Antibodies specific for DNA G-quadruplexes and uses

Abstract

The invention relates to an antibody or an antibody fragment which specifically recognizes telomeric DNA G-quadruplexes of antiparallel conformation and which comprises at least the six sequences SEQ ID NO: 21, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 22, SEQ ID NO: 3 and SEQ ID NO: 4, or at least the six sequences SEQ ID NO: 21, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 7 and SEQ ID NO: 8, its use in diagnostic or prognostic methods or as a medicament, in particular in the treatment of cancerous diseases, neurodegenerative diseases and genetic diseases. The invention also relates to a detection kit comprising such an antibody or such an antibody fragment, a nucleic acid encoding one of said peptide sequences and a recombinant or transforming vector containing the latter.

Description

Title of the invention: Antibodies specific for DNA G-quadruplexes and uses thereof

[0001] The invention relates to antibodies or antibody fragments which specifically recognize a determined topology of DNA G-quadruplex motifs, as well as their applications.

[0002] The DNA strands of chromosomes end with repetitive DNA sequences rich in guanine (G) and which do not contain genes, the telomeres. They protect the chromosomes against fusions by their ends and prevent the degradation of internal sequences. These sequences are capable of forming secondary structures called G-quadruplexes (or G4) observed in vitro, but still relatively unknown in vivo. Such structures have also been found at the level of gene promoter regions and replication origins. They can also be formed with sequences of other guanine-rich nucleic acids such as messenger RNA and non-coding RNA.

[0003] These G4 conformations comprise two or more guanine tetrads (or G-quartet), which result from the planar arrangement of four guanines, paired by hydrogen bonds. Their stacking is carried out in particular via hydrophobic interactions (ir-stacking, or ir stacking in French) and leads to G4 structures which exhibit great structural and topological diversity. Thus, depending on the orientation of each of the nucleic acid strands involved, the G4s can adopt conformations (or topologies) of the parallel, antiparallel or hybrid type. According to the invention, an antiparallel topology is of interest.

[0004] The likely involvement of G4s in several biological processes, notably in the regulation of transcription, in genomic rearrangements, in telomeric dysfunctions, with a role sometimes as activators or even inhibitors, makes these structures the target of numerous studies, and in particular with a view to therapeutic applications.

[0005] Thus, the thesis supported by Nasab REDA for “Selection of antibodies specific to an antiparallel topology of telomeric G-Quadruplex DNA” publicly defended on March 30, 2022 describes the development of a selection technique, called Phage Display (in French, phage expression), which aims to select antibody fragments directed against an antiparallel topology of telomeric G4 DNA. It uses a biomolecular system of G-quadruplex constrained in antiparallel topology on a cyclodecapeptide.

[0006] However, to date and despite the interest they arouse, such specific antibodies of a unique topology of the telomeric G-quadruplex have not yet been identified.

[0007] This is the objective that the invention addresses. It provides an antibody or an antibody fragment that specifically recognizes telomeric DNA G-quadruplexes of antiparallel conformation.

[0008] By antibody or antibody fragment which specifically recognizes telomeric DNA G4s of antiparallel conformation, it is understood that said antibody or said antibody fragment has a specificity with respect to a sequence in a unique topology, namely antiparallel. This property makes it possible to increase the detection of telomeric DNA G4s by a level of specificity in an attempt to elucidate the importance of the topology.

[0009] An antibody is a molecule consisting of four polypeptide chains, two identical heavy chains and two identical light chains, linked together by disulfide bridges. Each heavy chain is composed of a variable domain (VH) and at least 3 constant domains (CH1, CH2 and CH3, or even CH4), while each light chain is composed of a variable domain (VL) and a constant domain (CL). The constant domains determine the belonging of an antibody to a type of immunoglobulin and the variable domains, each consisting of three hypervariable regions (CDR), contain the binding site, or paratope, to the antigen via its epitope.

[0010] It is precisely the peptide sequences of CDR1, CDR2 and CDR3 of the light chains (VL) and heavy chains (VH) of two specific antibodies according to the invention which have been determined; these CDRs correspond to the following sequences:

[0011] for a first antibody, the CDR1, CDR2 and CDR3 of the heavy chain have the sequences SEQ ID NOs: 21, 1 and 2, respectively, and the CDR1, CDR2 and CDR3 of the light chain have the sequences SEQ ID NOs: 22, 3 and 4, respectively;

[0012] for a second antibody, the CDR1, CDR2 and CDR3 of the heavy chain have the sequences SEQ ID NOs: 21, 5 and 6, respectively, and the CDR1, CDR2 and CDR3 of the light chain have the sequences SEQ ID NOs: 22, 7 and 8, respectively.

[0013] Variable domains, even in the form of antibody fragments, retain their antigen-binding capacity. Among these, the main ones are the Fab, F(ab')2, Fv, scFv and VHH fragments. The Fab fragment, monovalent, comprises all or part of the constant domains CH1 and CL and the VH and VL domains of each of the heavy and light chains. The F(ab')2 fragment, divalent, corresponds to the association of two Fab fragments linked by the disulfide bridges of the hinge region of the heavy chains. The Fv fragment, monovalent, comprises at least the VH domain; it can also comprise the VL domain. The scFv fragment, monovalent, comprises all or part of the VH and VL domains linked by a very flexible peptide arm. The VHH fragment is composed of the three CDRs of the VL domain or the VH domain. By fragment of antibodies according to the invention, advantageously means an scFv fragment, or even a Fab fragment. The expression "antibody fragment" according to the invention nevertheless covers any fragment capable of specifically recognizing a G4 of telomeric DNA of antiparallel conformation and whose molecule comprises, for this purpose, at least the three CDRs of the VL domain and / or at least the three CDRs of the VH domain of an antibody of the invention. This means that it may result from a combination of one or more of the aforementioned fragments, it may also comprise amino acids or peptides, or even polypeptides, not present in the original structure of an antibody but originating from its production process or likely to be of interest, for example, in its production, purification, selection or use.For the purpose of a higher specificity of an antibody fragment, the latter comprises at least the three CDRs of the VL domain and at least the three CDRs of the VH domain of an antibody of the invention.

[0014] Thus, the invention relates to an antibody or an antibody fragment which specifically recognizes telomeric DNA G-quadruplexes and which comprises at least the six sequences SEQ ID NO: 21, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 22, SEQ ID NO: 3 and SEQ ID NO: 4 or which comprises at least the six sequences SEQ ID NO: 21, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 7 and SEQ ID NO: 8.

[0015] According to a variant of the invention, the antibody or antibody fragment which specifically recognizes telomeric DNA G-quadruplexes of antiparallel conformation comprises at least the six sequences SEQ ID NO: 21, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 22, SEQ ID NO: 3 and SEQ ID NO: 4 successively, or at least the six sequences SEQ ID NO: 21, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 7 and SEQ ID NO: 8 successively.

[0016] The authors of the invention have thus succeeded in obtaining an antibody or an antibody fragment which has a specificity with respect to G4 telomeric DNA of antiparallel topology, reproducible.

[0017] The choice of the antibody fragment is mainly determined by the selection technique implemented. Thus, according to a variant of the invention, it is the Phage Display technology which is used and the antibody fragment is preferably chosen from the Fab, Fv, scFv fragments, better still, it is a scFv fragment.

[0018] By antibody or antibody fragment which comprises at least the six sequences SEQ ID NO: 21, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 22, SEQ ID NO: 3 and SEQ ID NO: 4 successively, or at least the six sequences SEQ ID NO: 21, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 7 and SEQ ID NO: 8 successively, it is meant that it contains these six sequences in the order indicated, these sequences following each other, directly or indirectly, on the surface of the qua structure ternary of said antibody or said antibody fragment and not necessarily in their primary structure, it is an arrangement of a conformational nature. The six sequences may be consecutive, that is to say one after the other, or be separated by one or more amino acids, a peptide (oligopeptide or polypeptide), it being understood that, for example, among the 4 sequences, two may be consecutive and the others may be separated by one or more amino acids or by a peptide.It is also understood that it may contain, in addition to these six sequences, one or more amino acids, peptides (oligopeptides or polypeptides) or upstream of the first sequence (SEQ ID NO: 21 or 22) and / or downstream of the last sequence (SEQ ID NO: 4 or 8), one or more amino acids, peptides (oligopeptides or polypeptides), proteins, for example fusion proteins, provided that its specificity with respect to a G4 of antiparallel conformation is not significantly affected; preferably, it is improved. Thus, an antibody or an antibody fragment according to the invention may comprise, or consist of, a sequence chosen from SEQ ID NO: 9 and SEQ ID NO: 10, the presence of one and / or the other of these sequences making it possible to optimize the specificity of said antibody or said fragment.

[0019] By antibody fragment which consists of a sequence chosen from SEQ ID NO: 9 and SEQ ID NO: 10, it is meant that its sequence is identical to any one of the sequences SEQ ID NO: 9 or SEQ ID NO: 10; it is also meant that its sequence may consist of a series of at least two of said sequences, identical or different.

[0020] By antibody fragment which comprises a sequence chosen from SEQ ID NO: 9 and SEQ ID NO: 10, it is meant that its sequence is longer than any one of these sequences but that it comprises any one of these sequences, or several of these sequences, identical or different, consecutively or not.

[0021] According to the invention, an antibody is advantageously an IgG.

[0022] An antibody or antibody fragment of the invention constitutes a biological tool of major interest in order to explore the role of G4s and their different conformations, in vivo. It can thus supplant existing solutions which, although specific to G4 structures, do not allow their different topologies to be distinguished; for example, the BG4 antibody used in immunofluorescence can be cited.

[0023] Thus, within the framework of the invention, the antibodies or antibody fragments described above can be used in the stabilization of telomeric DNA G4s of antiparallel conformation. This is for the purposes of exploring and interpreting the function of G4s and their conformations, for detection or diagnosis purposes, or for therapeutic treatment purposes.

[0024] Within the scope of the invention, an antibody or an antibody fragment can therefore be used in diagnostic or prognostic methods, as well as as a medicament.

[0025] Since G4 structures are present in telomeres, they are associated with many tumor diseases. Antibodies or antibody fragments, thanks to their specificity, will stabilize the G4s and thus prevent the proliferation of tumor cells.

[0026] The invention further relates to an antibody or antibody fragment as defined above, for use in the treatment of cancerous diseases, neurodegenerative diseases and genetic diseases. Indeed, high and significant formation of G4s, as well as unbalanced dynamics of these structures, can be associated with such diseases. For example, an expansion of motifs capable of adopting a G4 structure has been associated with genetic disorders such as fragile X syndrome, and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) [Maizels N. G4-associated human diseases. EMBO reports. 2015;16(8):910-22]. According to a variant of the invention, said antibodies or said antibody fragments are used to treat amyotrophic lateral sclerosis or fragile X syndrome.

[0027] An antibody or a fragment of an antibody as defined above can be implemented in different forms. Thus, the invention relates to a composition containing at least one or more of said antibodies and / or antibody fragments and a pharmaceutically acceptable support. The invention further provides a kit for detecting and / or quantifying a DNA G4 of antiparallel conformation, said kit containing at least one or more of said antibodies and / or said antibody fragments and a detection and / or quantification reagent.

[0028] The invention also relates to a use of said antibody or said antibody fragment for preparing a reagent for detecting a DNA G4 of antiparallel conformation, in a biological sample or in vivo.

[0029] According to a variant, the aforementioned applications of an antibody or an antibody fragment are intended for human telomeric DNA G4s of antiparallel conformation. Of course, the invention is not restricted thereto.

[0030] The invention also relates to a nucleic acid coding for an antibody or an antibody fragment according to the invention. Thus, it may comprise at least the six sequences SEQ ID NO: 23, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 24, SEQ ID NO: 13 and SEQ ID NO: 14, said sequences coding respectively for SEQ ID NO: 21, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 22, SEQ ID NO: 3 and SEQ ID NO: 4. It may also comprise at least the six sequences SEQ ID NO: 23, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 17 and SEQ ID NO: 18, said sequences coding respectively for SEQ ID NO: 21, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 7 and SEQ ID NO: 8. According to one variant, this nucleic acid comprises or consists of a sequence chosen from SEQ ID NO: 19 and SEQ ID NO: 20. Another subject of the invention is a recombinant vector or transformant containing a nucleic acid as described above.

[0031] The invention is illustrated with the following examples and with the support of the figures according to which:

[0032] [Fig-1] represents the structure of the target, called Lat-H, of an antibody of the invention, namely a G4 DNA with an antiparallel Basket-type topology derived from the human telomeric DNA sequence and grafted onto a rigid cyclopeptide called RAFT (for Regioselectively Addressable Functionalized Template). The original sequence of 22 nucleotides is replaced by two sequences of 11 nucleotides each. These oligonucleotides are attached to the “RAFT” in a precise topology, thus constraining the three-dimensional structure.

[0033] [Fig.2] illustrates the mean values of 1 / KD (Mean 1 / KD) in 1 / nM of an IgG of the invention of SEQ ID NO: 9 for different DNA targets defined in Example 3, KD being the dissociation constant between said IgG and its target, measured by the optical technique of biolayer interferometry (BLI).

[0034] [Fig.3] illustrates the mean values of 1 / KD (Mean 1 / KD) in 1 / nM of an IgG of the invention of SEQ ID NO: 10 for different DNA targets defined in Example 3, KD being the dissociation constant between said IgG and its target, measured by the BLI technique.

[0035] Example 1: Obtaining two antibodies of the invention by the Phage Display method

[0036] 1.1) The Phage Display method

[0037] The Phage Display method was used to select antibodies directed against the constrained antiparallel G4 DNA structure as shown in [Fig.l] ( Lat-H ). This method is based on the use of M13 bacteriophages as tools for screening proteins of interest. This process is an in vitro selection.

[0038] The method consists of incubating a library of phages presenting a wide variety of antibodies on their surfaces with the target molecule immobilized on a solid support. The phages exposing the antibodies (in fusion with the PIII protein of the capsid) most specific to G4 DNA attach to the surface of the support on which the target molecules are attached. Several successive washes ensure selection of the antibodies most specific to the target and are retained on the solid support. The phages are then detached from the target and brought into contact with bacteria so that they are infected and can produce new phages containing the selected sequences. The new phage library obtained is more restricted in sequence diversity and is used for the next round of selection. After three rounds of selection, the specific phages are thus amplified, then isolated and recovered.Several completely independent experimental sessions resulted in a batch or panel of antibodies selected against the Lat-H target. This . The first screening resulted in about a hundred antibodies which were then tested by specificity analyses (ELISA). The most specific candidates for the Lat-H target were then produced and purified in order to perform characterization analyses by biolayer interferometry (BLI).

[0039] 1.2) Bank and bacteria used

[0040] The Tomlinson library was made available by the laboratory of G. Winter at MRC Cambridge and Geneservice (Source Bioscience, Nottingham, UK). The helper phage Ml3KO7 is used to produce the phages after infection of bacteria (E. coli TGI strains). The Tomlinson library is composed of 1.47x 109 ScFv cloned into an ampicillin-resistant phagemid vector and transformed into TGI cells. This semi-synthetic library is based on a single human framework for the variable heavy chain, VH, and the variable light chain, VL. The scFv antibodies are displayed on the minor coat protein PIII of M13 with 6xHistidine tags, which are useful for specific detection and purification by affinity chromatography.

[0041] 1.3) Production of M13 bacteriophages

[0042] TGI bacteria (containing the phagemid) are cultured in 2xTY medium (16g / L tryptone, 10g / L yeast extract and 5g / L NaCl) containing 100 pg / mL ampicillin and 1% glucose with shaking at 37°C. Once the optical density (OD) at 600 nm of 0.5 is reached, the bacteria are infected with the helper phage M13K07 (with a ratio of 1 bacterium to 20 helper phages) and incubated without shaking at 37°C for 30 min. Infected bacteria were centrifuged at 3300 xg for 10 min, resuspended in 2xTY medium in the presence of ampicillin (at 100 pg / mL) and kanamycin (at 25 pg / mL), and cultured overnight at 30°C. The culture was centrifuged at 10,000 xg for 10 min, and the supernatant containing the phages was collected.

[0043] 1.4) Selection

[0044] Phosphate buffered saline (PBS: KH2PO4 1.5mM; NaCl 155mM; Na2HPO4-7H2O 2.7mM; pH7.2) was used for selection incubations and then PBS-T (PBS with 0.1% Tween 20) was used for washes. The produced phages are first presented to the Lat-H target (0.4 nmol / mL) and then are brought into contact with magnetic beads functionalized with streptavidin on the surface. The Lat-H targets attach to the beads thanks to their biotin. The mixture is incubated at room temperature for 1h (with wheel shaking). The magnetic beads are subsequently washed 10 times, to eliminate all non-specific interactions, with PBS-T solution. Phages are eluted with triethylamine (TEA) solution and shaken for 10 min on a tray. The supernatant is removed and Tris-HCl solution (1M, pH 7) is added.TGI bacteria (at an OD of 0.5) are added to the eluted phages and the remaining beads, then incubated in a bath- . marie at 37°C for 30 min without shaking. Finally, these infected bacteria were spread on agar petri dishes (2xTY ampicillin / 1% glucose, agar) and incubated overnight at 30°C. The next day, the bacterial lawns from the agar dishes were collected to begin another round of selection. Several rounds of selection were necessary to obtain scFv sequences specific for the Lat-H target.

[0045] At the end of the selection rounds, a few bacterial clones (containing a phagemid with a scFv) are isolated and the portion of DNA of the phagemids corresponding to the scFv sequence is sequenced.

[0046] Example 2: Purification and selection of the two antibodies obtained in Example 1

[0047] 2.1) Expression and purification of ScFv antibodies

[0048] The scFvs were produced from E. coli TGI harboring the phagemid. Each TGI culture was incubated for 2 h at 37°C. Expression was then induced with isopropyl [3-Dl-thiogalactopyranoside (IPTG, final concentration 1 mM) to an optical density of 0.5 and the culture was incubated at 30°C for 18 to 24 h. The next day, the cultures were centrifuged at 10,000xg for 10 min. The bacterial pellets were resuspended in lysis buffer (with ImM protease inhibitors). The suspension was shaken on an ice bath for 10 min and on a wheel for 15 min; the supernatant was collected after centrifugation at 16,000xg for 30 min. The supernatants are pooled and used for affinity chromatography purification of hexahistidine-tagged scFv. The crude periplasmic fraction of E. coli is mixed with Ni-NTA resin, stirred at room temperature for a few minutes, and placed on the purification column.Elution is performed by adding 200 μL of 250 mM imidazole. The eluted scFv fractions are dialyzed against 1 liter of PBS for 24 h. The amount of dialyzed antibody is determined by an absorbance assay at a wavelength of 280 nm, as well as an alternative assay by Bradford staining. The dialyzed samples are incubated with Bradford dye for 10 min in the dark. The binding of this dye to proteins is detected by absorbance at 595 nm and compared to a standard range to determine the protein concentration value. The activity of the tested scFv antibodies is determined by ELISA. ScFv candidates with specific activity for the Lat-H target with the least activity against control targets (DNAs that are not anti-parallel G-quadruplex DNAs) are selected.

[0049] 2.2) Production of selected scFv candidates in IgG format

[0050] These antibodies are then produced from mammalian cells (CHO DG44) in IgG format (with human Fc, IgGl HC: Glm3). Purification is carried out by affinity chromatography (Ni-NTA). These produced and purified antibodies are then used in biolayer interferometry (BLI) studies.

[0051] Example 3: Specificity of the two antibodies of the invention selected in Example 2

[0052] [Fig.2] and [Fig.3] show the affinity (1 / KD) of the antibodies of SEQ ID NO: 9 and SEQ ID NO: 10 for different DNA and RNA targets. Tel 23 is the antiparallel telomeric G4 DNA target; 25 CEB, C-kit and C-myc correspond to parallel topology G-quadruplex DNAs; Terra corresponds to a parallel topology telomeric RNA; ss RNA corresponds to a single strand of RNA; and HP-ATT corresponds to a double strand of DNA.

[0053] The figures show the specificity of each of the antibodies of SEQ ID NO: 9 and SEQ ID NO: 10.

Claims

Claims

1. An antibody or antibody fragment specifically recognizing telomeric DNA G-quadruplexes of antiparallel conformation and comprising at least the six sequences SEQ ID NO: 21, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 22, SEQ ID NO: 3 and SEQ ID NO: 4, or at least the six sequences SEQ ID NO: 21, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 7 and SEQ ID NO:

8.

2. Antibody or antibody fragment according to claim 1, characterized in that it comprises or consists of a peptide sequence chosen from SEQ ID NO: 9 and SEQ ID NO:

10.

3. Antibody or antibody fragment according to claim 1 or 2, characterized in that it comprises or consists of a fragment chosen from scFv fragments and Fab fragments.

4. Antibody according to any one of claims 1 to 3, characterized in that it is an IgG.

5. Antibody or antibody fragment according to any one of claims 1 to 4, for use in diagnostic or prognostic methods.

6. Antibody or antibody fragment according to any one of claims 1 to 4, for use as a medicament.

7. An antibody or antibody fragment according to any one of claims 1 to 4, for use in stabilizing telomeric DNA G-quadruplexes of antiparallel conformation.

8. An antibody or antibody fragment according to any one of claims 1 to 4, for use in the treatment of cancerous diseases, neurodegenerative diseases and genetic diseases.

9. An antibody or antibody fragment for use according to claim 8, characterized in that the disease is amyotrophic lateral sclerosis or fragile X syndrome.

10. A composition containing at least one or more of the antibodies and / or antibody fragments according to any one of claims 1 to 4, and a pharmaceutically acceptable carrier.

11. Kit for detecting and / or quantifying a DNA G-quadruplex of antiparallel conformation, said kit containing at least one or more of the antibodies and / or antibody fragments according to any one of claims 1 to 4 and a detection and / or quantification reagent.

12. Use of an antibody or antibody fragment according to one of any of claims 1 to 4, for preparing a reagent for detecting and / or quantifying a DNA G-quadruplex of antiparallel conformation, in a biological sample or in vivo.

13. Nucleic acid encoding an antibody or antibody fragment according to claim 1 or 2.

14. Nucleic acid according to claim 13, characterized in that it comprises or consists of a sequence chosen from SEQ ID NO: 19 and SEQ ID NO: 20:

15. Recombinant or transforming vector containing the nucleic acid according to claim 13 or 14.