WO2023247766A1 - Anti-cng repeats antibodies and their diagnosis applications - Google Patents

Anti-cng repeats antibodies and their diagnosis applications Download PDF

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Publication number
WO2023247766A1
WO2023247766A1 PCT/EP2023/067146 EP2023067146W WO2023247766A1 WO 2023247766 A1 WO2023247766 A1 WO 2023247766A1 EP 2023067146 W EP2023067146 W EP 2023067146W WO 2023247766 A1 WO2023247766 A1 WO 2023247766A1
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seq
amino acid
antibody
single domain
hairpin
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PCT/EP2023/067146
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French (fr)
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Guy-Franck Richard
David VITERBO
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Institut Pasteur
Centre National De La Recherche Scientifique
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Publication of WO2023247766A1 publication Critical patent/WO2023247766A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • the present invention relates to antibody or antigen-binding fragment thereof that specifically binds to a nucleic acid molecule and their use in diagnostic methods.
  • DNA tandem repeats occur frequently in double-stranded DNAs of eukaryotic genomes, more particularly of the human genome. DNA tandem repeat units of 2, 3, 4, 5 or even more nucleotides can be observed in a genome at different frequencies and locations (exons, introns, intergenic regions). DNA tandem repeats are prone to recombination and/or random integration events, and are considered to be at the center of species evolution.
  • Such antigen based assays are simple, fast and less expensive than PCR assays. Moreover, given that the antibodies and antigen-binding fragment thereof of the invention binds specifically to hairpin, they keep their sensitivity and specificity even in sample with a high number of CNG repeats.
  • the present invention also relates to an isolated single domain antibody, comprising three complementary determining regions (CDR1 to CDR3, respectively);
  • amino acid sequence of the CDR1 is selected from the amino acid sequences of SEQ ID NOs: 23-33 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOs: 23-33;
  • - a complex comprising: the antibody or antigen-binding fragment thereof of the invention or the isolated single domain antibody of the invention and a nucleic acid hairpin comprising repeats of CNG nucleotide triplets;
  • the present invention also relates to a kit comprising the antibody or antigen-binding fragment of the invention and/or the single domain antibody of the invention.
  • the nucleic acid hairpin to which the antibody or antigen-binding fragment thereof of the application binds or specifically binds, is a hairpin from a nucleic acid molecule which comprises repeats of CNG nucleotide triplets.
  • Hairpin also called hairpin loop or stem-loop refers to the nucleic acid secondary structure.
  • the 4-nt loops are present in all (CNG)n hairpins with an even number of repeats, the 7-nt loop occurs in the (CAG)n and (CCG)n hairpins with an odd number of repeats and the 3-nt loops are present in the (CUG)n and (CGG)n hairpins with an odd number of repeats (Sobczak,K., de Mezer,M., Michlewski,G., Krol, J. and Krzyzosiak.W.J. (2003) RNA structure of trinucleotide repeats associated with human neurological diseases. Nucleic Acids Res., 31 , 5469-5482).
  • the nucleic acid hairpin is a 4-nucleotide loop hairpin.
  • the nucleic acid hairpin is a 3-nucleotide loop hairpin, preferably a 3-nucleotide loop hairpin comprising repeats of CUG, repeats of CTG or repeats of CGG, more preferably repeats of CUG or repeats of CTG, most preferably repeats of CTG.
  • the nucleic acid hairpin is a 7-nucleotide loop hairpin, preferably a 7-nucleotide loop hairpin comprising repeats of CAG or CCG, more preferably repeats of CAG.
  • nucleic acid When they relate to a nucleic acid, the phrases “repeat”, “triplet(s)”, “sequence unit(s)” and “unit(s)” (or equivalent or similar phrases) are given their respective general meaning of the field of nucleic acids.
  • the nucleic acid For example, the nucleic acid:
  • CNGi where i is a number means i repeats of the sequence CNG.
  • the copies of the CNG nucleotide triplet are adjacent to each other. They can either be spaced apart from each other by only a few nucleotides, e.g., by less than 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2 nucleotides, or can be directly adjacent to each other. According to an aspect of the application, said copies of CNG nucleotide triplet are spaced apart from each other by only a few nucleotides, e.g., by less than 6, 5, 4, 3, 2 nucleotides, or are directly adjacent to each other. In a preferred embodiment, said copies of DNA sequence unit are directly adjacent to each other. For example, in the above-mentioned nucleic acid of SEQ ID NO: 1 , said copies of CNG nucleotide triplets (i.e., the copies of the CTG triplet) are directly adjacent to each other.
  • a CNG repeat is a direct repeat, i.e., it is not an inverted repeat: the order in which the nucleotides are contained in one triplet is conserved throughout the CNG repeats.
  • a CNG repeat is a repeat of nucleotide triplets having the same CNG sequence.
  • the number of CNG nucleotide triplets that are repeated is of at least 3. According to an aspect of the application, said number is of at least 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52., 55, 100, 150, 200, 250, 300, 350, 500 or 1000.
  • the number of CNG nucleotide triplets that are repeated is at least 5, 10, 50 or 55. According to an aspect of the application, said number is of between 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 and 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 100, 150, 200, 250, 300, 350, 500, 1000, 1500 or 2500. In an embodiment, the number of CNG nucleotide triplets that are repeated is between 5, 10 or 25 and 250, 300, 400 or 500.
  • epitope means the part of an antigen to which the antibody binds.
  • the epitopes of protein antigens can be divided into two categories, conformational epitope and linear epitope.
  • a conformational epitope corresponds to discontinuous sections of the antigen's amino acid sequence.
  • a linear epitope corresponds to a continuous sequence of amino acids from the antigen.
  • the epitope is a conformational epitope.
  • the epitope may also be a linear epitope.
  • anti-CTG repeats antibody refers to an antibody which specifically binds to a nucleic acid hairpin comprising repeats of CTG nucleotide triplets
  • anti-CAG repeats antibody refers to an antibody which specifically binds to a nucleic acid hairpin comprising repeats of CAG nucleotide triplets etc.
  • the specific binding between the antibody or antigen-binding fragment thereof of the invention and the epitope (or the region comprising the epitope) implies that the antibody exhibits appreciable affinity for the epitope (or the region comprising the epitope) on a particular antigen (here a nucleic acid hairpin comprising repeats of CNG nucleotide triplets as defined above).
  • Appreciable affinity includes binding with an affinity of about 10 -9 M (KD) or stronger.
  • binding is considered specific when the binding affinity is between 10 -9 M and 10 -12 M, optionally between 10 -9 M and 10 -10 M, in particular 10 -10 M.
  • Whether a binding domain specifically reacts with or binds to a target can be tested readily by, inter alia, comparing the reaction of said binding domain with a target antigen with the reaction of said binding domain with antigens other than the target antigen.
  • the affinity can be determined by various methods well known from the one skilled in the art. These methods include, but are not limited to, Biacore Analysis, Blitz analysis and Scatchard plot.
  • the affinity is determined in non-denaturing conditions.
  • the invention also provides the nucleic acid having the nucleotide sequence SEQ ID NO: 4 or 5.
  • the anti-CNG repeats antibody or antigen-binding fragment thereof as defined above has a KD value inferior to 200x10 -9 M, preferably inferior to 10 -9 M, more preferably inferior to 10 -10 M for a nucleic acid having a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 - 3 or a nucleic acid comprising the nucleotide sequence selected from the group consisting of SEQ ID NO:6-1 1 , more preferably inferior to 1 .10 -11 M, particularly by Biacore Analysis.
  • the antibody, antigen-binding fragment thereof and/or the single domain antibody binds to the nucleic acid having the nucleotide sequence SEQ ID NO: 1 with a nanomolar KD.
  • the invention relates to an anti-CAG repeats antibody or antigen-binding fragment thereof as defined above that has a KD value inferior to 200x10 -9 M, preferably inferior to 10 -9 M, more preferably inferior to 10 -10 M for a nucleic acid hairpin comprising repeats of CAG nucleotide triplets, most preferably inferior to 1 .10 -11 M, particularly by Biacore Analysis.
  • the anti-CAG repeats antibody or antigen-binding fragment thereof as defined above has a KD value inferior to 200x10 -9 M, preferably inferior to 10 -9 M, more preferably inferior to 10 -10 M for a nucleic acid having a nucleotide sequence SEQ ID NO: 2, most preferably inferior to 1 .10 11 M, particularly by Biacore Analysis.
  • the invention relates to an anti-CGG repeats antibody or antigen-binding fragment thereof as defined above that has a KD value inferior to 200x10 -9 M, preferably inferior to 10 -9 M, more preferably inferior to 10 -10 M for a nucleic acid hairpin comprising repeats of CGG nucleotide triplets, most preferably inferior to 1 .10 -11 M, particularly by Biacore Analysis.
  • the anti-CGG repeats antibody or antigen-binding fragment thereof as defined above has a KD value inferior to 200x10 -9 M, preferably inferior to 10 -9 M, more preferably inferior to 10 -10 M for a nucleic acid having the nucleotide sequence SEQ ID NO: 3, most preferably inferior to 1 .10 -11 M, particularly by Biacore Analysis.
  • the anti-CCG repeats antibody or antigen-binding fragment thereof as defined above has a KD value inferior to 200x10 -9 M, preferably inferior to 10 -9 M, more preferably inferior to 10 -10 M for a nucleic acid having a nucleotide sequence SEQ ID NO: 60, most preferably inferior to 1 .10 11 M, particularly by Biacore Analysis.
  • the nucleic acid having the nucleotide sequence selected from the group consisting of SEQ ID NO: 4- 5 may be used as negative control to ensure that the binding is specific to the CNG repeats hairpin and not to the GC clamp.
  • the invention relates to an antibody or antigen-binding fragment thereof as defined above that has a KD value superior to 200x10 -9 M, preferably superior to 200x10 -8 M, more preferably superior to 200x10 7 M for nucleic acid having the nucleotide sequence selected from the group consisting of SEQ ID NO: 4-5 (respectively (GAA)so with GC clamp and GC clamp), particularly by Biacore Analysis.
  • Table 1 Positive and negative controls
  • antibody comprises polyclonal antibodies or monoclonal antibodies.
  • Monoclonal antibodies can be generated by several known technologies like phage, bacteria, yeast or ribosomal display, as well as by classical methods exemplified by hybridoma-derived antibodies.
  • the term "monoclonal” is used to refer to all antibodies derived from one nucleic acid clone.
  • the antibodies of the present invention include recombinant antibodies.
  • the term "recombinant antibody” refers to antibodies which are produced, expressed, generated or isolated by recombinant means, such as antibodies which are expressed using a recombinant expression vector transfected into a host cell; antibodies isolated from a recombinant combinatorial antibody library; antibodies isolated from an animal (e.g. a mouse) which is transgenic due to human immunoglobulin genes; or antibodies which are produced, expressed, generated or isolated in any other way in which particular immunoglobulin gene sequences (such as human immunoglobulin gene sequences) are assembled with other DNA sequences.
  • Recombinant antibodies include, for example, chimeric and humanized antibodies.
  • a “chimeric antibody” refers to an antibody in which the sequence of the variable domain derived from the germline of a mammalian species, such as a mouse, has been grafted onto the sequence of the constant domain derived from the germline of another mammalian species, such as a human.
  • a “humanized antibody” refers to an antibody in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • Antigen-binding fragments of antibodies are fragments which comprise their hypervariable domains designated CDRs (Complementary Determining Regions) or part(s) thereof encompassing the recognition site for the antigen, thereby defining antigen recognition specificity.
  • CDRs Complementary Determining Regions
  • the antibodies of the present invention include conventional antibodies made of dimers of heavy and light chains but also dimer of heavy chains only (such as camelid Hc-lgs or cartilaginous fish IgNAR).
  • each Light and Heavy chain variable domains (respectively VL and VH) of a four-chain immunoglobulin has three CDRs, designated VL-CDR1 (or LCDR1 ), VL-CDR2 (or LCDR2), VL-CDR3 (or LCDR3) and VH-CDR1 (or HCDR1 ), VH-CDR2 (or HCDR2), VH-CDR3 (or HCDR3), respectively.
  • each heavy chain variable domain has three CDRs: CDR1 , CDR2 and CDR3.
  • the skilled person is able to determine the location of the various regions/domains of antibodies by reference to the standard definitions in this respect set forth, including a reference numbering system, a reference to the numbering system of KABAT or by application of the IMGT “collier de perle” algorithm.
  • the delimitation of the regions/domains may vary from one reference system to another.
  • the regions/domains as defined in the present invention encompass sequences showing variations in length or localization of the concerned sequences within the full-length sequence of the variable domains of the antibodies, of approximately +/- 10%.
  • antigen binding fragments of a conventional antibody that contain the variable domains comprising the CDRs of said antibody encompass Fv, dsFv, scFv, Fab, Fab', F(ab')2.
  • Fv fragments consist of the VL and VH domains of an antibody associated together by hydrophobic interactions; in dsFv fragments, the VH:VL heterodimer is stabilised by a disulphide bond; in scFv fragments, the VL and VH domains are connected to one another via a flexible peptide linker thus forming a single-chain protein.
  • Fab fragments are monomeric fragments obtainable by papain digestion of an antibody; they comprise the entire L chain, and a VH-CH1 fragment of the H chain, bound together through a disulfide bond.
  • the F(ab')2 fragment can be produced by pepsin digestion of an antibody below the hinge disulfide; it comprises two Fab’ fragments, and additionally a portion of the hinge region of the immunoglobulin molecule.
  • the Fab' fragments are obtainable from F(ab')2 fragments by cutting a disulfide bond in the hinge region.
  • F(ab')2 fragments are divalent, i.e.
  • Fv a VHVL dimmer constituting the variable part of Fab
  • dsFv a VHVL dimmer constituting the variable part of Fab
  • dsFv a VHVL dimmer constituting the variable part of Fab
  • dsFv a VHVL dimmer constituting the variable part of Fab
  • dsFv a VHVL dimmer constituting the variable part of Fab
  • dsFv a VHVL dimmer constituting the variable part of Fab
  • dsFv a VHVL dimmer constituting the variable part of Fab
  • dsFv a VHVL dimmer constituting the variable part of Fab
  • dsFv a VHVL dimmer constituting the variable part of Fab
  • dsFv a VHVL dimmer constituting the variable part of Fab
  • dsFv a VHVL dimmer constituting the
  • Antigen binding fragments of a heavy-chain-only antibody that contain the variable domains comprising the CDRs of said antibody encompass single domain antibody (sdAb).
  • antigen binding fragments encompass variable region of camelid heavy-chain-only antibody (also called VHH) or variable region of cartilaginous fish heavy-chain-only antibody (also called VNAR).
  • the antibody or antigen-binding fragment thereof of the invention is a single domain antibody.
  • the single domain antibody is a camelid single domain antibody (VHH).
  • the antibody or antigen-binding fragment is a single domain antibody and comprises three complementary determining regions (CDR1 to CDR3, respectively);
  • amino acid sequence of the CDR1 is selected from the amino acid sequences of SEQ ID NOs: 23-33 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOs: 23-33;
  • amino acid sequence of the CDR2 is selected from the amino acid sequences of SEQ ID NOs: 34-44 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOs: 34-44;
  • amino acid sequence of the CDR3 is selected from the amino acid sequences of SEQ ID NOs: 45-55 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOs: 45-55.
  • a “modified antibody” corresponds to a molecule comprising an antibody or an antigenbinding fragment thereof, wherein said monoclonal antibody or functional fragment thereof is associated with a functionally different molecule.
  • a modified antibody of the invention may be either a fusion chimeric protein or a conjugate resulting from any suitable form of attachment including covalent attachment, grafting, chemical bonding with a chemical or biological group or with a molecule.
  • the invention relates to an antibody or antigen-binding fragment thereof as defined above that is modified.
  • nucleic acid hairpin comprising repeats of CNG nucleotide triplets are provided.
  • the single domain antibodies are ten times smaller than conventional antibodies and they present numerous advantages: they are well produced, they are easily engineered, they can bind to “non-conventional epitopes” or poorly accessible epitopes for conventional antibodies.
  • the single domain antibody is a VHH.
  • VHHs are variable domain of a camelid heavy-chain antibody. Indeed, in members of the family Camelidae a significant proportion of serum antibodies are homodimeric IgGs with a molecular weight of about 80 kD (Hamers-Casterman et al. 1993 Nature, 363, 446-448). These heavy chain immunoglobulins (Ig) contain three domains and their variable region is referred to as VHH, nanobodies or single domain VHH antibodies.
  • CDR region amino acid residues disclosed herein comply with the known CDR numbering criteria such as Kabat (Kabat, EA, etc. 1991 Sequences of Proteins of Immunological Interest, 5th Ed), IMGT (IMGT®: the international ImMunoGeneTics information system® http://www.imgt.org) or Chothia (Chothia C., Lesk A.M. Canonical structures for the hypervariable regions of immunoglobulins. Mol. Biol. 1987;196:901-917. doi: 10.1016/0022-2836(87)90412-8.), preferably IMGT.
  • Another aspect of the invention relates to an isolated single domain antibody, comprising three complementary determining regions (CDR1 to CDR3, respectively);
  • amino acid sequence of the CDR1 is selected from the amino acid sequences of SEQ ID NOs: 23-33 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOs: 23-33;
  • the single domain antibody also comprises four framework regions (FR1 to FR4, respectively).
  • the amino acid sequence of the CDR1 is selected from the amino acid sequences of SEQ ID NOs: 23-33; the amino acid sequence of the CDR2 is selected from the amino acid sequences of SEQ ID NOs: 34-44 and the amino acid sequence of the CDR3 is selected from the amino acid sequences of SEQ ID NOs: 45-55.
  • the single domain antibody consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 21 .
  • A02 comprises the following CDR domains:
  • CDR2 WSTSQTA (SEQ ID NO: 34);
  • this invention provides a single domain antibody that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12.
  • this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 23, a CDR2 domain having the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 34, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 45 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 45.
  • this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 23, a CDR2 domain having the amino acid sequence of SEQ ID NO: 34, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 45.
  • CDR1 to CDR3 complementary determining regions
  • the single domain antibody A02 or a variant thereof binds to a nucleic acid hairpin comprising repeats of CNG nucleotide triplets.
  • the nucleic acid hairpin comprising repeats of CNG nucleotide triplets comprises preferably at least 30 repeats of CNG nucleotide triplets.
  • the nucleic acid hairpin comprising repeats of CNG nucleotide triplets may be preferably a CTG hairpin or CAG hairpin, most preferably a CTG hairpin, such as the CTG hairpin of the nucleic acid having the nucleotide sequence SEQ ID NO: 1 .
  • the nucleic acid hairpin comprising repeats of CNG nucleotide triplets to which the single domain antibody binds may be on a 5’-3’ DNA strand or on a 3’-5’ DNA strand, preferably on the 5’-3’ DNA strand.
  • the single domain antibody B02 has the following amino acid sequence:
  • B02 comprises the following CDR domains:
  • CDR1 DSFKGYA (SEQ ID NO:24);
  • CDR2 SRGGGIS (SEQ ID NO: 35);
  • CDR3 KGGRHAAYTYPQ (SEQ ID NO: 46).
  • this invention provides a single domain antibody that consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13.
  • the nucleic acid hairpin comprising repeats of CNG nucleotide triplets may be preferably a CTG hairpin or CAG hairpin, most preferably a CTG hairpin, such as the CTG hairpin of the nucleic acid having the nucleotide sequence SEQ ID NO: 1 .
  • the nucleic acid hairpin comprising repeats of CNG nucleotide triplets to which the single domain antibody binds may be on a 5’-3’ DNA strand or on a 3’-5’ DNA strand, preferably on the 5’-3’ DNA strand.
  • the single domain antibody C04 has the following amino acid sequence: MAEVQLQASGGGFVQPGGSLRLSCAASGRGSNRTVMGWFRQAPGKEREFVSAISRGGTIAAYYAD SVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAPRGRWMAPDGPEHLQTEIYWGQGTQVTVSS (SEQ ID NO:14).
  • C04 comprises the following CDR domains:
  • CDR1 RGSNRTV (SEQ ID NO:25);
  • CDR2 RGGTIAA (SEQ ID NO: 36);
  • this invention provides a single domain antibody that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 14.
  • the nucleic acid hairpin comprising repeats of CNG nucleotide triplets to which the single domain antibody binds may be on a 5’-3’ DNA strand or on a 3’-5’ DNA strand, preferably on the 5’-3’ DNA strand.
  • C09 comprises the following CDR domains:
  • CDR2 RGENYHA (SEQ ID NO: 37);
  • this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 26 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 26, a CDR2 domain having the amino acid sequence of SEQ ID NO: 37 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 37, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 48 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 48.
  • this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 26, a CDR2 domain having the amino acid sequence of SEQ ID NO: 37, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 48.
  • CDR1 to CDR3 complementary determining regions
  • the nucleic acid hairpin comprising repeats of CNG nucleotide triplets to which the single domain antibody binds may be on a 5’-3’ DNA strand or on a 3’-5’ DNA strand, preferably on the 5’-3’ DNA strand.
  • CDR2 SGTGGAS (SEQ ID NO: 38);
  • this invention provides a single domain antibody that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 16.
  • this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 27, a CDR2 domain having the amino acid sequence of SEQ ID NO: 38 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 38, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 49 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 49.
  • this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 27, a CDR2 domain having the amino acid sequence of SEQ ID NO: 38, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 49.
  • CDR1 to CDR3 complementary determining regions
  • the single domain antibody D1 1 or a variant thereof binds to a nucleic acid hairpin comprising repeats of CNG nucleotide triplets.
  • the nucleic acid hairpin comprising repeats of CNG nucleotide triplets comprises preferably at least 30 repeats of CNG nucleotide triplets.
  • the nucleic acid hairpin comprising repeats of CNG nucleotide triplets may be preferably a CTG hairpin or CAG hairpin, most preferably a CTG hairpin, such as the CTG hairpin of the nucleic acid having the nucleotide sequence SEQ ID NO: 1 .
  • the nucleic acid hairpin comprising repeats of CNG nucleotide triplets to which the single domain antibody binds may be on a 5’-3’ DNA strand or on a 3’-5’ DNA strand, preferably on the 5’-3’ DNA strand.
  • the single domain antibody E02 has the following amino acid sequence:
  • E02 comprises the following CDR domains:
  • CDR1 RGSNRTS (SEQ ID NO:28);
  • CDR2 SLDDFKI (SEQ ID NO: 39);
  • CDR3 AASMWPGNWSPK (SEQ ID NO: 50).
  • this invention provides a single domain antibody that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 17.
  • this invention provides a single domain antibody that consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 17.
  • this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 28, a CDR2 domain having the amino acid sequence of SEQ ID NO: 39 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 39, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 50 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 50.
  • this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 28, a CDR2 domain having the amino acid sequence of SEQ ID NO: 39, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 50.
  • CDR1 to CDR3 complementary determining regions
  • the single domain antibody E02 or a variant thereof binds to a nucleic acid hairpin comprising repeats of CNG nucleotide triplets.
  • the nucleic acid hairpin comprising repeats of CNG nucleotide triplets comprises preferably at least 30 repeats of CNG nucleotide triplets.
  • the nucleic acid hairpin comprising repeats of CNG nucleotide triplets may be preferably a CTG hairpin or CAG hairpin, most preferably a CTG hairpin, such as the CTG hairpin of the nucleic acid having the nucleotide sequence SEQ ID NO: 1 .
  • the nucleic acid hairpin comprising repeats of CNG nucleotide triplets to which the single domain antibody binds may be on a 5’-3’ DNA strand or on a 3’-5’ DNA strand, preferably on the 5’-3’ DNA strand.
  • the single domain antibody F03 has the following amino acid sequence:
  • F03 comprises the following CDR domains:
  • CDR1 FTSRRSG (SEQ ID NO:29);
  • CDR2 WRASVRG (SEQ ID NO: 40);
  • CDR3 MRIRWGPQTKGDARW (SEQ ID NO: 51 ).
  • this invention provides a single domain antibody that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 18.
  • the single domain antibody F03 or a variant thereof binds to a nucleic acid hairpin comprising repeats of CNG nucleotide triplets.
  • the nucleic acid hairpin comprising repeats of CNG nucleotide triplets comprises preferably at least 30 repeats of CNG nucleotide triplets.
  • the nucleic acid hairpin comprising repeats of CNG nucleotide triplets may be preferably a CTG hairpin or CAG hairpin, most preferably a CTG hairpin, such as the CTG hairpin of the nucleic acid having the nucleotide sequence SEQ ID NO: 1 .
  • the single domain antibody F08 has the following amino acid sequence:
  • F08 comprises the following CDR domains:
  • CDR2 SSQTMGE (SEQ ID NO: 41 );
  • this invention provides a single domain antibody that consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 19.
  • this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 30, a CDR2 domain having the amino acid sequence of SEQ ID NO: 41 , and a CDR3 domain having the amino acid sequence of SEQ ID NO: 52.
  • CDR1 to CDR3 complementary determining regions
  • the single domain antibody F08 or a variant thereof binds to a nucleic acid hairpin comprising repeats of CNG nucleotide triplets.
  • the nucleic acid hairpin comprising repeats of CNG nucleotide triplets comprises preferably at least 30 repeats of CNG nucleotide triplets.
  • the nucleic acid hairpin comprising repeats of CNG nucleotide triplets may be preferably a CTG hairpin or CAG hairpin, most preferably a CTG hairpin, such as the CTG hairpin of the nucleic acid having the nucleotide sequence SEQ ID NO: 1 .
  • the nucleic acid hairpin comprising repeats of CNG nucleotide triplets to which the single domain antibody binds may be on a 5’-3’ DNA strand or on a 3’-5’ DNA strand, preferably on the 5’-3’ DNA strand.
  • G05 comprises the following CDR domains:
  • CDR1 TYSRNTI (SEQ ID NO:31 );
  • CDR2 SNPNHGL (SEQ ID NO: 42);
  • CDR3 AKLDRMSWYKQS (SEQ ID NO: 53).
  • this invention provides a single domain antibody that consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 20.
  • this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 31 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 31 , a CDR2 domain having the amino acid sequence of SEQ ID NO: 42 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 42, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 53 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 53.
  • this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 31 , a CDR2 domain having the amino acid sequence of SEQ ID NO: 42, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 53.
  • the single domain antibody G05 or a variant thereof binds to a nucleic acid hairpin comprising repeats of CNG nucleotide triplets.
  • the nucleic acid hairpin comprising repeats of CNG nucleotide triplets comprises preferably at least 30 repeats of CNG nucleotide triplets.
  • the nucleic acid hairpin comprising repeats of CNG nucleotide triplets may be preferably a CTG hairpin or CAG hairpin, most preferably a CTG hairpin, such as the CTG hairpin of the nucleic acid having the nucleotide sequence SEQ ID NO: 1 .
  • the nucleic acid hairpin comprising repeats of CNG nucleotide triplets to which the single domain antibody binds may be on a 5’-3’ DNA strand or on a 3’-5’ DNA strand, preferably on the 5’-3’ DNA strand.
  • the single domain antibody H01 has the following amino acid sequence: MAEVQLQASGGGFVQPGGSLRLSCAASGSTWASTIMGWFRQAPGKEREFVSAISYNDTNTRYYADS VKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAAKPHMRKWQTDGYWGQGTQVTVSS (SEQ ID NO: 21 ).
  • H01 comprises the following CDR domains:
  • CDR1 STWASTI (SEQ ID NO:32);
  • this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 32, a CDR2 domain having the amino acid sequence of SEQ ID NO: 43, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 54.
  • CDR1 to CDR3 complementary determining regions
  • the nucleic acid hairpin comprising repeats of CNG nucleotide triplets to which the single domain antibody binds may be on a 5’-3’ DNA strand or on a 3’-5’ DNA strand, preferably on the 5’-3’ DNA strand.
  • the single domain antibody H10 has the following amino acid sequence: MAEVQLQASGGGFVQPGGSLRLSCAASGRGSNWTDMGWFRQAPGKEREFVSAISRRDGGSPYYA DSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAKRAEWNIHYAADMQYYWGQGTQVTVSS (SEQ ID NO: 22).
  • CDR1 RGSNWTD (SEQ ID NO: 33);
  • CDR2 RRDGGSP (SEQ ID NO: 44);
  • CDR3 KRAEWNIHYAADMQY (SEQ ID NO: 55).
  • this invention provides a single domain antibody that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 22.
  • this invention provides a single domain antibody that consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 22.
  • this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 33 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 33, a CDR2 domain having the amino acid sequence of SEQ ID NO: 44 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 44, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 55 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 55.
  • this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 33, a CDR2 domain having the amino acid sequence of SEQ ID NO: 44, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 55.
  • CDR1 to CDR3 complementary determining regions
  • the single domain antibody H10 or a variant thereof binds to a nucleic acid hairpin comprising repeats of CNG nucleotide triplets.
  • the nucleic acid hairpin comprising repeats of CNG nucleotide triplets comprises preferably at least 30 repeats of CNG nucleotide triplets.
  • the nucleic acid hairpin comprising repeats of CNG nucleotide triplets may be preferably a CTG hairpin or CAG hairpin, most preferably a CTG hairpin, such as the CTG hairpin of the nucleic acid having the nucleotide sequence SEQ ID NO: 1 .
  • the nucleic acid hairpin comprising repeats of CNG nucleotide triplets to which the single domain antibody binds may be on a 5’-3’ DNA strand or on a 3’-5’ DNA strand, preferably on the 5’-3’ DNA strand.
  • the single domain antibody comprises a label.
  • the single domain antibody is covalently attached to a substrate.
  • the single domain antibody of the invention - binds to a nucleic acid hairpin comprising repeats of CNG nucleotide triplets;
  • - has a KD value inferior to 10 -9 M, preferably inferior to 10 -10 M, more preferably inferior to 1 .10 -11 M, particularly by Biacore Analysis for a nucleic acid hairpin comprising repeats of CNG nucleotide triplets, notably for a nucleic acid having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 , 6-1 1 , preferably for a nucleic acid having the nucleotide sequence SEQ ID NO: 1 ;
  • the nucleic acid hairpin may be a DNA and/or a RNA hairpin, preferably a DNA hairpin;
  • the CNG nucleotide triplet may have the nucleotide sequence selected from the group consisting of CAG, CGG, CTG, CUG and CCG, preferably selected from the group consisting of CTG, CUG and CAG, more preferably CTG and/or CUG, most preferably CTG;
  • nucleic acid hairpin is selected from the group consisting of a 4-nucleotide loop hairpin, a 3- nucleotide loop hairpin and a 7-nucleotide loop hairpin;
  • the number of CNG nucleotide triplets that are repeated is of at least 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52 units.
  • the antibody or antigen-binding fragment thereof as described in Section A above is an single domain antibody as defined above.
  • the present invention also relates to a complex comprising: the antibody or antigen-binding fragment as described in Section A above or the single domain antibody as described in Section B above and a nucleic acid hairpin comprising repeats of CNG nucleotide triplets as defined above.
  • Modified antibody shall be taken to mean a molecule comprising an antibody or an antigen-binding fragment thereof or a single domain antibody, wherein said antibody or functional fragment thereof or single domain antibody is associated with a functionally different molecule, chemical bonding with a chemical or biological group or with a molecule.
  • the present invention also relates to a method comprising a step of contacting the antibody or antigenbinding fragment as described in Section A above or the single domain antibody as described in Section B above with molecule(s) comprising a nucleic acid hairpin comprising repeats of CNG nucleotide triplets thereby forming such complex.
  • molecule(s) comprising a nucleic acid hairpin comprising repeats of CNG nucleotide triplets thereby forming such complex.
  • the invention relates to a vector comprising the polynucleotide as defined above.
  • the vectors are constructed and introduced into host cells by conventional recombinant DNA and genetic engineering methods which are known perse. Numerous vectors into which a polynucleotide of interest may be inserted in order to introduce it and to maintain it in a host cell are known per se; the choice of an appropriate vector depends on the use envisaged for this vector (for example replication of the sequence of interest, expression of this sequence), and on the nature of the host cell.
  • a recombinant cell comprising the polynucleotide or the vector as defined above is provided.
  • Suitable host cells include prokaryotic or eukaryotic cells, and also include but are not limited to bacteria, yeast cells, fungi cells, plant cells, and animal cells such as insect cells and mammalian cells, e.g., murine, rat, rabbit, macaque or human.
  • the methods may comprise culturing the recombinant cell comprising the polynucleotide that encodes the antibody or the antigen-binding fragment or the single domain antibody of the invention under conditions sufficient for the production thereof.
  • nucleic acid hairpin comprising repeats of CNG nucleotide triplets may be any of nucleic acid hairpin comprising repeats of CNG nucleotide triplets described in Section A above.
  • the methods comprise an ELISA, lateral flow immunoassay, bead-based immunoassay, or multiplex bead-based immunoassay.
  • the biological sample is obtained from a subject, preferably from a subject suspected to have a trinucleotide expansion disorder selected from the group consisting of myotonic dystrophy type 1 , spinocerebrallar ataxia 12, Huntington’s disease-like 2, spinal and bulbar muscular atrophy, Huntington’s disease, dentatorubral-pallidouysian atrophy, spinocerebrellar ataxia 1 , spinocerebrellar ataxia 2, spinocerebrellar ataxia 3, spinocerebrellar ataxia 6, spinocerebrellar ataxia 7, spinocerebrellar ataxia 17, fragile X syndrome, X tremor and/or ataxia syndrome and X-linked mental retardation.
  • a trinucleotide expansion disorder selected from the group consisting of myotonic dystrophy type 1 , spinocerebrallar ataxia 12, Huntington’s disease-like 2, spinal and bulbar muscular atrophy, Huntington’s disease,
  • Table 2 summarized the trinucleotide expansion disorder induced by repeats of CNG nucleotide triplets, their corresponding CNG nucleotide triplet and the normal average number of CNG triplets that are repeated. Table 2 - Trinucleotide expansion disorder induced by repeats of CNG nucleotide triplets
  • DM1 myotonic dystrophy type 1
  • DM1 is a dominant autosomal multisystemic disorder including myotonia, progressive weakness and wasting of muscle and extramuscular symptoms such as cataracts, cardiac conduction defects, testicular atrophy, endocrine and cognitive dysfunction. It is caused by an expansion of a CTG trinucleotide repeat in the 3’ untranslated region (UTR) or the DM protein kinase gene (DMPK) located on chromosome. In normal individuals, the CTG repeat is polymorphic and varies between 5 and 37 triplets, whereas 50 to 4 000 CTG repeats are observed in affected patients.
  • UTR untranslated region
  • DMPK DM protein kinase gene
  • the size of the repeat which usually increases from generation to generation, is generally correlated with the clinical severity and the age of the onset, providing a molecular basis of the phenomenon observed in DM1 families. Analyses of family pedigrees have shown that >90% of transmission result in the expansion of repeats.
  • the antibody or antigen-binding fragment and/or the single domain antibody preferably binds to a hairpin of an 5’-3’ DNA strand comprising repeats of CTG nucleotide triplets.
  • the subject is suspected to have a trinucleotide expansion disorder selected from the group consisting of spinocerebrallar ataxia 12, Huntington’s disease-like 2, spinal and bulbar muscular atrophy, Huntington’s disease, dentatorubral-pallidouysian atrophy, spinocerebrellar ataxia 1 , spinocerebrellar ataxia 2, spinocerebrellar ataxia 3, spinocerebrellar ataxia 6, spinocerebrellar ataxia 7 and spinocerebrellar ataxia 17.
  • the antibody or antigen-binding fragment and/or the single domain antibody binds preferably to a hairpin of an 5’-3’ DNA strand comprising repeats of CAG nucleotide triplets.
  • the subject is suspected to have a X-linked mental retardation.
  • the antibody or antigen-binding fragment and/or the single domain antibody binds preferably to a hairpin of an 5’-3’ DNA strand comprising repeats of CCG nucleotide triplets.
  • the antibody or antigen-binding fragment and/or the single domain antibody binds to the CNG nucleotide triplets of the complementary strand (3’-5’ strand).
  • the subject is suspected to have a myotonic dystrophy type 1 and the antibody or antigen-binding fragment and/or the single domain antibody preferably binds to a hairpin of an 3’-5’ DNA strand comprising repeats of CAG nucleotide triplets.
  • the subject is suspected to have a trinucleotide expansion disorder selected from the group consisting of spinocerebrallar ataxia 12, Huntington’s disease-like 2, spinal and bulbar muscular atrophy, Huntington’s disease, dentatorubral-pallidouysian atrophy, spinocerebrellar ataxia 1 , spinocerebrellar ataxia 2, spinocerebrellar ataxia 3, spinocerebrellar ataxia 6, spinocerebrellar ataxia 7 and spinocerebrellar ataxia 17 and the antibody or antigen-binding fragment and/or the single domain antibody binds preferably to a hairpin of an 3’-5’ DNA strand comprising repeats of CTG nucleotide triplets.
  • a trinucleotide expansion disorder selected from the group consisting of spinocerebrallar ataxia 12, Huntington’s disease-like 2, spinal and bulbar muscular atrophy, Huntington’s disease, dentatorubral-pal
  • the subject is suspected to have a X-linked mental retardation and the antibody or antigen-binding fragment and/or the single domain antibody binds preferably to a hairpin of an 3’-5’ DNA strand comprising repeats of CGG nucleotide triplets.
  • the method comprises comparing the results obtained with a subject sample to positive and negative controls.
  • the antibody or antigen-binding fragment and/or the single domain antibody is labeled and the step of visualizing the antigen-antibody complexes formed comprises visualizing the label.
  • Preferred labels include suitable enzymes such as horseradish peroxidase, alkaline phosphatase, betagalactosidase, luciferase or acetylcholinesterase; members of a binding pair that are capable of forming complexes such as streptavidin/biotin, avidin/biotin or an antigen/antibody complex including, for example, rabbit IgG and anti-rabbit IgG; fluorophores such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, tetramethyl rhodamine, eosin, green fluorescent protein, erythrosin, coumarin, methyl coumarin, pyrene, malachite green, stilbene, lucifer yellow, Cascade Blue, Texas
  • the antigen-antibody complexes are visualized by EIA, ELISA, RIA, or by immunofluorescence.
  • the method for detection and/or quantification may comprise: providing a first antibody attached to a solid support; providing a sample; contacting the solid support with the sample under conditions sufficient to allow formation of first antigenantibody complexes between the nucleic acid hairpin and the first antibody to the solid support; contacting the solid support with a second antibody, under conditions sufficient to allow formation of second antigen-antibody complexes between nucleic acid and the second antibody; and visualizing the second antigen-antibody complexes; the first antibody and/or the second antibody being an antibody or an antigen-binding fragment and/or a single domain antibody as defined above.
  • the second antibody comprises a label selected from a chemiluminescent label, an enzyme label, a fluorescence label, and a radioactive (e.g., iodine) label.
  • a label selected from a chemiluminescent label, an enzyme label, a fluorescence label, and a radioactive (e.g., iodine) label.
  • an in vitro method for diagnosing a trinucleotide expansion disorder selected from the group consisting of myotonic dystrophy type 1 , spinocerebrallar ataxia 12, Huntington’s disease-like 2, spinal and bulbar muscular atrophy, Huntington’s disease, dentatorubral-pallidouysian atrophy, spinocerebrellar ataxia 1 , spinocerebrellar ataxia 2, spinocerebrellar ataxia 3, spinocerebrellar ataxia 6, spinocerebrellar ataxia 7, spinocerebrellar ataxia 17, fragile X syndrome, X tremor ataxia syndrome and X-linked mental retardation wherein the antibody or antigen-binding fragment thereof as defined above or the single domain antibody as defined above is used for the detection and/or quantification of nucleic acid hairpin comprising repeats of CNG nucleotide triplets as defined above in a sample previously obtained from a subject.
  • the detection and/or quantification may be carried out with the method for detecting and/or quantifying as described above.
  • nucleic acid hairpin comprising repeats of CNG nucleotide triplets in the biological sample is indicative that the subject has a trinucleotide expansion disorder.
  • a trinucleotide expansion disorder selected from the group consisting of spinocerebrallar ataxia 12, Huntington’s disease-like 2, spinal and bulbar muscular atrophy, Huntington’s disease, dentatorubral-pallidouysian atrophy, spinocerebrellar ataxia 1 , spinocerebrellar ataxia 2, spinocerebrellar ataxia 3, spinocerebrellar ataxia 6, spinocerebrellar ataxia 7 and spinocerebrellar ataxia 17;
  • a trinucleotide expansion disorder selected from the group consisting of spinocerebrallar ataxia 12, Huntington’s disease-like 2, spinal and bulbar muscular atrophy, Huntington’s disease, dentatorubral-pallidouysian atrophy, spinocerebrellar ataxia 1 , spinocerebrellar ataxia 2, spinocerebrellar ataxia 3, spinocerebrellar ataxia 6, spinocerebrellar ataxia 7 and spinocerebrellar ataxia 17;
  • DNA strand comprising repeats of CCG nucleotide triplets is indicative that the subject has a trinucleotide expansion disorder being fragile X syndrome, X tremor and/or ataxia syndrome; and/or
  • the nucleic acid hairpin is quantified and the quantified nucleic acid hairpin are compared with a reference value and it is concluded that the subject has a trinucleotide expansion disorder as defined above when the quantified nucleic acid hairpin is higher than the reference value.
  • the reference value may be a sample from a healthy subject i.e. a subject who does not suffer from a trinucleotide triplet disorder.
  • the present invention also relates to the use of the antibody or antigen-binding fragment thereof as defined above or the single domain antibody as defined above in an in vitro diagnostic test of a trinucleotide expansion disorder selected from the group consisting of myotonic dystrophy type 1 , spinocerebrallar ataxia 12, Huntington’s disease-like 2, spinal and bulbar muscular atrophy, Huntington’s disease, dentatorubral-pallidouysian atrophy, spinocerebrellar ataxia 1 , spinocerebrellar ataxia 2, spinocerebrellar ataxia 3, spinocerebrellar ataxia 6, spinocerebrellar ataxia 7, spinocerebrellar ataxia 17, fragile X syndrome, X tremor, ataxia syndrome and X-linked mental retardation.
  • a trinucleotide expansion disorder selected from the group consisting of myotonic dystrophy type 1 , spinocerebrallar ataxia 12, Huntington’s
  • the invention in another aspect, relates to a kit comprising:
  • nucleic acid molecule coding for said antibody, antigen-binding fragment or single domain antibody
  • an antibody or antigen-binding fragment thereof or a single domain antibody as defined above that has a KD value inferior to 10 -9 M, preferably inferior to 10 -10 M, more preferably inferior to 1 .10 -11 M, particularly by Biacore Analysis for a nucleic acid hairpin comprising repeats of CNG nucleotide triplets, notably for a nucleic acid having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 , 6-1 1 , preferably for a nucleic acid having the nucleotide sequence SEQ ID NO: 1 ; and/or
  • amino acid sequence of the CDR1 is selected from the amino acid sequences of SEQ ID NOs: 23-33 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOs: 23-33;
  • an single domain antibody as defined above comprising an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-22, preferably with SEQ ID NOs: 17- 19 and 21 , more preferably with SEQ ID NO: 21 ; and/or
  • the antibody, the antigen-binding fragment thereof or the single domain antibody of the present invention can be provided in a kit, i.e., a packaged combination of reagents.
  • the kit may comprise one or more recipients (such as vials, ampoules, containers, syringes, bottles, bags) of any appropriate shape, size and material containing the antibody, the antigen-binding fragment thereof or the single domain antibody of the present invention.
  • the kit may additionally contain directions for use (e.g. in the form of a leaflet or instruction manual).
  • Figure 2 shows the migration of the (CTG)so oligonucleotide the GC clamp and a non structured (NS)
  • Figure 4 shows the spot test on minimal medium without histidine (SC-His) of the one-hybrid experiments.
  • FIG. 5 shows dot blots with purified camelid single domain antibodies (VHH) Top Nylon membranes on which 100 ng of the synthetic CTG hairpin (CTG) and of the GC clamp (GC) were spotted were incubated with one of the four VHH (E02, F03, F08 and H01 ). Three pictures were taken at different exposure times (labeled 1 , 2 or 3). Bottom: H01 was incubated with a membrane on which different amounts of the CTG hairpin (CTG), the GC clamp (GC) or a synthetic CAG hairpin (CAG) were spotted. Quantification of the positive signal detected with the CTG hairpin is shown to the right.
  • CTG hairpin CTG hairpin
  • GC GC clamp
  • CAG synthetic CAG hairpin
  • Figure 6 shows dot blots where different concentration of plasmids containing 98 or 255 CTG triplets were spotted and incubated with VHH H01 . Signal quantifications are shown to the right.
  • Figure 7 shows dot blots where heat denatured or non-heat denatured plasmids containing 98 CTG triplets, 255 CTG triplets or no triplets were spotted and incubated with VHH H01 .
  • Figure 8 shows dot blots where yeast total genomic DNA containing CTG trinucleotide repeats of different lengths (50, 100, 160 or 280 triplets), as well as human total genomic DNA of a DM1 patient were spotted and incubated with VHH H01 . Signal quantifications are shown to the right.
  • Plasmid pTRI148 was linearized with Hpal in the ADE2 gene and transformed in yeast strain FYEF01 - 7C, isogenic to FYAT01 carrying the ade2-opal mutation 1 (Table 4). The resulting [Ade+] transformants were analyzed by Southern blot, to check correct integration at the ADE2 locus as well as CTG repeat tract length. One clone, called GFY21 1 , was selected and crossed to all FYEF01 -9C transformants containing the pP9-VHH plasmids (pTRI136 to 146).
  • Hybrigenics synthetic hsd2Ab VHH library of 3.10 9 clones was expressed at the surface of M13 phage.
  • Hybrigenics’ Phage Display allowed to select VHHs recognizing the non-adsorbed antigen in a native form. A total of 90 VHHs were picked and analyzed after three rounds of Phage Display for binding to CTG hairpin, and 83 of these VHHs were positive in non-adsorbed phage ELISA. After sequencing of these 83 clones, 27 different clones were validated and positive in non-adsorbed phage ELISA.
  • HEK293 cells were plated in 6 well plates at 700 000 cells per well 2 days before transfection. Cells were transfected with 2.5 gg of Minibody DNA and 7.5 gL XtremeGene HP DNA. 24h post transfection the medium was replaced by medium without serum. After 6 days, the medium was collected, and the production was checked by Western blot with an anti-rabbit antibody coupled to HRP. The expected size for VHH-Fc is around 42 kDa.
  • VHH H01 Production and purification of VHH H01 was performed at the Institut Pasteur protein production facility, as follows.
  • the VHH H01 was expressed in Expi293F cells at 37 °C and 8% CO2 (Expi293, ThermoFisher Scientific). Cell culture supernatants were collected 4 days post transfection and purified by protein G chromatography (Cytiva). Washing step was done with DPBS 1 X pH 7.2 and VHH were eluted with Glycine 0.1 M pH 2.5 in wells containing TRIS 1 M pH 8.0.
  • Blocking was performed for one hour in TBST (20 mM Tris pH 7.5, 150 mM NaCI, 0.1 % Tween 20) supplemented with 5% dry milk.
  • VHH were diluted 1 /50th to 1 /500th in preliminary tests in TBST + 5% dry milk, and incubated overnight at 4°C.
  • Purified H01 VHH was diluted 1/1000th in TBST + 5% dry milk in subsequent experiments.
  • the secondary anti-rabbit antibody (Thermo Scientific) was diluted 1 /2000th, incubated for one hour in TBST + 5% dry milk at room temperature, and revealed by ECL Prime Western Blotting (Amersham). Signals were revealed on a Bio-Rad Chemidoc and quantifications performed using the Image Lab software (version 5.2). on HiTrap column
  • Plasmidic secondary structures were captured on an Akta start FPLC (Cytiva) at a flow rate of 1 mL/min for all buffers.
  • a 1 mL HiTrap protein G (Cytiva) was first equilibrated with 10 mL 20 mM sodium phosphate buffer (pH 7.0).
  • the VHH (20 gg) was loaded in 10 mL of the same buffer.
  • the column was saturated with 15 mL 1 % BSA in 20 mM sodium phosphate buffer.
  • the pRW3222 plasmid (Fabre et al.
  • T10N50 (10 mM Tris pH 8.0, 50 mM NaCI).
  • 1 mL fractions are collected until the end of the experiment (ca. 30 fractions).
  • the column is washed with 10 mL of the same buffer.
  • Elution of plasmidic DNA bound to the VHH is performed with 10 mL T10N200 (10 mM Tris pH 8.0, 200 mM NaCI, 10 mL).
  • the column was finally washed with 5 mL of T10N50.
  • a 130 nt DNA oligonucleotide was designed in such a way that its extremities are complementary and form a "GC clamp", that tolerate formation of an imperfect hairpin in only one conformation.
  • the GC clamp itself is synthesized to be used as a negative control in all experiments.
  • Both oligonucleotides are linked to a biotin at their 5' ends. Structures, free energy and Tm of both molecules were calculated using mFold version 3.6 ( Figure 1 ).
  • HIS3 gene As reporter was carried out.
  • the HIS3 gene under the control of a minimal promoter fused to a CTG trinucleotide repeat was integrated in the yeast genome.
  • the 1 1 VHH fused to the Gal4p activating domain were expressed in this yeast strain. If the fusion protein binds to the CTG repeat it will activate transcription of the HIS3 gene and cells will grow on minimal medium without histidine (SC-His).
  • SC-His minimal medium without histidine
  • a [His+] strain was used as positive control and the plasmid containing the GAL4 activating sequence but no VHH (Empty) allowed to determine the baseline of growth on SC-His. The score represents the difference of growth between each VHH-Gal4 fusion and the Empty vector.
  • VHH obtained a negative score, due to a worse growth than the Empty control.
  • Yeast plates were incubated at two different temperatures, 30°C which is the physiological temperature for budding yeast and 20°C. As expected, growth was slower at lower temperature, but it did not change the general outcome.
  • An one-hybrid experiment using a GFP reporter under the control of the minimal HIS3 promoter was carried out.
  • the minimal HIS3 promoter was fused to four different microsatellites: (CTG)so, (CAG)so, (GAA)so or (CGG)so.
  • CGG microsatellites

Abstract

The present invention provides an antibody or antigen-binding fragment thereof that specifically binds to a nucleic acid hairpin comprising repeats of CNG nucleotide triplets and their uses.

Description

ANTI-CNG REPEATS ANTIBODIES AND THEIR DIAGNOSIS APPLICATIONS
FIELD OF THE INVENTION
The present invention relates to antibody or antigen-binding fragment thereof that specifically binds to a nucleic acid molecule and their use in diagnostic methods.
BACKGROUND OF THE INVENTION
DNA tandem repeats occur frequently in double-stranded DNAs of eukaryotic genomes, more particularly of the human genome. DNA tandem repeat units of 2, 3, 4, 5 or even more nucleotides can be observed in a genome at different frequencies and locations (exons, introns, intergenic regions). DNA tandem repeats are prone to recombination and/or random integration events, and are considered to be at the center of species evolution.
However, expansion in the length of a DNA tandem repeat can result in deleterious effects on gene function, leading to disease or disorder (Brown, L.Y. and Brown, S.A. (2004) Alanine tracts: the expanding story of human illness and trinucleotide repeats. Trends Genet. 20, 1 -58, Gatchel, J.R. and Zoghbi, H.Y. (2005) Diseases of unstable repeat expansion: mechanisms and common principles. Nat. Rev. Genet. 6, 743-755, Orr, H.T. and Zoghbi, H.Y. (2007) Trinucleotide repeat disorders. Annu. Rev. Neurosci. 30, 575-621 ).
The most common of these diseases are trinucleotide expansion disorders (TREDs) induced by the expansion of trinucleotide repeats (TNRs). One major subset of pathogenic TNRs is the CNG repeats where N represents one of the four natural nucleotides (A. Kiliszesk et al., Nucleic Acids Research, 2014, Vol. 42, No. 13). CNG repeats are associated with at least 15 diseases such as myotonic dystrophy (DM), Huntington’s disease (HD), severe spinocerebellar ataxias and fragile X syndrome (S. Mirkin, Expandable DNA repeats and human disease, Nature 2007 Jun 21 ;447(7147):932-40).
At the present time, diagnostic tests for these diseases rely on PCR assays amplifying the expanded CNG region. However, these tests are tedious and need a special equipment and PCR protocol to be efficient, since expanded CNG repeats are impossible to amplify with standard PCR conditions, given the repeated and structured nature of these sequences. Indeed, expanded CNG trinucleotide repeats formed secondary structures (hairpins) on DNA. Thus, very large expansions may only be analyzed by Southern blot.
M. Tam et al. (J. Mol. Biol (2003 332, 585-600)) have studied the probing of models of slipped DNAs formed by disease associated trinucleotide repeats (CTG)x.(CAG)y with different anti-DNA antibodies raised against cruciform DNA (2D3), Z-DNA (Z22), individual bases (Ra492b, AD8, pGoat12B, DC4.1 and Goat4H) and native B-DNA (2C10). They have shown that the anti-cruciform antibody 2D3 bound both models of homoduplex slipped structures S-DNAs and heteroduplex slipped intermediate SI-DNAs. However, 2D3 had originally been raised against a stable cruciform DNA structure containing the 27- base pair palindrome of the SV40 origin of replication on one strand and an unrelated 26-base pair palindrome on the complementary strand and not against trinucleotide CNG repeats (Frappier L, et al. 1989). Despite many studies (Pearson CE et al. (1996) Biochemistry 35: 5041-5053. Liu G, et al. (2010) Nat Chem Biol 6: 652-659, J Biol Chem 264: 334-341 , Axford M. et al, PLoS Genet. 2013 Dec; 9(12)) the specific target of 2D3 on slipped DNA remains unknown. Therefore, given its established biochemical properties, 2D3 most probably binds to a CTG/CAG cruciform structure, but not specifically to a CTG or CAG imperfect hairpin (Axford M. et al, PLoS Genet. 2013 Dec; 9(12)).
There remains therefore a significant need in the art for new and improved tools and methods for detecting trinucleotide repeat expansions and diagnosing TREDs.
SUMMARY OF THE INVENTION
Now, the inventors have found antibodies and antigen-binding fragment thereof that specifically bind to repeats of CNG nucleotide triplets forming a hairpin secondary structure, thereby enabling to diagnose TRED induced by the expansion of CNG repeats.
Such antigen based assays are simple, fast and less expensive than PCR assays. Moreover, given that the antibodies and antigen-binding fragment thereof of the invention binds specifically to hairpin, they keep their sensitivity and specificity even in sample with a high number of CNG repeats.
A subject of the present invention is therefore an antibody or antigen-binding fragment thereof that specifically binds to a nucleic acid hairpin comprising repeats of CNG nucleotide triplets.
The present invention also relates to an isolated single domain antibody, comprising three complementary determining regions (CDR1 to CDR3, respectively);
- wherein the amino acid sequence of the CDR1 is selected from the amino acid sequences of SEQ ID NOs: 23-33 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOs: 23-33;
- wherein the amino acid sequence of the CDR2 is selected from the amino acid sequences of SEQ ID NOs: 34-44 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOs: 34-44; and
- wherein the amino acid sequence of the CDR3 is selected from the amino acid sequences of SEQ ID NOs: 45-55 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOs: 45-55.
Related aspects of the invention are:
- an isolated polynucleotide coding for the antibody or antigen-binding fragment of the invention and/or the single domain antibody of the invention;
- a vector comprising the polynucleotide coding for the antibody or antigen-binding fragment of the invention and/or the isolated single domain antibody of the invention;
- a cell containing the polynucleotide coding for the antibody or antigen-binding fragment of the invention and/or an isolated single domain antibody of the invention or the vector comprising the same;
- a method of producing the antibody or antigen-binding fragment of the invention and/or an isolated single domain antibody of the invention, comprising culturing the recombinant cell comprising the same under conditions sufficient for production of the antibody or antigen-binding fragment of the invention and/or the single domain antibody of the invention.
The present invention also relates to:
- a complex comprising: the antibody or antigen-binding fragment thereof of the invention or the isolated single domain antibody of the invention and a nucleic acid hairpin comprising repeats of CNG nucleotide triplets;
- the use of such complex;
- a method comprising a step of contacting the antibody or antigen-binding fragment thereof of the invention or the isolated single domain antibody of the invention with molecule(s) comprising a nucleic acid hairpin comprising repeats of CNG nucleotide triplets thereby forming a complex.
Another aspect of the invention is a method for detection of nucleic acid hairpin comprising repeats of CNG nucleotide triplets in a sample, comprising:
- providing the antibody or antigen-binding fragment of the invention and/or the single domain antibody of the invention;
- providing a sample;
- contacting the antibody or antigen-binding fragment of the invention and/or the single domain antibody of the invention with the sample; and
- visualizing the antigen-antibody complexes formed.
The present invention also relates to a kit comprising the antibody or antigen-binding fragment of the invention and/or the single domain antibody of the invention.
DETAILED DESCRIPTION OF THE INVENTION
A. ANTI-CNG REPEATS ANTIBODIES
In an aspect, the invention relates to an antibody or antigen-binding fragment thereof that specifically binds to a nucleic acid hairpin comprising repeats of CNG nucleotide triplets.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one skilled in the relevant art.
The nucleic acid hairpin, to which the antibody or antigen-binding fragment thereof of the application binds or specifically binds, is a hairpin from a nucleic acid molecule which comprises repeats of CNG nucleotide triplets.
Said nucleic acid molecule may be a RNA or a DNA molecule. Advantageously, the nucleic acid molecule is a DNA molecule. The DNA molecule can e.g., be a double-stranded DNA molecule or a strand of a double-stranded DNA molecule, more particularly a double-stranded DNA molecule, more particularly a chromosomal double-stranded DNA molecule. Said double-stranded DNA molecule can e.g., be a gene, more particularly a eukaryotic gene, more particularly a non-mammalian eukaryotic gene {e.g., a yeast gene) or a non-human mammalian gene {e.g., a rodent gene, a rat gene, a mouse gene, a pig gene, a rabbit gene) or a human gene.
Advantageously, the nucleic acid hairpin is a DNA and/or a RNA hairpin, preferably a DNA hairpin.
A CNG nucleotide triplet is a nucleic acid unit comprising 3 nucleotides and having the nucleotide acid sequence CNG wherein N is as defined in the sequence listing standards ST. 25 and ST. 26. Typically, N is selected from the group consisting of A, T, U, G and C, preferably wherein N is T/U or A, more preferably wherein N is T or U, most preferably where N is T. The CNG nucleotide triplet has the nucleotide sequence selected from the group consisting of CAG, CTG, CUG, CGG and CCG. The CNG nucleotide triplet is preferably selected from the group consisting of CTG, CUG and CAG, more preferably CTG and/or CUG, most preferably CTG.
Repeats of CNG nucleotide triplets (also called hereinafter CNG repeats) fold into hairpins in which the double stranded stems have non canonical N-N pairs that are stabilized by the sturdy C-G and G-C pairs (A. Kiliszesk et al., Nucleic Acids Research, 2014, Vol. 42, No. 13).
Hairpin also called hairpin loop or stem-loop refers to the nucleic acid secondary structure.
The 4-nt loops are present in all (CNG)n hairpins with an even number of repeats, the 7-nt loop occurs in the (CAG)n and (CCG)n hairpins with an odd number of repeats and the 3-nt loops are present in the (CUG)n and (CGG)n hairpins with an odd number of repeats (Sobczak,K., de Mezer,M., Michlewski,G., Krol, J. and Krzyzosiak.W.J. (2003) RNA structure of trinucleotide repeats associated with human neurological diseases. Nucleic Acids Res., 31 , 5469-5482).
In an embodiment, the nucleic acid hairpin is a 4-nucleotide loop hairpin.
In an embodiment, the nucleic acid hairpin is a 3-nucleotide loop hairpin, preferably a 3-nucleotide loop hairpin comprising repeats of CUG, repeats of CTG or repeats of CGG, more preferably repeats of CUG or repeats of CTG, most preferably repeats of CTG.
In another embodiment, the nucleic acid hairpin is a 7-nucleotide loop hairpin, preferably a 7-nucleotide loop hairpin comprising repeats of CAG or CCG, more preferably repeats of CAG.
When they relate to a nucleic acid, the phrases "repeat", "triplet(s)”, "sequence unit(s)" and "unit(s)" (or equivalent or similar phrases) are given their respective general meaning of the field of nucleic acids. For example, the nucleic acid:
GCG GGC GGG GCG GGG GCA CG CTG CTG CTG CTG CTG CTG CTG CTG CTG CTG CTG CTG CTG CTG CTG CTG CTG CTG CTG CTG CTG CTG CTG CTG CTG CTG CTG CTG CTG CTG CGT GCC CCC GCC CCG CCC GC [SEQ ID NO: 1 ] contains 30 repeats of the CTG nucleotide triplet (said 30 copies are shown underlined in SEQ ID NO: 1 above).
The abbreviation (CNG)i where i is a number means i repeats of the sequence CNG.
Within a nucleic acid hairpin, the copies of the CNG nucleotide triplet are adjacent to each other. They can either be spaced apart from each other by only a few nucleotides, e.g., by less than 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2 nucleotides, or can be directly adjacent to each other. According to an aspect of the application, said copies of CNG nucleotide triplet are spaced apart from each other by only a few nucleotides, e.g., by less than 6, 5, 4, 3, 2 nucleotides, or are directly adjacent to each other. In a preferred embodiment, said copies of DNA sequence unit are directly adjacent to each other. For example, in the above-mentioned nucleic acid of SEQ ID NO: 1 , said copies of CNG nucleotide triplets (i.e., the copies of the CTG triplet) are directly adjacent to each other.
In the application, a CNG repeat is a direct repeat, i.e., it is not an inverted repeat: the order in which the nucleotides are contained in one triplet is conserved throughout the CNG repeats.
In the application, a CNG repeat is a repeat of nucleotide triplets having the same CNG sequence.
The number of CNG nucleotide triplets that are repeated is of at least 3. According to an aspect of the application, said number is of at least 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52., 55, 100, 150, 200, 250, 300, 350, 500 or 1000.
In an embodiment, the number of CNG nucleotide triplets that are repeated is at least 5, 10, 50 or 55. According to an aspect of the application, said number is of between 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 and 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 100, 150, 200, 250, 300, 350, 500, 1000, 1500 or 2500. In an embodiment, the number of CNG nucleotide triplets that are repeated is between 5, 10 or 25 and 250, 300, 400 or 500.
As used herein, the term "epitope" means the part of an antigen to which the antibody binds. The epitopes of protein antigens can be divided into two categories, conformational epitope and linear epitope. A conformational epitope corresponds to discontinuous sections of the antigen's amino acid sequence. A linear epitope corresponds to a continuous sequence of amino acids from the antigen. Advantageously, given that the antibody or the antigen-binding fragment thereof binds to the hairpin secondary structure, the epitope is a conformational epitope. The epitope may also be a linear epitope. As used herein, the term “anti-CNG repeats antibody” refers to an antibody which specifically binds to a nucleic acid hairpin comprising repeats of CNG nucleotide triplets, in particular to a nucleic acid hairpin comprising repeats of CNG nucleotide triplets as defined above.
Mutatis mutandis, the term “anti-CTG repeats antibody” refers to an antibody which specifically binds to a nucleic acid hairpin comprising repeats of CTG nucleotide triplets; the term “anti-CAG repeats antibody” refers to an antibody which specifically binds to a nucleic acid hairpin comprising repeats of CAG nucleotide triplets etc.
The specific binding between the antibody or antigen-binding fragment thereof of the invention and the epitope (or the region comprising the epitope) implies that the antibody exhibits appreciable affinity for the epitope (or the region comprising the epitope) on a particular antigen (here a nucleic acid hairpin comprising repeats of CNG nucleotide triplets as defined above). “Appreciable affinity” includes binding with an affinity of about 10-9 M (KD) or stronger. Preferably, binding is considered specific when the binding affinity is between 10-9 M and 10-12 M, optionally between 10-9 M and 10-10 M, in particular 10-10 M. Whether a binding domain specifically reacts with or binds to a target can be tested readily by, inter alia, comparing the reaction of said binding domain with a target antigen with the reaction of said binding domain with antigens other than the target antigen.
The affinity can be determined by various methods well known from the one skilled in the art. These methods include, but are not limited to, Biacore Analysis, Blitz analysis and Scatchard plot. Advantageously, the affinity is determined in non-denaturing conditions.
In an embodiment, the invention relates to an anti-CNG repeats antibody or antigen-binding fragment thereof as defined above that has a KD value inferior to 10-9 M, preferably inferior to 10-10 M for a nucleic acid hairpin comprising repeats of CNG nucleotide triplets, more preferably inferior to 1 .10-11 M, particularly by Biacore Analysis.
The nucleic acid having the nucleotide sequence selected from the group consisting of SEQ ID NO: 1 - 3 and 60 (respectively (CTG)so hairpin with GC clamp, (CAG)so hairpin with GC clamp, (CGG)so hairpin with GC clamp and (CCG)so hairpin with GC clamp) or nucleic acid comprising the nucleotide sequence selected from the group consisting of SEQ ID NO: 6-1 1 (respectively (CTG)so , (CTG)98, (CTG)ioo, (CTG) o, (CTG)255 and (CTG)2so) may be used as positive control to identify anti-CNG repeats antibody or antigen-binding fragment thereof.
An aspect of the invention is the nucleic acid having the nucleotide sequence selected from the group consisting of SEQ ID NO: 1 -3 and 60. These 130 nt DNA nucleic acid were designed in such a way that their extremities are complementary and form a "GC clamp", that tolerate formation of a hairpin in only one conformation.
The invention also provides the nucleic acid having the nucleotide sequence SEQ ID NO: 4 or 5.
In an embodiment, the anti-CNG repeats antibody or antigen-binding fragment thereof as defined above has a KD value inferior to 200x10-9 M, preferably inferior to 10-9 M, more preferably inferior to 10-10 M for a nucleic acid having a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 - 3 or a nucleic acid comprising the nucleotide sequence selected from the group consisting of SEQ ID NO:6-1 1 , more preferably inferior to 1 .10-11 M, particularly by Biacore Analysis.
In an embodiment, the anti-CTG repeats antibody or antigen-binding fragment thereof as defined above has a KD value inferior to 200x10-9 M, preferably inferior to 10-9 M, more preferably inferior to 10-10 M for a nucleic acid having a nucleotide sequence SEQ ID NO: 1 or nucleic acid comprising the nucleotide sequence selected from the group consisting of 6-1 1 , more preferably inferior to 1 .10 11 M, particularly by Biacore Analysis.
In an embodiment, the antibody, antigen-binding fragment thereof and/or the single domain antibody binds to the nucleic acid having the nucleotide sequence SEQ ID NO: 1 with a nanomolar KD.
In a preferred embodiment, the anti-CTG repeats antibody or antigen-binding fragment thereof as defined above has a KD value inferior to 200x10-9 M, preferably inferior to 10-9 M, more preferably inferior to I O 10 M for a nucleic acid having a nucleotide sequence SEQ ID NO: 1 , most preferably inferior to 1.10'11 M, particularly by Biacore Analysis.
In an embodiment, the invention relates to an anti-CAG repeats antibody or antigen-binding fragment thereof as defined above that has a KD value inferior to 200x10-9 M, preferably inferior to 10-9 M, more preferably inferior to 10-10 M for a nucleic acid hairpin comprising repeats of CAG nucleotide triplets, most preferably inferior to 1 .10-11 M, particularly by Biacore Analysis.
In an embodiment, the anti-CAG repeats antibody or antigen-binding fragment thereof as defined above has a KD value inferior to 200x10-9 M, preferably inferior to 10-9 M, more preferably inferior to 10-10 M for a nucleic acid having a nucleotide sequence SEQ ID NO: 2, most preferably inferior to 1 .10 11 M, particularly by Biacore Analysis.
In an embodiment, the invention relates to an anti-CGG repeats antibody or antigen-binding fragment thereof as defined above that has a KD value inferior to 200x10-9 M, preferably inferior to 10-9 M, more preferably inferior to 10-10 M for a nucleic acid hairpin comprising repeats of CGG nucleotide triplets, most preferably inferior to 1 .10-11 M, particularly by Biacore Analysis.
In an embodiment, the anti-CGG repeats antibody or antigen-binding fragment thereof as defined above has a KD value inferior to 200x10-9 M, preferably inferior to 10-9 M, more preferably inferior to 10-10 M for a nucleic acid having the nucleotide sequence SEQ ID NO: 3, most preferably inferior to 1 .10-11 M, particularly by Biacore Analysis. In an embodiment, the invention relates to an anti-CCG repeats antibody or antigen-binding fragment thereof as defined above that has a KD value inferior to 200x10-9 M, preferably inferior to 10-9 M, more preferably inferior to 10-10 M for a nucleic acid hairpin comprising repeats of CCG nucleotide triplets, most preferably inferior to 1 .10-11 M, particularly by Biacore Analysis. In an embodiment, the anti-CCG repeats antibody or antigen-binding fragment thereof as defined above has a KD value inferior to 200x10-9 M, preferably inferior to 10-9 M, more preferably inferior to 10-10 M for a nucleic acid having a nucleotide sequence SEQ ID NO: 60, most preferably inferior to 1 .10 11 M, particularly by Biacore Analysis.
The nucleic acid having the nucleotide sequence selected from the group consisting of SEQ ID NO: 4- 5 (respectively (GAA)so with GC clamp and GC clamp), may be used as negative control to ensure that the binding is specific to the CNG repeats hairpin and not to the GC clamp.
In an embodiment, the invention relates to an antibody or antigen-binding fragment thereof as defined above that has a KD value superior to 200x10-9 M, preferably superior to 200x10-8 M, more preferably superior to 200x107 M for nucleic acid having the nucleotide sequence selected from the group consisting of SEQ ID NO: 4-5 (respectively (GAA)so with GC clamp and GC clamp), particularly by Biacore Analysis.
Figure imgf000008_0001
Figure imgf000009_0001
Figure imgf000010_0001
Table 1 : Positive and negative controls
As used herein, the term “antibody” comprises polyclonal antibodies or monoclonal antibodies.
Monoclonal antibodies can be generated by several known technologies like phage, bacteria, yeast or ribosomal display, as well as by classical methods exemplified by hybridoma-derived antibodies. Thus, the term "monoclonal" is used to refer to all antibodies derived from one nucleic acid clone.
The antibodies of the present invention include recombinant antibodies. As used herein, the term "recombinant antibody" refers to antibodies which are produced, expressed, generated or isolated by recombinant means, such as antibodies which are expressed using a recombinant expression vector transfected into a host cell; antibodies isolated from a recombinant combinatorial antibody library; antibodies isolated from an animal (e.g. a mouse) which is transgenic due to human immunoglobulin genes; or antibodies which are produced, expressed, generated or isolated in any other way in which particular immunoglobulin gene sequences (such as human immunoglobulin gene sequences) are assembled with other DNA sequences. Recombinant antibodies include, for example, chimeric and humanized antibodies.
As used herein, a “chimeric antibody” refers to an antibody in which the sequence of the variable domain derived from the germline of a mammalian species, such as a mouse, has been grafted onto the sequence of the constant domain derived from the germline of another mammalian species, such as a human.
As used herein, a “humanized antibody” refers to an antibody in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
As used herein, an “antigen-binding fragment of an antibody” means a part of an antibody, i.e. a molecule corresponding to a portion of the structure of the antibody of the invention, that exhibits antigen-binding capacity for nucleic acid hairpin comprising CNG repeats. The antigen-binding capacity can be determined by measuring the affinity between the antibody and its target.
Antigen-binding fragments of antibodies are fragments which comprise their hypervariable domains designated CDRs (Complementary Determining Regions) or part(s) thereof encompassing the recognition site for the antigen, thereby defining antigen recognition specificity.
The antibodies of the present invention include conventional antibodies made of dimers of heavy and light chains but also dimer of heavy chains only (such as camelid Hc-lgs or cartilaginous fish IgNAR).
In conventional antibodies, each Light and Heavy chain variable domains (respectively VL and VH) of a four-chain immunoglobulin has three CDRs, designated VL-CDR1 (or LCDR1 ), VL-CDR2 (or LCDR2), VL-CDR3 (or LCDR3) and VH-CDR1 (or HCDR1 ), VH-CDR2 (or HCDR2), VH-CDR3 (or HCDR3), respectively.
In heavy-chain-only antibodies, each heavy chain variable domain has three CDRs: CDR1 , CDR2 and CDR3.
The skilled person is able to determine the location of the various regions/domains of antibodies by reference to the standard definitions in this respect set forth, including a reference numbering system, a reference to the numbering system of KABAT or by application of the IMGT “collier de perle” algorithm. In this respect, for the definition of the sequences of the invention, it is noted that the delimitation of the regions/domains may vary from one reference system to another. Accordingly, the regions/domains as defined in the present invention encompass sequences showing variations in length or localization of the concerned sequences within the full-length sequence of the variable domains of the antibodies, of approximately +/- 10%.
For illustration purpose of specific embodiments of the invention, antigen binding fragments of a conventional antibody that contain the variable domains comprising the CDRs of said antibody encompass Fv, dsFv, scFv, Fab, Fab', F(ab')2. Fv fragments consist of the VL and VH domains of an antibody associated together by hydrophobic interactions; in dsFv fragments, the VH:VL heterodimer is stabilised by a disulphide bond; in scFv fragments, the VL and VH domains are connected to one another via a flexible peptide linker thus forming a single-chain protein. Fab fragments are monomeric fragments obtainable by papain digestion of an antibody; they comprise the entire L chain, and a VH-CH1 fragment of the H chain, bound together through a disulfide bond. The F(ab')2 fragment can be produced by pepsin digestion of an antibody below the hinge disulfide; it comprises two Fab’ fragments, and additionally a portion of the hinge region of the immunoglobulin molecule. The Fab' fragments are obtainable from F(ab')2 fragments by cutting a disulfide bond in the hinge region. F(ab')2 fragments are divalent, i.e. they comprise two antigen binding sites, like the native immunoglobulin molecule; on the other hand, Fv (a VHVL dimmer constituting the variable part of Fab), dsFv, scFv, Fab, and Fab' fragments are monovalent, i.e. they comprise a single antigen-binding site.
Antigen binding fragments of a heavy-chain-only antibody that contain the variable domains comprising the CDRs of said antibody encompass single domain antibody (sdAb). In particular, antigen binding fragments encompass variable region of camelid heavy-chain-only antibody (also called VHH) or variable region of cartilaginous fish heavy-chain-only antibody (also called VNAR). In a preferred embodiment, the antibody or antigen-binding fragment thereof of the invention is a single domain antibody. In a preferred embodiment, the single domain antibody is a camelid single domain antibody (VHH).
In a most preferred embodiment, the antibody or antigen-binding fragment is a single domain antibody and comprises three complementary determining regions (CDR1 to CDR3, respectively);
- wherein the amino acid sequence of the CDR1 is selected from the amino acid sequences of SEQ ID NOs: 23-33 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOs: 23-33;
- wherein the amino acid sequence of the CDR2 is selected from the amino acid sequences of SEQ ID NOs: 34-44 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOs: 34-44; and
- wherein the amino acid sequence of the CDR3 is selected from the amino acid sequences of SEQ ID NOs: 45-55 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOs: 45-55.
As used herein, a “modified antibody” corresponds to a molecule comprising an antibody or an antigenbinding fragment thereof, wherein said monoclonal antibody or functional fragment thereof is associated with a functionally different molecule. A modified antibody of the invention may be either a fusion chimeric protein or a conjugate resulting from any suitable form of attachment including covalent attachment, grafting, chemical bonding with a chemical or biological group or with a molecule.
In an embodiment, the invention relates to an antibody or antigen-binding fragment thereof as defined above that is modified.
In an embodiment, the antibody or antigen-binding fragment thereof as defined above comprises a label. In an embodiment, the antibody or antigen-binding fragment thereof as defined above is covalently attached to a substrate.
B. Single domain antibodies
As reported in the examples, single domain antibodies that bind to nucleic acid hairpin comprising repeats of CNG nucleotide triplets are provided.
The single domain antibodies, especially VHH, are ten times smaller than conventional antibodies and they present numerous advantages: they are well produced, they are easily engineered, they can bind to “non-conventional epitopes” or poorly accessible epitopes for conventional antibodies.
Preferably, the single domain antibody is a VHH. VHHs are variable domain of a camelid heavy-chain antibody. Indeed, in members of the family Camelidae a significant proportion of serum antibodies are homodimeric IgGs with a molecular weight of about 80 kD (Hamers-Casterman et al. 1993 Nature, 363, 446-448). These heavy chain immunoglobulins (Ig) contain three domains and their variable region is referred to as VHH, nanobodies or single domain VHH antibodies.
Specifically, the examples describe isolation and characterization of the following single domain VHH antibodies: VHH-A02, VHH-B02, VHH-C04, VHH-C09, VHH-D1 1 , VHH-E02, VHH-F03, VHH-F08, VHH- G05, VHH-H01 and VHH-H10. All these VHH bind to the hairpin comprising 30 repeats of the CTG nucleotide triplets having the nucleotide sequence SEQ ID NO: 1 .
The amino acid sequences of the VHHs are provided below and the CDR1 , CDR2, and CDR3 domains are indicated. The following table indicates the sequence identification numbers of each disclosed sequence.
Figure imgf000013_0001
Figure imgf000014_0001
The number and location of CDR region amino acid residues disclosed herein comply with the known CDR numbering criteria such as Kabat (Kabat, EA, etc. 1991 Sequences of Proteins of Immunological Interest, 5th Ed), IMGT (IMGT®: the international ImMunoGeneTics information system® http://www.imgt.org) or Chothia (Chothia C., Lesk A.M. Canonical structures for the hypervariable regions of immunoglobulins. Mol. Biol. 1987;196:901-917. doi: 10.1016/0022-2836(87)90412-8.), preferably IMGT.
Thus, another aspect of the invention relates to an isolated single domain antibody, comprising three complementary determining regions (CDR1 to CDR3, respectively);
- wherein the amino acid sequence of the CDR1 is selected from the amino acid sequences of SEQ ID NOs: 23-33 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOs: 23-33;
- wherein the amino acid sequence of the CDR2 is selected from the amino acid sequences of SEQ ID NOs: 34-44 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOs: 34-44; and
- wherein the amino acid sequence of the CDR3 is selected from the amino acid sequences of SEQ ID NOs: 45-55 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOs: 45-55.
Typically, the single domain antibody also comprises four framework regions (FR1 to FR4, respectively). In some embodiments, the amino acid sequence of the CDR1 is selected from the amino acid sequences of SEQ ID NOs: 23-33; the amino acid sequence of the CDR2 is selected from the amino acid sequences of SEQ ID NOs: 34-44 and the amino acid sequence of the CDR3 is selected from the amino acid sequences of SEQ ID NOs: 45-55.
In some embodiments, the single domain antibody comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-19 and 21 .
In some embodiments, the single domain antibody consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-19 and 21 .
In some embodiments, the single domain antibody comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 21 .
In some embodiments, the single domain antibody consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 21 .
The single domain antibody is preferably a VHH.
A02
The single domain antibody A02 has the following amino acid sequence:
MAEVQLQASGGGFVQPGGSLRLSCAASGRGSNRTTMGWFRQAPGKEREFVSAISWSTSQTAYYAD SVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAIKGRGPVPFYNPESPYWGQGTQVTVSS (SEQ ID NO: 12).
A02 comprises the following CDR domains:
CDR1 : RGSNRTT (SEQ ID NO:23);
CDR2: WSTSQTA (SEQ ID NO: 34);
CDR3: IKGRGPVPFYNPESP (SEQ ID NO: 45).
Therefore, in some embodiments, this invention provides a single domain antibody that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12. In some embodiments, this invention provides a single domain antibody that consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12.
In some embodiments, this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 23, a CDR2 domain having the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 34, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 45 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 45.
In some embodiments, this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 23, a CDR2 domain having the amino acid sequence of SEQ ID NO: 34, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 45.
In an embodiment, the single domain antibody A02 or a variant thereof binds to a nucleic acid hairpin comprising repeats of CNG nucleotide triplets. The nucleic acid hairpin comprising repeats of CNG nucleotide triplets comprises preferably at least 30 repeats of CNG nucleotide triplets. The nucleic acid hairpin comprising repeats of CNG nucleotide triplets may be preferably a CTG hairpin or CAG hairpin, most preferably a CTG hairpin, such as the CTG hairpin of the nucleic acid having the nucleotide sequence SEQ ID NO: 1 . The nucleic acid hairpin comprising repeats of CNG nucleotide triplets to which the single domain antibody binds may be on a 5’-3’ DNA strand or on a 3’-5’ DNA strand, preferably on the 5’-3’ DNA strand.
B02
The single domain antibody B02 has the following amino acid sequence:
MAEVQLQASGGGFVQPGGSLRLSCAASGDSFKGYAMGWFRQAPGKEREFVSAISSRGGGISYYAD SVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAKGGRHAAYTYPQYWGQGTQVTVSS (SEQ ID NO: 13).
B02 comprises the following CDR domains:
CDR1 : DSFKGYA (SEQ ID NO:24);
CDR2: SRGGGIS (SEQ ID NO: 35);
CDR3: KGGRHAAYTYPQ (SEQ ID NO: 46).
Therefore, in some embodiments, this invention provides a single domain antibody that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13. In some embodiments, this invention provides a single domain antibody that consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13.
In some embodiments, this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 24, a CDR2 domain having the amino acid sequence of SEQ ID NO: 35 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 35, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 46 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 46.
In some embodiments, this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 24, a CDR2 domain having the amino acid sequence of SEQ ID NO: 35, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 46. In an embodiment, the single domain antibody B02 or a variant thereof binds to a nucleic acid hairpin comprising repeats of CNG nucleotide triplets. The nucleic acid hairpin comprising repeats of CNG nucleotide triplets comprises preferably at least 30 repeats of CNG nucleotide triplets. The nucleic acid hairpin comprising repeats of CNG nucleotide triplets may be preferably a CTG hairpin or CAG hairpin, most preferably a CTG hairpin, such as the CTG hairpin of the nucleic acid having the nucleotide sequence SEQ ID NO: 1 . The nucleic acid hairpin comprising repeats of CNG nucleotide triplets to which the single domain antibody binds may be on a 5’-3’ DNA strand or on a 3’-5’ DNA strand, preferably on the 5’-3’ DNA strand.
C04
The single domain antibody C04 has the following amino acid sequence: MAEVQLQASGGGFVQPGGSLRLSCAASGRGSNRTVMGWFRQAPGKEREFVSAISRGGTIAAYYAD SVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAPRGRWMAPDGPEHLQTEIYWGQGTQVTVSS (SEQ ID NO:14).
C04 comprises the following CDR domains:
CDR1 : RGSNRTV (SEQ ID NO:25);
CDR2: RGGTIAA (SEQ ID NO: 36);
CDR3: PRGRWMAPDGPEHLQTEI (SEQ ID NO: 47).
Therefore, in some embodiments, this invention provides a single domain antibody that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 14. In some embodiments, this invention provides a single domain antibody that consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 14.
In some embodiments, this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 25, a CDR2 domain having the amino acid sequence of SEQ ID NO: 36 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 36, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 47 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 47.
In some embodiments, this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 25, a CDR2 domain having the amino acid sequence of SEQ ID NO: 36, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 47.
In an embodiment, the single domain antibody C04 or a variant thereof binds to a nucleic acid hairpin comprising repeats of CNG nucleotide triplets. The nucleic acid hairpin comprising repeats of CNG nucleotide triplets comprises preferably at least 30 repeats of CNG nucleotide triplets. The nucleic acid hairpin comprising repeats of CNG nucleotide triplets may be preferably a CTG hairpin or CAG hairpin, most preferably a CTG hairpin, such as the CTG hairpin of the nucleic acid having the nucleotide sequence SEQ ID NO: 1 . The nucleic acid hairpin comprising repeats of CNG nucleotide triplets to which the single domain antibody binds may be on a 5’-3’ DNA strand or on a 3’-5’ DNA strand, preferably on the 5’-3’ DNA strand.
C09
The single domain antibody C09 has the following amino acid sequence:
MAEVQLQASGGGFVEPGGSLRLSCAASGRGSNLTTMGWFRQAPGKEREFVSAISRGENYHAYYAD SVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAKWSGTKAHHVMRYWGQGTQVTVSS (SEQ ID NO: 15).
C09 comprises the following CDR domains:
CDR1 : RGSNLTT (SEQ ID NO:26);
CDR2: RGENYHA (SEQ ID NO: 37);
CDR3: KWSGTKAHHVMR (SEQ ID NO: 48).
Therefore, in some embodiments, this invention provides a single domain antibody that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 15. In some embodiments, this invention provides a single domain antibody that consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 15.
In some embodiments, this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 26 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 26, a CDR2 domain having the amino acid sequence of SEQ ID NO: 37 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 37, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 48 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 48.
In some embodiments, this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 26, a CDR2 domain having the amino acid sequence of SEQ ID NO: 37, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 48.
In an embodiment, the single domain antibody C09 or a variant thereof binds to a nucleic acid hairpin comprising repeats of CNG nucleotide triplets. The nucleic acid hairpin comprising repeats of CNG nucleotide triplets comprises preferably at least 30 repeats of CNG nucleotide triplets. The nucleic acid hairpin comprising repeats of CNG nucleotide triplets may be preferably a CTG hairpin or CAG hairpin, most preferably a CTG hairpin, such as the CTG hairpin of the nucleic acid having the nucleotide sequence SEQ ID NO: 1 . The nucleic acid hairpin comprising repeats of CNG nucleotide triplets to which the single domain antibody binds may be on a 5’-3’ DNA strand or on a 3’-5’ DNA strand, preferably on the 5’-3’ DNA strand.
D11
The single domain antibody D1 1 has the following amino acid sequence:
MAEVQLQASGGGFVQPGGSLRLSCAASGRGSNWTAMGWFRQAPGKEREFVSAISSGTGGASYYA DSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAPKNRTTTKPNEAMEKYWGQGTQVTVSS (SEQ ID NO: 16).
D1 1 comprises the following CDR domains:
CDR1 : GSNWTA (SEQ ID NO:27);
CDR2: SGTGGAS (SEQ ID NO: 38);
CDR3: PKNRTTTKPNEAMEKY (SEQ ID NO: 49).
Therefore, in some embodiments, this invention provides a single domain antibody that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 16. In some embodiments, this invention provides a single domain antibody that consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 16.
In some embodiments, this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 27, a CDR2 domain having the amino acid sequence of SEQ ID NO: 38 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 38, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 49 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 49.
In some embodiments, this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 27, a CDR2 domain having the amino acid sequence of SEQ ID NO: 38, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 49.
In an embodiment, the single domain antibody D1 1 or a variant thereof binds to a nucleic acid hairpin comprising repeats of CNG nucleotide triplets. The nucleic acid hairpin comprising repeats of CNG nucleotide triplets comprises preferably at least 30 repeats of CNG nucleotide triplets. The nucleic acid hairpin comprising repeats of CNG nucleotide triplets may be preferably a CTG hairpin or CAG hairpin, most preferably a CTG hairpin, such as the CTG hairpin of the nucleic acid having the nucleotide sequence SEQ ID NO: 1 . The nucleic acid hairpin comprising repeats of CNG nucleotide triplets to which the single domain antibody binds may be on a 5’-3’ DNA strand or on a 3’-5’ DNA strand, preferably on the 5’-3’ DNA strand.
E02
The single domain antibody E02 has the following amino acid sequence:
MAEVQLQASGGGFVQPGGSLRLSCAASGRGSNRTSMGWFRQAPGKEREFVSAISSLDDFKIYYADS VKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAAASMWPGNWSPKYWGQGTQVTVSS (SEQ ID NO: 17).
E02 comprises the following CDR domains:
CDR1 : RGSNRTS (SEQ ID NO:28);
CDR2: SLDDFKI (SEQ ID NO: 39);
CDR3: AASMWPGNWSPK (SEQ ID NO: 50).
Therefore, in some embodiments, this invention provides a single domain antibody that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 17. In some embodiments, this invention provides a single domain antibody that consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 17.
In some embodiments, this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 28, a CDR2 domain having the amino acid sequence of SEQ ID NO: 39 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 39, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 50 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 50.
In some embodiments, this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 28, a CDR2 domain having the amino acid sequence of SEQ ID NO: 39, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 50.
In an embodiment, the single domain antibody E02 or a variant thereof binds to a nucleic acid hairpin comprising repeats of CNG nucleotide triplets. The nucleic acid hairpin comprising repeats of CNG nucleotide triplets comprises preferably at least 30 repeats of CNG nucleotide triplets. The nucleic acid hairpin comprising repeats of CNG nucleotide triplets may be preferably a CTG hairpin or CAG hairpin, most preferably a CTG hairpin, such as the CTG hairpin of the nucleic acid having the nucleotide sequence SEQ ID NO: 1 . The nucleic acid hairpin comprising repeats of CNG nucleotide triplets to which the single domain antibody binds may be on a 5’-3’ DNA strand or on a 3’-5’ DNA strand, preferably on the 5’-3’ DNA strand. F03
The single domain antibody F03 has the following amino acid sequence:
MAEVQLQASGGGFVEPGGSLRLSCAASGFTSRRSGMGWFRQAPGKEREFVSAISWRASVRGYYAD SVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAMRIRWGPQTKGDARWYWGQGTQVTVSS (SEQ ID NO: 18).
F03 comprises the following CDR domains:
CDR1 : FTSRRSG (SEQ ID NO:29);
CDR2: WRASVRG (SEQ ID NO: 40);
CDR3: MRIRWGPQTKGDARW (SEQ ID NO: 51 ).
Therefore, in some embodiments, this invention provides a single domain antibody that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 18. In some embodiments, this invention provides a single domain antibody that consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 18.
In some embodiments, this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 29, a CDR2 domain having the amino acid sequence of SEQ ID NO: 40 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 40, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 51 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 51 .
In some embodiments, this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 29, a CDR2 domain having the amino acid sequence of SEQ ID NO: 40, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 51 .
In an embodiment, the single domain antibody F03 or a variant thereof binds to a nucleic acid hairpin comprising repeats of CNG nucleotide triplets. The nucleic acid hairpin comprising repeats of CNG nucleotide triplets comprises preferably at least 30 repeats of CNG nucleotide triplets. The nucleic acid hairpin comprising repeats of CNG nucleotide triplets may be preferably a CTG hairpin or CAG hairpin, most preferably a CTG hairpin, such as the CTG hairpin of the nucleic acid having the nucleotide sequence SEQ ID NO: 1 . The nucleic acid hairpin comprising repeats of CNG nucleotide triplets to which the single domain antibody binds may be on a 5’-3’ DNA strand or on a 3’-5’ DNA strand, preferably on the 5’-3’ DNA strand. F08
The single domain antibody F08 has the following amino acid sequence:
MAEVQLQASGGGFVQPGGSLRLSCAASGRGSNRTTMGWFRQAPGKEREFVSAISSSQTMGEYYAD SVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCARAYRKSNSPDAYAFREIMYWGQGTQVTVSS (SEQ ID NO: 19).
F08 comprises the following CDR domains:
CDR1 : RGSNRTT (SEQ ID NO:30);
CDR2: SSQTMGE (SEQ ID NO: 41 );
CDR3: RAYRKSNSPDAYAFREIM (SEQ ID NO: 52).
Therefore, in some embodiments, this invention provides a single domain antibody that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 19. In some embodiments, this invention provides a single domain antibody that consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 19.
In some embodiments, this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 30 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 30, a CDR2 domain having the amino acid sequence of SEQ ID NO: 41 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 41 , and a CDR3 domain having the amino acid sequence of SEQ ID NO: 52 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 52.
In some embodiments, this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 30, a CDR2 domain having the amino acid sequence of SEQ ID NO: 41 , and a CDR3 domain having the amino acid sequence of SEQ ID NO: 52.
In an embodiment, the single domain antibody F08 or a variant thereof binds to a nucleic acid hairpin comprising repeats of CNG nucleotide triplets. The nucleic acid hairpin comprising repeats of CNG nucleotide triplets comprises preferably at least 30 repeats of CNG nucleotide triplets. The nucleic acid hairpin comprising repeats of CNG nucleotide triplets may be preferably a CTG hairpin or CAG hairpin, most preferably a CTG hairpin, such as the CTG hairpin of the nucleic acid having the nucleotide sequence SEQ ID NO: 1 . The nucleic acid hairpin comprising repeats of CNG nucleotide triplets to which the single domain antibody binds may be on a 5’-3’ DNA strand or on a 3’-5’ DNA strand, preferably on the 5’-3’ DNA strand.
G05 The single domain antibody G05 has the following amino acid sequence:
MAEVQLQASGGGFVQPGGSLRLSCAASGTYSRNTIMGWFRQAPGKEREFVSAISSNPNHGLYYADS VKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAAKLDRMSWYKQSYWGQGTQVTVSS (SEQ ID NO: 20).
G05 comprises the following CDR domains:
CDR1 : TYSRNTI (SEQ ID NO:31 );
CDR2: SNPNHGL (SEQ ID NO: 42);
CDR3: AKLDRMSWYKQS (SEQ ID NO: 53).
Therefore, in some embodiments, this invention provides a single domain antibody that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 20. In some embodiments, this invention provides a single domain antibody that consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 20.
In some embodiments, this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 31 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 31 , a CDR2 domain having the amino acid sequence of SEQ ID NO: 42 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 42, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 53 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 53.
In some embodiments, this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 31 , a CDR2 domain having the amino acid sequence of SEQ ID NO: 42, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 53.
In an embodiment, the single domain antibody G05 or a variant thereof binds to a nucleic acid hairpin comprising repeats of CNG nucleotide triplets. The nucleic acid hairpin comprising repeats of CNG nucleotide triplets comprises preferably at least 30 repeats of CNG nucleotide triplets. The nucleic acid hairpin comprising repeats of CNG nucleotide triplets may be preferably a CTG hairpin or CAG hairpin, most preferably a CTG hairpin, such as the CTG hairpin of the nucleic acid having the nucleotide sequence SEQ ID NO: 1 . The nucleic acid hairpin comprising repeats of CNG nucleotide triplets to which the single domain antibody binds may be on a 5’-3’ DNA strand or on a 3’-5’ DNA strand, preferably on the 5’-3’ DNA strand.
H01
The single domain antibody H01 has the following amino acid sequence: MAEVQLQASGGGFVQPGGSLRLSCAASGSTWASTIMGWFRQAPGKEREFVSAISYNDTNTRYYADS VKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAAKPHMRKWQTDGYWGQGTQVTVSS (SEQ ID NO: 21 ).
H01 comprises the following CDR domains:
CDR1 : STWASTI (SEQ ID NO:32);
CDR2: YNDTNTR (SEQ ID NO: 43);
CDR3: AKPHMRKWQTDG (SEQ ID NO: 54).
Therefore, in some embodiments, this invention provides a single domain antibody that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 21 . In some embodiments, this invention provides a single domain antibody that consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 21 .
In some embodiments, this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 32, a CDR2 domain having the amino acid sequence of SEQ ID NO: 43 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 43, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 54 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 54.
In some embodiments, this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 32, a CDR2 domain having the amino acid sequence of SEQ ID NO: 43, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 54.
In an embodiment, the single domain antibody H01 or a variant thereof binds to a nucleic acid hairpin comprising repeats of CNG nucleotide triplets. The nucleic acid hairpin comprising repeats of CNG nucleotide triplets comprises preferably at least 30 repeats of CNG nucleotide triplets. The nucleic acid hairpin comprising repeats of CNG nucleotide triplets may be preferably a CTG hairpin or CAG hairpin, most preferably a CTG hairpin, such as the CTG hairpin of the nucleic acid having the nucleotide sequence SEQ ID NO: 1 . The nucleic acid hairpin comprising repeats of CNG nucleotide triplets to which the single domain antibody binds may be on a 5’-3’ DNA strand or on a 3’-5’ DNA strand, preferably on the 5’-3’ DNA strand.
H10
The single domain antibody H10 has the following amino acid sequence: MAEVQLQASGGGFVQPGGSLRLSCAASGRGSNWTDMGWFRQAPGKEREFVSAISRRDGGSPYYA DSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAKRAEWNIHYAADMQYYWGQGTQVTVSS (SEQ ID NO: 22).
H10 comprises the following CDR domains:
CDR1 : RGSNWTD (SEQ ID NO: 33);
CDR2: RRDGGSP (SEQ ID NO: 44);
CDR3: KRAEWNIHYAADMQY (SEQ ID NO: 55).
Therefore, in some embodiments, this invention provides a single domain antibody that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 22. In some embodiments, this invention provides a single domain antibody that consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 22.
In some embodiments, this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 33 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 33, a CDR2 domain having the amino acid sequence of SEQ ID NO: 44 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 44, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 55 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 55.
In some embodiments, this invention provides a single domain antibody that comprises three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain antibody comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 33, a CDR2 domain having the amino acid sequence of SEQ ID NO: 44, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 55.
In an embodiment, the single domain antibody H10 or a variant thereof binds to a nucleic acid hairpin comprising repeats of CNG nucleotide triplets. The nucleic acid hairpin comprising repeats of CNG nucleotide triplets comprises preferably at least 30 repeats of CNG nucleotide triplets. The nucleic acid hairpin comprising repeats of CNG nucleotide triplets may be preferably a CTG hairpin or CAG hairpin, most preferably a CTG hairpin, such as the CTG hairpin of the nucleic acid having the nucleotide sequence SEQ ID NO: 1 . The nucleic acid hairpin comprising repeats of CNG nucleotide triplets to which the single domain antibody binds may be on a 5’-3’ DNA strand or on a 3’-5’ DNA strand, preferably on the 5’-3’ DNA strand.
In an embodiment, the single domain antibody comprises a label.
In an embodiment, the single domain antibody is covalently attached to a substrate.
The embodiments recited for the antibodies and antigen-binding fragment as defined above are repeated mutadis mutandis io the single domain antibody of the invention.
In some embodiments, the single domain antibody of the invention: - binds to a nucleic acid hairpin comprising repeats of CNG nucleotide triplets;
- has a KD value inferior to 10-9 M, preferably inferior to 10-10 M, more preferably inferior to 1 .10-11 M, particularly by Biacore Analysis for a nucleic acid hairpin comprising repeats of CNG nucleotide triplets, notably for a nucleic acid having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 , 6-1 1 , preferably for a nucleic acid having the nucleotide sequence SEQ ID NO: 1 ;
- the nucleic acid hairpin may be a DNA and/or a RNA hairpin, preferably a DNA hairpin;
- the CNG nucleotide triplet may have the nucleotide sequence selected from the group consisting of CAG, CGG, CTG, CUG and CCG, preferably selected from the group consisting of CTG, CUG and CAG, more preferably CTG and/or CUG, most preferably CTG;
- the copies of CNG nucleotide triplets are preferably directly adjacent to each other;
- the nucleic acid hairpin is selected from the group consisting of a 4-nucleotide loop hairpin, a 3- nucleotide loop hairpin and a 7-nucleotide loop hairpin;
- the number of CNG nucleotide triplets that are repeated is of at least 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52 units.
In some embodiments, the antibody or antigen-binding fragment thereof as described in Section A above is an single domain antibody as defined above.
C. Complex
The present invention also relates to a complex comprising: the antibody or antigen-binding fragment as described in Section A above or the single domain antibody as described in Section B above and a nucleic acid hairpin comprising repeats of CNG nucleotide triplets as defined above.
The antibody or antigen-binding fragment as described in Section A or the single domain antibody as described in Section B may be modified. “Modified antibody” shall be taken to mean a molecule comprising an antibody or an antigen-binding fragment thereof or a single domain antibody, wherein said antibody or functional fragment thereof or single domain antibody is associated with a functionally different molecule, chemical bonding with a chemical or biological group or with a molecule.
In one embodiment, the complex consists of: the antibody or antigen-binding fragment or the single domain antibody, optionally the modified antibody or modified antigen-binding fragment or the modified single domain antibody, and a nucleic acid hairpin comprising repeats of CNG nucleotide triplets or a molecule comprising the same. Another aspect of the invention is the use of such complex.
The present invention also relates to a method comprising a step of contacting the antibody or antigenbinding fragment as described in Section A above or the single domain antibody as described in Section B above with molecule(s) comprising a nucleic acid hairpin comprising repeats of CNG nucleotide triplets thereby forming such complex. D. Polynucleotides and vectors
In another aspect an isolated polynucleotide that encodes the antibody, the antigen-binding fragment thereof or the single domain antibody is provided. The antibody or the antigen-binding fragment thereof may be any antibody or antigen-binding fragment thereof described in Section A above. The single domain antibody is any single domain antibody described in Section B above.
In another aspect, the invention relates to a vector comprising the polynucleotide as defined above.
As used herein, a “vector” is a nucleic acid molecule used as a vehicle to transfer genetic material into a cell. The term “vector” encompasses plasmids, viruses, cosmids and artificial chromosomes. The vector can be a vector for eukaryotic or prokaryotic expression, such as a plasmid, a phage for bacterium introduction, a YAC able to transform yeast, a viral vector and especially a retroviral vector, or any expression vector.
In general, engineered vectors comprise an origin of replication, a multicloning site and a selectable marker. The vector itself is generally a nucleotide sequence, commonly a DNA sequence, that comprises an insert (transgene) and a larger sequence that serves as the “backbone” of the vector. Modern vectors may encompass additional features besides the transgene insert and a backbone: promoter, genetic marker, antibiotic resistance, reporter gene, targeting sequence, protein purification tag. Vectors called expression vectors (expression constructs) specifically are for the expression of the transgene in the target cell, and generally have control sequences.
A vector as defined herein may be chosen to enable the production of the antibody or the antigenbinding fragment thereof, notably single domain antibody, either in vitro or in vivo.
The polynucleotides according to the invention can be obtained by conventional methods, known per se, following standard protocols such as those described in Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc., Library of Congress, USA). For example, they may be obtained by amplification of a nucleic sequence by PCR or RT-PCR or alternatively by total or partial chemical synthesis.
The vectors are constructed and introduced into host cells by conventional recombinant DNA and genetic engineering methods which are known perse. Numerous vectors into which a polynucleotide of interest may be inserted in order to introduce it and to maintain it in a host cell are known per se; the choice of an appropriate vector depends on the use envisaged for this vector (for example replication of the sequence of interest, expression of this sequence), and on the nature of the host cell.
E. Cells
In another aspect a recombinant cell comprising the polynucleotide or the vector as defined above is provided.
As used herein, the term “host cell” is intended to include any individual cell or cell culture that can be or has been recipient of vectors, exogenous nucleic acid molecules, and polynucleotides encoding the antibody, the antigen-binding fragment thereof or the single domain antibody of the present invention; and/or recipients of the antibody, the antigen-binding fragment thereof or the single domain antibody itself. The introduction of the respective material into the cell can be carried out by way of transformation, transfection and the like. The term “host cell” is also intended to include progeny or potential progeny of a single cell. Suitable host cells include prokaryotic or eukaryotic cells, and also include but are not limited to bacteria, yeast cells, fungi cells, plant cells, and animal cells such as insect cells and mammalian cells, e.g., murine, rat, rabbit, macaque or human.
F. Method of producing the anti-CNG repeats antibodies
In another aspect, the invention provides methods of producing the antibody or the antigen-binding fragment or the single domain antibody of the invention.
The methods may comprise culturing the recombinant cell comprising the polynucleotide that encodes the antibody or the antigen-binding fragment or the single domain antibody of the invention under conditions sufficient for the production thereof.
G. Methods of Use, Detection methods, Diagnostic methods
Unless otherwise specified, the antibody or the antigen-binding fragment thereof may be any antibody or antigen-binding fragment thereof described in Section A above. The single domain antibody may be any single domain antibody described in Section B above.
In another aspect of the invention, it is provided a method for detecting and/or quantifying nucleic acid hairpin comprising repeats of CNG nucleotide triplets in a sample, comprising: providing the antibody or antigen-binding fragment thereof as defined above or the single domain antibody as defined above; providing a sample; contacting the antibody or the antigen-binding fragment or the single domain antibody with the sample; and visualizing the antigen-antibody complexes formed.
Advantageously, the nucleic acid hairpin comprising repeats of CNG nucleotide triplets may be any of nucleic acid hairpin comprising repeats of CNG nucleotide triplets described in Section A above.
In the embodiment wherein the method is a method for quantifying nucleic acid hairpin, the method may comprise a step of comparison with a standard curve.
In some embodiments the methods comprise an ELISA, lateral flow immunoassay, bead-based immunoassay, or multiplex bead-based immunoassay.
Advantageously, the sample is a biological sample. The biological sample may be blood, plasma, serum, saliva, peripheral blood mononuclear cells (PBMCs), gametes or other body fluids or tissues.
In an embodiment, the biological sample is obtained from a subject, preferably from a subject suspected to have a trinucleotide expansion disorder selected from the group consisting of myotonic dystrophy type 1 , spinocerebrallar ataxia 12, Huntington’s disease-like 2, spinal and bulbar muscular atrophy, Huntington’s disease, dentatorubral-pallidouysian atrophy, spinocerebrellar ataxia 1 , spinocerebrellar ataxia 2, spinocerebrellar ataxia 3, spinocerebrellar ataxia 6, spinocerebrellar ataxia 7, spinocerebrellar ataxia 17, fragile X syndrome, X tremor and/or ataxia syndrome and X-linked mental retardation. The table 2 below summarized the trinucleotide expansion disorder induced by repeats of CNG nucleotide triplets, their corresponding CNG nucleotide triplet and the normal average number of CNG triplets that are repeated.
Figure imgf000029_0001
Table 2 - Trinucleotide expansion disorder induced by repeats of CNG nucleotide triplets
In a preferred embodiment, the subject is suspected to have a myotonic dystrophy type 1 (DM1 ). DM1 is a dominant autosomal multisystemic disorder including myotonia, progressive weakness and wasting of muscle and extramuscular symptoms such as cataracts, cardiac conduction defects, testicular atrophy, endocrine and cognitive dysfunction. It is caused by an expansion of a CTG trinucleotide repeat in the 3’ untranslated region (UTR) or the DM protein kinase gene (DMPK) located on chromosome. In normal individuals, the CTG repeat is polymorphic and varies between 5 and 37 triplets, whereas 50 to 4 000 CTG repeats are observed in affected patients. The size of the repeat which usually increases from generation to generation, is generally correlated with the clinical severity and the age of the onset, providing a molecular basis of the phenomenon observed in DM1 families. Analyses of family pedigrees have shown that >90% of transmission result in the expansion of repeats.
Thus, in the embodiment where the subject is suspected to have a myotonic dystrophy type 1 , the antibody or antigen-binding fragment and/or the single domain antibody preferably binds to a hairpin of an 5’-3’ DNA strand comprising repeats of CTG nucleotide triplets. In another embodiment, the subject is suspected to have a trinucleotide expansion disorder selected from the group consisting of spinocerebrallar ataxia 12, Huntington’s disease-like 2, spinal and bulbar muscular atrophy, Huntington’s disease, dentatorubral-pallidouysian atrophy, spinocerebrellar ataxia 1 , spinocerebrellar ataxia 2, spinocerebrellar ataxia 3, spinocerebrellar ataxia 6, spinocerebrellar ataxia 7 and spinocerebrellar ataxia 17. In this embodiment, the antibody or antigen-binding fragment and/or the single domain antibody binds preferably to a hairpin of an 5’-3’ DNA strand comprising repeats of CAG nucleotide triplets.
In another embodiment, the subject is suspected to have a trinucleotide expansion disorder being fragile X syndrome, X tremor and/or ataxia syndrome. In this embodiment, the antibody or antigen-binding fragment and/or the single domain antibody binds preferably to a hairpin of an 5’-3’ DNA strand comprising repeats of CGG nucleotide triplets.
In another embodiment, the subject is suspected to have a X-linked mental retardation. In this embodiment, the antibody or antigen-binding fragment and/or the single domain antibody binds preferably to a hairpin of an 5’-3’ DNA strand comprising repeats of CCG nucleotide triplets.
In an alternative embodiment, the antibody or antigen-binding fragment and/or the single domain antibody binds to the CNG nucleotide triplets of the complementary strand (3’-5’ strand).
Thus, in an alternative embodiment, the subject is suspected to have a myotonic dystrophy type 1 and the antibody or antigen-binding fragment and/or the single domain antibody preferably binds to a hairpin of an 3’-5’ DNA strand comprising repeats of CAG nucleotide triplets.
In another alternative embodiment, the subject is suspected to have a trinucleotide expansion disorder selected from the group consisting of spinocerebrallar ataxia 12, Huntington’s disease-like 2, spinal and bulbar muscular atrophy, Huntington’s disease, dentatorubral-pallidouysian atrophy, spinocerebrellar ataxia 1 , spinocerebrellar ataxia 2, spinocerebrellar ataxia 3, spinocerebrellar ataxia 6, spinocerebrellar ataxia 7 and spinocerebrellar ataxia 17 and the antibody or antigen-binding fragment and/or the single domain antibody binds preferably to a hairpin of an 3’-5’ DNA strand comprising repeats of CTG nucleotide triplets.
In another alternative embodiment, the subject is suspected to have a trinucleotide expansion disorder being fragile X syndrome, X tremor and/or ataxia syndrome and the antibody or antigen-binding fragment and/or the single domain antibody binds preferably to a hairpin of an 3’-5’ DNA strand comprising repeats of CCG nucleotide triplets.
In another alternative embodiment, the subject is suspected to have a X-linked mental retardation and the antibody or antigen-binding fragment and/or the single domain antibody binds preferably to a hairpin of an 3’-5’ DNA strand comprising repeats of CGG nucleotide triplets.
Preferably, the method comprises comparing the results obtained with a subject sample to positive and negative controls.
In an embodiment, the antibody or antigen-binding fragment and/or the single domain antibody is labeled and the step of visualizing the antigen-antibody complexes formed comprises visualizing the label. Preferred labels include suitable enzymes such as horseradish peroxidase, alkaline phosphatase, betagalactosidase, luciferase or acetylcholinesterase; members of a binding pair that are capable of forming complexes such as streptavidin/biotin, avidin/biotin or an antigen/antibody complex including, for example, rabbit IgG and anti-rabbit IgG; fluorophores such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, tetramethyl rhodamine, eosin, green fluorescent protein, erythrosin, coumarin, methyl coumarin, pyrene, malachite green, stilbene, lucifer yellow, Cascade Blue, Texas Red, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin, fluorescent lanthanide complexes such as those including Europium and Terbium, cyanine dye family members, such as Cy3 and Cy5, molecular beacons and fluorescent derivatives thereof, as well as others known in the art; a luminescent material such as luminol; light scattering or plasmon resonant materials such as gold or silver particles or quantum dots; or radioactive material including 14C, 123l, 124l, 125l, 32P, 33P, 35S, or 3H.
Preferably, the antigen-antibody complexes are visualized by EIA, ELISA, RIA, or by immunofluorescence.
The method for detection and/or quantification may comprise: providing a first antibody attached to a solid support; providing a sample; contacting the solid support with the sample under conditions sufficient to allow formation of first antigenantibody complexes between the nucleic acid hairpin and the first antibody to the solid support; contacting the solid support with a second antibody, under conditions sufficient to allow formation of second antigen-antibody complexes between nucleic acid and the second antibody; and visualizing the second antigen-antibody complexes; the first antibody and/or the second antibody being an antibody or an antigen-binding fragment and/or a single domain antibody as defined above.
Preferably, the second antibody comprises a label selected from a chemiluminescent label, an enzyme label, a fluorescence label, and a radioactive (e.g., iodine) label.
It is also provided an in vitro method for diagnosing a trinucleotide expansion disorder selected from the group consisting of myotonic dystrophy type 1 , spinocerebrallar ataxia 12, Huntington’s disease-like 2, spinal and bulbar muscular atrophy, Huntington’s disease, dentatorubral-pallidouysian atrophy, spinocerebrellar ataxia 1 , spinocerebrellar ataxia 2, spinocerebrellar ataxia 3, spinocerebrellar ataxia 6, spinocerebrellar ataxia 7, spinocerebrellar ataxia 17, fragile X syndrome, X tremor ataxia syndrome and X-linked mental retardation wherein the antibody or antigen-binding fragment thereof as defined above or the single domain antibody as defined above is used for the detection and/or quantification of nucleic acid hairpin comprising repeats of CNG nucleotide triplets as defined above in a sample previously obtained from a subject.
The detection and/or quantification may be carried out with the method for detecting and/or quantifying as described above.
In an embodiment, the presence of nucleic acid hairpin comprising repeats of CNG nucleotide triplets in the biological sample is indicative that the subject has a trinucleotide expansion disorder. For example:
- the presence of hairpin of an 5’-3’ DNA strand comprising repeats of CTG nucleotide triplets is indicative that the subject has a myotonic dystrophy type 1 ;
- the presence of a hairpin of an 5’-3’ DNA strand comprising repeats of CAG nucleotide triplets is indicative that the subject has a trinucleotide expansion disorder selected from the group consisting of spinocerebrallar ataxia 12, Huntington’s disease-like 2, spinal and bulbar muscular atrophy, Huntington’s disease, dentatorubral-pallidouysian atrophy, spinocerebrellar ataxia 1 , spinocerebrellar ataxia 2, spinocerebrellar ataxia 3, spinocerebrellar ataxia 6, spinocerebrellar ataxia 7 and spinocerebrellar ataxia 17;
- the presence of a hairpin of an 5’-3’ DNA strand comprising repeats of CGG nucleotide triplets is indicative that the subject has a trinucleotide expansion disorder being fragile X syndrome, X tremor and/or ataxia syndrome; and/or
- the presence of a hairpin of an 5’-3’ DNA strand comprising repeats of CCG nucleotide triplets is indicative that the subject has a X-linked mental retardation,
- the presence of hairpin of an 3’-5’ DNA strand comprising repeats of CAG nucleotide triplets is indicative that the subject has a myotonic dystrophy type 1 ;
- the presence of a hairpin of an 3’-5’ DNA strand comprising repeats of CTG nucleotide triplets is indicative that the subject has a trinucleotide expansion disorder selected from the group consisting of spinocerebrallar ataxia 12, Huntington’s disease-like 2, spinal and bulbar muscular atrophy, Huntington’s disease, dentatorubral-pallidouysian atrophy, spinocerebrellar ataxia 1 , spinocerebrellar ataxia 2, spinocerebrellar ataxia 3, spinocerebrellar ataxia 6, spinocerebrellar ataxia 7 and spinocerebrellar ataxia 17;
- the presence of a hairpin of an 3’-5’ DNA strand comprising repeats of CCG nucleotide triplets is indicative that the subject has a trinucleotide expansion disorder being fragile X syndrome, X tremor and/or ataxia syndrome; and/or
- the presence of a hairpin of an 3’-5’ DNA strand comprising repeats of CGG nucleotide triplets is indicative that the subject has a X-linked mental retardation.
In an embodiment, the nucleic acid hairpin is quantified and the quantified nucleic acid hairpin are compared with a reference value and it is concluded that the subject has a trinucleotide expansion disorder as defined above when the quantified nucleic acid hairpin is higher than the reference value. The reference value may be a sample from a healthy subject i.e. a subject who does not suffer from a trinucleotide triplet disorder.
The present invention also relates to the use of the antibody or antigen-binding fragment thereof as defined above or the single domain antibody as defined above in an in vitro diagnostic test of a trinucleotide expansion disorder selected from the group consisting of myotonic dystrophy type 1 , spinocerebrallar ataxia 12, Huntington’s disease-like 2, spinal and bulbar muscular atrophy, Huntington’s disease, dentatorubral-pallidouysian atrophy, spinocerebrellar ataxia 1 , spinocerebrellar ataxia 2, spinocerebrellar ataxia 3, spinocerebrellar ataxia 6, spinocerebrellar ataxia 7, spinocerebrellar ataxia 17, fragile X syndrome, X tremor, ataxia syndrome and X-linked mental retardation.
H. Kits
In another aspect, the invention relates to a kit comprising:
- an antibody or antigen-binding fragment thereof or a single domain antibody as defined above,
- a nucleic acid molecule coding for said antibody, antigen-binding fragment or single domain antibody,
- a vector comprising said nucleic acid molecule, and/or
- a cell comprising said vector.
In particular, in some embodiments, the kit comprises: - an antibody or antigen-binding fragment thereof or a single domain antibody as defined above that specifically binds to a nucleic acid hairpin comprising repeats of CNG nucleotide triplets, notably the CNG nucleotide triplet having the nucleotide sequence selected from the group consisting of CAG, CGG, CTG, CUG and CCG, preferably selected from the group consisting of CTG, CUG and CAG, more preferably CTG and/or CUG, most preferably CTG; and/or
- an antibody or antigen-binding fragment thereof or a single domain antibody as defined above that has a KD value inferior to 10-9 M, preferably inferior to 10-10 M, more preferably inferior to 1 .10-11 M, particularly by Biacore Analysis for a nucleic acid hairpin comprising repeats of CNG nucleotide triplets, notably for a nucleic acid having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 , 6-1 1 , preferably for a nucleic acid having the nucleotide sequence SEQ ID NO: 1 ; and/or
- a single domain antibody as defined above comprising three complementary determining regions (CDR1 to CDR3, respectively);
- wherein the amino acid sequence of the CDR1 is selected from the amino acid sequences of SEQ ID NOs: 23-33 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOs: 23-33;
- wherein the amino acid sequence of the CDR2 is selected from the amino acid sequences of SEQ ID NOs: 34-44 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOs: 34-44; and
- wherein the amino acid sequence of the CDR3 is selected from the amino acid sequences of SEQ ID NOs: 45-55 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOs: 45-55; and/or
- an single domain antibody as defined above comprising an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-22, preferably with SEQ ID NOs: 17- 19 and 21 , more preferably with SEQ ID NO: 21 ; and/or
- an antibody or antigen-binding fragment thereof or a single domain antibody as defined above comprising a label
- an antibody or antigen-binding fragment thereof or a single domain antibody as defined above covalently attached to a substrate.
As a matter of convenience, the antibody, the antigen-binding fragment thereof or the single domain antibody of the present invention can be provided in a kit, i.e., a packaged combination of reagents.
In the context of the present invention, the term “kit” means two or more components (one of which corresponding to the antibody or antigen-binding thereof or a single domain antibody, the polynucleotide, the vector or the cell of the invention) packaged together in a container, recipient or otherwise. A kit can hence be described as a set of products and/or utensils that are sufficient to achieve a certain goal, which can be marketed as a single unit.
The kit may comprise one or more recipients (such as vials, ampoules, containers, syringes, bottles, bags) of any appropriate shape, size and material containing the antibody, the antigen-binding fragment thereof or the single domain antibody of the present invention. The kit may additionally contain directions for use (e.g. in the form of a leaflet or instruction manual).
In an embodiment, the invention relates to a kit as defined above for a single-dose administration unit.
The following Figures and Examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.
FIGURES
Figure 1 shows a schematic drawing of the synthetic CTG hairpin and the synthetic GC clamp.
Figure 2 shows the migration of the (CTG)so oligonucleotide the GC clamp and a non structured (NS)
1 11 nt long oligonucleotide on, non-denaturing (left) and denaturing (right) agarose gels.
Figure 3 shows a schematic drawing of the one hybrid experiments.
Figure 4 shows the spot test on minimal medium without histidine (SC-His) of the one-hybrid experiments.
Figure 5 shows dot blots with purified camelid single domain antibodies (VHH) Top Nylon membranes on which 100 ng of the synthetic CTG hairpin (CTG) and of the GC clamp (GC) were spotted were incubated with one of the four VHH (E02, F03, F08 and H01 ). Three pictures were taken at different exposure times (labeled 1 , 2 or 3). Bottom: H01 was incubated with a membrane on which different amounts of the CTG hairpin (CTG), the GC clamp (GC) or a synthetic CAG hairpin (CAG) were spotted. Quantification of the positive signal detected with the CTG hairpin is shown to the right.
Figure 6 shows dot blots where different concentration of plasmids containing 98 or 255 CTG triplets were spotted and incubated with VHH H01 . Signal quantifications are shown to the right.
Figure 7 shows dot blots where heat denatured or non-heat denatured plasmids containing 98 CTG triplets, 255 CTG triplets or no triplets were spotted and incubated with VHH H01 . Figure 8 shows dot blots where yeast total genomic DNA containing CTG trinucleotide repeats of different lengths (50, 100, 160 or 280 triplets), as well as human total genomic DNA of a DM1 patient were spotted and incubated with VHH H01 . Signal quantifications are shown to the right.
Figure 9 shows VHH H01 hybridization on DM1 human DNA and DNA of transgenic mouse models of myotonic dystrophy type 1 . Transgenic mouse models are as disclosed in M. Gomes-Pereira et al., CTG trinucleotide repeat "big jumps": large expansions, small mice, PLoS Genet . 2007 Apr 6;3(4):e52. The dot blot was performed in the same conditions as in figure 5, 300 ng of whole genomic DNA was denatured, crosslinked and hybridized with VHH H01 on each spot. The two human genomic DNAs (#8 and #9) were spotted only once, the transgenic mice DNAs were spotted as duplicates.
Figure 10 shows the signal quantification as a function of CTG repeat length as the dot blot of figure 9. Each dot corresponds to one spot in figure 9. Dots #8 and #9 are human DNA and the other dots are mice DNA.
Figure 1 1 shows the secondary structures capture on HiTrap column. On the top of figure 1 1 : Fractions collected from the FPLC were loaded on a non-denaturing agarose gel (Top), run at 90V in 1 X TBE buffer in the presence of 0.02 pg/mL ethidium bromide. The gel was transferred and hybridized with radioactively labeled pUC19 DNA. The different supercoiled and relaxed forms of the pRW3222 plasmid are visible as discrete bands. The total signals detected in each lane are quantified on a Fujifilm FLA- 9000, as previously described (Viterbo et al. 2018). On the bottom of figure 1 1 : Quantification results are given in arbitrary units. The amount of structured plasmid eluted from the VHH after increasing salt concentration (T 10N200) corresponds to 7% of the total plasmid loaded on the column, in this particular experiment.
EXAMPLES
Materials and Methods
Plasmids
VHH sequences were PCR amplified with Phusion polymerase (NEBiolabs), with primers VHHup and VHHdown (Table 3).
Figure imgf000035_0001
Homology with pP9 vector in bold
Table 3: List of primers used They were cloned into the pP9 plasmid (Hybrigenics) digested by Ncol and Notl, by gap repair in the FYEF01 -9C yeast strain, a MATa derivative of the FYAT01 strain (Table 4).
Figure imgf000036_0001
Table 4: yeast strains used pP9 contains the strong constitutive ADH1 promoter driving the expression of the GAL4 activation domain to which VHH were cloned in frame. The resulting plasmids pTRI136 to pTRI146 were sequenced (Eurofins) to verify the absence of any mutation and the integrity of each fusion gene.
Figure imgf000036_0002
Table 5: VHH clones identified by phage display To clone CTG repeats in the pB300 one-hybrid reporter plasmid (Hybrigenics), the repeats were gel purified from plasmid pRW3216 digested with BamHI and Pstl, and ligated to pB300 digested with BamHI and Sall, using complementary oligonucleotides pB300-Sal and pB300-Pst as adapters. The resulting pTRI148 plasmid was sequenced (Eurofins) and contains 99 uninterrupted CTG triplets, upstream the minimal HIS3 promoter driving the expression of the HIS3 gene.
One-hybrid GFP reporters were ordered as synthetic pieces of DNA from Thermo Fisher Scientific for CAG-GFP, CTG-GFP and GAA-GFP and from Eurofins for CGG-GFP. Each sequence contains exactly 30 triplets, followed by the minimal HIS3 promoter and the eGFP gene. Each synthetic gene was cloned at Apal and BamHI in pTRI109, to give plasmids pTRI149 (CAG), pTRI150 (CTG), pTRI151 (GAA) and pTRI152 (CGG), that contain flanking homologous regions with the ARG2 gene. Plasmid pTRI150 was subsequently digested with BamHI and Eagl, to remove the CTG repeat tract, treated with T4 DNA polymerase to blunt the extremities and religated to make pTRI153, which contains eGFP under the control of the HIS3 minimal promoter and no repeat tract (A in FACS analyses).
Yeast strains
Plasmid pTRI148 was linearized with Hpal in the ADE2 gene and transformed in yeast strain FYEF01 - 7C, isogenic to FYAT01 carrying the ade2-opal mutation 1 (Table 4). The resulting [Ade+] transformants were analyzed by Southern blot, to check correct integration at the ADE2 locus as well as CTG repeat tract length. One clone, called GFY21 1 , was selected and crossed to all FYEF01 -9C transformants containing the pP9-VHH plasmids (pTRI136 to 146).
Plasmids pTRI149-152 containing eGFP constructs were digested by Xhol, transformed into FYEF01 - 7C and transformants were selected on SC-Trp. Since the plasmid is targeted to the ARG2 gene, [Arg- ] transformants were analyzed by Southern blot to check correct integration and repeat length. pTRI153 was digested with Xhol, transformed into FYBL2-7B, and [Trp+, Arg-] transformants were kept for further experiments. Strains GFY212 to 215 were subsequently switched to MATa using pJH132, as previously described 4. Ultra Low Range DNA ladder (Invitrogen) was used as molecular weight marker.
Phage display
Synthetic CAG hairpin, CTG hairpin and GC clamp were ordered as PAGE Ultramer deoxynucleotides (IDT), linked to a 5' biotin (Figure 1 ). A non-related antigen, GC Clamp-Biotin was used in an initial round of Phage Display to deplete the library from unspecific binders. Prior to the Phage Display selection, CTG Hairpin-Biotin and GC Clamp-Biotin were bound to Streptavidin Magnetic Beads (Dynabeads® M- 280 Streptavidin, Life Technologies) with a 50 nM final concentration of biotinylated antigen for the first round and 10 nM final concentration of biotinylated antigen for the second and third rounds.
Three rounds of Phage Display selection were carried out using biotinylated CTG Hairpin. Hybrigenics’ synthetic hsd2Ab VHH library of 3.109 clones was expressed at the surface of M13 phage. Hybrigenics’ Phage Display allowed to select VHHs recognizing the non-adsorbed antigen in a native form. A total of 90 VHHs were picked and analyzed after three rounds of Phage Display for binding to CTG hairpin, and 83 of these VHHs were positive in non-adsorbed phage ELISA. After sequencing of these 83 clones, 27 different clones were validated and positive in non-adsorbed phage ELISA. Among those, 1 1 clones were selected from the screen according to their criterion of redundancy, ELISA assay results and the sequencing results. The 1 1 clones selected are Nali_PA00301_C09_woAmber, H01 , A02, G05, E02, F03, F08, H10, C04, D1 1 and B02.
Production and purification of VHH
Figure imgf000038_0001
For miniproduction of the four VHHs (E02, F03, F08 and H01 ), HEK293 cells were plated in 6 well plates at 700 000 cells per well 2 days before transfection. Cells were transfected with 2.5 gg of Minibody DNA and 7.5 gL XtremeGene HP DNA. 24h post transfection the medium was replaced by medium without serum. After 6 days, the medium was collected, and the production was checked by Western blot with an anti-rabbit antibody coupled to HRP. The expected size for VHH-Fc is around 42 kDa.
Production and purification of VHH H01 was performed at the Institut Pasteur protein production facility, as follows. The VHH H01 was expressed in Expi293F cells at 37 °C and 8% CO2 (Expi293, ThermoFisher Scientific). Cell culture supernatants were collected 4 days post transfection and purified by protein G chromatography (Cytiva). Washing step was done with DPBS 1 X pH 7.2 and VHH were eluted with Glycine 0.1 M pH 2.5 in wells containing TRIS 1 M pH 8.0.
Dot blot experiments with purified VHH proteins
Hybond-XL nylon membrane (Amersham-GE Healthcare) was soaked in 2X SSC for 20 minutes. Synthetic oligonucleotides, plasmidic or genomic DNA were denatured in 50-75 gL 0.4 M NaOH, incubated at 95°C for 2 minutes, and rapidly put on ice. They were transferred on the nylon membrane under limited vaccum, using a homemade dot blotter. For each sample, 500 gL of 0.4 M NaOH were added in each well, three times. After disassembly of the dot blotter, the membrane was incubated one hour in PBS. Blocking was performed for one hour in TBST (20 mM Tris pH 7.5, 150 mM NaCI, 0.1 % Tween 20) supplemented with 5% dry milk. VHH were diluted 1 /50th to 1 /500th in preliminary tests in TBST + 5% dry milk, and incubated overnight at 4°C. Purified H01 VHH was diluted 1/1000th in TBST + 5% dry milk in subsequent experiments. The secondary anti-rabbit antibody (Thermo Scientific) was diluted 1 /2000th, incubated for one hour in TBST + 5% dry milk at room temperature, and revealed by ECL Prime Western Blotting (Amersham). Signals were revealed on a Bio-Rad Chemidoc and quantifications performed using the Image Lab software (version 5.2).
Figure imgf000038_0002
on HiTrap column
Plasmidic secondary structures were captured on an Akta start FPLC (Cytiva) at a flow rate of 1 mL/min for all buffers. A 1 mL HiTrap protein G (Cytiva) was first equilibrated with 10 mL 20 mM sodium phosphate buffer (pH 7.0). The VHH (20 gg) was loaded in 10 mL of the same buffer. To lower nonspecific binding, the column was saturated with 15 mL 1 % BSA in 20 mM sodium phosphate buffer. The pRW3222 plasmid (Fabre et al. 2002) was heat denatured for 10' at 95°C before being loaded on the column in 5 mL T10N50 (10 mM Tris pH 8.0, 50 mM NaCI). Right after plasmid injection, 1 mL fractions are collected until the end of the experiment (ca. 30 fractions). The column is washed with 10 mL of the same buffer. Elution of plasmidic DNA bound to the VHH is performed with 10 mL T10N200 (10 mM Tris pH 8.0, 200 mM NaCI, 10 mL). The column was finally washed with 5 mL of T10N50.
For each fraction, 100 pL were concentrated using a Concentrator plus (Eppendorf) and loaded on a 1 % agarose gel. Transfer and hybridization with the radioactively labeled pUC19 plasmid were performed as previously described (Viterbo et al. 2018). Signal quantification was performed as previously (Viterbo et al. 2016).
Results
Design of the synthetic CTG hairpin
A 130 nt DNA oligonucleotide was designed in such a way that its extremities are complementary and form a "GC clamp", that tolerate formation of an imperfect hairpin in only one conformation. The GC clamp itself is synthesized to be used as a negative control in all experiments. Both oligonucleotides are linked to a biotin at their 5' ends. Structures, free energy and Tm of both molecules were calculated using mFold version 3.6 (Figure 1 ).
The (CTG)so oligonucleotide, the GC clamp and a non structured (NS) 1 1 1 nt long oligonucleotide has been migrated on non-denaturing and denaturing agarose gels (Figure 2). On the non-denaturing gel (3% Metaphor agarose (Lonza), TBE 1 X), both the CTG hairpin and the GC clamp migrate as predicted for double-stranded DNA of these expected molecular weights. Note that the ethidium bromide signal is much stronger than for the NS oligonucleotide. On the denaturing gel (3% Metaphor agarose (Lonza), 50 mM NaOH, 1 mM EDTA) all three species migrate at their expected molecular weight. The ethidium bromide signal is stronger for the CTG hairpin, suggesting the possibility that at least part of the oligonucleotide may be double-stranded. Amounts loaded on each gel are approximately: NS control oligonucleotide: 20 pmoles, CTG hairpin: 5 pmoles, GC clamp: 28 pmoles.
Figure imgf000039_0001
An one-hybrid experiment using HIS3 gene as reporter was carried out. The HIS3 gene under the control of a minimal promoter fused to a CTG trinucleotide repeat was integrated in the yeast genome. The 1 1 VHH fused to the Gal4p activating domain were expressed in this yeast strain. If the fusion protein binds to the CTG repeat it will activate transcription of the HIS3 gene and cells will grow on minimal medium without histidine (SC-His). A [His+] strain was used as positive control and the plasmid containing the GAL4 activating sequence but no VHH (Empty) allowed to determine the baseline of growth on SC-His. The score represents the difference of growth between each VHH-Gal4 fusion and the Empty vector. Note than some VHH obtained a negative score, due to a worse growth than the Empty control. Yeast plates were incubated at two different temperatures, 30°C which is the physiological temperature for budding yeast and 20°C. As expected, growth was slower at lower temperature, but it did not change the general outcome. An one-hybrid experiment using a GFP reporter under the control of the minimal HIS3 promoter was carried out. The minimal HIS3 promoter was fused to four different microsatellites: (CTG)so, (CAG)so, (GAA)so or (CGG)so. An isogenic construct in which no microsatellite was present was used as a control of baseline fluorescence. The table 6 shows the average ratio of the GFP fluorescence of each repeat tract divided by the baseline fluorescence (A), at the two time points after alpha factor release. Values above 1 indicate a higher fluorescence with a repeat tract than with no repeat (A) and are shown in bold. The best one-hybrid results both with the HIS3 reporter and the GFP reporter are E02, F03, F08, H01 and were produced as proteins.
Figure imgf000040_0001
Table 6 Dot blots with purified VHHs.
100 ng of the synthetic CTG hairpin (CTG) and of the GC clamp (GC)Top: Nylon membranes on were spotted on nylon membranes and were incubated with one of the four VHH (E02, F03, F08 and H01 ). Three pictures were taken at different exposure times (labeled 1 , 2 or 3). Only H01 gave a significant positive signal with the CTG hairpin and no signal with the GC clamp (Figure 5). When H01 was incubated with a membrane on which different amounts of the CTG hairpin (CTG), the GC clamp (GC) or a synthetic CAG hairpin (CAG) were spotted, a positive signal was detected only with the CTG hairpin, and its quantification is shown to the right (Figure 5).
The VHH H01 reveals the presence of CTG hairpins on plasmidic DNA containing 98 or 255 CTG triplets on dot blot (Figure 6). Heat denaturation of plasmidic DNA creates additional CTG hairpins that are detected by the VHH H01 . No signal is visible with the same plasmid containing no repeat tract (Figure 7). The response of VHH on denatured then renatured DNA is greater. Indeed, thermodynamics predicts that the formation of a hairpin on single-stranded DNA is faster than the renaturation of the plasmid to its native doublestranded form without a hairpin.
Yeast total genomic DNA containing CTG trinucleotide repeats of different lengths (50, 100, 160 or 280 triplets), as well as human total genomic DNA of a DM1 patient contain different amounts of CTG hairpins, has been revealed by the VHH H01 . Signal quantifications show that genomic DNA containing 160 and 280 triplets exhibit a lower signal than genomic DNA containing shorter repeat lengths. The DM1 genome contains 2600 CTG triplets at the DMPK locus (Figure 8). confirms the ability of VHH H01 to hybridize on DM1 human DNA (patient #8 301 triplets, patient #9 - 371 triplets) and transgenic mice DNA (mouse 351 and mouse 352 - 130 triplets, mouse
506 and mouse 515 - 250 triplets, mouse 4431 and 4432 - 1000 triplets). The signal revealed by the VHH H01 is linearly correlated to CTG repeat length (Figure 10). This shows that the VHH may be used to quantitatively estimate CTG repeat length from whole genomic DNA.
Figure imgf000041_0001
on HiTrap column
As shown on figure 1 1 , ca. 7% of the repeat-containing supercoiled plasmid is bound by the VHH on the HiTrap column.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to a nucleic acid hairpin comprising repeats of CNG nucleotide triplets.
2. The antibody or antigen-binding fragment thereof according to claim 1 , wherein the CNG nucleotide triplet has the nucleotide sequence selected from the group consisting of CAG, CCG, CTG, CUG and CCG, preferably selected from the group consisting of CTG, CUG and CAG, more preferably CTG and/or CUG, most preferably CTG.
3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the number of CNG nucleotide triplets that are repeated is at least 30.
4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the hairpin is selected from the group consisting of a 4-nucleotide loop hairpin, a 3-nucleotide loop hairpin and a 7-nucleotide loop hairpin.
5. The antibody or antigen-binding fragment thereof according to claim 4, wherein the antibody or antigen-binding fragment is a single domain antibody and comprises three complementary determining regions (CDR1 to CDR3, respectively);
- wherein the amino acid sequence of the CDR1 is selected from the group of amino acid sequences consisting of SEQ ID NO: 32, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 33 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 32, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO:33;
- wherein the amino acid sequence of the CDR2 is selected from the group of amino acid sequences consisting of SEQ ID NO: 43, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 44 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 43, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42 or SEQ ID NO 44; and
- wherein the amino acid sequence of the CDR3 is selected from the group of amino acid sequences consisting of SEQ ID NO: 54, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 55 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 54, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53 or SEQ ID NO: 55.
6. An isolated single domain antibody, comprising three complementary determining regions (CDR1 to CDR3, respectively);
- wherein the amino acid sequence of the CDR1 is selected from the group of amino acid sequences consisting of SEQ ID NO: 32, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO:33 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 32, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO:33;
- wherein the amino acid sequence of the CDR2 is selected from the group of amino acid sequences consisting of SEQ ID NO: 43, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42 and SEQ ID NO: 44 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 43, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42 or SEQ ID NO: 44; and
- wherein the amino acid sequence of the CDR3 is selected from the group of amino acid sequences consisting of SEQ ID NO: 54, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 55 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 54, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53 or SEQ ID NO: 55.
7. The antibody or antigen-binding fragment thereof according to claim 5 and/or the single domain antibody according to claim 6, wherein the single domain antibody comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 21 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 22.
8. The antibody or antigen-binding fragment thereof according to claim 5 or 7 and/or the single domain antibody according to claim 6 or 7, wherein the single domain antibody comprises an amino acid sequence selected from SEQ ID NO: 21 and 17-19.
9. The antibody or antigen-binding fragment thereof according to any one of claims 5 and 7-8, and/or the single domain antibody according to any one of claims 6-8, wherein the single domain antibody consists of an amino acid sequence of SEQ ID NO: 21 .
10. The antibody or antigen-binding fragment thereof according to any one of claims 1 -5 and, 7- 9, and/or the single domain antibody according to any one of claims 6-9, wherein the antibody, the antigen-binding fragment thereof and/or the single domain antibody binds to the nucleic acid having the nucleotide sequence SEQ ID NO: 1 with a KD value inferior to 200 nM.
11. A complex comprising: the antibody or antigen-binding fragment thereof according to any one of claims 1 -5 and 7-10 or the single domain antibody according to any one of claims 6-10 and a nucleic acid hairpin comprising repeats of CNG nucleotide triplets as defined in any one of claims 1 -4.
12. The use of the complex according to claim 1 1 .
13. A method comprising a step of contacting the antibody or antigen-binding fragment thereof according to any one of claims 1 -5 and 7-10 or the single domain antibody according to any one of claims 6-10 with molecule(s) comprising a nucleic acid hairpin comprising repeats of CNG nucleotide triplets as defined in any one of claims 1 -4 thereby forming a complex.
14. A method for detecting and/or quantifying nucleic acid hairpin comprising repeats of CNG nucleotide triplets as defined in any one of claims 1 -4 in a sample, comprising: providing the antibody or antigen-binding fragment thereof according to any one of claims 1 -5 and 7-10 or the single domain antibody according to any one of claims 6-10; providing a sample; contacting the antibody or the antigen-binding fragment or the single domain antibody with the sample; and visualizing the antigen-antibody complexes formed.
15. An in vitro method for diagnosing a trinucleotide expansion disorder selected from the group consisting of myotonic dystrophy type 1 , spinocerebrallar ataxia 12, Huntington’s disease-like 2, spinal and bulbar muscular atrophy, Huntington’s disease, dentatorubral-pallidouysian atrophy, spinocerebrellar ataxia 1 , spinocerebrellar ataxia 2, spinocerebrellar ataxia 3, spinocerebrellar ataxia 6, spinocerebrellar ataxia 7, spinocerebrellar ataxia 17, fragile X syndrome, X tremor, ataxia syndrome and X-linked mental retardation wherein the antibody or antigen-binding fragment thereof according to any one of claims 1 -5 and 7-10 or the single domain antibody according to any one of claims 6-10 is used for the detection and/or quantification of nucleic acid hairpin comprising repeats of CNG nucleotide triplets as defined in any one of claims 1 -4 in a sample previously obtained from a subject.
16. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 -5 and 7-10 or the single domain antibody according to any one of claims 6-10 in an in vitro diagnostic test of a trinucleotide expansion disorder selected from the group consisting of myotonic dystrophy type 1 , spinocerebrallar ataxia 12, Huntington’s disease-like 2, spinal and bulbar muscular atrophy, Huntington’s disease, dentatorubral-pallidouysian atrophy, spinocerebrellar ataxia 1 , spinocerebrellar ataxia 2, spinocerebrellar ataxia 3, spinocerebrellar ataxia 6, spinocerebrellar ataxia 7, spinocerebrellar ataxia 17, fragile X syndrome, X tremor, ataxia syndrome and X-linked mental retardation.
17. A kit for detection of a nucleic acid hairpin comprising repeats of CNG nucleotide triplets as defined in any one of claims 1 -4 in a biological sample, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 -5 and 7-10 or the single domain antibody according to any one of claims 6-10.
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