody (nucleic acids) abolishes mutant SODl aggregate formation in NSC34 cells Transfection of the SODl nanobody abolishes mutant SODl aggregate formation in NSC-34 cells We investigated the effect of the expression of the HA-tagged anti-SODl nanobody on the formation of mutant SODl-induced aggregates in motor neuronal NSC-34 cells. Transfection of NSC-34 cells with mutant (AV4 and G93A) but not WT SODl-eGFP induced the formation of cytoplasmic mutant SODl aggregates, as has been described before (Figure 3A, B and C). First we tested the nanobody expression after transfection by immunocitochemistry and western blot (Figure 3D and E). We assessed the effect of nanobody expression on aggregate formation by co-transfecting NSC-34 cells with SODl-eGFP and either the SODl nanobody or control nanobody (Figure 4). The number of NSC-34 cells containing aggregates was quantified 24, 48 and 72 hours after transfection (Figure 4A and B). Expression of the anti-SODl nanobody, but not of the control nanobody, significantly decreased the number of cells with aggregates induced by both S0D1G93A-EGFP (Figure 4A) and SODlA4V-EGFP (Figure 4B). Increasing the ratio of SODl nanobody/mutant SOD1-EGFP cDNA, demonstrated this effect to be dose-dependent (Figure 4C).
By performing immunoprecipitation on cell lysates from the NSC-34 transfections we demonstrated that the anti-SODl nanobody, but not the control nanobody, binds to SODl in co-transfected cells, as is shown in Figures 5A-B. The anti-SODl nanobody was not specific for mutant SODl as also SODlWT and endogenous SODl was immunoprecipitated.
We also checked SODl levels after SODl nanoboby and eGFP-SODl co-transfection and we could see that protein levels remained unaltered despite the reduction of the number of cells with aggregates and the recognition and specificity of the SODl nanobody for SODl (Figure 5C). Chaperone levels were also checked but no variation was observed (data not shown).
Example 3. The SODl nanobody (nucleic acids) clears SODl and rescues the axonopathy in zebrafish in vivo.
The SODl nanobody clears SODl and rescues the axonopathy in zebrafish in vivo
Expression of human mutant SODl or mutant TDP-43 induces axon outgrowth defects and aberrant branching in zebrafish 22 23. This model has been used for identification of disease modifiers in animal models and humans24. In order to investigate the effect of the anti-SODl nanobody on the mutant SODl-induced axonopathy, zebrafish embryos were injected with mutant SODlA4V mRNA with anti- SODl nanobody or control nanobody mRNA. First, nanobody expression was checked after nanobody mRNA injection in zebrafish by western blot (Figure 6A).
SODl immunoblot analysis revealed that co-expression of the anti-SODl nanobody decreased SODl levels in a dose-dependent manner (Figure 6B), with 25ng^l achieving a significantly decreased SODl level (p=0.022). Injection of the control nanobody had no effect on SODl levels.
To evaluate whether the SODl nanobody rescued the axonal phenotype induced by mutant SODl, we measured the axonal length of motor neurons at 30 hpf. Overexpression of SODlA4V induced clear motor axonal abnormalities compared to overexpression of SODlWT (p=0.0011), as described previously 21. This axopathy was rescued when fish were co-injected with the SODl nanobody (2.5- 25ng/ul, p<0.0031), but not the control nanobody (Figure 6C). Example 4. The SODl nanobody (protein) can enter the cell, reduce the number of transfected NSC-34 cells with SODl aggregates and rescue the axonopathy in zebrafish in vivo.
The SODl nanobody can enter the cell and reduce the number of cells with SODl aggregates
Next, we wanted to test the SODl nanobody in its protein form. First we tested the detection of the SODl nanobody through its His tag. Blotting pure SODl nanobody with an anti-His tag antibody we could see a thick band at the predicted molecular weight (~15KDa) and also some other thinner bands corresponding to the dimerization or trimerization of the nanobody (Figure 7A). To assess whether the SODl nanobody could cross the cell membrane and enter the cell, we cultured NSC-34 cells and we added different doses of SODl nanobody to the medium. After blotting the cells and the concentrated medium with the anti-His antibody, we could detect the nanobody in the medium and also associated with the cells (Figure 7B).
To confirm that the SODl nanobody was really inside the cell, we transfected NSC-34 cells with eGFP- S0D1G93A and then we added 50μg/μ\ of nanobody to the culture medium. After processing the samples for immunocytochemistry, we could observe by fluorescence (Figure 7C) and confocal (Figure 7D) microscopy that the SODl nanobody, but not the control nanobody (nanobody protein against lisozyme), was colocalizing with mutant SODl aggregates.
When we fractionated and blotted the transfected cells, immunoreactivity for the anti-His antibody appeared in the cytosolic fraction only in treated cells (Figure 8A), and SODl and SODl nanobody (through the His-tag) were co-immunoprecipitated (Figure 8B).
To demonstrate the effect of the SODl nanobody in vitro, we transfected NSC-34 cells with eGFP- S0D1G93A and then we added different doses of the SODl nanobody to the medium. 48 hours after transfection and treatment, we quantified the number of cells with aggregates (Figure 8C). We could see a reduction of the number of transfected cells with aggregates in a dose dependent way only when
the cells were treated with the S0D1 nanobody, but not when the cells were not treated or treated with the control nanobody. By contrast, when the SOD1 nanobody was added to the medium 48 hours after transfection, once the aggregates were formed, the SOD1 nanobody could not decrease the number of cells with aggregates (data not shown). As the SOD1 nanobody has a mCaspase-3 sequence to remove the fusion tag from the nanobody, we incubate the SOD1 nanobody with Caspase-3 and then we separate the His tag from the pure nanobody and we treated transfected cells. 24 hours after, we quantified the number of cells with aggregates. When the cells were treated with SOD1 nanobody pre incubated with Caspase-3, with the His fraction or with the nanobody fraction, the number of cells with aggregates did not decrease (Figure 8D), showing that the entrance of the nanobody to the cell was due to the His tag.
The SOD1 nanobody reduces SOD activity in transfected NSC-34 cells
The assay principle, as described by the manufacturer, is that Superoxide anions (02-) are generated by a Xanthine/Xanthine Oxidase (XOD) system, and then detected with a Chromagen Solution, provided by the kit. However, in the presence of SOD, these superoxide anion concentrations are reduced, yielding less colorimetric signal. Thus, more colorimetric signal means less SOD activity. When NSC-34 cells where transfected with eGFP-SODl, those that were treated with the SOD1 nanobody showed less SOD activity (Figure 9), giving evidence for the binding of the SOD1 nanobody and the SOD1 protein. Effect of the SOD1 nanobody in the zebrafish model for ALS
As we described previously, zebrafish is a good model to investigate ALS pathogenesis and possible treatments. We injected zebrafish with mutant SOD1 and 3 hours later we added SOD1 nanobody to the tank water. 30hpf we fixed the zebrafish and then we measured axonal length. SOD1 nanobody could rescue the axonal length in injected zebrafish when added directly to the tank water (Figure 10). Example 5. The SOD1 nanobody (protein) prevents motorneuron death, delays disease onset and prolongs lifespan in a mouse model for ALS.
Effect of the SOD1 nanobody in a mouse model for ALS
As mutant SOD1 has been described as a cause of ALS, next we wanted to test our SOD1 nanobody in a well-established mouse model for ALS. Therefore, we used the ALS mouse model overexpressing human mutant SOD1 (S0D1G93A). The administration of the nanobody was performed intracerebroventricular through an implanted cannula (Figure 11A).
To test whether the nanobody was correctly injected and could reach different parts of the central nervous system (CNS), we injected 2 μΙ (7μ /μΙ), we wait for 2 hours and we dissect different parts of the brain and spinal cord. As a negative control, we used the whole brain and spinal cord of a non- injected mouse. As shown in Figure 11B and C, the nanobody was detectable in different structures of the brain (Figure 11B) and spinal cord (Figure 11C) whereas no signalling was detected in the negative control.
The nanobody was also detected 6, 12 and 24 hours after injection in both brain and spinal cord (Figure 11D).
To assess whether the SODl nanobody could bind overexpressed mutant SODl, we injected the SODl nanobody to a hSODlG93A mouse, we dissect the brain and we perform an immunoprecipitation using an anti-SODl antibody. The blot with an anti-His antibody showed that the nanobody and SODl were associated and that the nanobody could recognize and bind SODl (Figure HE).
Last, we want to test the biological effect of the SODl nanobody in this mouse model for ALS. To do so, we injected 2 different groups of transgenic mice with both control and SODl nanobody starting at different time points (Figure 12) testing motor performance and survival. The nanobody was administrated 3 times a week starting in a pre-symptomatic, symptomatic and end-stage and also daily starting in a symptomatic stage. When the injections started at P60 (Figure 12A) and P90 (Figure 12B) and P120 (Figure 12C) the survival was increased, and the disease onset was delayed also starting at P60 (Figure 12D).
In all the cases, SODl levels or proteins associated with autophagy or the ubiquitin-proteosome system remained the same (data no shown).
We also measured the number of MN and neuromuscular junctions (NMJ) of the mice treated from P60 with the control and the SODl nanobody. When the countings were done at P145, the number of small MN (250-400 μιτι2) in the group treated with the SODl nanobody was significantly higher than for the control group (Figure 13A). The number of big MN (>400 μιτι2) was not significantly different. Besides this fact, the number of NMJ remained the same at this age (Figure 13B). No toxicity due to the treatment was observed during the experiments.
During all the experiments, the compound muscle axonal potential (CMAP) did not vary between the two groups of injected mice.
As it was described previously, the antibody against the ATP receptor P2X4 recognizes mutant misfolded forms of SODl25. It was also described that these neurotoxic conformers of SODl induce astroglia and microglia activation. When we immunoprecipitate brains from mice treated with the control and SODl nanobody with the P2X4 antibody, we could detect the SODl nanobody associated with neurotoxic forms of mutant SODl (Figure 14).
DISCUSSION
ALS is a neurodegenerative disease where around 90% of patients have no familial history and are considered to have the sporadic form. ALS is familial in 10% of patients, and in about 20% of FALS patients the disease is caused by mutations in the gene encoding SODl (see Bento-Abreu26 and obberecht and Philips 27 for review). The elimination of mutant SODl, the primary cause of motor neuron toxicity, is an obvious therapeutic strategy. This has been achieved previously by the viral delivery of RNAi against SODl28 29 , by intracerebroventricular administration of antisense oligonucleotides 30 and by crossbreeding mutant SODl mice with mice that express a shRNA against mutant SODl31. Hence, gene silencing holds great promise as a therapy for ALS 32 (and in fact for many neurodegenerative diseases). The first clinical studies investigating the feasibility of these approaches in humans are currently underway.
In this study we aimed to test the potential of nanobodies as a method of decreasing toxicity of mutant SODl for the treatment of ALS.
Nanobodies are a novel form of antibodies developed from the discovery that antibodies within camelids can function without light chains and can bind antigens through a single N-terminal (VHH) domain. Harnessing this fact, dromedary and alpaca were immunized with human SODl and the VHH of the resulting camelid antibodies was cloned to produce an anti SODl nanobody. In this study we have identified four different isotypes of nanobodies that were selected after consecutive rounds of phage display. Based on preliminary selection, we have chosen two nanobodies (one from alpaca and one from dromedary) for further characterization in vitro. The one that had higher affinity was selected for further characterization in vivo.
Although our anti-SODl nanobody is not specific for mutant SODl, here we demonstrate the effect of the anti-SODl nanobody at disrupting formation of high molecular weight species by mutant SODl in vitro, that nanobodies can be expressed in mammalian cells, reducing mutant SODl aggregation in different cell lines, that they can rescue related axonopathy induced by mutant SODl in zebrafish and also its beneficial effect in delaying onset and extending lifespan in a mouse model for ALS.
When different cells lines transiently overexpress mutant SODl, cytoplasmic inclusions are formed. This fact correlates with cell toxicity 33. After testing SODl nanobody expression in transfected cells, we have found that the aggregation, due to transfection with mutant SODl, is reduced when co- transfecting or treating with the SODl nanobody in a dose-dependent way, but not when using a control nanobody. As we can find SODl nanobody in the cytosol, also associated with SODl and the SOD activity of the cells is decreased when treating with the SODl nanobody, we can conclude that SODl nanobody binds SODl and prevents it from aggregation. However, further experiments are
needed to identify the epitope that the nanobody binds. SODl and chaperone levels remain the same, meaning that the SODl expression is not altered and the effect is mostly due to the binding of SODl. We could not see interactions with other elements involved in protein elimination pathways. In our in vitro experiments, once the aggregates are formed, the presence of the SOD nanobody cannot decrease the number of cells with aggregates, meaning that it could only reduce toxicity when binds oligomeric forms of SODl.
To test the benefit of the anti-SODl nanobody in vivo we injected mutant SODl into zebrafish embryos. Zebrafish is a model of ALS where a toxic gain of function of a mutated SODl protein results in a neuronal phenotype22. The embryonic nature of this model for a neurodegenerative disease has several major advantages. Treatment of embryos with small compound libraries is more feasible when compared with ALS rodent models and is likely to have more potential than the in vitro models currently used for chemical screening. Another advantage is that drug testing can be performed within 2 days.
We have found that the SODl nanobody could decrease levels of human SODl protein following mRNA injection in zebrafish. The fact that in zebrafish embryos the proteosome system is upregulated might explain why mSODl protein levels decreases in the zebrafish model. The beneficial effect of the SODl nanobody, injected or added to the water, can be observed in the rescue of the axonal length induced by the injection of mutant SODl.
One disadvantage of the nanobodies in general is that they are not able to cross the cell membrane. Our experiments showed that the SODl nanobody is able to enter the cell when it is added to the cell medium. We could demonstrate that this characteristic is due to the His tag attached to the nanobody, because when we removed it, we did not find inhibition of aggregate formation. Although we did not investigate the mechanism further, we hypothesize that the high positive charge of the molecule could favour the interaction with the cell membrane and its internalization.
Next we used the SODl nanobody to treat hSODlG93A mice, a well-established model to investigate ALS. To optimize the treatment and make possible that small volumes of SODl nanobody could reach the CNS with high concentrations, we administered it intracerebroventriculary through a cannula. With one single injection, we could detect it in different areas of the CNS. The detection was possible also after several hours. The injected SODl nanobody was able to bind SODl, although we cannot distinguish between cytoplasmic or extracellular SODl34 or between cell types.
The treatment with SODl nanobody significantly inhibits MN death, increases survival and delays disease onset in hSODlG93A mice. We have demonstrated that SODl nanobody binds mutant SODl, which is associated with ALS35, including specific neurotoxic conformers25. As we could not observe any decrease in SODl levels or changes in other proteins like chaperones, proteasome or autophagy-
related proteins, the binding of mutant SODl and the blocking of its toxic effect appears to be cause of the neuroprotection in these transgenic animals. Since toxicity is thought to be related to the formation of high-molecular-weight complexes and in a final stage, the formation of aggregates36 37 , we can hypothesize that the SODl nanobody could inhibit the toxicity and the aggregate formation also in vivo.
We have also shown that the SODl nanobody is not selective for mutant human SODl but can also bind WT human SODl and endogenous SODl. However, this feature does not imply any apparent toxicity or side effects in our experiments, as it was described before38 39.
Nanobodies have a wide range of advantages and applications compared with conventional antibodies. Besides the size and the stability, they are easy to produce and also to modify in order to change their properties. The intracerebroventricular infusion of the Fab fragment of a monoclonal antibody against misfolded forms of SODl40, the immunization with mutant SODl 41 and the isolation of single chain fragments of variable regions (scFvs) of antibodies directed against SODl and its expression as intrabodies42 have been published already and they imply a therapeutic approach, but our promising findings could have a direct application in ALS therapeutics, not only in SODl-linked familial cases, where they could have an obvious role, but also in sporadic cases1,43,44, where it has been demonstrated the implication of WT SODl in the pathogenesis of ALS. Obviously some modifications would be needed in order to make them suitable for patients, but SODl nanobodies could be a novel and real therapy for ALS patients.
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Claims
1. A single domain antibody against SOD1.
2. The single domain antibody according to claim 1, wherein SOD1 is human SOD1.
3. The single domain antibody according to claim 1 or 2, wherein the single domain antibody binds mutant SOD1.
4. The single domain antibody according to claim 3, wherein the mutant SOD1 is characterized by a mutation of amino acids at position 4, 93 and/or 113, particularly by A4V, G93A, and/or G113W mutation.
5. The single domain antibody according to any one of claims 1 to 4, wherein the single domain antibody binds both wildtype and mutant SOD1.
6. The single domain antibody according to any one of claims 1 to 5, wherein the single domain antibody is an inhibitory single domain antibody against SOD1.
7. The single domain antibody according to any one of claims 1 to 6, which has a sequence selected from the group of SEQ ID NO: 1-14, or wherein the CD s are selected from SEQ ID NO: 15-28.
8. The single domain antibody according to any one of claims 1 to 7, which is fused to a tag, such as a His-tag, HA-tag, or Myc-tag
9. The single domain antibody according to any one of claims 1 to 8, which is able to enter cells.
10. A nucleic acid encoding a single domain antibody according to any one of claims 1 to 9.
11. A vector comprising a nucleic acid according to claim 10.
12. A host cell comprising a nucleic acid according to claim 10 or a vector according to claim 11.
13. A method of treating ALS or improving symptoms of ALS in a subject, comprising administering a single domain antibody against SOD1 to said subject.
14. The method according to claim 13, wherein the single domain antibody is administered intracerebroventricularly, such as through injection or pump.
15. The method according to claim 13, wherein the single domain antibody is administered through gene therapy.
16. The single domain antibody according to any one of claims 1 to 9, nucleic acid according to claim 10 or vector according to claim 11 for use as a medicament.
17. The single domain antibody according to any one of claims 1 to 9, nucleic acid according to claim 10 or vector according to claim 11 for use in treatment of ALS.
18. A kit comprising a single domain antibody according to any one of claims 1 to 9, nucleic acid according to claim 10 or vector according to claim 11, and a pharmaceutically acceptable excipient.
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Cited By (8)
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WO2016124512A1 (en) | 2015-02-02 | 2016-08-11 | Georg Bauer | Antigen-binding constructs, namely single domain vhh fragments which bind to and inhibit catalase and/or superoxide dismutase as well as pharmaceutical compositions containing them for tumor therapy |
WO2017007825A1 (en) * | 2015-07-06 | 2017-01-12 | Rxi Pharmaceuticals Corporation | Methods for treating neurological disorders using a synergistic small molecule and nucleic acids therapeutic approach |
US9745574B2 (en) | 2009-02-04 | 2017-08-29 | Rxi Pharmaceuticals Corporation | RNA duplexes with single stranded phosphorothioate nucleotide regions for additional functionality |
US9862777B2 (en) | 2013-05-28 | 2018-01-09 | Vib Vzw | Single domain antibodies against SOD1 and their use in medicine |
US10131904B2 (en) | 2008-02-11 | 2018-11-20 | Rxi Pharmaceuticals Corporation | Modified RNAi polynucleotides and uses thereof |
WO2020092937A1 (en) | 2018-11-02 | 2020-05-07 | Annexon, Inc. | Compositions and methods for treating brain injury |
US10774330B2 (en) | 2008-09-22 | 2020-09-15 | Phio Pharmaceuticals Corp. | Reduced size self-delivering RNAI compounds |
US11001845B2 (en) | 2015-07-06 | 2021-05-11 | Phio Pharmaceuticals Corp. | Nucleic acid molecules targeting superoxide dismutase 1 (SOD1) |
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SI3322734T1 (en) | 2015-07-16 | 2021-02-26 | Inhibrx, Inc. | Multivalent and multispecific dr5-binding fusion proteins |
CN111153997B (en) * | 2020-01-16 | 2022-06-24 | 佛山汉腾生物科技有限公司 | anti-CTLA-4 nano antibody and application thereof in tumor treatment |
CN116284422B (en) * | 2023-05-25 | 2023-07-21 | 北京和颐林生物科技有限公司 | Anti-superoxide dismutase antibody and application thereof |
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EP3004170A1 (en) | 2016-04-13 |
US9862777B2 (en) | 2018-01-09 |
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