WO2021214343A1 - Adp-ribosylation utilisée en tant que marqueur pronostique dans le cancer - Google Patents

Adp-ribosylation utilisée en tant que marqueur pronostique dans le cancer Download PDF

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WO2021214343A1
WO2021214343A1 PCT/EP2021/060856 EP2021060856W WO2021214343A1 WO 2021214343 A1 WO2021214343 A1 WO 2021214343A1 EP 2021060856 W EP2021060856 W EP 2021060856W WO 2021214343 A1 WO2021214343 A1 WO 2021214343A1
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adp
adpr
tag
ribosyl
ribosylated
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PCT/EP2021/060856
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English (en)
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Michael Hottiger
Kathrin NOWAK
Fabio AIMI
Florian ROSENTHAL
Andreas Plückthun
Birgit Dreier
Holger Moch
Peter Schraml
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Universität Zürich
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2497—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing N- glycosyl compounds (3.2.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]

Definitions

  • the present invention relates to a diagnostic method for cancer based on an ADP-ribosylation signal in an isolated sample of tissue of the patient.
  • the present invention further relates to a method for detection of ADP-ribosylation in a cell and to a polypeptide, which binds to ADP- ribosylation.
  • ADP-ribosylation is prognostic marker in cancer diagnosis.
  • the present application shows that this marker is useful in several different cancer models. The data are gained from several hundreds of patients, which makes them statistically more relevant than many other studies.
  • ADP-ribosylation The detection of ADP-ribosylation is performed with immunohistochemistry on histological tissue slides.
  • the pipeline for cancer diagnosis nearly always goes via a histological analysis performed by a pathologist who scores the tissue biomarker stained by immunohistochemistry.
  • protein ADP-ribosylation was first described in the early 1960s, ADP-ribosylation was traditionally studied and identified in vitro via the incorporation of radioactive ADPr or ADPr-analogs. For a long time, only antibodies recognizing poly-ADP-ribosylation (PAR) were available, which restricted the ability to detect only PAR events by immunoblotting or immunofluorescence.
  • PAR poly-ADP-ribosylation
  • the inventors’ methodology offers the possibility to the pathologist to directly observe and then score the ADPR signal in the tissue biopsies. Moreover, the methodology can colocalize the ADPR signal with other biomarkers by immunofluorescence-multiplexing, which is the new frontier of cancer diagnosis (with detection of currently up to 7 biomarkers at the same time on a tissue biopsy, saving time and patient tissues).
  • a first aspect of the invention relates to a method for diagnosis of cancer in a patient, or a method of determining the prognosis of a cancer patient, or a method of assigning a patient to an outcome group, or a method of assigning a patient to a treatment regimen, said method comprising the steps of a. providing an isolated tissue sample of said patient, wherein the isolated tissue sample comprises a plurality of cells; b. fixating said plurality of cells, thereby permeabilizing the cells’ membrane; c. contacting, i.e.
  • ADPR binder capable to specifically and selectively non-covalently bind ADP-ribosylated biomolecules, particularly wherein the biomolecules are peptides or polypeptides or nucleic acid molecules, more particularly wherein the biomolecules are polypeptides, and wherein the ADPR binder comprises a detectable label, or a binding moiety allowing specific labelling of the ADPR binder with a second binder, which comprises a detectable label; and optionally, washing off excess first ADPR binder and contacting the sample with the second binder; d. detecting the amount and location of ADP-ribosylated biomolecules inside the isolated tissue sample; e.
  • a second aspect of the invention relates to a method for detection of ADP-ribosylated peptides or polypeptides in a cell.
  • the method comprises the steps of a. providing an isolated tissue sample of said patient, wherein the isolated tissue sample comprises a plurality of cells; b. permeabilizing the cells’ membrane; c. in a labelling step, contacting said cells with an ADPR binder, wherein the ADPR binder is capable to specifically bind ADP-ribosylated biomolecules; d. in a detection step, detecting the amount and location of ADP-ribosylated biomolecules inside the isolated tissue sample.
  • the method comprises the steps of a. providing a cell from an isolated tissue sample or from cell culture; b. contacting said cell with an ADPR binder, wherein said ADPR binder comprises SEQ ID NO 001 or said ADPR binder comprises an ADP-ribosyl-binding sequence having 390%, particularly >92%, or 394%, more particularly 396%, even more particularly 398%, most particularly 399% identity to SEQ ID NO 001, wherein said ADP-ribosyl-binding sequence contains the residues Glu35 and Arg145, and wherein said ADP-ribosyl- binding sequence has at least 85% (particularly 90%, 92%, 94%, 96% most particularly 398%) of the ADP-ribosyl-binding activity of SEQ ID NO 001 ; c. detecting ADP-ribosylated peptides or polypeptides.
  • a third aspect of the invention relates to a polypeptide comprising a. a polypeptide sequence of SEQ ID NO 001 or a polypeptide sequence having >90%, particularly 392%, more particularly >94%, even more particularly >96%, more particularly >98%, most particularly 399% identity to SEQ ID NO 001 and containing the residues Glu35 and Arg145, and wherein said polypeptide sequence has at least 85% (particularly 90%, 92%, 94%, 96% most particularly 398%) of the ADP-ribosyl-binding activity of SEQ ID NO 001, and b. a detectable label, particularly a detectable label selected from a tag sequence, a fluorescent or luminescent protein moiety and a fluorescent dye.
  • a fourth aspect of the invention relates to a system to carry out the method of any one of aspects 1 or 2, particularly a system for determining the content of ADP-ribosylated biomolecules in a sample obtained from a patient.
  • references to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”
  • polypeptide in the context of the present specification relates to a molecule consisting of 50 or more amino acids that form a linear chain wherein the amino acids are connected by peptide bonds.
  • the amino acid sequence of a polypeptide may represent the amino acid sequence of a whole (as found physiologically) protein or fragments thereof.
  • polypeptides and protein are used interchangeably herein and include proteins and fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences.
  • peptide in the context of the present specification relates to a molecule consisting of up to 50 amino acids, in particular 8 to 30 amino acids, more particularly 8 to 15amino acids, that form a linear chain wherein the amino acids are connected by peptide bonds.
  • Amino acid residue sequences are given from amino to carboxyl terminus.
  • Capital letters for sequence positions refer to L-amino acids in the one-letter code (Stryer, Biochemistry, 3 rd ed. p. 21).
  • Lower case letters for amino acid sequence positions refer to the corresponding D- or (2R)-amino acids. Sequences are written left to right in the direction from the amino to the carboxy terminus.
  • amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (lie, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V).
  • variant refers to a polypeptide that differs from a reference polypeptide, but retains essential properties.
  • a typical variant of a polypeptide differs in its primary amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions).
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.
  • sequence identity and percentage of sequence identity refer to a single quantitative parameter representing the result of a sequence comparison determined by comparing two aligned sequences position by position.
  • Methods for alignment of sequences for comparison are well-known in the art. Alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the global alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci.
  • sequence identity values refer to the value obtained using the BLAST suite of programs (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) using the above identified default parameters for protein and nucleic acid comparison, respectively.
  • antibody refers to whole antibodies including but not limited to immunoglobulin type G (IgG), type A (IgA), type D (IgD), type E (IgE) or type M (IgM), any antigen binding fragment or single chains thereof and related or derived constructs.
  • a whole antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • VH heavy chain variable region
  • CH heavy chain constant region
  • the heavy chain constant region of IgG is comprised of three domains, CH1 , CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL).
  • the light chain constant region is comprised of one domain, CL.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system.
  • the term encompasses a so- called nanobody or single domain antibody, an antibody fragment consisting of a single monomeric variable antibody domain.
  • fragment crystallizable (Fc) region is used in its meaning known in the art of cell biology and immunology; it refers to a fraction of an antibody comprising, if applied to IgG, two identical heavy chain fragments comprised of a CH2 and a CH3 domain, covalently linked by disulfide bonds.
  • specific binding in the context of the present invention refers to a property of ligands that bind to their target with a certain affinity and target specificity.
  • the affinity of such a ligand is indicated by the dissociation constant of the ligand.
  • a specifically reactive ligand has a dissociation constant of £ 10 7 mol/L when binding to its target, but a dissociation constant at least three orders of magnitude higher in its interaction with a molecule having a globally similar chemical composition as the target, but a different three-dimensional structure.
  • a polymer of a given group of monomers is a homopolymer (made up of a multiple of the same monomer); a copolymer of a given selection of monomers is a heteropolymer constituted by monomers of at least two of the group.
  • a first aspect of the invention relates to a method for diagnosis of cancer in a patient, or a method of determining the prognosis of a cancer patient, or a method of assigning a patient to an outcome group, or a method of assigning a patient to a treatment regimen.
  • the method comprises the steps of a. providing an isolated tissue sample of said patient, wherein the isolated tissue sample comprises a plurality of cells; b. in a fixating step, fixating said plurality of cells, thereby permeabilizing the cells’ membrane; c. in a contacting step, contacting, i.e.
  • ADPR binder capable to specifically and selectively non-covalently bind ADP-ribosylated biomolecules
  • the ADPR binder comprises a detectable label, or a binding moiety allowing specific labelling of the ADPR binder with a second binder, which comprises a detectable label; and optionally, washing off excess first ADPR binder and contacting the sample with the second binder; d. in a detection step, detecting the amount and location of ADP-ribosylated biomolecules inside the isolated tissue sample; e.
  • assigning in an assigning step, assigning, as a function of where and/or how much ADPR binder is detected in the isolated tissue sample o a likelihood of having or developing cancer to said patient, or o assigning a likelihood of prognosis to said patient, or o assigning the patient to an outcome group or o assigning the patient to treatment with an anticancer treatment.
  • the ADPR binder is capable of binding mono-ADP-ribosylated biomolecules.
  • the ADPR binder does not specifically react to poly-ADP-ribosylated biomolecules.
  • ADPR binders described here have the capacity to bind both mono- and poly-ADP- ribosylation structures (MAR and PAR, respectively), it is their capacity to bind mono-ADP- ribosylated (MARylated) biomolecules which renders them superior to PAR-specific regents.
  • MAR and PAR structures are generated by different enzymes within the cells and these enzymes have different subcellular localization. The inventors found that it is the cytoplasmic MAR signal which correlates with different patient outcome data.
  • the ADP-ribosylated biomolecules are ADP-ribosylated peptides or ADP-ribosylated polypeptides or ADP-ribosylated nucleic acid molecules. In certain embodiments, the ADP-ribosylated biomolecules are ADP-ribosylated polypeptides.
  • o a high likelihood of having or developing cancer is assigned to said patient, or o a more severe prognosis is assigned to said patient; or o treatment with an anticancer treatment is assigned to said patient; if said cells in said isolated tissue sample of said patient are stained weakly with said ADPR binder in comparison to control tissue (healthy tissue of the same tissue origin) as judged by a blinded experienced pathologist.
  • the isolated tissue sample is compared with healthy tissue on the same array. Quantification is performed by a trained pathologist or by an image-evaluation software.
  • a particular advantage of the method of the first aspect is that tumor cells can be identified independently of their cell cycle status and their proliferation activity.
  • the method of the first aspect is a general classification method for the existence and severity of a tumor, independently of its origin. This is in contrast to the main markers used in histochemistry.
  • the method of the first aspect is a useful addition to current standards or even a stand-alone tool.
  • the method of the first aspect can be used if only a very small amount of cell material is available. Many other methods highly depend on the cells’ growth pattern, which is not needed for the method of the invention.
  • said ADPR binder comprises SEQ ID NO 001. In certain embodiments, said ADPR binder comprises an ADP-ribosyl-binding sequence having 390% identity to SEQ ID NO 001 , wherein said ADP-ribosyl-binding sequence contains the residues Glu35 and Arg145. In certain embodiments, said ADPR binder comprises an ADP-ribosyl-binding sequence having 392% identity to SEQ ID NO 001, wherein said ADP- ribosyl-binding sequence contains the residues Glu35 and Arg145.
  • said ADPR binder comprises an ADP-ribosyl-binding sequence having 394% identity to SEQ ID NO 001 , wherein said ADP-ribosyl-binding sequence contains the residues Glu35 and Arg145. In certain embodiments, said ADPR binder comprises an ADP-ribosyl-binding sequence having 396% identity to SEQ ID NO 001, wherein said ADP-ribosyl-binding sequence contains the residues Glu35 and Arg145. In certain embodiments, said ADPR binder comprises an ADP-ribosyl-binding sequence having 398% identity to SEQ ID NO 001, wherein said ADP-ribosyl-binding sequence contains the residues Glu35 and Arg145.
  • said ADPR binder comprises an ADP-ribosyl-binding sequence having 399% identity to SEQ ID NO 001 , wherein said ADP-ribosyl-binding sequence contains the residues Glu35 and Arg145.
  • said ADP-ribosyl-binding sequence additionally contains one residue selected from Arg15, Cys74, Leu97, Val103, Gly105, Gly110, and Asp162. In certain embodiments, said ADP-ribosyl-binding sequence additionally contains two residues selected from Arg15, Cys74, Leu97, Val103, Gly105, Gly110, and Asp162. In certain embodiments, said ADP-ribosyl-binding sequence additionally contains three residues selected from Arg15, Cys74, Leu97, Val103, Gly105, Gly110, and Asp162.
  • said ADP- ribosyl-binding sequence additionally contains four residues selected from Arg15, Cys74, Leu97, Val103, Gly105, Gly110, and Asp162. In certain embodiments, said ADP-ribosyl- binding sequence additionally contains five residues selected from Arg15, Cys74, Leu97, Val103, Gly105, Gly110, and Asp162. In certain embodiments, said ADP-ribosyl-binding sequence additionally contains six residues selected from Arg15, Cys74, Leu97, Val103, Gly105, Gly110, and Asp162. In certain embodiments, said ADP-ribosyl-binding sequence additionally contains all residues Arg15, Cys74, Leu97, Val103, Gly105, Gly110, and Asp162.
  • the above-mentioned ADP-ribosyl-binding sequence has a certain ADP-ribosyl-binding activity. In certain embodiments, the above-mentioned ADP-ribosyl-binding sequence has at least 85% of the ADP-ribosyl-binding activity of SEQ ID NO 001. In certain embodiments, the above-mentioned ADP-ribosyl-binding sequence has at least 90% of the ADP-ribosyl-binding activity of SEQ ID NO 001. In certain embodiments, the above-mentioned ADP-ribosyl-binding sequence has at least 92% of the ADP-ribosyl-binding activity of SEQ ID NO 001.
  • the above-mentioned ADP-ribosyl-binding sequence has at least 94% of the ADP-ribosyl-binding activity of SEQ ID NO 001. In certain embodiments, the above-mentioned ADP-ribosyl-binding sequence has at least 96% of the ADP-ribosyl-binding activity of SEQ ID NO 001. In certain embodiments, the above-mentioned ADP-ribosyl-binding sequence has at least 98% of the ADP-ribosyl-binding activity of SEQ ID NO 001.
  • the method of the first aspect is even more reliable in detecting and classifying tumors when used with the ADPR binder comprising SEQ ID NO 001, as shown in Example 8.
  • the cancer is selected from renal cell carcinoma, breast cancer, ovarian cancer and colon cancer. In certain embodiments, the cancer is renal cell carcinoma.
  • the cells are contacted with an additional binder.
  • the additional binder is selected from GLUT1, CA IX, Ki-67, Mib-1, and p53.
  • GLUT 1 is aberrantly expressed in several tumor types, as e.g. in some types of colon, ovarian or breast cancer.
  • CA IX is over-expressed in VHL mutated clear cell renal cell carcinoma (ccRCC) and hypoxic solid tumors, but is low-expressed in normal kidney and most other normal tissues. It may be involved in cell proliferation and transformation.
  • ccRCC clear cell renal cell carcinoma
  • Ki-67 is a cellular marker for proliferation. During interphase, the Ki-67 antigen can be exclusively detected within the cell nucleus, whereas in mitosis most of the protein is relocated to the surface of the chromosomes. Ki-67 protein is present during all active phases of the cell cycle (G1, S, G2, and mitosis), but is absent in resting (quiescent) cells (GO).
  • Mib-1 is a cellular marker for proliferation. It is directed at the same antigen as Ki-67. p53 is upregulated in many types of cancer and thus, it serves as a marker for malignancy.
  • the isolated tissue sample is a biopsy of a neoplasm of said patient. In certain embodiments, the isolated tissue sample is a biopsy of a solid neoplasm of said patient.
  • a second aspect of the invention relates to a method for detection of ADP-ribosylated peptides or polypeptides in a cell, comprising the steps of a. providing a cell from an isolated tissue sample or from cell culture; b. contacting said cell with an ADPR binder; c. detecting ADP-ribosylated peptides or polypeptides.
  • said ADPR binder comprises SEQ ID NO 001. In certain embodiments, said ADPR binder comprises an ADP-ribosyl-binding sequence having 390% identity to SEQ ID NO 001, wherein said ADP-ribosyl-binding sequence contains the residues Glu35 and Arg145. In certain embodiments, said ADPR binder comprises an ADP-ribosyl-binding sequence having 392% identity to SEQ ID NO 001 , wherein said ADP- ribosyl-binding sequence contains the residues Glu35 and Arg145.
  • said ADPR binder comprises an ADP-ribosyl-binding sequence having 394% identity to SEQ ID NO 001 , wherein said ADP-ribosyl-binding sequence contains the residues Glu35 and Arg145. In certain embodiments, said ADPR binder comprises an ADP-ribosyl-binding sequence having 396% identity to SEQ ID NO 001, wherein said ADP-ribosyl-binding sequence contains the residues Glu35 and Arg145. In certain embodiments, said ADPR binder comprises an ADP-ribosyl-binding sequence having 398% identity to SEQ ID NO 001, wherein said ADP-ribosyl-binding sequence contains the residues Glu35 and Arg145.
  • said ADPR binder comprises an ADP-ribosyl-binding sequence having 399% identity to SEQ ID NO 001 , wherein said ADP-ribosyl-binding sequence contains the residues Glu35 and Arg145.
  • said ADP-ribosyl-binding sequence additionally contains one residue selected from Arg15, Cys74, Leu97, Val103, Gly105, Gly110, and Asp162. In certain embodiments, said ADP-ribosyl-binding sequence additionally contains two residues selected from Arg15, Cys74, Leu97, Val103, Gly105, Gly110, and Asp162. In certain embodiments, said ADP-ribosyl-binding sequence additionally contains three residues selected from Arg15, Cys74, Leu97, Val103, Gly105, Gly110, and Asp162.
  • said ADP- ribosyl-binding sequence additionally contains four residues selected from Arg15, Cys74, Leu97, Val103, Gly105, Gly110, and Asp162. In certain embodiments, said ADP-ribosyl- binding sequence additionally contains five residues selected from Arg15, Cys74, Leu97, Val103, Gly105, Gly110, and Asp162. In certain embodiments, said ADP-ribosyl-binding sequence additionally contains six residues selected from Arg15, Cys74, Leu97, Val103, Gly105, Gly110, and Asp162. In certain embodiments, said ADP-ribosyl-binding sequence additionally contains all residues Arg15, Cys74, Leu97, Val103, Gly105, Gly110, and Asp162.
  • the above-mentioned ADP-ribosyl-binding sequence has a certain ADP-ribosyl-binding activity. In certain embodiments, the above-mentioned ADP-ribosyl-binding sequence has at least 85% of the ADP-ribosyl-binding activity of SEQ ID NO 001. In certain embodiments, the above-mentioned ADP-ribosyl-binding sequence has at least 90% of the ADP-ribosyl-binding activity of SEQ ID NO 001. In certain embodiments, the above-mentioned ADP-ribosyl-binding sequence has at least 92% of the ADP-ribosyl-binding activity of SEQ ID NO 001.
  • the above-mentioned ADP-ribosyl-binding sequence has at least 94% of the ADP-ribosyl-binding activity of SEQ ID NO 001. In certain embodiments, the above-mentioned ADP-ribosyl-binding sequence has at least 96% of the ADP-ribosyl-binding activity of SEQ ID NO 001. In certain embodiments, the above-mentioned ADP-ribosyl-binding sequence has at least 98% of the ADP-ribosyl-binding activity of SEQ ID NO 001.
  • the detection of ADP-ribosylated biomolecules or peptides or polypeptides is non-nuclear, which means that only the signal outside of the nucleus is considered.
  • the detection of ADP-ribosylated biomolecules or peptides or polypeptides is in the cytoplasm. Cytoplasm includes all organelles outside of the nucleus.
  • the detection of ADP-ribosylated biomolecules or peptides or polypeptides is in mitochondria.
  • the detection of ADP- ribosylated biomolecules or peptides or polypeptides is in the endoplasmatic reticulum (ER). In certain embodiments of the first or second aspect, the detection of ADP-ribosylated biomolecules or peptides or polypeptides is in stress granules. Stress granules are dense aggregations in the cytosol composed of proteins and RNAs that appear when the cell is under stress.
  • the ADPR signal in the experiments of the Examples showed a signal in the cytoplasm, not in the nucleus, which presumably stems from mitochondria based on immunofluorescence co- stainings with mitochondrial markers using TMA. It is assumed that the majority of the signal results from mono-ADPR, because enzymes catalysing mono-APDR were reported to be in the cytoplasm and in mitochondria (Hopp et al., Cells. 2019 Aug 13;8(8).), while poly-ADPR was reported to be mainly in the nucleus (Ray Chaudhuri et al., Nat Rev Mol Cell Biol. 2017 Oct; 18(10):610-621.).
  • a third aspect of the invention relates to a polypeptide comprising a. a polypeptide sequence comprising or essentially consisting of the sequence denoted as SEQ ID NO 001, or a variant thereof retaining its essential binding qualities and showing the key residues Glu35 and Arg145, and b. a detectable label.
  • the inventors have surprisingly found that the variant of the ADP binding moiety given as SEQ ID NO 001 , having the residues Glu35 and Arg145, has far superior binding characteristics than previously characterized ADPR binding domains, and enables diagnostic procedures detecting ADPR modification of biomolecules, particularly of proteins, with far higher sensitivity and specificity than previously known ligands.
  • the polypeptide In order to enable detection of the binder, the polypeptide needs to be modified. Any label allowing detection may be considered.
  • the most salient examples of a detectable label include, but are not necessarily limited to: a tag sequence, allowing the specific and selective attachment of a secondary binder that in turn is labelled in a way to enable detection, for example by optical means; an enzymatic activity such as a fluorescent or luminescent protein moiety allowing direct detection, and a fluorescent dye attached to the polypeptide.
  • Tag sequences are of particular interest, as specific, labelled binders are readily available for a number of such tags.
  • the polypeptide comprises a tag sequence selected from an Fc-tag (a fragment crystallizable derived from an immunoglobulin), a Strep-tag, a glutathione-S- transferase-tag, a green fluorescent protein-tag, a BCCP-tag, a FI_AG-tag, an HA-tag, a Myc- tag, a maltose binding protein-tag, a Nus-tag, a thioredoxin-tag, a CRDSAT-tag, a poly- glutamate-tag, a calmodulin-tag, an ALFA-tag, an Avi-tag, a C-tag, an E-tag, an NE-tag, an S- tag, an SBP-tag, aSpot-tag, a T7-tag, a Ty-tag, a V5-tag, a VSV-tag, an Xpress-tag, and a TC- tag, particularly said tag sequence is an Fc-tag.
  • Fc-tag
  • the polypeptide comprises a fluorescent protein tag.
  • GFP green fluorescent protein
  • EBFP enhanced blue fluorescent protein
  • EBFP2 enhanced blue fluorescent protein 2
  • EGFP sirius enhanced green fluorescent protein
  • EYFP enhanced yellow fluorescent protein
  • citrine venus, YPet, topaz
  • SYFP mAmetrine enhanced cyan fluorescent protein
  • ECFP mTurquoise
  • mTurquoise2 cerulean
  • CyPet SCFP.
  • a fluorescent protein for practicing the invention may also be selected from the group comprising fluorescent protein from Discosoma striata and derivatives thereof: mTagBFP,
  • TagCFP AmCyan, Midoriishi Cyan, mTFP1
  • Fluorescent proteins also comprise proteins derived from alpha-allophycocyanin from the cyanobacterium Trichodesmium erythraeum such as small ultra-red fluorescent protein.
  • the polypeptide comprising SEQ ID NO 001 or a variant thereof comprises a dye molecule, exemplified but not limited to, octadecyl rhodamine B, 7-nitro-2- 1,3-benzoxadiazol-4-yl, 4-acetamido-4'-isothiocyanatostilbene-2,2' disulfonic acid, acridine and derivatives, 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 4-amino-N-(3- [vinylsulfonyl]phenyl)naphthalimide-3, 6-disulfonate dilithium salt, N-(4-anilino-1- naphthyl)maleimide, anthranilamide, BODIPY, Brilliant Yellow, coumarin and derivatives, cyanine dyes, cyanosine, 4',6-diaminidino-2-pheny
  • said polypeptide sequence comprises SEQ ID NO 001. In certain embodiments, said polypeptide sequence comprises an ADP-ribosyl-binding sequence having 390% identity to SEQ ID NO 001, wherein said ADP-ribosyl-binding sequence contains the residues Glu35 and Arg145. In certain embodiments, said polypeptide sequence comprises an ADP-ribosyl-binding sequence having >92% identity to SEQ ID NO 001 , wherein said ADP-ribosyl-binding sequence contains the residues Glu35 and Arg145.
  • said polypeptide sequence comprises an ADP-ribosyl-binding sequence having 394% identity to SEQ ID NO 001 , wherein said ADP-ribosyl-binding sequence contains the residues Glu35 and Arg145. In certain embodiments, said polypeptide sequence comprises an ADP-ribosyl-binding sequence having 396% identity to SEQ ID NO 001, wherein said ADP-ribosyl-binding sequence contains the residues Glu35 and Arg145. In certain embodiments, said polypeptide sequence comprises an ADP-ribosyl-binding sequence having 398% identity to SEQ ID NO 001, wherein said ADP-ribosyl-binding sequence contains the residues Glu35 and Arg145.
  • said polypeptide sequence comprises an ADP-ribosyl-binding sequence having 399% identity to SEQ ID NO 001, wherein said ADP- ribosyl-binding sequence contains the residues Glu35 and Arg145.
  • said ADP-ribosyl-binding sequence additionally contains one residue selected from Arg15, Cys74, Leu97, Val103, Gly105, Gly110, and Asp162.
  • said ADP-ribosyl-binding sequence additionally contains two residues selected from Arg15, Cys74, Leu97, Val103, Gly105, Gly110, and Asp162.
  • said ADP-ribosyl-binding sequence additionally contains three residues selected from Arg15, Cys74, Leu97, Val103, Gly105, Gly110, and Asp162. In certain embodiments, said ADP- ribosyl-binding sequence additionally contains four residues selected from Arg15, Cys74, Leu97, Val103, Gly105, Gly110, and Asp162. In certain embodiments, said ADP-ribosyl- binding sequence additionally contains five residues selected from Arg15, Cys74, Leu97, Val103, Gly105, Gly110, and Asp162.
  • said ADP-ribosyl-binding sequence additionally contains six residues selected from Arg15, Cys74, Leu97, Val103, Gly105, Gly110, and Asp162. In certain embodiments, said ADP-ribosyl-binding sequence additionally contains all residues Arg15, Cys74, Leu97, Val103, Gly105, Gly110, and Asp162. The above-mentioned ADP-ribosyl-binding sequence has a certain ADP-ribosyl- binding activity. In certain embodiments, the above-mentioned ADP-ribosyl-binding sequence has at least 85% of the ADP-ribosyl-binding activity of SEQ ID NO 001.
  • the above-mentioned ADP-ribosyl-binding sequence has at least 90% of the ADP-ribosyl-binding activity of SEQ ID NO 001. In certain embodiments, the above-mentioned ADP-ribosyl-binding sequence has at least 92% of the ADP-ribosyl-binding activity of SEQ ID NO 001. In certain embodiments, the above-mentioned ADP-ribosyl-binding sequence has at least 94% of the ADP-ribosyl-binding activity of SEQ ID NO 001.
  • the above-mentioned ADP-ribosyl-binding sequence has at least 96% of the ADP-ribosyl-binding activity of SEQ ID NO 001. In certain embodiments, the above-mentioned ADP-ribosyl-binding sequence has at least 98% of the ADP-ribosyl-binding activity of SEQ ID NO 001.
  • the polypeptide of the third aspect is particularly useful for carrying out the methods of the first and second aspect.
  • a fourth aspect of the invention relates to a system to carry out the method of any one of aspects 1 or 2.
  • the system according to the invention is designed and configured to the method for determining the content of ADP-ribosylated biomolecules in a sample obtained from a patient, wherein an isolated tissue sample of the patient, the isolated tissue sample comprising a plurality of cells having a permeabilized cell membrane is contacted with an ADPR binder capable to specifically bind ADP-ribosylated biomolecules, and the system is fitted to detect the amount and location of ADP-ribosylated biomolecules inside the isolated tissue sample.
  • Fig. 1 Characterization of evolved Af1521 macro domain
  • A Pull-down experiment of unmodified and ADP-ribosylated H2B peptides (biotinylated and bound to streptavidin Sepharose beads) of WT Af1521 with the sequence: MEVLFEAKVGDITLKLAQGDITQYPAKAIVNAANKRLEHGGGVAYAIAKACAGDA GLYTEISKKAMREQFGRDYIDHGEVVVTPAMNLEERGIKYVFHTVGPICSGMWS EELKEKLYKAFLGPLEKAEEMGVESIAFPAVSAGIYGCDLEKVVETFLEAVKNFK GSAVKEVALVIYDRKSAEVALKVFERSL (SEQ ID NO 022) and eAf1521 with the sequence:
  • the substrate binding region is underlined. K35E and Y145R substitutions are marked in grey.
  • C Structural comparison of the evolved macro domains eAf1521 (mustard) and WT Af1521 (turquoise; PDB ID: 2BFQ).
  • D upper panel
  • D, lower panel left Surface rendering of the WT Af1521 ADPr binding site. The 1141 sidechain is highlighted.
  • FIG. 2 Stronger enrichment of global ADP-ribosylome by eAf1521 compared to WT Af1521
  • A, B Volcano plots comparing ADP-ribosylated peptides enriched from untreated and H2C>2-treated HeLa cells with either WT Af1521 oreAf1521.
  • C Venn diagrams displaying the overlap of ADP-ribosylated proteins enriched from untreated (left panel) or hhC reated HeLa cells (right panel) with either WT Af1521 or eAf1521.
  • D Distribution of ADPr on different amino acid acceptor sites (D, E, K, R, S, Y).
  • E Venn diagram representing the overlap of ADP-ribosylated proteins enriched with eAf1521 using different starting material amounts (5 mg, 10 mg and 20 mg).
  • F Scatter plot comparing the number of ADP-ribosylated proteins identified via eAf1521 enrichment using different starting material (5 mg, 10 mg and 20 mg).
  • FIG. 3 eAf1521 more strongly enriches for Serine-ADP-ribosylation
  • A Box plot representing MS1 intensities of all ADPr peptides of untreated and H2C>2-treated HeLa cell lysate enriched with either WT Af1521 or eAf2151.
  • B Scatter plots depicting MS1 ADPr peptide intensities of selected S-ADPr modified peptides (PARP1: SKGQVKEEGINKSEK (SEQ ID NO 002), HNRNPA1:
  • Fc-eAf1521 is more sensitive than Fc-WT Af1521 in detecting mono- and poly-ADP-ribosylated target proteins
  • A Immunoblot analyses of different substrates with either Fc-eAf1521 or Fc-WT Af1521 (untreated and H 2 0 2 -treated HeLa cells including PJ34 treatment as control for ADP-ribosylation inhibition; unmodified, poly-ADP-ribosylated and mono-ADP-ribosylated ARTD1 and unmodified and mono-ADP-ribosylated ARTD8cat). Detection was performed with IRDye 680 goat anti-mouse antibody.
  • Fig. 5 ADPR signal in kidney tissues.
  • B ADPR staining of healthy kidney epithelial cells on the left and of RCC tissue on the right. On the top, the overview (scale bar 100 pm, 10x), on the bottom a ROI (scale bar 10 pm, 40x).
  • D RCC tissues stained with anti-ADPR antibody. Scores: 1 (negative), 2 (weak), 3 (moderate), 4 (elevate) and 5 (strong).
  • Fig. 6 Mitochondrial ADPR (mtADPR) signal in papillary RCC subtype.
  • A ADPR staining in papillary RCC subtype and related scores. Upper pictures overview (scale bar 100 pm, 10x), lower pictures ROI (scale bar 10 pm, 40x).
  • Fig. 7 mtADPR signal in breast cancer (BrCa) and its subtypes.
  • Fig. 8 mtADPR signal in high grade serous ovarian cancer.
  • A OvCa TMA stained with anti-ADPR antibody overview.
  • B High grade serous OvCa tissues stained with anti-ADPR antibody. Scores: negative/weak (1- blue), moderate (2 - yellow), strong (3 - red). On the top the overview of the tissue spot in the TMA (scale bar 100 urn, 10x), on the bottom the ROI (scale bar 10 urn, 40x).
  • Fig. 9 mtADPR signal in colon tissues.
  • A Colon tissues stained with anti-ADPR antibody. Scores: negative/weak (1- blue), moderate (2 - yellow), strong (3 - red).
  • colon cancer CoCa
  • Colon cancer CoCa
  • Colon epithelium with tissue overview scale bar 100 pm, 10x
  • ROI scale bar 10 pm, 40x
  • (C) Kaplan-Meier overall survival plot of RCC patients stratified based on eAf1521 (mtADPR) signal intensity as follows: strong (3 - red), moderate (2 - yellow) and weak/negative (1 - blue). Mantel-Cox test, p-value ⁇ 0.0001, N 293.
  • FIG. 13 Correlation ADPR and eAf1521 in RCC.
  • Ribosome display evolves macro domain eAf1521.
  • A ELISA analysis of eAf1521 variants against the unmodified and ADPr H2B peptide. The ELISA was performed after the 4 th round of error-prone PCR followed by selection using ribosome display. AU, absorbance unit.
  • B Exemplary SDS-PAGE gel of pull down experiment using unmodified and ADP-ribosylated H2B peptides (biotinylated and bound to streptavidin Sepharose beads) of Af1521 WT. The proteins were analyzed by SDS-PAGE followed by Coomassie staining.
  • FIG. 15 Comparison of MS-based identification of ADP-ribosylated peptides and proteins using WT Af1521 or eAf1521. Venn diagram depicting the overlap of ADP-ribosylated proteins in H2C>2-treated HeLa cells lysate in different ADPr proteomic studies. For comparison with Hendriks et al. 2019 dataset only the closest experimental conditions Trypsin and EThcD were included.
  • Fig. 16 Fc-WT Af1521 and eAf1521 as a tool for Western Blot analysis. Immunoblot analyses to be compared were performed at the same time and with the same exposure.
  • A Dot blot analyses of free PAR chains with either Fc-eAf1521 or Fc- WT Af1521. Immunoblot analyses to be compared were performed at the same time and with the same exposure. Detection was performed with IRDye 680 goat anti-mouse antibody.
  • B Immunoblot analyses of oligo-ADP-ribosylated and poly- ADP-ribosylated ARTD1 with either Fc-eAf1521 or Fc-WT Af1521.
  • ARTD1 The automodification reaction of ARTD1 was performed with either 3 mM or 300 pM NAD + for 30 min at 37°C. Immunoblot analyses to be compared were performed at the same time and with the same exposure. Detection was performed with IRDye 680 goat anti-mouse antibody.
  • C Pull-down experiment using GST-Af1521 WT, GST-eAf1521 and GST of poly-ADP-ribosylated ARTD1. Only 10% of input and unbound fractions were loaded. The proteins were detected by immunoblot analysis stained with 10H antibody (PAR antibody) and followed by IRDye 680 goat anti-mouse antibody.
  • Fig. 17 shows panADPR staining (Millipore) staining in RCC.
  • A IHC staining of kidney tissues with anti-panADPR binding reagent (Millipore) and related intensity scores: strong (red, 3), moderate (yellow, 2) and weak/negative (blue, 1).
  • selected TMA cores scale bar 100 pm, 10x
  • ROIs scale bar 20 pm, 40x.
  • Fig. 18 shows PAR staining (Enzo) in RCC.
  • A IHC staining of kidney tissues with anti- PAR antibody (Enzo) and related intensity scores: strong (red, 3), moderate (yellow, 2) and weak/negative (blue, 1). On the top, selected TMA cores (scale bar 100 pm, 10x), on the bottom, ROIs (scale bar 20 pm, 40x).
  • Example 1 In vitro selection of an AH521 macro domain with 1000-fold increased affinity for
  • MS mass spectrometry
  • the inventors used an H2B peptide that was synthetically ADP-ribosylated with a N-glycosidic linkage on Q at position 2 of the peptide (Methods Mol Biol, 687, 283-306; Angew Chem Int Ed Engl, 55:10634-10638).
  • the Af1521 mutants were in vitro transcribed, translated (such that they do not leave the ribosome) and subsequently selected using the ADP- ribosylated peptide.
  • the enriched mRNA was isolated and amplified by RT-PCR.
  • the enriched pools were recloned into the ribosome display vector pRDV which served as template for the next round of selection.
  • Four rounds of selection using the modified peptide coupled either to streptavidin-coated plates (for rounds 1-3) or magnetic beads (for round 4) were performed with increasing stringency. This was achieved by extending the washing times in round 2, 3 and 4, reducing the target concentration from 200 nM ADPr H2B peptide to 100 nM in round 2 and to 20 nM in round 3 and 4.
  • Off-rate selection was also implemented using unmodified H2B peptide for round 3 and auto-ADP- ribosylated ARTD10 for round 4 (Ahmad, S. et al. (2016), Sci Rep, 6, 28922; Zahnd, C. et al. (2010) Protein Eng Des Sel, 23, 175-184; Dreier, B. et al. (2011) J Mol Biol, 405, 410-426.)
  • the inventors compared the catalytic activities of WT Af1521 and eAf1521 using an in vitro ADP-ribosylation assay.
  • the catalytic domain of ARTD8 (ARTD8cat) was auto-modified using radiolabeled 32 P-NAD + , residual NAD + removed and the resulting mono-ADP-ribosylated ARTD8cat used to define the hydrolytic activities of WT Af1521 or eAf1521.
  • nicotinamide-linked ribose moiety of the eAf1521-bound ADPr was rotated such that the ribose C-T oxygen, which serves as the anchor point for the target side chain, resided closer to the macro domain surface (Fig. 1D). This change was facilitated by the K35E and Y145R amino acid substitutions.
  • the R145 sidechain formed a salt bridge with the E35 carboxyl and thus provides a rigid boundary for terminal ribose binding while allowing hydrogen bond formation between the arginine Ns atom and the C-4’ (ring-forming) oxygen.
  • the terminal ribose site is delineated by the Y145 side chain, which is situated between the ribose carbon ring on one face, and 1102 side chain on the other.
  • this novel eAf1521 amino acid arrangement appears to contribute directly to strengthening the interaction with the proximal ribose.
  • the hydrogen bonding interaction between R145 of eAf1521 and the bound proximal ribose also changes the rotamer of the 1144 side chain.
  • the 1144 sidechain adopts a rotamer that bridges the central phosphates of ADPr and apparently appears to trap the ADPr (Fig. 1D and Fig. 14D) but may not energetically contribute to the binding.
  • Example 3 eAf1521 enriches ADP-ribosylation present in oenotoxic stressed HeLa cells to a larger extent than its WT counterpart
  • Example 4 Modified peptide and ADP-ribose acceptor site identifications were strongly improved due to higher MS spectral intensities
  • S-ADPr peptides shown here were also identified in the untreated HeLa cell lysates, indicating that ARTD1 regulates cellular processes via ADP- ribosylation also under basal conditions using the same modification sites.
  • the intensities of the detected R-ADPr peptides did not change after H 2 0 2 -treatment (Fig. 3C).
  • the R-ADPr modified proteins are located in the cytoplasm, suggesting that these targets are not modified by ARTD1 and are not affected by H 2 0 2 -treatment.
  • the inventors compared the ability of WT Af1521 and eAf1521 to enrich S- ADPr versus R-ADPr peptides. Most of the S-ADPr modified peptide intensities were significantly enhanced using eAf1521 compared to WT Af1521 , which was not the case for the R-ADPr peptides (Fig. 3B, C). Nevertheless, eAf1521 was still capable of enriching R-ADPr modified peptides to the same relative extent than Af1521 WT.
  • Example 5 eAf1521 improves mono- and oolv-ADP-ribosylated protein detection via immunoblotting
  • the fusion constructs were first characterized by immunoblots, testing against three different extracts: i) a HeLa cell lysate that was treated with H2O2 alone or together with the broad PARP-inhibitor PJ34, ii) in vitro auto-modified poly-ADP-ribosylated ARTD1 that was subsequently left untreated or treated with the enzyme poly(ADP-ribose)-glycohydrolase PARG to reduce PARylation to mono-ADP-ribosylated ARTD1 , or iii) in vitro auto-modified mono-ADP-ribosylated ARTD8cat (Fig. 4A).
  • eAf1521 recognized the modified proteins to a stronger extent irrespective of whether the proteins were mono- or poly- ADP-ribosylated.
  • Treatment of samples with PJ34 reduced the ADP-ribosylation signal after H2O2 treatment, indicating that the signals detected were indeed dependent on ADP- ribosylation.
  • the same experiment was performed with different concentrations of the purified Af1521-Fc constructs. The detection differences between WT Af1521 or eAf1521 could be confirmed at all concentrations; at the highest concentration used (500 ng/mL) eAf1521 signals were stronger than WT.
  • the inventors aimed to determine the enrichment capability of eAf1521 towards poly-ADP-ribosylated proteins. Therefore, the inventors performed for poly-ADP-ribosylated ARTD1 pulldown assays using our GST-fusion constructs (Fig. 16B). Poly-ADP-ribosylated ARTD1 was enriched comparably using both WT Af1521 and eAf1521 GST-fusion proteins compared to the control of GST alone. Finally, the inventors tested whether eAf1521 also detects free PAR chains that were isolated from poly- ADP-ribosylated ARTD1 digested with Proteinase K.
  • the inventors performed a co-incubation experiment of WT Af1521 and eAf1521 in the presence of either 1 mM free ADPr or another nucleotide (e.g. GTP) on H2C>2-treated HeLa cells.
  • the detected nuclear signal was reduced by co-incubation of both our Fc fusion proteins (i.e. Fc- WT Af1521 and -eAf1521) with ADPr but not with GTP, further confirming the detection specificity towards ADP-ribosylation.
  • the inventors observed also a weak nuclear and extranuclear signal with eAf1521 in untreated HeLa cells that was barely detectable by WT AH521 (Fig. 4B).
  • kidney epithelial cells that normally give rise to the tumor
  • mtADPR mitochondrial ADP- ribosylation
  • the inventors report the case of papillary RCC, the second most frequent RCC subtype, which is very aggressive. By re-stratifying the patients, the inventors could observe that a high mtADPR signal associates with a better patient prognosis, therefore with a longer overall survival.
  • the inventors focus on breast cancer and its subtypes invasive lobular and invasive ductal. Strong mtADPR signal associates with a better overall survival of the patients in this cancer type and its subtypes.
  • the inventors focus on ovarian cancer, particularly in its subtype high grade serous. Also in this tumor type, the inventors observe that a strong tADPR signal is a favorable factor that is associated with a better patient prognosis.
  • the inventors focus on colon tissues. As in kidney, the inventors observed that healthy colon epithelial cells display generally a stronger mtADPR signal intensity than colon cancer tissues. In colon cancer tissues, since the inventors did not have patient survival data, they could associate the mtADPR signal intensity to the tumor stage, a parameter which indicates the development of the tumor, and that it is considered favorable when low and a bad prognostic factor when it is high. In colon cancer, the inventors observed that that a strong mtADPR signal is a characteristic of most of the early stages colon cancer, and therefore mtADPR has to be still considered a favorable prognostic factor also for this tumor type.
  • Example 8 eAf1521 in cancer prognosis
  • the inventors used another tool to stain ADPR: instead of the anti-ADPR antibody, the inventors stained the same RCC TMA with eAf1521. The inventors repeated the same staining and scoring as performed previously with the anti-ADPR antibody.
  • the inventors stained RCC TMA with eAf1521 and they quantified the mitochondrial ADPR (mtADPR) signal intensity.
  • mtADPR mitochondrial ADPR
  • the inventors observe that mtADPR signal intensity is generally stronger in healthy kidney epithelium than in RCC.
  • mtADPR signal intensity correlates with a good prognosis.
  • the patient stratification seems to be more homogeneous than what observed previously with the anti-ADPR antibody.
  • eAf1521 the inventors are also able to stratify the patients according to the tumor stage and ISUP grade, which are two of the most important traditional prognostic markers: low grade and early stage RCCs are frequently characterized by a weak/negative mtADPR signal intensity.
  • the ability of stratifying patients based on overall survival, tumor stage and ISUP grade makes eAf1521 a very promising tool for prognostic.
  • the inventors show that also in the second most common RCC subtype, papillary RCC (papRCC), a strong mtADPR signal intensity observed with eAf1521 correlates with a better patient overall survival, as observed after staining with anti-ADPR antibody. Due to the small patient cohort, the inventors stratified the patients in two groups (strong, 3, red - low, 1- 2, green).
  • papRCC papillary RCC
  • the inventors show that in RCC the signal intensity obtained with anti-ADPR antibody correlates with the one given by the eAf1521. Tumors scored as strong ADPR are mostly scored as strong eAf1521, and tumors scored as weak ADPR are very frequently scored as weak with eAf1521.
  • the inventors stained the same RCC TMA with an antibody that recognizes only poly-ADPR (Enzo (Kawamitsu et al. (1984), Biochemistry, 23, 3771-3777), anti-PAR) and blindly scored the extranuclear PAR signal intensity.
  • the IHC stainings performed with the anti-ADPR antibody show a clear prognostic potential in several cancer types.
  • the signal intensity observed in the stainings represent an indication of the cytoplasmic/mitochondrial ADPR (mtADPR) levels in the tissues, therefore the stronger the signal, the higher the ADPR levels, and vice versa.
  • mtADPR cytoplasmic/mitochondrial ADPR
  • Associating the mtADPR levels (1- weak, 2- moderate, 3- strong) to the patients’ survival rate the inventors observed that patients with a longer life expectation are also characterized by a stronger mtADPR signal in the cancer biopsies, and vice versa.
  • the inventors observed a clear correlation between mtADPR levels and patient overall survival in: renal cell carcinoma (RCC), breast cancer, and ovarian cancer (high grade serous).
  • RCC renal cell carcinoma
  • breast cancer breast cancer
  • ovarian cancer high grade serous.
  • colon cancer patient survival data were missing
  • the inventors could associate strong mtADPR signal to early stage tumors, indicating that mtADPR levels may decrease with tumor development (and therefore decreasing in patients characterized by a lower overall survival).
  • kidney and colon the inventors could observe that stronger mtADPR signals were also characteristic of healthy epithelium (epithelial cells are the source of cancer in these organs) whereas cancer tissues display also weak signals.
  • eAf1521 (ADPR) and anti-ADPR antibody can both identify healthy kidney epithelial tissues as characterized by a more frequent strong signal.
  • eAf1521 (ADPR) and anti-ADPR antibody both indicate a strong mtADPR signal as a favourable prognostic factor, thus high ADPR levels are associated with a higher patient overall survival.
  • eAf1521 (ADPR) and anti-ADPR antibody signals correlate.
  • ADPR eAf1521
  • anti-ADPR antibody signals correlate.
  • eAf1521 (ADPR) staining allows for a better patient distribution/stratification, with more linear curves in the survival plot.
  • clear cell RCC ccRCC
  • the most frequent RCC subtype only eAf1521 (ADPR) can display a statistically significant difference in patient survival associated to staining intensity.
  • eAf1521 shows that the higher is the mtADPR signal intensity, the better is the prognosis for the patient.
  • eAf1521 shows a correlation between mtADPR signal intensity and tumor stage and ISUP grade (tumor grade and stage are both traditional prognostic markers).
  • anti-ADPR antibody can identify an association between mtADPR signal intensity and ISUP grade in papRCC.
  • HeLa cells Kerbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal calf serum (FCS) and 1% pen ici 11 i n/strepta vi d i n at 37 °C with 5% CO2.
  • DMEM Dulbecco's modified Eagle's medium
  • FCS fetal calf serum
  • pen ici 11 i n/strepta vi d i n at 37 °C with 5% CO2.
  • HeLa cells were either untreated or treated with 1 mM H2O2 in PBS containing 1 mM MgCh for 10 min.
  • Pretreatment with ADP-ribosylation inhibitors e.g. Olaparib
  • PCR products were cloned into the pRDV vector using the restriction enzymes BamHI and EcoRI (NEB). Respective bands were gelpurified using the Gel Purification Kit (QIAGEN) and cDNA fragments were digested with SamHI and EcoRI followed by a clean-up using the PCR purification kit (QIAGEN) and ligated into pRDV.
  • the resulting enriched pool served as template for the next round of selection using error-prone PCR and the primers T7B and TolAk. In total four rounds of selection were performed. After the second round of selection the washes were prolonged (2 direct washes, 4x 10 min). In round 3 and 4, an off-rate selection was implemented (Dreier and Pluckthun (2012) Methods Mol Biol, 805, 261-286).
  • round 3 error-prone PCR was applied to increase diversity, but no error-prone PCR was used in round 4, which instead served to efficiently enrich for generated high-affinity H2B binders. While round 1-3 were carried out with target immobilized on plates, in round 4 the selection was performed in solution MyOne T 1 streptavidin coated magnetic beads (Thermo Scientific) as previously described (Dreier and Pluckthun (2012) Methods Mol Biol, 805, 261- 286). For round 3, a completion with a 1000-fold access of competitor was performed. Afterwards, the wells were directly washed twice and additionally 4 times for 10 min.
  • Af 1521 mutants still binding to the biotinylated H2B peptide were eluted with EB buffer followed by RNA purification and RT-PCR. After performing the last iteration, the recovered mRNA was reversed transcribed and subsequently amplified by PCR. The PCR products were cloned into the expression vector pDST67 a pQE30 (QIAGEN) derivative (Steiner et al. (2008) J Mol Biol, 382, 1211-1227.) containing a MRGS-FMag using the restriction enzymes BamHI and Pstl. After transformation of E.
  • coli XL-1 Blue 92 single colonies were picked from selection plates and expressed in a 96 well format in 2xYT media containing 100 pg/ml ampicillin and 1% glucose for 4 h at 37°C after induction using 0.5 mM IPTG. Cells were harvested by centrifugation for 10 min at 4000 rpm and cells were lysed by addition of 50 pi B-PERII cell lysis buffer (Pierce) as previously described (Dreier and Pluckthun (2012) Methods Mol Biol, 805, 261-286).
  • the lysate was diluted with 950 mI PBS- TB (PBS: 137 mM NaCI, 30 mM KCI, 80 mM Na 2 HP0 4 , 15 mM KH 2 P0 4, pH 7.4 containing 0.1 (v/v) Tween20 and 0.2% (w/v) BSA) and cleared by centrifugation for 10 min at 4000 rpm.
  • PBS 137 mM NaCI, 30 mM KCI, 80 mM Na 2 HP0 4 , 15 mM KH 2 P0 4, pH 7.4 containing 0.1 (v/v) Tween20 and 0.2% (w/v) BSA) and cleared by centrifugation for 10 min at 4000 rpm.
  • an ELISA was performed against the biotinylated and ADP- ribosylated H2B peptide and unmodified peptide as control. The binding was detected using the MRGS-FMag (Dreier and Pluckthun (2011) Methods
  • Randomly mutagenized Af1521 candidates were cloned into pGEX6P-1 (Addgene) using the restriction enzymes BamHI and EcoRI (NEB). Mutants of eAf1521 and WT Af1521 were obtained by site-directed mutagenesis (G42E, forward primer GAGCACGGCGAAGGGGTGGC (SEQ ID NO 006), reverse primer GCCACCCCTTCGCCGTGCTC (SED ID NO 007); R145K: forward primer
  • CAGATCACAGCCTTTTATCCCAGC (SEQ ID NO 009)).
  • AH521-K35E forward primer 5’- GCCAACGAGAGGCTGG-3’ (SEQ ID 011), reverse primer 5’-CCAGCCTCTCGTTGGC-3’ (SEQ ID 012); eAf1521-E35E, forward primer 5’-GCCAACAAGAGGCTGG-3’ (SEQ ID 013), reverse primer 5’-CCAGCCTCTTGTTGGC-3’ (SEQ ID 14); eAf1521-H44G, forward primer 5’- CTGGGGGACGCGGC-3’ (SEQ ID 015), reverse primer 5’-GCCGCGTCCCCCAG-3’ (SEQ ID 016), WT AH521-Y145R forward primer 5’-CTGGGATACGCGGCTGTG-3’ (SEQ ID 017), reverse primer 5’-CACAGCCGCGTATCCCAG-3’ (SEQ ID 018); eAf1521-R145Y, forward primer 5’
  • His-tagged WT eAf1521 (with a N-terminal His tag) were constructed in the bacterial expression vector pET19b by GenScript (Piscataway, NJ, USA).
  • GenScript Procataway, NJ, USA.
  • fragments encoding the IL-2 secretion signal and an engineered mouse lgG2a Fc domain (described in (Gortz et ai.
  • the fragment containing the IL-2 secretion signal and the Fc domain with HA and His tags was cloned into pcDNA5/FRT/TO (Invitrogen) using the restriction enzymes Hindi 11 and Xhol (NEB).
  • the Af1521-encoding fragments were cloned in between the secretion signal and the Fc tag via Kpnl and BamHI sites.
  • Bacterial expression vectors were transformed into E. coli BL21 , and protein expression was induced by adding 1 mM IPTG at O ⁇ boo 0.4-0.6 for 3 h at 30 °C. Batch purification of GST- tagged or His-tagged proteins was carried out using glutathione Sepharose 4B beads (GE Healthcare) or ProBondTM Nickel-Chelating Resin (Thermo Fisher Scientific) according to the manufacturer's manual. Fc fusion domains were expressed by transfecting HEK293T cells with the mammalian expression vectors using calcium phosphate. Roughly 6 hrs after transfection, the medium was removed and replaced with fresh DM EM containing 1% of FCS.
  • biotinylated ADP-ribosylated or non-modified H2B peptides were bound to streptavidin Sepharose high-performance beads (GE Healthcare).
  • streptavidin Sepharose high-performance beads GE Healthcare
  • 5 pL of beads were washed three times in binding buffer (50 mM NaCI, 50 mM Tris-HCI pH 8, 0.05% NP-40) and incubated overnight at 4 °C in 1 ml_ binding buffer with 2 pg of the modified or unmodified H2B peptide.
  • beads were washed three times with 1 ml_ of in incubation buffer (1x protease inhibitor cocktail (Roche), 50 mM Tris-HCI pH 8, 0.05% NP-40) containing different salt concentration (50 mM, 200 mM and 400 mM NaCI). 2 pg of the recombinant protein was incubated with the beads in 1 ml_ of incubation buffer for 3 hrs at 4 °C. After centrifugation at 1,500 g for 5 min, the supernatant containing unbound protein was added on 5 pL prewashed glutathione Sepharose 4B beads (GE Healthcare) and additionally incubated for 2 hrs at 4 °C. Subsequently, all beads were washed three times with incubation buffer before analysis by SDS-PAGE followed by Coomassie blue staining.
  • IMAC-purified N-terminal His-tagged eAf1521 was further purified by SEC (Sephacryl-100; GE Healthcare) in 20 mM Tris pH 7.5, 300 mM NaCI, 10% glycerol, 2 mM TCEP.
  • SEC Sephacryl-100; GE Healthcare
  • the main peak fractions were concentrated by ultrafiltration in Vivaspin cartridges (Sartorius). Crystallization conditions were identified using the JCSG+ crystal screen (Qiagen) and sitting drop vapor diffusion.
  • Crystals grew at 4 °C in droplets consisting of 0.1 pL protein solution (23.1 mg/ml_ including 2 mM ADP-ribose) and 0.2 pL of well solution (25% w/v PEG3350, 0.1 M Bis-Tris, pH 5.5, and either 0.2 M (NH 4 ) 2 S0 4 or 0.2 M NaCI). Crystals were briefly transferred to cryo solution (well solution supplemented with 15% glycerol, 0.2 M NaCI, and 5 mM ADPr) and then stored under liquid nitrogen. Data collection, structure solution, and refinement
  • a 0.5 M EDTA solution (pH 8.5) was injected for 300 s, followed by a 120 s buffer injection, a surface activation step with 5 mM NiCL for 60 s, and a 120 s injection of eAf1521 (cone) and 150 s WT Af1521 (cone) respectively, both containing a hexa-His tag.
  • eAf1521 measurements were conducted with dilution series of only 3 concentrations (25 nM-6.25 nM).
  • 3 flow cells with immobilized eAf1521 at densities of 520 RU, 500 RU, and 390 RU, resp., were used in parallel.
  • WT Af1521 was measured under the conditions used for eAf1521.
  • the chip surface was regenerated by injection of 0.5 M EDTA for 5 min.
  • the chip was regenerated by a 5 min injection of 0.5 M EDTA in water.
  • Uncoated flow cell 1 of the sensor chip was used as a reference. Data were evaluated using Biacore software version 2.0.3. Sensorgrams were fitted using a 1 :1 kinetic model. Sensorgrams of Af1521 were additionally evaluated using a steady-state model. The equilibrium dissociation constant of WT Af1521 calculated for steady state (ss) conditions was 4 times larger, and an overestimation of the affinity in kinetic experiments is possible, presumably caused by rebinding as a common surface effect often observed in SPR measurements. The binding behavior of eAf1521 was governed by a 600 times slower dissociation and a 2 times faster association step compared to WT AH521. Calculation of the dissociation rate constant using KD (SS) resulted in a more reliable value (Tab. 1).
  • ARTD1 recombinant ARTD1 (10 pmol) was incubated in reaction buffer (RB; 50 mM Tris-HCI pH 7.4, 4 mM MgCh and 250 mM dithiothreitol (DTT)) with 100 mM NAD + and 200 nM of double-stranded annealed 40 bp long oligomer (5'-TGCGACAACGATGAGATTGCCACT ACTTGAACCAGTGCGG-3' (SEQ ID NO 010), 5’-CCGCACTGGTTCAAGTAGTGGCAATCTCATCGTTGTCGCA-3’ (SEQ ID 021)) for 15 min at 37 °C.
  • Recombinant ARTD8cat was incubated in RB buffer with either 100 pM NAD + or 200 nM [ 32 P] NAD + (Perkin Elmer) for 30 min at 37 °C. These reactions were stopped via the addition of SDS-buffer or by filtering through an lllustra MicroSpin G-50 column (GE Healthcare) according to the manufacturer’s protocol. De-modification assays were performed in RB buffer. For de-modification of ARTD1, the auto-modified recombinant proteins were incubated with 10 pmol PARG for 30 min at 37 °C.
  • ADPr-Peptide enrichments were carried out as previously described (Leutert etai. (2016) Cell Rep, 24, 1916-1929 e1915.) with the following protocol modifications. After reducing potentially PARylated peptides to MARylated peptides using the enzyme PARG, the affinity enrichment of ADP-ribosylated peptides was either performed using either WT Af1521 or eAf1521 for 2 hrs at 4 °C. Both enriched samples were then prepared for MS analysis as described previously (Martello et al. (2016) Nat Commun, 7, 12917.).
  • HCD-PP-EThcD ADPr product-dependent analysis
  • MS1 -based label-free quantification was performed by applying Progenesis Ql for Proteomics software (v. 3.0.6039.34628, Nonlinear Dynamics, Purham, NC) with default settings and the following exceptions.
  • Peptide ions were filtered for charges ranging from +2 - +5.
  • a maximum of the top 5 ranked MS/MS spectra per peptide ion were exported with the most intense 200 peaks per spectrum with activated charge-deconvolution and deisotoping option as a Mascot generic formatted file (MGF).
  • MS/MS spectra were searched with Mascot for each type of fragmentation (HCD and EThcD). Mascot searches were carried out as previously described (Leutert et al.
  • the MGFs were searched against the target-decoy UniProtKB human database (taxonomy 9606, canonical sequences and reviewed entries only, downloaded on 2019/07/06).
  • N-Terminal protein acetylation was set as a variable modification.
  • S, R, K, D, E and Y residues were set as variable ADPr acceptor sites.
  • the neutral losses from the ADPr 249.0862 Da, 347.0631 Da, and 583.0829 Da were scored in HCD fragment ion spectra (Gehrig et al., manuscript in preparation).
  • the Mascot search results were imported into Scaffold and filtered for protein and peptide FDR values of 2% and 1% respectively. When multiple precursors were observed for the same peptide, the values were summed up to obtain the total intensity level of the peptide.
  • MS and MS/MS spectra were converted to Mascot generic format (MGF) using Proteome Discoverer, v2.1 (Thermo Fisher Scientific, Bremen, Germany). Separate MGF files were created from the raw file for each type of fragmentation (HCD and EThcD) using a dedicated rule in the converter control (Barkow-Oelaborer et al. (2013) Source Code Biol Med, 8:3). . Mascot was used as described above and the Mascot search results were imported into Scaffold 4 software (version 4.8.4). Peptides were considered correctly identified when a Mascot score >15 and a Mascot delta score >5 were obtained. These settings ensured a FDR lower than 1% at the PSM level.
  • MGF Mascot generic format
  • ARTD1 was in vitro poly-ADP-ribosylated as described above. 25 pmol of automodified ARTD1 was incubated with 125 pmol of either WT Af 1521 , eAf1521 or GST in 1 ml binding buffer (1% BSA,
  • Sepharose 4B beads (GE Healthcare) were added and additionally incubated for 1 h at 4°C.
  • ARTD1 was added on 10 pL prewashed ProBondTM Nickel-Chelating Resin (Thermo Fisher
  • Untreated or treated HeLa cells were lysed with RIPA buffer (50 mM Tris-HCI pH 7.4, 400 mM NaCI, 1% NP-40, 0.1% Na-deoxycholate, 1x protease inhibitor cocktail (Roche), 10 mM PJ- 34), sonicated and centrifuged at 16,000 g for 10 min.
  • RIPA buffer 50 mM Tris-HCI pH 7.4, 400 mM NaCI, 1% NP-40, 0.1% Na-deoxycholate, 1x protease inhibitor cocktail (Roche), 10 mM PJ- 34
  • HeLa lysates or recombinant proteins were mixed with SDS-buffer, boiled at 95 °C for 5 min. After separation by SDS-PAGE, a wet-transfer onto PVDF membrane was performed. The membranes were blocked with 5% milk in TBS-T for 1 hr at room temperature. Primary antibodies were diluted in 5% milk in TBS-T and incubated at 4 °C overnight. After three washes with TBS-T for 5 min, the secondary antibody (in TBS-T) was incubated for 1 hr at RT and the membranes were additionally washed 3x with TBS-T. For dot blot analysis, proteins were vacuum blotted onto a nitrocellulose membrane that was further blocked in milk and stained with antibodies as described above.
  • the bands or dots were visualized using the Odyssey infrared imaging system (LICOR).
  • LICOR Odyssey infrared imaging system
  • the following primary and secondary antibodies were used for immunoblot and dotblot analyses: anti-tetra-His (1:1000, Qiagen), Fc-WT Af1521 (1:400, 500 ng/mL,), Fc- eAf1521 (1:400, 500 ng/mL), IRDye 800CW goat anti-rabbit IgG (1 :15,000, LI-COR, P/N 925- 32211), and IRDye 680RD goat anti-mouse IgG (1:15,000, LI-COR, P/N 925-68070).
  • Molecular weights are indicated by the PageRuler Plus Prestained Protein Ladder (Thermo Scientific).
  • HeLa cells were grown on glass coverslips. After treatment, HeLa cells were fixed with 4% PFA for 15 min at room temperature and permeabilized for 10 min at room temperature in PBS supplemented with 0.2% Triton-X100 (Sigma Aldrich). After blocking the cells with PBS supplemented with 10% of goat serum for 1 hr, the cells were incubated with the primary antibody (diluted in blocking solution) overnight at 4 °C. The cells were washed 2x with PBS and subsequently incubated with the secondary antibody (diluted in blocking solution) for 2 h at room temperature. After two 5 min washes with PBS, the cells were incubated with 0.1 pg/mL DAPI in PBS for 20 min at room temperature.
  • PBS primary antibody
  • secondary antibody diluted in blocking solution
  • TMAs Tumor Micro Arrays
  • TMA Human cancer tissue microarrays containing breast carcinomas, ovarian carcinomas, renal cell carcinomas, colorectal carcinomas (one punch of 0.6 mm diameter per sample) were constructed as described (Kononen et al. , Nat Med. 1998 Jul;4(7):844-7.). TMA sections (2.5 pm) were on glass slides. The samples were retrieved from the archives of the Department of Pathology and Molecular Pathology, University Hospital Zurich (Zurich, Switzerland) between the years 1993 to 2013. All patients were patients of the USZ. For each tumor, one representative tumor tissue block, with a minimum of 1 cm tumor diameter, was re-evaluated using hematoxylin and eosin-stained sections.
  • Tumor samples with necrosis and high content of inflammatory cells were excluded. Only those cases with representative tumor regions that contained at least 70% tumor cells were selected for the TMA construction. This study was approved by the local commission of ethics (BASEC-Nr_2016-00811). All tumors were reviewed by pathologists of the Department of Pathology and Molecular Pathology specialized in their field. Classification, grading and staging was performed according to current TNM and WHO classification. Tissue sections were pre-treated with Tris-EDTA-Borate Buffer at 100°C for 60 min (CC1 standard protocol, Ventana), incubated with ADPR antibody for 44 min at 37°C. Tumors were considered strong (+3), moderate (+2) or weak/negative (+1) for cytoplasmic ADP ribosylation in the tumor cells.
  • TMAs were stained in University Hospital Zurich (USZ) with Ultra Discovery Ventana machine (using the standard protocol) by using the anti-ADPR antibody.
  • eAf1521 was run on the Leica Bond machine.
  • the scoring of the immunohistochemistry (IHC) signal was based on the following parameters:
  • Tumor tissue micro-arrays containing formalin-fixed paraffin-embedded tissue blocks were cut and then stained in an Ultra Discovery device from Ventana.
  • the tissue was pre-treated using the CC1 standard protocol (60 min at 100°C in Tris-EDTA/borate buffer).
  • the polyclonal rabbit anti-ADPR antibody was used at 1 pg/ml (1:500 dilution). Incubation time was 30 min.
  • the staining was revealed using the UltraMap-Rabbit DAB kit.
  • the eAf1521-Fc was used at 0.8 pg/ml. Stained slides were scanned using a NanoZoomer (Hamamatsu Photonics, Shizuoka, Japan).
  • rabbits were immunized with a terminal peptide from histone H2B tail chemically modified at one amino acid side chain to carry an analogue of ADP-ribose. Similar syntheses were used in e.g. (Liu et al. Org Biomol Chem. 2019 Jun 5 ; 17(22) : 5460-5474 ; Liu et al. Angew Chem Int Ed Engl. 2018 Feb 5;57(6):1659- 1662; and van der Heden van Noort et al., J Am Chem Soc. 2010 Apr 14;132(14):5236-40.). The immune serum was then negatively purified on an affinity column carrying the unmodified peptide and subsequently positively purified on an affinity column carrying the modified peptide.

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Abstract

La présente invention concerne un procédé de diagnostic du cancer basé sur un signal d'ADP-Ribosylation dans un échantillon isolé de tissu du patient. La présente invention concerne en outre un procédé de détection de l'ADP-ribosylation dans une cellule et un polypeptide, qui se lie à l'ADP-ribosylation.
PCT/EP2021/060856 2020-04-24 2021-04-26 Adp-ribosylation utilisée en tant que marqueur pronostique dans le cancer WO2021214343A1 (fr)

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US20150355172A1 (en) * 2014-06-10 2015-12-10 The Board Of Regents Of The University Of Texas System Adp-ribose detection reagents
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