Patent Application
20240272176
The present invention relates to an antibody specific for γ-H2AX and its uses as a laboratory tool.
Histones constitute the core proteins of chromatin and their post-translational modifications (PTMs) contribute to the molecular basis of epigenetic gene regulation and cellular memory. In humans, several variant forms of histones have been described and this is particularly relevant for the H2A histone. The H2A variants represent the largest and most diverse family of histones; there is overwhelming evidence that their unstructured N- and C-termini, which protrude out of the core structure of the nucleosome, harbor several sites for PTMs in response to varying stimuli. The H2AX variant shares high amino acid similarity with H2A and is characterized by an extended C-terminus, which is phosphorylated when the cells become injured by agents that provoke DNA replication stress (RS) and genome instability. The phosphorylation of serine at position 139 (S139) of H2AX has been particularly well studied and represents a key event in the detection and response to DNA damage.
Phosphorylation of histone H2AX at S139, which gives rise to what is generally referred to as γ-H2AX, is in fact a very early step in the DNA damage response (DDR) and an essential signal for the recruitment and retention of DDR complexes at the site of damage. Three different phosphatidylinositol 3 kinase (PI3K)-related kinases mediate S139 phosphorylation on H2AX: ATM (ataxia-telangiectasia mutated), ATR (ATM and Rad3-related), and DNA-PK (DNA-dependent protein kinase). ATM and DNA-PK share functional redundancy upon ionizing radiation, while ATR may preferentially phosphorylate H2AX during RS. This PTM of H2AX is highly dynamic and a number of phosphatases, including those of the PPP family and Wip1, are able to dephosphorylate γ-H2AX to fine-tune the duration and intensity of the DDR signaling. It has also been found that H2AX can be phosphorylated at the threonine residue at position 136 (T136) and at the C-terminal tyrosine residue at position 142 (Y142) to facilitate DNA repair, whereas the persistency of the latter PTM may also trigger apoptosis. Nevertheless, S139 phosphorylation is regarded as the main PTM of H2AX since it is specifically recognized by the adaptor protein MDC1, which further recruits several E3 ubiquitin ligases to favor DNA repair and/or restart of the halted forks during RS.
Because γ-H2AX is involved in the DDR, it is generally considered a biomarker of DNA double-strand breaks (DSBs) and its relevance as read-out of sustained RS is well accepted. In addition, H2AX is also phosphorylated in the absence of DNA breakage, likely during replication fork arrest and subsequent single-stranded DNA accumulation, and this early event upon insult induces the formation of discrete nuclear foci of γ-H2AX, which can be visualized with specific antibodies under the microscope. The formation of γ-H2AX, which can spread progressively over the whole nucleus (pan-nuclear γ-H2AX) following chromatin modification by loop extrusion, gives in fact an estimate of the severity of the RS. γ-H2AX is considered nowadays a universal bio-indicator of the severity of genotoxic compounds that interfere with DNA replication in vitro and in vivo. γ-H2AX is also an early biomarker in clinics to check for tissue health status after radiotherapy, chemotherapy or radiation treatment. Indeed, almost all studies aiming at selecting small molecules triggering irreversible genome instability refer to γ-H2AX formation and retention to assess their potency. In particular, tracking γ-H2AX is of high interest for validating chemotherapeutics and for controlling the carcinogenic properties of chemicals present in biological samples. From a drug discovery point of view, this biomarker is of great interest to screen for efficacy and toxicity of therapeutic treatments. Immunofluorescence with validated antibodies remains so far the method of choice for accurately determining γ-H2AX levels; however, currently, there is no simple tool available for monitoring γ-H2AX turn-over and for measuring its direct impact on cell viability.
In a previous study, the inventors have shown that delivery in cells of antigen-binding fragments (Fabs) derived from an anti-γ-H2AX monoclonal antibody (mAb) allows following the fate of cancer cells after treatment with varying RS-inducing drug combinations (Moeglin et al, 2019, Cancers. 11:355. doi:10.3390/cancers11030355; Conic et al, 2018, J. Cell Biol. 217:1537-1552. doi:10.1083/jcb.201709153). Although it was possible to show with this method that extensive γ-H2AX phosphorylation is indicative of commitment to irreversible cell death, the inventors could not clearly identify the dynamic changes in the levels of γ-H2AX during the treatment. Indeed, conventional antibodies cannot be easily delivered in living cells, and therefore require fixation of the samples.
Recently, it has been shown that single-domain antibody fragments of camelids, generally termed nanobodies, represent exquisite tools for tracking intracellular molecules. Nanobodies correspond to the variable domain (VHH) of the heavy chain-only antibodies (HcAb) expressed in these animals. They can be cloned as VHH repertoires with minimal modification from total RNA of peripheral blood mononuclear cells (PBMCs) obtained after immunization, thus presenting an authentic picture of the in vivo-maturated heavy chain repertoire diversity. Moreover, their small size (˜15 kDa) compared to conventional antibodies (˜150 kDa) and, especially, their capacity to fold stably in a reducing environment make them excellent binding molecules in cells. While alpaca-derived nanobodies against γ-H2AX have already been generated (Rajan et al, 2015, FEBS Open Bio. 5:779-788. doi:10.1016/j.fob.2015.09.005), these tools didn't allow for the specific unambiguous detection of γ-H2AX in irradiated cancer cells.
In conclusion, Histone H2AX phosphorylated at serine 139 (γ-H2AX) is a hallmark of DNA damage, signaling the presence of DNA double-strand breaks and global replication stress in mammalian cells. While γ-H2AX can be visualized with antibodies in fixed cells, its detection in living cells was so far not possible. Therefore, there is still a need for tools specific for γ-H2AX suitable for in vivo use in living cells for detecting DNA Damage and replication stress.
More specifically, since upon insult, γ-H2AX levels vary from one cell to another, a reagent that would consent monitoring in individual cells both γ-H2AX levels and their fate after treatment with varying doses of genotoxic agents would be useful. Indeed, classical antibodies (i.e., IgG) have the disadvantages to be relatively expensive reagents and only the monovalent Fab format of IgG, that can be obtained following digestion with papain, diffuses freely into the nucleus upon delivery. In addition, detection of γ-H2AX with complete antibodies can only be carried out in fixed cells (end-point experiments) and thus does not allow to study transient dynamic states of the chromatin following damage. When compared to classical antibodies, single-domain antibodies can be easily produced in bacteria and the methodologies used for the selection gives access to their DNA sequence.
DETAILED DESCRITION OF THE INVENTION To develop an immunological probe able to detect and track γ-H2AX in living cells, the inventors had to perform numerous selection attempts to isolate one clone that could specifically interact with the peptide used for immunization, confirming that single-domain antibodies cannot easily bind to small linear epitopes. This might also be the reason why another group was unsuccessful in selecting an anti-γ-H2AX nanobody following immunization of a lama with the same peptide (Jullien et al, J. Cell Sci. 2016, 129, 2673-2683, doi:10.1242/jcs.183103). Finally, they have isolated single domain antibodies (called nanobodies) that are easily expressed as functional recombinant proteins and report the extensive characterization of a novel nanobody that specifically recognizes γ-H2AX. The interaction of this nanobody with the C-terminal end of γ-H2AX was solved by X-ray crystallography. Moreover, the inventors engineered a bivalent version of this nanobody and showed that bivalency is essential to quantitatively visualize γ-H2AX in fixed drug-treated cells. After labelling with a chemical fluorophore, the inventors were able to detect γ-H2AX in a single-step assay with the same sensitivity as with validated antibodies that are used with an assay having several steps. Then, the use of the nanobodies identified by the inventors allows an improved assay which is more cost-effective. Moreover, the inventors produced fluorescent nanobody fusion proteins and applied a transduction strategy to visualize with precision γ-H2AX foci present in intact living cells following drug treatment. Together, this novel tool allows performing fast screenings of genotoxic drugs and enables to study the dynamics of this particular chromatin modification in individual cells under a variety of conditions.
Accordingly, the present invention relates to a single domain antibody directed against H2AX with a phosphorylation of serine at position 139 (γ-H2AX) comprising a variable domain comprising three CDRs (complementarity determining regions), namely CDR1, CDR2 and CDR3, consisting in the amino acid sequence of SEQ ID NO: 1: GLT(L/F)SRYA for CDR1, the amino acid sequence of SEQ ID NO: 2: ITASGRTT for CDR2, and the amino acid sequence of SEQ ID NO: 3: AADYGX1X2X3YTRRQSEYX4Y for CDR3, wherein X1 and X2 are any amino acid, X3 is K or R, and X4 is D or E.
Optionally, CDR1 is GLTLSRYA. Preferably, CDR1 is GLTFSRYA.
Optionally, X1 and X2 are independently selected in the group consisting of A, V, S, N, K, R, T and G, especially of S, N, K, R, T and G. Preferably, X1 is selected in the group consisting of S, N, T and G. Preferably, X2 is selected in the group consisting of G, K and S.
Optionally, X1 is selected in the group consisting of S, N, T and G; X2 is selected in the group consisting of G, K and S; X3 is K or R; and X4 is D or E. Preferably, X3 is R. Preferably, X4 is E. Especially, X3 is R and X4 is E. Alternatively, X3 is K and X4 is D.
Optionally, the amino acid sequence of CDR3 can be selected in the following group:
In a particular aspect, the amino acid sequence of CDR3 is selected from the group consisting of
In a more particular aspect, the amino acid sequence of CDR3 is selected from the group consisting of
and more particularly of
In a very particular aspect, the amino acid sequence of CDR3 is AADYGTSRYTRRQSEYEY (SEQ ID NO: 6).
In a particular aspect, the single domain antibody is a VHH, preferably from Camelidae, more preferably from Llama species, or camelized framework regions of a human VH.
In a particular aspect, the single domain antibody is an antibody that comprises, consists in, or consists essentially in, the amino acid sequence of SEQ ID NO: 20 or a variant amino acid sequence having no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid additions, deletions, substitutions, or combinations thereof within the sequence of SEQ ID NO: 20, said addition, deletion, or substitution being outside of CDR1, CDR2 and CDR3 (underlined in the sequence for convenience), wherein the amino acid sequence of SEQ ID NO: 20 is
L)SRYAMGWFRQAPGNEREFVAVITASGRTTLYADS(V/L)KGRFTISR
with n being 0-10, preferably being 0 or 9, more preferably being 0; X being any amino acid; X1 and X2 being any amino acid, X3 being K or R, and X4 being D or E. In a particular aspect, X1, X2, X3 and X4 can be as defined in any particular aspect as described above.
In a particular aspect, the single domain antibody is an antibody that comprises, consists in, or consists essentially in, the amino acid sequence of SEQ ID NO: 21 or a variant amino acid sequence having no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid additions, deletions, substitutions, or combinations thereof within the sequence of SEQ ID NO: 21, said addition, deletion, or substitution being outside of CDR1, CDR2 and CDR3 (underlined in the sequence for convenience), wherein the amino acid sequence of SEQ ID NO: 21 is
with X1 and X2 are any amino acid and X4 being D or E. In a particular aspect, X1 and X2 can be as defined in any particular aspect as described above.
In a particular aspect, the single domain antibody is an antibody that comprises, consists in, or consists essentially in, the amino acid sequence of any one of SEQ ID NOs: 22, 23, 24 and 25 or a variant amino acid sequence having no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid additions, deletions, substitutions, or combinations thereof within the sequence of any one of SEQ ID NOs: 22, 23, 24 and 25, said addition, deletion, or substitution being outside of CDR1, CDR2 and CDR3 (underlined in the sequence for convenience), wherein SEQ ID NOs: 22, 23, 24 and 25 are as following
AADYGSGKYTRRQSEYDYWGQGTQVTVSSEPKTPKPQPAAA;
AADYGNKRYTRRQSEYEYWGQGTQVTVSSAAA;
AADYGGGRYTRRQSEYEYWGQGTQVTVSSAAA
In a particular aspect, the present invention relates to a bivalent molecule comprising two single domain antibodies directed against γ-H2AX as described herein. The two single domain antibodies can be the same or different.
Optionally, the bivalent molecule is a bivalent protein in which the two single domain antibodies are connected as a protein fusion. Optionally, the two single domain antibodies are connected via a peptide linker.
The linker is usually 3-44 amino acid residues in length. Preferably, the linker has 3-30 amino acid residues in length. Examples of linker sequences are Gly/Ser linkers of different length including (Gly4Ser)4, (Gly4Ser)3, (Gly4Ser)2, Gly4Ser, Gly3Ser, Gly3, Gly2Ser and (Gly3Ser2)3. In a particular aspect, the linker is (Gly4Ser)3.
In a particular aspect, the bivalent protein can comprise, essentially consist in or consist in an amino acid sequence selected from the group consisting of
RTTLYADS(V/L)KGRFTISRDNAKNTVALQMQSLKPEDTAVYYCAADYGX1X2X3YTRRQSEYX4YWGQGTQV
RTTLYADS(V/L)KGRFTISRDNAKNTVALQMQSLKPEDTAVYYCAADYGX1X2X3YTRRQSEYX4YWGQGTQV
wherein “linker” is a peptide linker, n is 0-10, preferably 0 or 9, more preferably 0; X is any amino acid; X1 and X2 are any amino acid, X3 is K or R, and X4 is D or E
RTTLYADS(V/L)KGRFTISRDNAKNTVALQMQSLKPEDTAVYYCAADYGX1X2X3YTRRQSEYX4YWGQGTQV
SEYX4YWGQGTQVTVSS(X)nAAA,
wherein n is 0-10, preferably 0 or 9, more preferably 0; X is any amino acid; X1 and X2 are any amino acid, X3 is K or R, and X4 is D or E;
wherein “linker” is a peptide linker, and X1 and X2 are any amino acid and X4 being D or E;
with X1 and X2 being any amino acid and X4 being D or E;
wherein “linker” is a peptide linker;
DYGSGKYTRRQSEYDYWGQGTQVTVSSEPKTPKPQPAA (A/-) GGGSG
DYGNKRYTRRQSEYEYWGQGTQVTVSSAA (A/-) - linker - MAE
TASGRTTLYADSVKGRFTISRDNAKNTVALQMQSLKPEDTAVYYCAADYG
NKRYTRRQSEYEYWGQGTQVTVSSAAA,
wherein “linker” is a peptide linker;
DYGNKRYTRRQSEYEYWGQGTQVTVSSAA (A/-) GGGSGGGSGGGSMA
ITASGRTTLYADSVKGRFTISRDNAKNTVALQMQSLKPEDTAVYYCAADY
GNKRYTRRQSEYEYWGQGTQVTVSSAAA;
DYGTSRYTRRQSEYEYWGQGTQVTVSSAA (A/-) - linker - MAD
TASGRTTLYADSVKGRFTISRDNAKNTVALQMQSLKPEDTAVYYCAADYG
TSRYTRRQSEYEYWGQGTQVTVSSAAA,
wherein “linker” is a peptide linker;
DYGTSRYTRRQSEYEYWGQGTQVTVSSAA (A/-) GGGSGGGSGGGSMA
ITASGRTTLYADSVKGRFTISRDNAKNTVALQMQSLKPEDTAVYYCAADY
GTSRYTRRQSEYEYWGQGTQVTVSSAAA;
DYGGGRYTRRQSEYEYWGQGTQVTVSSAA (A/-) - linker - MAE
TASGRTTLYADSVKGRFTISRDNAKNTVALQMQSLKPEDTAVYYCAADYG
GGRYTRRQSEYEYWGQGTQVTVSSAAA,
wherein “linker” is a peptide linker; and
DYGGGRYTRRQSEYEYWGQGTQVTVSSAA (A/-) GGGSGGGSGGGSMA
ITASGRTTLYADSVKGRFTISRDNAKNTVALQMQSLKPEDTAVYYCAADY
GGGRYTRRQSEYEYWGQGTQVTVSSAAA;
or a variant amino acid sequence having no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid additions, deletions, substitutions, or combinations thereof, said addition, deletion, or substitution being outside of CDR1, CDR2 and CDR3 (underlined in the sequence for convenience). In a particular aspect, the (A/−) is no amino acid.
Is also contemplated herein a protein with higher valance than a bivalent protein. The present disclosure may relate to a protein (monomeric or polymeric) comprising 2, 3, 4, 5 or 6 single domain antibody as described herein.
Optionally, the single domain antibody or the bivalent molecule is labelled with a detectable entity (“label”). The term “label”, as used herein, refers to any atom or molecule that can be used to provide a quantifiable signal and that can be attached to a single domain antibody or bivalent molecule as disclosed herein via a covalent bond or a noncovalent interaction (e.g., through ionic or hydrogen bonding, or via immobilization, adsorption, or the like). A label may be selected from the group consisting in a radiolabel, an enzyme label, a fluorescent label, a bioluminescent molecule, a biotin-avidin label, a chemiluminescent label, and a detectable entity. Optionally, the detectable entity can be a tag that can be detected by an antibody specific for the tag.
Optionally, the detectable label is selected from the group consisting of: a hapten, a fluorescent dye, a fluorescent protein, a chromophore, a metal ion, a gold particle, a silver particle, a magnetic particle, a polypeptide, an enzyme, a luminescent compound, or an oligonucleotide.
In a preferred aspect, the detectable label is a fluorescent protein. For instance, the fluorescent protein can be selected in the non-exhaustive list comprising Green Fluorescent Protein, Enhanced Green Fluorescent Protein (EGFP), Enhanced Yellow Fluorescent Protein (EYFP), Venus, mVenus, Citrine, mCitrine, Cerulean, mCerulean, Orange Fluorescent Protein (OFP), mNeonGreen, moxNeonGreen, mCherry, mTagBFP, mTurquoise, mScarlet, mWasabi, mOrange, mStrawberry and dTomato.
In a very specific aspect, the fluorescent protein is dTomato and has the following sequence:
The detectable label can be a fluorescent dye, for instance selected in the non-exhaustive list including Oregon Green®, Pacific Blue™, Pacific Orange™, Pacific Green™, Cascade Blue™, Cascade Yellow™, Lucifer Yellow™, Marina Blue™, and Texas Red® (TxRed); an AlexaFluor®(AF) dye such as AF350, AF405, AF488, AF500, AF514, AF532, AF546, AF555, AF568, AF594, AF610, AF633, AF635, AF647, AF680, AF700, AF710, AF750, AF790, and AF800; a Cy dye such as Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, and Cy 7.5; Atto 390, Atto 425, Atto 465, Atto 488, Atto 495, Atto 514Atto 520, Atto 532, Atto 550, Atto 565, Atto 590, Atto 594, Atto 610, Atto 620, Atto 633, Atto 647, Atto 655, Atto 665, Atto 680, Atto 700, Atto 725, Atto 740, Super Bright™ 436, Super Bright™ 600, Super Bright™ 645, Super Bright™ 702, Super Bright™ 780, Brilliant™ Violet 421, Brilliant™ Violet 480, Brilliant™ Violet 510, Brilliant™ Violet 605, Brilliant Violet™ 650, Brilliant Violet™ 711, Brilliant Violet™ 786, Brilliant™ Ultraviolet 395 (BUV395), Brilliant™ Ultraviolet 496 (BUV496), Brilliant™ Ultraviolet 563 (BUV563), Brilliant™ Ultraviolet 661 (BUV661), Brilliant™ Ultraviolet 737 (BUV737), Brilliant™ Ultraviolet 805 (BUV805), Brilliant™ Blue 515 (BB515), Brilliant™ Blue 700 (BB700) and IR Dye 680, IR Dye 680LT, IR Dye 700, IR Dye 700DX, IR Dye 800, IR Dye 800RS, and IR Dye 800CW.
The detectable label can be a hapten such as a fluorescein or a derivative thereof, fluorescein isothiocyanate, carboxyfluorescein, dichlorotriazinylamine fluorescein, digoxigenin, dinitrophenol (DNP), trinitrophenol (TNP), and biotin.
Optionally, the detectable molecule can be a detectable tag, preferably a peptide detectable tag. Non-limiting example of tag includes E6 tag (for instance of sequence TSMFQDPQERPRASA).
Alternatively, the detectable label can be a bioluminescent molecule or an enzyme such as luciferase, β-galactosidase, β-lactamase, peroxidase, alkaline phosphatase, β-glucuronidase, and β-glucosidase.
The detectable label can be a radiolabel, such as a radionuclide selected from the group consisting of: carbon (14C), chromium (5ICr), cobalt (57Co), fluorine (18F), gadolinium (I53Gd, I59Gd), germanium (68Ge), holmium (166H0), indium (1151h, 1131h, 1121h, min), iodine (1251, 1231, 1211), lanthanium (I40La), lutetium (I77Lu), manganese (54Mn), molybdenum (99 Mo), palladium (103 Pd), phosphorous (32 P), praseodymium (142 Pr), promethium (149Pm), rhenium (I86Re, I88Re), rhodium (I05Rh), rutheroium (97Ru), samarium (I53Sm), scandium (47Sc), selenium (75Se), (85Sr), sulphur (35S), technetium (99Tc), thallium (20ITi), tin (H3Sn, H7Sn), tritium (3H), xenon (I33Xe), ytterbium (I69Yb, I75Yb), and yttrium (90Y).
Accordingly, the present disclosure relates to a single domain antibody or bivalent protein as disclosed herein conjugated to a detectable label. The methods for preparing such a conjugate are well-known in the art. Optionally, the sequence of the single domain antibody or the bivalent protein can be modified by a substitution or addition of a residue suitable for the conjugation of the detectable label. Then, the amino acid sequence of the single domain antibody or bivalent protein includes a substitution or addition of a residue, preferably a cysteine, preferably near to the C-terminal end, for instance within the 2-10 most C-terminal amino acids of the single domain antibody or bivalent protein as described herein, more preferably within the 3-5 most C-terminal amino acids. For instance, the additional residue, preferably a cysteine residue, is added before the stretch of three A (i.e., AAA replaced by CAAA). This additional residue, preferably a cysteine residue, allows the introduction of a covalent link with a detectable label, in particular a fluorescent molecule, for instance by thiol maleimide reaction.
Optionally, the label is a protein and is fused or linked to the single domain antibody or the bivalent molecule, thereby forming a protein fusion. The label can be fused or linked at the N-terminal end of the single domain antibody or the bivalent molecule, or at the C-terminal end of the single domain antibody or the bivalent molecule or, in the context of the bivalent molecule, between the two single domain antibodies. In a particular aspect, the label is fused or linked at the C-terminal end of the single domain antibody or the bivalent molecule.
Optionally, the single domain antibody or the bivalent protein can further comprise tag sequence, such as a histidine tag, useful for the purification of the recombinant protein. Optionally, the single domain antibody or the bivalent protein can further comprise a NLS sequence (nuclear localization signal).
The present invention further relates to a nucleic acid sequence encoding the single domain antibody or the bivalent protein as disclosed above, an expression cassette comprising such a nucleic acid sequence, a vector comprising such a nucleic acid sequence or expression cassette, and a host cell comprising such a nucleic acid sequence, expression cassette or vector.
Optionally, the promoter used to control the expression of the single domain antibody or the bivalent protein is a weak promoter. Optionally, the expression vector is a low copy number vector. Optionally, the expression vector may comprise a restriction site allowing the insertion of a detectable label so as to obtain a protein fusion comprising the single domain antibody or the bivalent protein and the detectable protein.
The present disclosure also relates to a method for producing the single domain antibody or the bivalent protein as described herein comprising expressing the single domain antibody or the bivalent protein in a host cell and recovering the produced single domain antibody or bivalent protein.
The present disclosure relates to the single domain antibody or the bivalent protein or a nucleic acid, expression cassette or vector encoding it as a research tool. For instance, it relates to a kit comprising the single domain antibody or the bivalent protein as described herein or an expression vector encoding the single domain antibody or the bivalent protein and a leaflet for the use of this reagent. Preferably, the single domain antibody or the bivalent protein comprises a detectable label as detailed above.
The present disclosure relates to the use of the single domain antibody or the bivalent protein as described herein or a nucleic acid, expression cassette or vector encoding it for detecting and/or quantifying and/or monitoring γ-H2AX in a cell or a cellular extract thereof, especially γ-H2AX foci. It relates to the use of the single domain antibody or the bivalent protein as described herein or a nucleic acid, expression cassette or vector encoding it for detecting or monitoring DNA damage or Replication stress in a cell or a cellular extract thereof. The use is a non-therapeutic use. The use can be an in vitro use, an in cellulo use or an ex vivo use (on isolated cells). In particular, the in vivo use can be excluded.
Optionally, the single domain antibody or bivalent protein is used in one of the following assays: ELISA, flow cytometry, immunofluorescence, live cell imaging (non fixed), immunoprecipitation, in particular Chromatin immunoprecipitation, and Western blot.
The present disclosure further relates to a method for detecting and/or quantifying and/or monitoring γ-H2AX in a cell, comprising contacting the cell with a single domain antibody or a bivalent protein as described herein or with a nucleic acid, expression cassette or vector encoding said single domain antibody or bivalent protein, and detecting and/or quantifying and/or monitoring the single domain antibody or bivalent protein in the cell or a cellular extract thereof. The method can be for detecting or quantifying or monitoring DNA damage or Replication stress in a cell. The method is a non-therapeutic method. The method can be an in vitro method, an in cellulo method or an ex vivo method (on isolated cells). In particular, the in vivo method can be excluded. The method allows to study the dynamics of the chromosome modification in an individual cell.
Optionally, the cell is a cancer cell. Optionally, the cell is a living cell. Optionally, the cell is a fixed cell. Preferably, the cell is an eukaryotic cell, more preferably a mammalian cell.
Optionally, the cell is contacted or has been contacted or will be contacted with a test compound or molecule simultaneously or before the contacting step with the single domain antibody or bivalent protein. The test compound or molecule can be any compound or molecule, especially can be a compound or molecule known or suspected to be a genotoxic agent. Optionally, the use and method as disclosed above is preferable after induction of DNA damage or replication stress.
The present disclosure may relate to the use of the single domain antibody or the bivalent protein as described herein or a nucleic acid, expression cassette or vector encoding it for screening or identifying a compound or a molecule having a genotoxic effect; or to a method for screening or identifying a compound or a molecule having a genotoxic effect, the method comprising contacting a eukaryotic cell with a compound or a molecule, the cell expressing the single domain antibody or the bivalent protein as described herein or the cell being contacted with the single domain antibody or the bivalent protein as described herein, and detecting and/or quantifying and/or monitoring the single domain antibody or the bivalent protein in the cell, thereby determining the effect of the compound or molecule on DNA damage or replication stress or determining the genotoxic effect of the compound or molecule. In one aspect, the compound or molecule is selected if no genotoxic effect is detected. In an alternative aspect, the compound or molecule is selected if a genotoxic effect is detected. Optionally, the effect observed for the compound or molecule can be compared with one or several compounds or molecules of reference for which the genotoxic effect or the absence of genotoxic effect is well-documented.
Preferably, the single domain antibody or the bivalent protein is detected, quantified or monitored in the nucleus of the cell. Preferably, the single domain antibody or the bivalent protein is use for detecting, quantifying or monitoring the γ-H2AX foci.
Preferably, the single domain antibody or the bivalent protein is linked to a fluorescent label as detailed above and the single domain antibody or the bivalent protein is detected, quantified or monitored by the fluorescence of the fluorescent label. The advantage is that the detection, quantification or monitoring can be carried in a one-step process.
Optionally, the single domain antibody or the bivalent protein is monitored for a period of time, for instance by video recording, to follow the event occurring in the living cell after induction of DNA damage or replication stress.
Optionally, other kind of detectable label can be used and the method may comprise the detection of the detectable label through the addition or the use of a mean specific for the detectable label. For instance, if the detectable label is a tag, an antibody specific for this tag can be used to detect the detectable label.
As used herein, the term “H2AX” refers to H2A histone family member X (H2AX). It is described in UniProtKB under reference P16104 for human and P27661 for mouse. Human sequence of H2AX is the following
Mouse sequence of H2AX is the following
When the serine residue is phosphorylated, the protein is called gammaH2AX or γ-H2AX.
As used herein, the terms “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that specifically binds an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also antigen-binding antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments. In particular, the term antibody refers to heavy-chain only antibodies, VHH, fragments and derivatives thereof such (VHH)2 fragments and single domain antibodies.
As used herein, the terms “Heavy-chain only antibody” or “HCAbs” refer to immunoglobulins which are devoid of light chains and consist in two heavy chains. These antibodies do not rely upon the association of heavy and light chain variable domains for the formation of the antigen-binding site but instead the variable domain of the heavy polypeptide chains alone naturally forms the complete antigen binding site. Each heavy chain comprises a constant region and a variable domain which enables the binding to a specific antigen, epitope or ligand. As used herein, HCAbs encompass heavy chain antibodies of the camelid-type in which each heavy chain comprises a variable domain called VHH and two constant domains. Such heavy-chain antibodies directed against a specific antigen can be obtained from immunized camelids. Camelids encompass dromedary, camel, lama and alpaca. Camelid HCAbs have been described by Hamers-Casterman et al., Nature, 1993, 363:446. Other examples of HCAb are immunoglobulin-like structures (Ig-NAR) from cartilaginous fishes. Heavy-chain antibodies can be humanized using well-known methods.
The terms “single domain antibody”, “sdAb” and “nanobody” are used interchangeably and have the same meaning. As used herein, the term single domain antibody refers to a single variable domain derived from a heavy chain antibody, which is able to bind an antigen, an epitope or a ligand alone, that is to say, without the requirement of another binding domain. A single domain antibody may be or may derive from VHH and V-NAR. V-NAR refers to the variable domain found in immunoglobulin-like structures (Ig-NAR) discovered in cartilaginous fishes such as sharks. For review about single domain antibodies, one may refer to Saerens et al., Current Opinion in Pharmacology, 2008, 8:600-608, the disclosure of which being incorporated by reference. In a preferred aspect, the single domain antibody according to the present disclosure is a synthetic single domain antibody. As used herein, the term “synthetic” means that such antibody has not been obtained from fragments of naturally occurring antibodies but produced from recombinant nucleic acids comprising artificial coding sequences (cf. WO 2015/063331).
The term “VHH”, as used herein, refers to an antibody fragment consisting of the VH domain of camelid heavy-chain antibody. VHH fragments can be produced through recombinant DNA technology in a number of microbial hosts (bacterial, yeast, mould), as described in WO 94/29457. Alternatively, binding domains can be obtained by modification of the VH fragments of classical antibodies by a procedure termed “camelization”, described by Davies et al, 1995. Dimers of VHH fragments, i.e. (VHH)2, can be generated by fusing two sequences encoding VHH fragments, end to end, e.g., by PCR. Preferably, the (VHH)2 fragment is monospecific.
The variable domain of an antibody of the present disclosure comprises at least three complementarity determining region (CDR) which determine its binding specificity. Preferably, in a variable domain, the CDRs are distributed between framework regions (FRs). The variable domain thus contains at least 4 framework regions interspaced by 3 CDR regions, resulting in the following typical antibody variable domain structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. CDRs and/or FRs of the single domain antibody of the present disclosure may be fragments or derivatives from a naturally-occurring antibody variable domain or may be synthetic.
As used herein, the terms “Amino acid modification”, “amino acid change”, and “mutation” are used interchangeably and refer to a change in an amino acid sequence such as a substitution, an insertion, and/or a deletion. By “amino acid substitution” or “substitution” herein is meant the replacement of an amino acid at a particular position in a parent amino acid sequence with another amino acid. By “amino acid insertion” or “insertion” is meant the addition of an amino acid at a particular position in a parent amino acid sequence. By “amino acid deletion” or “deletion” is meant the removal of an amino acid at a particular position in a parent amino acid sequence.
The amino acid substitutions may be conservative. A conservative substitution is the replacement of a given amino acid residue by another residue having a side chain (“R-group”) with similar chemical properties (e.g., charge, bulk and/or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. Conservative substitutions and the corresponding rules are well-described in the state of the art. For instance, conservative substitutions can be defined by substitutions within the groups of amino acids reflected in the following tables:
As used herein, the term “fusion protein” or “protein fusion” are equivalent and refers to protein created through the joining of two or more genes that originally coded for separate proteins. Translation of this fusion gene results in a single polypeptide with functional properties derived from each of the original proteins. Preferably, the fusion protein of the invention is a recombinant fusion protein created artificially by recombinant DNA technology.
As used herein, (AA1/AA2) refers to the choice between the residue AA1 or the residue AA2. For instance, (E/D) means E or D; (A/T) means A or T; (G/D) means G or D; (S/A) means S or A; (F/L) means F or L; (V/L) means V or L; (A/−) means A or no amino acid.
As used herein, the term “consists essentially in” is intended to refer to an amino acid sequence that differs from that of a parent amino acid sequence by virtue of 1, 2, or 3 substitutions, additions, deletions or combination thereof.
As used herein, the terms “variant amino acid sequence”, variant polypeptide” or “variant” are equivalent and refer to an amino acid sequence that differs from that of a parent amino acid sequence by virtue of at least one amino acid modification. In the context of the invention, a variant is a variant of a variable domain, a CDR or a FR. Typically, a variant comprises from 1 to 40 amino acid modifications, preferably from 1 to 30 amino acid modifications, more preferably 1 to 20 amino acid modifications. In particular, the variant may have from 1 to 15 amino acid changes, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 amino acid changes as compared to its parent amino acid sequence. In a specific aspect, the variant may have from 1 to 3 amino acid changes, e.g., 1, 2, or 3 amino acid changes as compared to its parent amino acid sequence. The variants may comprise one or several amino acid substitutions, and/or, one or several amino acid insertions, and/or one or several amino acid deletions. In some embodiments, the variant may comprise one or several conservative substitutions, e.g., as shown here above. In some other embodiments, the variant comprises one or several amino acid modifications in the framework domains.
As used herein, the term “expression cassette” refers to a nucleic acid construction comprising a coding region and regulatory regions necessary for expression, operably linked to the coding region. The expression “operably linked” indicates that the elements are combined in such a way that the expression of the coding region is under the control of the regulatory regions. Typically, a regulatory region is located upstream of the coding region at a distance compatible with the control of its expression. The regulatory region can include promoters, enhancers, silencers, attenuators, and internal ribosome entry sites (IRES). Spacer sequences may also be present between regulatory elements and the coding region, as long as they don't prevent its expression. An expression cassette may also include a start codon in front of a protein-encoding gene, splicing signals for introns, and stop codons, transcription terminators, polyadenylation sequences.
As used herein, the terms “promoter” and “transcriptional promoter” are equivalent and refer to a region of DNA that is part of the regulatory region of an expression cassette. The promoter is the regulatory element that initiates the transcription of a particular gene. Promoters are located near the transcription start site of genes, on the same strand and upstream on the DNA (towards the 5′ region of the sense strand).
As used herein, the term “expression vector” refers to a vector designed for gene expression in cells. An expression vector allows to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. An expression vector comprises expression elements including, for example, a promoter, the correct translation initiation sequence such as a ribosomal binding site and a start codon, a termination codon, and a transcription termination sequence. An expression vector may also comprise other regulatory regions such as enhancers, silencers and boundary elements/insulators to direct the level of transcription of a given gene. The expression vector can be a vector for stable or transient expression of a gene.
To produce a nanobody to track γ-H2AX in cells under DNA replication stress (RS), the inventors immunized alpacas with the phosphorylated peptide CKATQA(p) SQEY corresponding to the C-terminal end of γ-H2AX (residues 134-142). This peptide has been used in a previous study to generate monoclonal antibodies that are suitable for detecting γ-H2AX in various immunoassays (Moeglin, E. et al.; Cancers 2019, 11, 355, doi:10.3390/cancers11030355). Following immunization, the PBMCs were collected and VHH libraries of approximately 107 independent clones were constructed. The phage display technology, which consist in displaying the VHH molecules on the tip of M13-based phages, allows selecting those that bind to the phospho-peptide immobilized on plate. This method of antigen display was preferred to other methods such as immobilization on magnetic beads since it previously allowed successful screening of cell culture supernatants containing monoclonal antibodies. Colony counting following the first round of panning (R1) showed that phages expressing a VHH against the phospho-peptide were only present in the repertoire of one animal (alpaca 2) (
The inventors tested the binding specificity of the VHH-displaying phages selected from library 2 by comparing their reactivity against the phosphorylated and non-phosphorylated peptides coated on plate (phage-ELISA). The selected populations bound preferentially to the phospho-peptide, which suggests that the phosphate group at 5139 is important for the recognition (
Individual clones of the positive phage population (R1 and R2 from library 2) were amplified and subjected to DNA sequencing. The alignment of 165 different sequences showed that almost all analyzed VHH clones have a similar amino acid composition, except at positions 98 and 99 (Kabat numbering) in the complementary-determining region 3 (CDR3) (
To test whether the four identified VHH variants could be used as nanobodies in immunoassays and cells, the inventors first sub-cloned their coding regions into a bacterial vector equipped with the relevant tags for detection and purification, then expressed them as single polypeptides in the cytoplasm of E. coli cells. SDS-PAGE analysis of cell extracts showed that the four nanobodies behave differently, despite their high amino acid sequence homology: C6 and A9 are soluble, whereas A4 and G2 are mostly insoluble after cytoplasmic expression (
To understand the interaction between the nanobodies and the phospho-peptide at the atomic level, the inventors solved the crystal structure of the complex at 1.8 A resolution. The inventors selected the C6 nanobody due to its higher stability upon storage and overall better performance compared to A9. The crystals belonged to space group P31, with 6 equivalent copies of the complex in the asymmetric unit where significant electron density is observed for the last five residues of the peptide (
The inventors examined the behavior of the C6 nanobody when expressed in mammalian cells. The inventors cloned the coding region of C6 fused in frame to mCherry to generate a chromobody (Panza, P. et al.; Development 2015, 142, 1879-1884, doi:10.1242/dev.118943) expressed under the control of the R-actin promoter and transiently transfected it into H1299 cells. The C6 chromobody was located in the nucleus of the treated cells as well as the untreated cells after analysis with either a widefield (
In a previous work the inventors have shown that antibodies and fragments thereof can be efficiently transduced into cultured cells by electroporation (Muyldermans, S.; Annu. Rev. Biochem. 2013, 82, 775-797, doi:10.1146/annurev-biochem-063011-092449; Conic, S. et al.; J. Cell Biol. 2018, 217, 1537-1552, doi:10.1083/jcb.201709153). Given their small size (15-20 kDa), nanobodies can theoretically easily diffuse into the nucleus after delivery in the cytoplasm. Therefore, the inventors transduced the purified C6 nanobody in H1299 cells subsequently treated with H and imaged them after 24 or 48 hours of incubation. As shown in
The inventors constructed a bivalent C6 nanobody (called C6B hereafter) and a mutated version of it (called C6BM), where the two R residues at positions 100C and 100D, which are critical for binding (see
To assess whether the purified C6B molecules harbor two functional binding sites, the inventors performed quantitative binding assays using the surface plasmon resonance (SPR) technology. To calculate the affinity of C6 for the antigen (KD value) the inventors used purified monovalent molecules and found that it lies in the low nanomolar range (11+/−4 nM;
To examine if this property of binding by avidity represents an advantage for the detection of γ-H2AX in drug-treated H1299 cells, the inventors performed IF experiments as done for the monovalent molecules (
To test if C6B could be used as a single-step reagent to detect γ-H2AX foci in fixed cells, the inventors added a cysteine residue in the coding region of C6B between the C-terminus of the second VHH and the E6 tag. The purified protein (C6BC) was labelled with Alexa-Fluor 568-maleimide and used in IF (
To investigate whether C6B could be used in cells, the inventors modified the previously constructed chromobody C6-mCherry to add a second VHH copy thus creating C6B-mCherry. Upon transfection of H1299 cells with this construct, a strong nuclear mCherry signal was observed (
Next, the inventors investigated the performance of the bivalent C6B and C6BM nanobodies by transduction since the corresponding polypeptides showed single bands on gel after purification (
To further analyze whether the inventors could use the C6B chromobody in transduction experiments, the inventors produced C6B-mCherry and C6B-dTomato fusion proteins in E. coli. dTomato protein was tested because its intrinsic fluorescence brightness is approximately 3 times higher than that of mCherry. The expected structures of these molecules are schematically depicted in
To follow the fate of γ-H2AX foci in live cells, the inventors took advantage of the strong fluorescence signal emitted by the dTomato protein and the fact that precise low amounts of C6B-dTo molecules can be delivered in cells via our electroporation method. Preliminary experiments showed that almost all of the internalized molecules accumulated in the nucleus when 0.5 to 2 μg of purified fusion protein were used.
To assess if the delivered C6B nanobody interferes with the cell response to genotoxic drugs, the inventors performed cell survival assays with transduced H1299 cells and monitored the γ-H2AX levels following pulse-treatment with H for 24 hours. Cells transduced with either PBS, C6B or C6B-dTo grew similarly at day 1, 2 and 3 post-treatment with H (
Alpacas (Llama pacos) were immunized at days 0, 21 and 35 with the synthetic phosphorylated peptide CKATQA(p)SQEY corresponding to the C-terminus of H2AX after covalent cross-linking to ovalbumin (150 μg). The immunogen was mixed with Freund complete adjuvant for the first immunization and with Freund incomplete adjuvant for the following immunizations. The immune response was monitored by titration of serum samples by ELISA with immunizing peptide on plate. Bound antibodies were detected with anti-alpaca rabbit IgG (Lafaye, P. et al.; Mol. Immunol. 2009, 46, 695-704, doi:10.1016/j.molimm.2008.09.008). For the construction of the libraries, blood samples (200 ml) of the immunized animals were collected under strict veterinary control and the PBMCs were isolated by Ficoll gradient centrifugation (GE Healthcare, Vélizy-Villacoublay, France). For the preparation of total RNA, approximately 107 cells were lysed with the TRlzol reagent (ThermoFisher Scientific, Grand Island, NY, USA). Complementary DNA (cDNA) was amplified using either SuperScript IV reverse transcriptase (ThermoFischer Scientific) or the BD Smart RACE kit (BD Biosciences). The VHH repertoires were amplified from the cDNA by two successive PCR reactions using 3 different primer pairs (PCR1, PCR2; Table D) and the VHH fragments were cloned into the SfiI/NotI restriction sites of the pHEN1 phagemid vector. After transformation into either E. coli TG1 or XL1-blue cells by electroporation, the bacterial colonies (approximately 4×107 independent transformants per library) were infected with M13KO7 helper phage to produce the phage libraries. The recombinant phages of each library were purified by PEG 8,000/NaCl precipitation and aliquots were stored at −80° C. after addition of 15% glycerol. Biopanning was performed with the phospho-peptide (0.5-5 μg/ml) coated on microtiter wells (ThermoFisher Scientific). Briefly, approximately 1011 phages in PBS containing 5% nonfat-dried milk were added to uncoated wells for 1 h and subsequently transferred to the peptide-coated wells. After incubation at 20° C. for 1 hour, the wells were extensively washed with PBS containing 0.05% Tween 20. Bound phages were eluted with trypsin and amplified in growing TG1 cells for the next round of selection. The amount of phospho-peptide coated on plate was lowered to 0.5 μg/ml in the second round of selection. Phage titers and enrichment after each panning round were determined by infecting TG1 cells with 10-fold serial dilutions of the collected phages and plating on LB agar plates containing 100 μg/mL ampicillin and 1% glucose. Where indicated, binding of the phages to antigen on plate was revealed with an anti-M13 monoclonal antibody conjugated to horse radish peroxidase (HRP; Abcam, Cambridge, UK). The VHH nucleotide sequences were determined using the M13-RP primer (GATC-Eurofins, Ebersberg, Germany).
The coding region of the selected VHHs in the pHEN1 vector were amplified by PCR with primers VHH-BspHI-Deg and VHH-NotI-short and subcloned into the pET-E6T-6H expression plasmid, a derivative of pETOM (Desplancq, D. et al.; J. Immunol. Methods 2011, 369, 42-50, doi:10.1016/j.jim.2011.04.001), which contains in frame at the NotI site the E6 epitope tag recognized by the 4C6 mAb and a His6 tag. To generate the bivalent VHH constructs, the coding region of the VHH was amplified by SOE-PCR with the primer pairs pETOM-For/G4S-Rev and G4S-For/E6T-Rev. The G4S-Rev and G4S-For are the annealing primers to add the (G4S)3 linker region. The recombinant fragment was cloned into the NcoI-digested pET-C6-E6T-6H plasmid after digestion with NcoI restriction enzyme, thus generating pET-C6B-E6T-6H. To generate the C6 mutant construct, which harbors an Ala residue and an Ile residue instead of the two Arg residues at position 100C and 100D in the CDR3 region, the inventors amplified by SOE-PCR the coding region of the C6 with primers pETOM-For and pETOM-Rev, in combination with C6-Mut-Rev and C6-Mut-For as annealing primers. The resulting PCR fragment was sub-cloned into the NcoI/NotI-digested pET-C6-E6T-6H to obtain pET-C6M-E6T-6H. The plasmid pET-C6BM-E6T-6H, which encodes the bivalent form of the mutated C6 coding region was constructed as described above. The additional Cys residue in the coding region of the bivalent C6 was obtained by amplification of the C6 coding region with primers VHH-BspHI-For and C6-Cys-Rev and sub-cloned into the pET-C6B-E6T-6H.
The pET-C6B-mCherry and pET-C6B-dTomato plasmids were constructed by inserting in frame the coding regions of mCherry protein or dTomato protein in the unique BamHI located in the E6 tag region. The dTomato coding region was subcloned from the ptdTomato-N1 vector (Clontech, Mountain View, USA). All primers used to generate the above-described plasmids are listed in Table D.
The VHH variants were expressed in E. coli BL21(DE3) plysS cells by addition of IPTG (1 mM) and incubation overnight at 20° C. The expressed polypeptides were purified as previously described (Desplancq, D. et al.; J. Immunol. Methods 2011, 369, 42-50, doi:10.1016/j.jim.2011.04.001), except that IMAC chromatography was performed on a HITRAP™ IMAC HP 1 ml column (GE Healthcare) loaded with nickel ions. The C6 variants with a cysteine residue at the C-terminal end of the second VHH coding region were purified on HIS TRAP™ Excel columns (GE Healthcare) in the presence of 2 mM TCEP. All buffers used in the process were supplemented with 1 mM EDTA and 0.2 mM PMSF. Where indicated, the eluted samples were further purified by size exclusion chromatography on a Superdex 75 10/300 GL column equilibrated in 20 mM Hepes buffer pH 7.2 containing 50 mM NaCl, 1 mM EDTA, 0.1 mM PMSF and 2 mM TCEP (optional). The C6B-mCherry and C6B-dTomato fusion proteins were purified by IMAC chromatography on HITRAP™ columns as above and subsequently polished by size exclusion chromatography on a HILOAD 16/600 Superdex 200 PG column (GE Healthcare) equilibrated in PBS. All purified proteins were stored at −80° C. after addition of 10% glycerol.
For the ELISA assays, microtiter wells (ThermoFisher Scientific) were coated with 1 μg/mL of phosphorylated or non-phosphorylated peptide CKATQASQEY in PBS overnight at 4° C. The purified VHH preparations were diluted in PBS containing 0.2% non-fat died milk and following incubation at RT for 1 hour they were revealed with mAb 4C6 and subsequent addition of HRP-conjugated rabbit anti-mouse IgG (GE Healthcare). After several washes with PBS containing 0.1% NP40 and addition of 3′,3′,5′,5′-tetramethylbenzidine (Sigma-Aldrich), the optical density was measured at 450 nm in an ELISA reader. The data were processed with R software using the drc package (Ritz, C. et al.; PLoS ONE 2015, 10, e0146021, doi:10.1371/journal.pone.0146021).
All experiments were performed on a Biacore T200 instrument at 25° C. in HBS-P buffer containing 10 mM HEPES (pH 7.4), 150 mM NaCl, 0.05% P20 surfactant. The phospho-peptide CKATQA(p)SQEY was immobilized on the biosensor surface (BR-1005-30; GE healthcare) through the SH group of the N-terminal cysteine using thiol coupling chemistry. The reference surface was treated similarly except that peptide injection was omitted. The purified VHH samples were serially injected in duplicate for 120 seconds over reference and peptide surfaces. Each sample injection was followed by a wash with HBS-P buffer during 600 sec. Sensorgrams were corrected for signals from the reference flow cell as well as after running buffer injections. The Kd was determined by fitting the equilibrium response (Req) versus the concentration curve to a 1:1 interaction model with the Biacore 2.0.2 evaluation software (GE Healthcare). Responses were normalized relative to phospho-peptide density as fractional occupancy (FO) of target sites (Zeder-Lutz, G. et al.; Anal. Biochem. 2012, 421, 417-427, doi:10.1016/j.ab.2011.09.015).
Purified C6 protein was incubated for 1 hour with a 1.3-fold excess of phospho-peptide treated with 2 mM N-ethyl maleimide to prevent dimerization. The complexes were subjected to size exclusion chromatography on a Superdex 75 10/300 GL column (GE Healthcare) equilibrated in 20 mM Hepes buffer pH 7.2, 150 mM NaCl. The peak fractions were concentrated to 5.1 mg/ml with a Amicon Ultra 3K filter (Merck-Millipore). The crystallization experiments were carried out by the sitting-drop vapor diffusion method at 20° C. using a Mosquito Crystal dispensing robot (TTP Labtech) for mixing equal volumes (200 nL) of the C6-peptide sample and reservoir solutions in 96-well 2-drop MRC crystallization plates (Molecular Dimensions). Crystallization conditions were tested using commercially available screens (Qiagen, Molecular Dimensions). Several wells were found positive after about 1 week of incubation and crystals obtained with 25% PEG 3350, 0.2M sodium acetate. The crystals were transferred to 35% PEG 3350, 0.2M sodium acetate before being flash cooled in liquid nitrogen. The data were collected at the Proxima 2A beamline of the synchrotron Soleil at a wavelength 0.98 Å (12.65 keV) on an EIGER X 9M detector (Dectris) with 20% transmission. 360° of data were collected using 0.1° oscillation and 0.025 s exposure per image, with a crystal to detector distance of 134.25 mm. The data were indexed, integrated, and scaled using XDS (Kabsch, W.; Acta Crystallogr. D Biol. Crystallogr. 2010, 66, 125-132, doi:10.1107/S0907444909047337). The 3D structure of the C6/phosphopeptide complex was solved by molecular replacement using the PHASER module of PHENIX (Liebschner, D. et al.; Acta Crystallogr. D Struct. Biol. 2019, 75, 861-877, doi:10.1107/S2059798319011471) with the structure of VHH PorM_01 (PDB ID: 5LZO) edited to remove water molecules and the CDR loops, being used as a search model. Refinement was performed using the refine module of PHENIX followed by iterative model building in COOT (Emsley, P. et al.; Acta Crystallogr. D Biol. Crystallogr. 2010, 66, 486-501, doi:10.1107/S0907444910007493). The structural figures were prepared with Chimera-X software (http://www.rbvi.ucsf.edu/chimerax).
The H1299 and U2OS cells (laboratory stocks) were maintained in Dulbecco's modified Eagle's tissue culture medium (DMEM; Life Technologies, Carlsbad, USA) supplemented with L-glutamine (2 mM), gentamicin (50 μg/mL) and 10% heat inactivated fetal calf serum at 37° C. in a humidified 5% CO2 atmosphere. Fresh cells were thawed from frozen stocks after 10 passages and mycoplasma contamination was tested by DAPI staining. Counting of the cells was performed with the automated cell counter LUNA-II (Logos Biosystems, Villeneuve d'Ascq, France). Where indicated, the cells were treated with either hydroxyurea (H; 2 mM), gemcitabine (G; 0.1 μM), AZD-7762 (A; 0.1 μM), clofarabine (C; 0.3 μM), triapine (T; 2 μM), camptothecin (CPT, 1 μM), epirubicin (EPI, 0.5 μM), etoposide (ETO, 10 μM), cisplatin (CIS, 10 μM), oxaliplatin (OXA, 10 μM) or combinations of two drugs at the same concentration as indicated. All drugs were purchased from Sigma-Aldrich.
Transduction experiments with purified C6, C6B, C6B-dTomato proteins or Fab 3F4 were performed essentially as previously described (Freund, G. et al.; mAbs 2013, 5, 518-522, doi:10.4161/mabs.25084). Briefly, 105 cells in PBS were mixed with the protein sample (0.5-2 μg) and subjected to electroporation (1550 V, 10 msec, 3 pulses) using the Neon transfection device (Life Technologies). The treated cells were incubated for 1 hour at 37° C. in medium and, after centrifugation for 5 min at 100 g, the pelleted cells were seeded and allowed to recover overnight in complete medium without antibiotics before addition of the drugs.
For the purification of the histone proteins, the harvested cells (approximately 107/ml) were lysed for 10 minutes at 4° C. in PBS supplemented with 0.5% Triton X100, 2 mM PMSF, 0.02% NaN3 and 1 mM Na3VO4. After centrifugation for 10 minutes at 6500 g at 4° C., the recovered nuclei were acid extracted overnight at 4° C. in 0.2 M HCl. The histone proteins present in the clarified lysate were stored at −20° C.
For the analysis of the H1299 proteins by Western blotting, soluble extracts (60 μg/lane) in RIPA buffer were used. γ-H2AX and β-actin were revealed with monoclonal antibody 3F4 (0.1 μg/mL) and rabbit polyclonal serum A2066 (Sigma-Aldrich), respectively. Bound secondary HRP-labeled antibodies were revealed with ECL reagent (GE Healthcare) and analyzed with the Image QuantLAS 4000 imager (GE Healthcare).
Construction of the pβ3-actin plasmids and transient transfection
The pβA-scFv-eGFP, a derivative of pDRIVE-hβ-actin (Rinaldi, A.-S. et al.; Exp. Cell Res. 2013, 319, 838-849, doi:10.1016/j.yexcr.2013.01.011) was modified by PCR to insert in frame to the scFv the E6 tag and the mCherry protein using E6T-HindIII-For/E6T-HindIII-Rev and mCherry-For/mCherry-Rev primer pairs, respectively. This vector which carries unique NcoI and SpeI restriction sites was used to sub-clone the VHH variants as described above, thereby generating pβA-C6-E6T-mCherry, pβA-C6M-E6T-mCherry, pβA-C6B-E6T-mCherry and pβA-C6BM-E6T-mCherry. All oligonucleotides used to construct these expression vectors are listed in Table D.
The day before transfection, 8×104 cells were plated in 12-well culture plates containing glass coverslips. Transient DNA transfection was performed using jetPRIME (Polyplus Transfection, Illkirch, France) according to manufacturer's instructions. The culture medium was replaced with fresh medium after 4-24 hours of incubation with the polymer/plasmid mixtures. Cells were incubated (37° C., 5% CO2) for 40 hours (H-treated cells) or 24 hours (G+A-treated cells), followed by microscopic analysis.
For the analysis by classical immunofluorescence microscopy, the transfected or transduced cells were fixed with 4% paraformaldehyde for 20 minutes and, after permeabilization with 0.2% Triton X100 for 5 min, they were incubated with mAb 3F4 or VHH preparations diluted in PBS containing either 10% fetal calf serum or 2% BSA. Where indicated, the cells were treated with CSK-100 modified buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 10 mM HEPES pH 6.8, 1 mM EGTA, and 0.2% Triton X-100) for 5 minutes prior to fixation. The VHH molecules were revealed by addition of mAb 4C6 which binds to the E6 tag and bound antibodies were detected with Alexa Fluor 488 or 568 labelled-anti-mouse immunoglobulins (Life Technologies). Where indicated, Alexa 568 labelled-C6B molecules were used. The labelling of the purified bivalent C6 equipped with a cysteine residue at the C-terminus was performed essentially as previously described (Shaner, N.C. et al.; Nat. Biotechnol. 2004, 22, 1567-1572). Briefly, purified protein in 0.1 M KH2PO4 pH 6.5, 150 mM NaCl, 1 mM EDTA, 250 mM sucrose was mixed with a 1.2 molar amount of Alexa Fluor 568 maleimide derivative (ThermoFisher Scientific). After adjustement of the pH at 7.5 and subsequent incubation for 1 hour at room temperature, the chemical reaction was blocked with N-ethylmaleimide in excess. The mixture was centrifuged through either a Zeba spin column with a cut-off of 7 kDa (GE Healthcare) or the fluorescent dye removal column provided by Thermofischer Scientific. The amount of fluorophore per bivalent C6 in the flow-through was calculated by spectrophotometry with a Nanodrop 2000 device (ThermoFisher Scientific). After incubation of the cells with the different reagents and several washes with PBS, the coverslips were mounted with 4′,6′-diamino-2phenyl-indole (DAPI) Fluoromount-G (Southern Biotech, Birmingham, USA) and imaged with a Leica DM5500 microscope (Leica, Wetzlar, Germany) equipped with 20× and 63× objectives. The signal was recorded with a Leica DFC350FX camera. Confocal microscopy was performed as previously described (Conic, S. et al.; J. Cell Biol. 2018, 217, 1537-1552). All microscopy images were processed using the Fiji/Image J software. For the measurement of the nuclear fluorescence intensity, the images of microscopy fields were acquired with the 20× objective. The nuclei were set with the DAPI channel acquisition as regions of interest (ROI) and the mean fluorescence intensity in each ROI was measured using the Fiji built-in-tool and data were processed with the R software.
Widefield fluorescence microscopy was performed on a home-built system composed by a Nikon TiE inverted microscope coupled to a high-numerical aperture (NA) TIRF objective (Apo TIRF 100X, oil, NA 1.49, Nikon). Live-sample were illuminated with a laser diode at 561 nm (10 W/cm2, Oxxius) at 37° C. Real-time imaging was performed by introducing a single edge dichroic mirror and a bandpass filter in the emission path of the microscope (Semrock, 560 nm edge BrightLine single-edge imaging-flat dichroic beamsplitter, 593/40 nm BrightLine single-band bandpass filter) and by using an EM-CCD camera (ImagEM, Hamamatsu, 0.106 μm pixel size) with a typical integration time of 100 ms. The videos were recorded using the perfect focus system of the microscope to avoid z-drift during the acquisition (1 image recorded every minute during 10 minutes). Images were processed using Fiji. To visualize the movement of the foci, the inventors used a filtering procedure in which two different Gaussian blurs (A=1.3 pixel and B=2 pixels) were applied to each image of the stack. The improved stack was obtained by computing the difference between A and B. The Mosaic plugin was then used on the final stack to reconstruct the single foci trajectories over the whole acquisition.
Statistical analysis was performed using R software version 3.6.1. Averages are represented as means+/−SD and the number of replicates is indicated in the figure legends. In the boxplots (
| Number | Date | Country | Kind |
|---|---|---|---|
| 21305627.8 | May 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/062831 | 5/11/2022 | WO |
