WO2014096386A1

WO2014096386A1 - Anti-hdac11 antibodies

Info

Publication number: WO2014096386A1
Application number: PCT/EP2013/077750
Authority: WO
Inventors: Marie Catherine SCHIER; Hedwig Deubzer; Olaf Witt
Original assignee: Deutsches Krebsforschungszentrum; Ruprecht-Karls-Universität Heidelberg
Priority date: 2012-12-20
Filing date: 2013-12-20
Publication date: 2014-06-26

Abstract

The present invention relates to an antibody specifically recognizing histone deacetylase 11 (HDAC11), wherein said antibody recognizes native HDAC11 or native and denatured HDAC11. The present invention also relates to a cell line producing said antibody. It further relates to methods for identifying a subject afflicted with cancer as being susceptible to treatment with an HDAC11 inhibitor, for identifying HDAC11 recruitment sites, for identifying a subject afflicted with cancer, for identifying an inhibitor of HDAC11 recruitment, and for identifying an HDAC11 binding compound, said methods making use of the antibody of the present invention. The invention further relates to the use of the antibody of the present invention in said methods and to devices and kits comprising the antibody of the present invention.

Description

anti-HDACll antibodies

The present invention relates to an antibody specifically recognizing histone deacetylase 11 (HDAC11), wherein said antibody recognizes native HDAC11 or native and denatured HDAC11. The present invention also relates to a cell line producing said antibody. It further relates to methods for identifying a subject afflicted with cancer as being susceptible to treatment with an HDAC 11 inhibitor, for identifying HDAC 11 recruitment sites, for identifying a subject afflicted with cancer, for identifying an inhibitor of HDAC 11 recruitment, and for identifying an HDAC 11 binding compound, said methods making use of the antibody of the present invention. The invention further relates to the use of the antibody of the present invention in said methods and to devices and kits comprising the antibody of the present invention.

Histone deacetylases (HDAC, EC number 3.5.1) are a group of hydrolases removing the acetyl group from an ε-Ν-acetyl lysine amino acid of a histone. Depending on sequence identity and domain organization, HDACs have been classified into class I (including HDAC1 - 3 and 8), class Ila (HDAC4, 5, 7, 9), class lib (HDAC 6 and 10), class III (including sirtuins) and class IV (HDAC11) (Dokmanovic et al, 2007, Mol Cancer Res October 5; 981-989). It has become clear over recent years that HDACs are not restricted to histones as substrates and that they have functions in several regulative pathways of the cell (Choudhary, C, Kumar, C, Gnad, F, Nielsen, ML, Rehman, M, Walther, TC et al, (2009) Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325: 834-40.). HDAC inhibitors have been traditionally used in psychiatry and neurology as mood stabilizers and anti-epileptics (e.g. valproic acid). More recently, they have been studied for their effects in neurodegenerative diseases, but also in a supportive manner in cancer therapy (Bolden, JE, Peart, MJ and Johnstone, RW, (2006) Anticancer activities of histone deacetylase inhibitors. Nat Rev Drug Discov 5 : 769-84.). Despite the fact that several non- histone substrates have been identified for the various HDAC, information is scarce as to which molecular pathways mediate the effects of HDAC inhibitor treatment in cancer; even less is known as to if all cancer patients or just certain cohorts would benefit from HDAC treatment (Witt, O, Deubzer, HE, Milde, T and Oehme, I, (2009) HDAC family: What are the cancer relevant targets? Cancer Lett 277: 8-21.). Moreover, it is unknown, how a potential cohort of patients who would benefit from treatment with a HDAC inhibitor could be identified (Witt, O, Deubzer, HE, Milde, T and Oehme, I, (2009) HDAC family: What are the cancer relevant targets? Cancer Lett 277: 8-21.). HDAC1, -2, and 8 have been suggested to play a role in reduction of multi-drug resistance, apoptosis, and differentiation of tumor cells (see Oehme 2009, Expert Opin Investig Drugs 18: 1605-1617; Witt 2009, Curr Pharm Des 15:436-447; Keshelva 2007, J Natl Cancer Inst 99: 1107-1119; Oehme 2009, Clin Cancer Res 15 : 91-99; Witt 2009, Cancer Lett 277: 8- 21). HDAC 10 has been merely reported to be involved in deacetylation and thioredoxin regulation (Lai 2010, J Biol Chem 285: 7187-7196; Lee 2010, Mol Cells 30: 107-112).

HDACl l is the most recently identified HDAC family member with, as yet, poorly understood function. The catalytic domain of HDACl l is located at the amino terminus, and contains residues conserved in class I and Ila/b HDACs (Gao L, Cueto MA, Asselbergs F, Atadja P. Cloning and functional characterization of HDACl l, a novel member of the human histone deacetylase family. J Biol Chem. 2002;277(28):25748- 25755). Yet, amino acid sequences are so dissimilar that HDACl l was not grouped into classes I or Ila/b, but designated as a separate novel class IV (Yang XJ, Seto E. The Rpd3/Hdal family of lysine deacetylases: from bacteria and yeast to mice and men. Nat Rev Mol Cell Biol. 2008;9(3):206-218). Phylogenetic analysis has shown that HDAC 11 is most closely related to HDACs 3 and 8 (de Ruijter AJ, van Gennip AH, Caron HN, Kemp S, van Kuilenburg AB. Histone deacetylases (HDACs): characterization of the classical HDAC family. Biochem J. 2003;370:737-749). HDACl l was shown to negatively regulate IL10 expression in human antigen-presenting cells (Villagra A, Cheng F, Wang HW, Suarez I, Glozak M, Maurin M, Nguyen D, Wright KL, Atadja PW, Bhalla K, Pinilla-Ibarz J, Seto E, Sotomayor EM. The histone deacetylase HDACl l regulates the expression of interleukin 10 and immune tolerance. Nat Immunol. 2009;10(1):92-100.) and to regulate OX40 ligand expression in Hodgkin lymphoma (Buglio D, Khaskhely NM, Voo KS, Martinez- Valdez H, Liu YJ, Younes A. HDACl l plays an essential role in regulating OX40 ligand expression in Hodgkin lymphoma. Blood 201 1;117(10):2910-2917. ). So far, HDACl l has not been detected in any of the multi-protein HDAC complexes (Gao L, Cueto MA, Asselbergs F, Atadja P. Cloning and functional characterization of HDAC11 , a novel member of the human histone deacetylase family. J Biol Chem. 2002;277(28):25748-25755), but was shown to interact with the Cdtl replication licensing factor (Glozak MA, Seto E. Acetylation/deacetylation modulates the stability of DNA replication licensing factor Cdtl . J Biol Chem. 2009;284: 11446-11453). Transfected into 293 cells, HDACl l co-immunoprecipitates with HDAC6, suggesting they share common binding partners or associate directly (Gao et al, loc. cit.). Antibodies are an indispensable tool for elucidating the role of HDACl l in different tissues. Anti-HDACl l antibodies available at present were found to give results with varying consistency in most of the assays tested. There is thus a need in the art for reliable anti-HDACl l antibodies. Specifically, antibodies recognizing the native form of HDAC 11 are required. Therefore, the technical problem underlying the present invention is to provide means and methods for complying with the aforementioned needs. Thus, the present invention relates to an antibody specifically recognizing histone deacetylase 11 (HDACl l), wherein said antibody recognizes native HDAC 11 or native and denatured HDAC 11. As used herein, the term "histone deacetylase" or "HDAC" relates to a member of the family of enzymes removing acetyl groups from an ε-Ν-acetyl lysine amino acid on a histone (EC 3.5.1) and, preferably, thereby controlling gene expression. The term "histone deacetylase 11" or "HDAC 11" relates to members of the family of HDAC enzymes similar to the polypeptide encoded by the human HDACl l gene (human gene: SEQ ID NO: 1, Genbank Acc No : NC 000003.11 GI :224589815 ; transcript variant 1 of the human gene : SEQ ID NO:2, NM_024827.3 GL227430412; transcript variant 2 of the human gene: SEQ ID NO:3, NM 001136041.2 GL227430411) classified as the hitherto sole members of class IV of histone deacetylases. Preferably, HDACl l relates to the human HDACl l isoform 1 and / or isoform 2 polypeptides (isoform 1 : SEQ ID NO: 4, Genbank Acc No: NP_079103.2 GL217272877; isoform 2: SEQ ID NO:5, Genbank Acc NO: NP 001129513.1 GL209862883). It is understood by the skilled person that HDACl l binds to proteins other than histones in order to fulfill its function, e.g. HDAC6 or proteins recruiting HDAC 11 to its recruitment sites. The terms "specifically recognizing", as used herein, relates to binding of an antibody specific to HDACl l . Specific recognition, preferably, requires binding to HDACl l with an affinity at least 5fold, at least lOfold, at least 25fold, at least 50fold higher, or at least lOOfold higher than for polypeptides or peptides which are not variants or peptide fragments of HDAC11. More preferably, specifically recognizing relates to a lack of cross reaction with other polypeptides or peptides which are not variants or peptide fragments of HDACl l, i.e. to binding to any of said other polypeptides or peptides with neglegible affinity. Specific binding can be tested by various well known techniques. The term "native" HDAC11, as used herein, relates to a non-denatured HDAC11 molecule. Preferably, the native HDACl l has the conformation, preferably tertiary structure, assumed by HDAC11 in the normal living cell, as can be isolated by the methods described herein below. More preferably, the native HDAC 11 is the polypeptide having at least one enzymatic or binding activity of HDACl l, most preferably having all measurable enzymatic and binding activities of HDACl l . It is understood by the skilled artisan that the binding activity of HDACl l does not necessarily relate to DNA binding but may, preferably, also relate to binding of other interaction partners, e.g. polypeptides or other molecules, said other interaction partners themselves binding to DNA. The term "denatured" HDACl l, as used herein, relates to an HDACl l molecule wherein the tertiary structure has been caused at least partially to unfold, e.g. by treatment with one of heat, detergent, alkali, and acid. Preferably, in the the denatured HDACl l some of its original properties, preferably its biological activity, i.e. enzymatic and / or binding activities, are diminished or eliminated. More preferably, in the the denatured HDAC 11 all of its biological activities, i.e. enzymatic and / or binding activities, are diminished. Most preferably, in the denatured HDAC 11 all of its biological activities, i.e. enzymatic and / or binding activities, are eliminated. Preferably, in the denatured HDACl l the amino acid chain of HDACl l is essentially linear as is e.g. induced by heating to more than 90°C in the presence of at least 0.1% sodium dodecylsulfate as described herein below.

The term "antibody" as used in this specification refers to a molecule from the subgroup of gamma globulin proteins which is also referred to as the immunoglobulins (Ig). Antibodies can, preferably, be of any subtype, i.e. IgA, IgD, IgE, IgM or, more preferably, IgG. Antibodies against polypeptides encoded by target genes of the invention can be prepared by well known methods using a purified polypeptide or a suitable fragment derived therefrom as an antigen. A fragment which is suitable as an antigen may be identified by antigenicity determining algorithms well known in the art. Such fragments may be obtained either by proteolytic digestion from polypeptides encoded by target genes or may be synthetic peptides. Preferably, the antibody of the present invention is a monoclonal antibody, a polyclonal antibody, a single chain antibody, a human or humanized antibody or primatized, chimerized or fragment thereof. Also comprised as antibodies by the present invention are a bispecific antibody, a synthetic antibody, an antibody fragment, such as Fab, Fv or scFv fragments etc., or a chemically modified derivative of any of these. Antibodies or fragments thereof can be obtained by using methods which are described, e.g., in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. Preferably, the antibody of the present invention is a monoclonal antibody, which can, e.g. be prepared by the techniques originally described in Kohler and Milstein, Nature 256 (1975), 495, and Galfre, Meth. Enzymol. 73 (1981), 3, which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals. More preferably, the antibody of the present invention is an antibody recognizing an epitope comprising an amino acid sequence as depicted in Fig. 2a) (SEQ ID NO:6), Fig. 2b) (SEQ ID NO:7), Fig. 2c) (SEQ ID NO:8), or Fig. 2d) (SE ID NO:9); or the antibody of the present invention is an antibody recognizing a discontinuous and/or conformational epitope of HDAC11. Most preferably, the antibody of the present invention is an antibody produced by one of the cell lines deposited at the Leibnitz-Institut DSMZ-Deutsche Sammlung fur Mikroorganismen und Zellkulturen GmbH (DSMZ) under No DSM ACC3170 and No DSM ACC3171. The antibody of the present invention recognizes native HDAC11. It is, however, also envisaged by the present invention that the antibody recognizes native and denatured HDAC11. In a preferred embodiment, the antibody of the present invention recognizes HDAC11 in immune cytochemistry. Preferably, the antibody of the present invention recognizes HDAC11 also in immune histochemistry. The term "cytochemistry" relates to the biochemistry of cells, especially to the macromolecules determining cell structure and function. The term "immune cytochemistry" or "immunocytochemistry" relates to determining the presence and / or the amount of at least one macromolecule in a cell of interest by immunological means, e.g. by use of an antibody specific for said macromolecule. The term "immune histochemistry" or immunohistochemistry" refers to detecting a macromolecule (e.g., proteins) in cells of a tissue section by immunological means, e.g. by use of an antibody specific for said macromolecule.

In a further preferred embodiment, the antibody of the present invention recognizes HDAC11 in chromatin immunoprecipitation. The term "chromatin immunoprecipitation" or "ChIP" is understood by the skilled artisan and relates to a method wherein a protein of interest and associated sheared chromatin are bound by an agent specific for said protein, preferably an antibody, and by this means isolated, allowing a detection of polynucleotides associated with said protein of interest. More preferably, the ChIP is native ChIP, wherein native chromatin sheared machanically and / or digested by micrococcal nuclease is used. It is understood by the skilled artisan that the protein of interest is required to be native in native ChIP, since at least binding of the protein of interest to said polynucleotide or to a protein mediating its recruitment is required for the method to work. The definitions made above apply mutatis mutandis to the following:

The present invention also relates to a cell line producing the monoclonal antibody of the present invention. As used herein, the term "cell line" relates to cells, preferably mammalian cells, more preferably mouse or rat cells, grown in tissue culture and consisting of, preferably, clonal descendants of a single cell. Preferably, the cell line is a hybridoma cell line, more preferably a mouse hybridoma cell line, even more preferably a mouse hybridoma cell line prepared by fusion of mouse cell line Sp 2/0-AG14 and mouse lymphocytes, most preferably a hybridoma cell line deposited at the Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ) under No DSM ACC3170 and No DSM ACC3171. Preferably, the hybridoma cell line is obtained from lymphocytes obtained from a mammal, preferably a non- human mammal, immunized with HDAC 11 , more preferably immunized with a polypeptide having an amino acid as depicted in Fig. 1 (SEQ ID NO:4). Preferably, the cell line produces an antibody of the present invention. More preferably, the cell line secretes an antibody of the present invention into the culture medium at a concentration of at least 25 μg/ml, at least 50 μg/ml, at least 75 μg/ml, at least 100 μg/ml, at least 125 μg/ml, or at least 150 μg/ml. The present invention further relates to a method for identifying a subject afflicted with cancer as being susceptible to treatment with an HDAC11 inhibitor, comprising (a) contacting a sample from said subject with the antibody of the present invention, (b) determining the amount of HD AC 11 /antibody complexes formed in the mixture of (a), and (c) comparing said amount with a reference amount, thereby identifying a subject afflicted with cancer as being susceptible to treatment with an HDAC11 inhibitor.

The method for identifying a subject afflicted with cancer as being susceptible to treatment with an HDAC11 inhibitor, preferably, is an in vitro method. Moreover, it may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate to cell and/or compound pre-treatments or evaluation of the results obtained by the method. The method may be carried out manually or assisted by automation. Preferably, steps a) and/or b) and/or c) or parts thereof may in total or in part be assisted by automation, e.g., by a suitable robotic equipment for the determining the amount of HDAC11/antibody complexes in step b), or a computer-implemented calculation or comparison step in step c). Preferably, the method for identifying a subject susceptible to treatment with an HDAC11 inhibitor is comprised in a method for initiating treatment in a subject afflicted with cancer with an HDAC11 inhibitor, comprising the further step of initiating treatment with a pharmaceutical composition comprising at least one HDAC11 inhibitor in case said subject afflicted with cancer was identified as being susceptible to treatment with an HD AC 11 inhibitor. Also preferably, the method for identifying a subject susceptible to treatment with an HDAC11 inhibitor is comprised in a method of treament of a subject afflicted with cancer with an HDAC11 inhibitor, comprising the further step of initiating treatment with a pharmaceutical composition comprising at least one HDAC11 inhibitor in case said subject afflicted with cancer was identified as being susceptible to treatment with an HD AC 11 inhibitor.

The term "identifying" as used herein refers to assessing the probability according to which a subject afflicted with cancer is susceptible to treatment with an HDAC11 inhibitor. As will be understood by those skilled in the art, such an assessment is usually not intended to be correct for 100% of the subjects to be identified. The term, however, requires that a statistically significant portion of subjects afflicted with cancer can be correctly identified to be susceptible to treatment with an HDAC 11 inhibitor. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student^'s t-test, Mann- Whitney test etc.. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99 %. The p-values are, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the probability envisaged by the present invention allows that the identification will be correct for at least 60%, at least 70%, at least 80%, or at least 90% of the subjects of a given cohort or population. "Cancer" in the context of this invention refers to a disease of an animal, including man, characterized by uncontrolled growth by a group of body cells ("cancer cells"). This uncontrolled growth may be accompanied by intrusion into and destruction of surrounding tissue and possibly spread of cancer cells to other locations in the body (metastasis). Preferably, the cancer is selected from the list consisting of acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, aids-related lymphoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid, basal cell carcinoma, bile duct cancer, bladder cancer, brain stem glioma, breast cancer, burkitt lymphoma, carcinoid tumor, cerebellar astrocytoma, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngioma, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal stromal tumor, gestational trophoblastic tumor, hairy cell leukemia, head and neck cancer, hepatocellular cancer, hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, kaposi sarcoma, laryngeal cancer, medulloblastoma, medulloepithelioma, melanoma, merkel cell carcinoma, mesothelioma, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, papillomatosis, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sezary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, testicular cancer, throat cancer, thymic carcinoma, thymoma, thyroid cancer, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macro globulinemia, and wilms tumor. More preferably, the cancer is a neuroblastoma.

The term "subject afflicted with cancer" as referred to herein encompasses animals, preferably mammals, and, more preferably, humans. Preferably, said subject is afflicted with a cancer as specified herein above. Subjects afflicted with cancer can be identified by the accompanying symptoms known for the disease. These symptoms are known in the art.

The term "HDACl l inhibitor" encompasses compounds which are capable of specifically inhibiting at least one biological activity of HDACl l . Biological activities of HDACl l which are preferably inhibited by the inhibitor according to the invention are specified herein above. The inhibitor may directly or indirectly inhibit the at least one biological activity of HDACl l . Also included are compounds developing inhibitory activity after transformation in or by the cell, like, e.g. prodrugs or polynucleotides encoding inhibitory polypeptides. A direct inhibition is achieved by a compound which binds to HDACl l and thereby inhibits the said biological activity. Compounds which directly inhibit HDACl l in this sense are, preferably, compounds which block the interaction of HDACl l with other proteins or with its ligands. Alternatively, but nevertheless preferred, a direct inhibitor of HDACl l may induce an allosteric change in the conformation of the HDACl l polypeptide. The allosteric change may subsequently block the interaction of HDACl l with other proteins or with its ligands and, thus, interfere with the biological activity of HDACl l . Compounds which are suitable as direct inhibitors of HDACl l encompass antibodies, aptameres, soluble mutants or variants of the HDACl l polypeptide, and a small molecule antagonist. Small molecule antagonists of HDACl l activity are known to the skilled artisan and are selected from hydroxamic acids (e.g. Trichostatin A), cyclic peptides (such as Trapoxin B, HC toxin) and depsipeptides, benzamides (e.g. entinostat, mocetinostat (MGCD0103; Fournel M, Bonfils C, Hou Y, Yan PT, Trachy-Bourget MC, Kalita A, Liu J, Lu AH, Zhou NZ, Robert MF, Gillespie J, Wang JJ, Ste-Croix H, Rahil J, Lefebvre S, Moradei O, Delorme D, Macleod AR, Besterman JM, Li Z. MGCD0103, a novel isotype-selective histone deacetylase inhibitor, has broad spectrum antitumor activity in vitro and in vivo. Mol Cancer Ther. 2008; 7(4):759-68)), or aliphatic acids (such as phenylbutyrate or valproic acid). The small molecule antagonists of HDACl l known at present are inhibitors of the deacetylase activity of HDACl l . It is known to the skilled person that the known inhobotors of HDACl l are not specific for HDACl l, though molecular modeling has been proposed for the development of iso form-selective HDAC inhibitors(Sundarapandian et al., Molecular Modeling Study on Tunnel Behavior in Different Histone Deacetylase Isoforms. PLoS One. 2012;7(1 l):e49327).. The term "treatment with an HDACl l inhibitor" relates to a treatment comprising the application of at least one HDACl l inhibitor to a subject. It is to be understood that the term "treatment with an HDAC 11 inhibitor" may relate to the treament with an HDAC 11 inhibitor alone or in combination with other one or more pharmaceutically active compounds, i.e. the treament preferably is an HDACl l inhibitor single therapy, or, more preferably, a co-treatment comprising the application of at least one second compound, said second compound, most preferably, not being an HDAC 11 inhibitor.

As used herein, a "subject afflicted with cancer being susceptible to treatment with an HDACl l inhibitor" is a subject wherein treatment with an HDACl l inhibitor, preferably in monotherapy or in combination with, e.g. chemotherapy, leads to an amelioration of the diseases or disorders referred to herein or the symptoms accompanied therewith to a significant extent. Whether an amelioration is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools described herein above. Said amelioration as used herein also includes an entire restoration of the health with respect to the diseases or disorders referred to herein.

The term "contacting" as used in the context of the methods of the present invention is understood by the skilled person. Preferably, the term relates to bringing an antibody of the present invention in physical contact with a sample and thereby, e.g. allowing the sample and the composition to interact. Contacting may further involve lysing cells present in the sample and/or the extraction or purification of DNA and / or R A and / or proteins.

The term "sample" refers to a sample of a body fluid, to a sample of separated cells or to a sample from a tissue or an organ or to a sample of wash/rinse fluid obtained from an outer or inner body surface. Samples can be obtained by well known techniques and include, preferably, scrapes, swabs or biopsies. Samples can be obtained by use of brushes, (cotton) swabs, spatula, rinse/wash fluids, punch biopsy devices, puncture of cavities with needles or surgical instrumentation. Tissue, organ or tumor samples may be obtained from any tissue or organ by, e.g., biopsy or other surgical procedures. Preferably, a sample is a tissue or a tumor obtained from brain, bone, sceletal or smooth muscle, the digestive tract, oropharynx, tongue, oesophagus, stomach, duodenum, ileum, colon, rectum, lung, liver, spleen, kidney, bladder, skin, a lymph node, ovary, oviduct, uterus, vagina, prostate, or testis. More preferably, the sample is a sample from a brain tumor, most preferably a neuroblastoma. Separated cells may be obtained from the body fluids or the tissues or organs by separating techniques such as filtration, centrifugation or cell sorting. Preferably, cell, tissue or organ samples are obtained from those cells, tissues or organs, which are known or suspected to contain HDAC 1 1. Preferably, the sample is known or suspected to be comprising HDACl 1.

The term "HDACl 1/antibody complex" is understood by the skilled person and relates to a complex formed between at least one molecule of the HDAC 1 1 polypeptide and at least one molecule of the antibody of the present invention. Preferably, the HDACl 1/antibody complex is a complex between HDAC 1 1 and an antibody according to the present invention recognizing native, but not denatured HDAC 1 1 , meaning, preferably, that the amount of said native HDACl 1/antibody complex is a measure for HDACl 1 activity in a sample.

The term "determining" relates to the quantification of the amount of HDACl 1/antibody complex present in a sample, i.e. measuring the amount or concentration of said HDACl 1 - antibody complex, preferably semi-quantitatively or quantitatively. Measuring can be done directly or indirectly. The determining of the amount of immune complexes can be accomplished in a variety of ways known to the skilled person, e.g. gel filtration chromatography followed by western blotting, co-immunoprecipitation, ELISA, or the like. In accordance with the present invention, determining the amount of the HDACl 1 - antibody complexes can be achieved by all known means for determining the amount of a polypeptide or peptide in a sample, provided that they are adapted to specifically detect the HDACl 1 -antibody complexes of the present invention. Preferably, detection agents are to be used which specifically bind to and, thus, allow for the detection of the HDACl 1 - antibody complexes. Detection agents, preferably, encompass antibodies or fragments thereof that specifically bind to the complexes, aptameres, anticalins, or Designed Ankyrin Repeat Proteins (DARPins) that specifically bind to the complexes. Preferably, double- specificity immunoassays are applied, i.e. assays wherein the presence or the intensity of a signal will depend on the presence of both kinds of molecules comprised in the HDACl 1- antibody complexes, i.e. the HDACl 1 polypeptide and the anti-HDACl 1 immunoglobulin. Said means comprise immunoassay devices and methods which may utilize labeled molecules in various sandwich, competition, or other assay formats. Said assays will develop a signal which is indicative of the presence or absence of the HDACl 1 -antibody complexes. Moreover, the signal strength can, preferably, be correlated directly or indirectly (e.g. reverse-proportional) to the amount of HDACl 1 -antibody complexes present in a sample. Said methods comprise, preferably, biosensors, optical devices coupled to immunoassays, biochips, analytical devices such as mass-spectrometers, NMR- analyzers, or chromatography devices. Further, methods include micro-plate ELISA-based methods, fully-automated or robotic immunoassays (available for example on multi parameter biochip platforms), CBA (an enzymatic Cobalt Binding Assay), and latex agglutination assays.

The term "amount" as used herein encompasses the absolute amount of the HDACl 1 /antibody complexes referred to herein, the relative amount or concentration of the HDACl 1 /antibody complexes referred to herein as well as any value or parameter which correlates thereto. Such values or parameters comprise intensity signal values from all specific physical or chemical properties obtained from the HDACl 1 /antibody complexes referred to herein by measurements, e.g., expression levels determined from biological read out systems in response to the polypeptides referred to herein or intensity signals obtained from specifically bound ligands. It is to be understood that values correlating to the aforementioned amounts or parameters can also be obtained by all standard mathematical operations. "Comparing" as used herein encompasses comparing the amount of the HDACl 1/antibody complexes referred to herein which are comprised by the sample to be analyzed with an amount of the said HDACl 1/antibody complexes in a suitable reference sample as specified elsewhere herein. It is to be understood that comparing as used herein refers to a comparison of corresponding parameters or values, e.g., an absolute amount of the HDAC 11 /antibody complexes as referred to herein is compared to an absolute reference amount of said HDAC 11 /antibody complexes; a concentration of the HDAC 11 /antibody complexes as referred to herein is compared to a reference concentration of said HDAC 11 /antibody complexes; an intensity signal obtained from the HDAC 11 /antibody complexes as referred to herein in a test sample is compared to the same type of intensity signal of said HDAC 11 /antibody complexes in a reference sample. The comparison referred to in the methods of the present invention may be carried out manually or computer assisted. For a computer assisted comparison, the value of the determined amount or ratio may be compared to values corresponding to suitable reference values which are stored in a database by a computer program. The computer program may further evaluate the result of the comparison by means of an expert system. Accordingly, the result of the identification referred to herein may be automatically provided in a suitable output format. The terms "reference value" or "reference amount" as used herein refer to an amount of HDAC 11 /antibody complexes, which allows assessing if being susceptible to HDAC 11 inhibitor treatment or not being susceptible to HDAC 11 inhibitor treatment is to be assumed for the subject from which the sample is derived. A suitable reference value may be determined from a reference sample to be analyzed together, i.e. simultaneously or subsequently, with the sample.

Reference amounts can, in principle, be calculated for a group or cohort of subjects as specified herein based on the average or mean values for a given HDAC 11 /antibody complex by applying standard methods of statistics. In particular, accuracy of a test such as a method aiming to diagnose an event, or not, is best described by its receiver-operating characteristics (ROC) (see especially Zweig 1993, Clin. Chem. 39:561-577). The ROC graph is a plot of all of the sensitivity versus specificity pairs resulting from continuously varying the decision threshold over the entire range of data observed. The clinical performance of a diagnostic method depends on its accuracy, i.e. its ability to correctly allocate subjects to a certain prognosis or diagnosis. The ROC plot indicates the overlap between the two distributions by plotting the sensitivity versus 1 -specificity for the complete range of thresholds suitable for making a distinction. On the y-axis is sensitivity, or the true-positive fraction, which is defined as the ratio of number of true-positive test results to the product of number of true-positive and number of false-negative test results. This has also been referred to as positivity in the presence of a disease or condition. It is calculated solely from the affected subgroup. On the x-axis is the false-positive fraction, or 1 -specificity, which is defined as the ratio of number of false-positive results to the product of number of true-negative and number of false-positive results. It is an index of specificity and is calculated entirely from the unaffected subgroup. Because the true- and false- positive fractions are calculated entirely separately, by using the test results from two different subgroups, the ROC plot is independent of the prevalence of the event in the cohort. Each point on the ROC plot represents a sensitivity/-specificity pair corresponding to a particular decision threshold. A test with perfect discrimination (no overlap in the two distributions of results) has an ROC plot that passes through the upper left corner, where the true-positive fraction is 1.0, or 100% (perfect sensitivity), and the false-positive fraction is 0 (perfect specificity). The theoretical plot for a test with no discrimination (identical distributions of results for the two groups) is a 45° diagonal line from the lower left corner to the upper right corner. Most plots fall in between these two extremes. If the ROC plot falls completely below the 45° diagonal, this is easily remedied by reversing the criterion for "positivity" from "greater than" to "less than" or vice versa. Qualitatively, the closer the plot is to the upper left corner, the higher the overall accuracy of the test. Dependent on a desired confidence interval, a threshold can be derived from the ROC curve allowing for the diagnosis or prediction for a given event with a proper balance of sensitivity and specificity, respectively. Accordingly, the reference to be used for the methods of the present invention can be generated, preferably, by establishing a ROC for said cohort as described above and deriving a threshold amount there from. Dependent on a desired sensitivity and specificity for a diagnostic method, the ROC plot allows deriving suitable thresholds.

Preferably, the reference amount as used herein is derived from samples for which it is known if their donors responded to treatment or not. This reference amount level may be a discrete figure or may be a range of figures. Evidently, the reference level or amount may vary between individual species of HD AC 11 /antibody complexes. The measuring system therefore, preferably, is calibrated with a sample or with a series of samples comprising known amounts of HD AC 11 /antibody complex or HDAC11 /antibody complexes. It is understood by the skilled person that in such case the amount of HDAC11 /antibody complex will preferably be expressed as arbitrary units (AU). Thus, preferably, the amounts of HDAC11 /antibody complex or HDAC11 /antibody complexes is or are determined by comparing the signal obtained from the sample to signals comprised in a calibration curve.

The reference amount applicable for an individual subject may vary depending on various physiological parameters such as age, gender, or subpopulation. Thus, a suitable reference amount may be determined by the methods of the present invention from a reference sample to be analyzed together, i.e. simultaneously or subsequently, with the test sample. Moreover, a threshold amount can be preferably used as a reference amount. Preferably, an amount of HDACl 1 /antibody complexes which is above the threshold amount is indicative of a subject susceptible to treatment with an HDAC11 inhibitor; and an amount of HDAC 11 /antibody complexes which is equal or below the threshold amount will be indicative for a subject not susceptible to treatment with an HDAC 11 inhibitor. It is to be understood that the aforementioned amounts may vary due to statistics and errors of measurement. Preferably, the threshold amount is equal to the detection limit of the method applied to detect the HDAC 11 /antibody complexes, i.e. a detectable amount of HDAC 11 /antibody complexes is indicative of a subject susceptible to treatment with an HDAC 11 inhibitor, and an amount of HDACl 1/antibody complexes below the detection limit is indicative of a subject not susceptible to treatment with an HDACl 1 inhibitor. The present invention also relates to a method for identifying HDAC 11 recruitment sites, comprising (a) contacting a mixture comprising HDAC 11 and polynucleotides with an antibody of the present invention, and (b) detecting polynucleotides specifically bound to HDACl 1/antibody complexes formed in the mixture of (a), thereby identifying HDAC 11 recruiting sites.

The present invention also relates to a method for identifying a subject afflicted with cancer, comprising (a) contacting a sample from said subject comprising HDAC 11 and polynucleotides with the antibody of the present invention, and (b) detecting polynucleotides specifically bound to HDACl 1/antibody complexes formed in the mixture of (a), and (c) comparing the polynucleotides specifically bound to HDACl 1/antibody complexes to a reference, thereby identifying a subject afflicted with cancer.

The method for identifying HDAC 11 recruitment sites and the method for identifying a subject afflicted with cancer, preferably, are in vitro methods. Moreover, they may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate to cell and/or sample pre-treatments or evaluation of the results obtained by the method. The method may be carried out manually or, preferably, assisted by automation. Preferably, all steps or parts thereof may in total or in part be assisted by automation, e.g., by a suitable robotic equipment for detecting polynucleotides. Preferably, the method for identifying a subject afflicted with cancer is comprised in a method for initiating treatment in a subject afflicted with cancer, comprising the further step of initiating treatment in case said subject was identified as being afflicted with cancer. Also preferably, the method for identifying a subject afflicted with cancer is comprised in a method of treament of a subject afflicted with cancer, comprising the further step of initiating treatment in case said subject was identified as being afflicted with cancer.

As used herein, the term "HDAC11 recruitment site" relates to a section of a genome bound by HDACl 1. It is to be understood that binding by HDAC11 does not necessarily relate to direct binding between HDAC11 and DNA. Preferably, binding relates to any kind of interaction leading to an accumulation of HDACl 1 in the vincinity of a section of the genome. Thus, indirect binding of HDACl 1 mediated by one or more macromolecules of the cell, e.g. macromolecular HDAC11 binding compounds as specified herein below, e.g. HDACl 1 binding proteins, is also encompassed by the term binding. Given the role of HDAC11 in transcription activation and/ or repression, it is to be understood that the pattern of HDACl 1 recruitment sites is, preferably, dependent on the kind and state of cell or cells analyzed. E.g., preferably, in a tumor cell HDACl 1 may be recruited to at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 15, at least 20 HDAC11 recruitment sites not used by a normal cell. Also preferably, in a tumor cell HDACl 1 is not recruited to at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 15, at least 20 HDACl 1 recruitment sites used by a normal cell. I.e., preferably, tumor cells and normal cell may have differential patterns of HDACl 1 recruitment sites.

As used herein, the term "mixture" relates to a composition comprising at least the compounds described. It is, however, clear from the above that the mixture preferably comprises macromolecules, more preferably polypeptides, in addition to HDAC11 and polynucleotides. Most preferably, the mixture comprises chromatin, in which case the polynucleotides can be comprised in the chromatin. The skilled artisan knows that the mixture may also comprise low-molecular weight substances mediating stabilization of polypeptides and / or their interactions and providing an environment suitable for the macro molecules to interact, like salts, buffer compounds, and the like.

The term "detecting polynucleotides specifically bound to HDACl l/antibody complexes", as used herein, relates to identifying polynucleotide molecules, preferably by their nucleotide sequences, bound to HDACl l/antibody complexes formed in the mixture in step a) in a specific manner, specific binding relating to binding under conditions under which the binding affinity of polynucleotides not comprising HDAC 11 recruitment sites is very low or neglegible. The skilled person knows conditions permitting specific binding, e.g. Lee TI, Johnstone SE, Young RA. Chromatin immunoprecipitation and microarray- based analysis of protein location. Nat Protoc. 2006;l(2):729-48; Schmidt D, Wilson MD, Spyrou C, Brown GD, Hadfield J, Odom DT. ChlP-seq: using high-throughput sequencing to discover protein-DNA interactions. Methods. 2009;48(3):240-8 or the conditions provided in the Examples below. Methods of detecting polynucleotides are well known in the art and include, e.g. cloning followed by sequencing after ChIP, ChlP-PCR (direct sequencing after PCR amplification), ChlP-Seq (direct sequencing after chromatin immunoprecipitation); ChlP-ChIP (hybridization to microarrays after chromatin immunoprecipitation); ChlP-qRT-PCR (quantitative realt-time PCR after chromatin immunoprecipitation), see, e.g. Kaufmann K, Muino JM, 0steras M, Farinelli L, Krajewski P, Angenent GC. Chromatin immunoprecipitation (ChIP) of plant transcription factors followed by sequencing (ChlP-SEQ) or hybridization to whole genome arrays (ChlP- CHIP). Nat Protoc. 2010; 5(3):457-72.

The sample of the method for identifying a subject afflicted with cancer, preferably, is a sample comprising, more preferably essentially consisting of, tumor cells. Preferably, the sample is a tumor biopsy. The term "reference", as used in conjunction with the method for identifying a subject afflicted with cancer, relates to a compilation of polynucleotide species, preferably polynucleotide sequences, being HDAC 11 recruitment sites in normal cells. In such case, detecting only the polynucleotides comprised in said compilation in a sample from a subject is indicative of a subject not afflicted with cancer, whereas detecting at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 15, at least 20 polynucleotides not comprised in said compilation is indicative of a subject afflicted with cancer. It is, however, also envisaged by the method for identifying a subject afflicted with cancer that the reference is a compilation of polynucleotide species, preferably polynucleotide sequences, being HDAC11 recruitment sites in cells known to be tumor cells, but not in normal cells. In such case, detecting at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 15, at least 20 polynucleotides comprised in said compilation in a sample from a subject is indicative of a subject afflicted with cancer, whereas not detecting a significant number of polynucleotides comprised in said compilation is indicative of a subject not afflicted with cancer.

The present invention also relates to a method for identifying an inhibitor of HD AC 11 recruitment, comprising (a) contacting a mixture comprising HDAC 11 and polynucleotides with a compound suspected to be an inhibitor of HDAC 11 recruitment, (b) contacting the mixture of (a) with the antibody of any one of claims 3 to 5, and (c) determining the polynucleotide content of HDAC 11 /antibody complexes formed in the mixture of (a), thereby identifying an inhibitor of HDAC 11 recruitment.

The method for identifying an inhibitor of HDAC 11 recruitment, preferably, is an in vitro method. Moreover, the method may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate to cell and/or compound pre- treatments or evaluation of the results obtained by the method. The method may be carried out manually or, preferably, assisted by automation. Preferably, all steps or parts thereof may in total or in part be assisted by automation, e.g., by a suitable robotic equipment for determining the polynucleotide content of HDAC 11 /antibody complexes.

As used herein, an "inhibitor of HDAC 11 recruitment" is an HDAC 11 inhibitor as described herein above inhibiting the interaction of HDAC 11 with chromatin and / or chromatin components, or with other compounds, preferably polypeptides, said compounds mediating accumulation of HDAC 11 in the vincinity of a section of the genome. It is thus clear that small molecule HDAC 11 inhibitors specifically inhibiting the deacetylase activity of HDAC 11 described herein above are not inhibitors of HDAC 11 recruitment. It is also clear for the skilled person that an antibody of the present invention may be, but need not necessarily be, an inhibitor of HDACl 1 recruitment, and that the antibodies being inhibitors of HDACl 1 recruitment are identified by the method for identifying an inhibitor of HD AC 11 recruitment.

The "compound suspected to be an inhibitor of HDAC11 recruitment" of the present invention can be of any molecular species, e.g. a small molecule compound, a polypeptide or protein, a poly- or oligonucleotide, an antibody, and the like. Preferably, the compound is able to penetrate a cell either by itself or in combination with other chemical or biological agents, such as penetratins, lipofection or other transfection reagents, recombinant viruses or bacteria. Preferably, the compound is a polypeptide, more preferably a protein, most preferably an antibody. Also included are compounds developing inhibitory activity after transformation in or by the cell, like, e.g. prodrugs or polynucleotides encoding inhibitory polypeptides. The term "suspected to be an inhibitor of HDACl 1 recruitment" relates to the absence of knowledge to the contrary, i.e., preferably, any substance not known to be ineffective in inhibiting HDAC11 recruitment can be a compound suspected to be an inhibitor of HDACl 1 recruitment.

The skilled artisan knows that identifying a specific compound as an inhibitor of HDACl 1 recruitment according to the method of the present invention can be accomplished by determining that the HDACl 1/antibody complexes formed in the presence of said compound do not comprise significant amounts of polynucleotides. It is, however, also envisaged by the method for identifying an inhibitor of HDACl 1 recruitment, that the polynucleotide content of the HDACl 1/antibody complexes formed in the presence of said compound is compared to the polynucleotide content of the HDACl 1/antibody complexes formed in the absence of said compound, wherein a decreased polynucleotide content of the HDACl 1/antibody complexes formed in the presence of said compound being indicative of said compound being an inhibitor of HDACl 1 recruitment. The present invention also relates to a method for identifying an HDAC11 binding compound, comprising (a) providing a mixture comprising HDAC11 and a compound suspected to be an HDACl 1 binding compound, (b) contacting the mixture of (a) with the antibody of the present invention, and (c) determining the amount of compound suspected to be an HDACl l binding compound in HDAC11 /antibody complexes formed in the mixture of (a), thereby identifying an HDAC11 binding compound.

The method for identifying an HDACl l binding compound, preferably, is an in vitro method. Moreover, the method may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate to cell and/or compound pre- treatments or evaluation of the results obtained by the method. The method may be carried out manually or, preferably, assisted by automation. Preferably, all steps or parts thereof may in total or in part be assisted by automation, e.g., by a suitable robotic equipment for determining the amount of compound suspected to be an HDAC 11 binding compound.

The "HDACl l binding compound" of the present invention can be of any molecular species, e.g. a small molecule compound, a polypeptide or protein, a poly- or oligonucleotide, an antibody, and the like. Preferably, the compound is able to penetrate a cell either by itself or in combination with other chemical or biological agents, such as penetratins, lipofection or other transfection reagents, recombinant viruses or bacteria. Preferably, the compound is a polypeptide, more preferably a protein. The term "suspected to be an HDAC 11 binding compound" relates to the absence of knowledge to the contrary, i.e., preferably, any substance not known to be ineffective in binding HDACl l can be a compound suspected to be an HDACl l binding compound. Methods of determining the amount of a specific compound comprised in HDAC 11 /antibody complexes will depend on the nature of said compound. The skilled artisan knows how to select the appropriate method of determining a compound, said methods preferably including, e.g. mass spectrometry and other spectrometric methods, preferably for small molecule compounds, quantitative PCR and other amplification methods, preferably for polynucleotides, and immunological or mass spectrometry methods, preferably for polypeptides.

The present invention also relates to the use of the antibody of present invention for identifying HDAC recruitment sites, for identifying an inhibitor of HDACl l recruitment, or for identifying HDAC 11 binding compounds.

The present invention further relates to a device comprising the antibody of the present invention. Preferably, the device comprises an analyzing unit comprising a) the antibody of the present invention for determining the amount of HDAC11 in a sample and b) an evaluation unit comprising a data processor having tangibly embedded an algorithm for comparing the amount determined by said analyzing unit to a reference amount and which is capable of generating an output file containing a result or diagnosis established based on said comparison. The term "device" as used herein relates to a system comprising the aforementioned units operatively linked to each other as to allow the determination according to the methods of the invention. The analyzing unit, preferably, comprises said antibodies in immobilized form on a solid support which is to be contacted to the sample wherein the amount is to be determined. Moreover, the analyzing unit can also comprise a detector, which determines the amount of antibody which is specifically bound to HDAC11. The determined amount can be transmitted to the evaluation unit. Said evaluation unit comprises a data processing element, such as a computer, with an implemented, tangibly embedded algorithm for carrying out a comparison between the determined amount and a suitable reference. Suitable references depend on the method the device is used in and are described elsewhere herein. The results may be given as output of parametric diagnostic raw data, preferably, as absolute or relative amounts. It is to be understood that these data will need interpretation a human being, preferably by a clinician. However, also envisaged are expert system devices wherein the output comprises processed diagnostic raw data the interpretation of which does not require human intervention and may directly serve as an aid for diagnosis.

Finally, the invention relates to a kit for carrying out at least one of the methods of the present invention, wherein said kit comprises instructions for carrying out said method, an antibody of the present invention, and standards for a reference.

The term "kit" as used herein refers to a collection of the aforementioned components, preferably, provided in separately or within a single container. The container also comprises instructions for carrying out the method of the present invention. These instructions may be in the form of a manual or may be provided by a computer program code, which is capable of carrying out the comparisons referred to in the methods of the present invention and to establish a diagnosis accordingly when implemented on a computer or a data processing device. The computer program code may be provided on a data storage medium or device such as an optical storage medium (e.g., a Compact Disc) or directly on a computer or data processing device. Moreover, the kit may, preferably, comprise standards for reference amounts as described elsewhere herein in detail.

All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.

Figure Legends:

Fig. 1 represents the amino acid sequence of HDAC1 1 used as an antigen for mice immunization (SEQ ID NO:4). Fig. 2 represents the amino acid sequence of the epitope recognized by the antibody produced by the hybridoma cell line deposited at DSMZ under No DSM ACC3170 (SEQ ID NO:6).

Fig. 3 shows a western blot analysis of HDAC1 1 protein in human neuroblastoma BE(2)-C cell line. Cells were transfected either with empty vector or plasmid that encodes wild type HDAC1 1 flanked by Myc tag. After 48 hours, the cells were harvested and lysed in urea- lysis-buffer. Whole cell lysate of 50μg was loaded per lane, transferred on PVDF membrane. The upper panel details the western blot analysis using purified antibody produced by the hybridoma cell line deposited at DSMZ under No DSM ACC3170.

Fig. 4 shows a western blot analysis in human neuroblastoma BE(2)-C cell line as in Fig. 3 where the antibody binding was diminished by a blocking peptide.

Fig. 5 shows immunocytochemical analysis of HD AC 1 1 protein in human neuroblastoma BE(2)-C cell line using the antibody produced by the hybridoma cell line deposited at DSMZ under No DSM ACC3170. FIG. 6 shows immunocytochemical analysis of HD AC 1 1 protein in human neuroblastoma BE(2)-C cell line using the antibody produced by the hybridoma cell line deposited at DSMZ under No DSM ACC3171. Fig. 7: Chromatin-Immunoprecipitation (ChIP) using antibodies produced by the hybridoma cell line deposited at DSMZ under No DSM ACC3170 and No DSM ACC3171. ChlP-PCR shows HDACI I protein recruitment to the BMP4 promoter site in BE(2)-C neuroblastoma cells. No HDAC11-recruitmat was observed to the Rassfla gene and to the house keeping genes Gapdh and Actb.

Fig 8: Summary of characteristic and applications of the antibodies produced by the hybridoma cell line deposited at DSMZ under No DSM ACC3170 and No DSM ACC3171. Abbreviations IC: Immunocytochemistry, IHC: Immunohistochemistry, WB: Western Blot, ChIP: Chromatine Immunoprecipitation; n.d.: not determined, - : antibody not suited for application, + : antibody suited for application, +++: : antibody preferred for application.

Fig. 9: Plasmid map of the pQcH6-HDACl 1 vector The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

Examples Example 1: HDACII antigen production

HDACII Antigen preparation and purification

The recombinant histidine-tagged HDACI I variant 1 protein (amino acids 1-347) was used as an antigen. pENTR™221 clone encoding HDACI I variant 1 (Ultimate™ ORFCard for Clone ID IOH9974) was purchased from Invitrogen. Antigen encoding cDNA corresponding to the full-length sequence of human HDACII variant 1, Genbank accession number NM 024827.3, was cloned via TA- vector (Invitrogen) into the pQcH6- vector to yield the expression vector pQcH6-HDACl l as depicted in Figure 9 (original vector pQE8 tagged with six histidine residues at C-terminus, Qiagen), according to the manufacturer's instructions. The construct was confirmed by sequencing and transformed into competent Escherichia coli M15[pREP4] host strain (Qiagen).

The recombinant HDAC11 protein fused to a hexa-histidine tag was expressed by competent Ml 5 bacteria and purified by Ni-NTA-agarose affinity chromatography column according to the manufacturer's conditions (Qiagen). Shortly, the bacterial pellet was resuspended in buffer containing 6M guanidine hydrochloride, lOOmM NaH₂P0₄ x 2H₂0, lOmM Tris/HCl and slowly loaded onto the Ni-NT A- Agarose column. The protein was eluted with a pH gradient in the buffer containing 8M urea, lOOmM NaH₂P0₄ x 2H₂0, lOmM Tris/HCl; the pH value was adjusted to 8.0, 6.3, 5.9, and 4.5, respectively. After elution, the pH was adjusted by NaOH to the value of 7. The purified recombinant HDAC11 protein fused to a hexa-histidine tag was further used as antigen for mice immunization.

Example 2: Generation of hybridomas Mice immunization

Two female BALB/c mice were immunized with recombinant HDAC11 protein of whole HDAC11 variant 1 sequence (Sequence: Accession number NP 079103.2) fused to a hexa- histidine tag by s.c. injections according to the standard protocols. The injection sites were situated in the left and right leg. The immunization scheme was as follows: the primary dose of l(^g of antigen in Freund's adjuvant Complete for stimulating the animals' immune system (Sigma) was followed by a dose of l(^g of antigen with Freund's adjuvant Incomplete (Sigma). The immunization followed with two to five doses of l(^g of antigen in 4M urea buffer. Blood taken from the submandibular vein was used for an assessment of antiserum titre via enzyme-linked immunosorbent assay (direct binding ELISA). Test bleeds were additionally evaluated by immunocytochemistry and western blot analyses.

Fusion

The monoclonal antibodies were raised from successfully immunized mice according to the technique developed by Kohler and Milstein (Kohler, G. and C. Milstein (1975). "Continuous cultures of fused cells secreting antibody of predefined specificity." Nature 256(5517): 495-497). The hybridoma fusion of mouse lymphocytes from lymph nodes and mouse Sp2/0-AG14 myeloma cells (Kohler, G. and C. Milstein (1976). "Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion." Eur J Immunol 6(7): 511-519.) was done by use of polyethylene glycol (PEG). Hybridomas were grown in RPMI1640 medium supplemented by 10% fetal calf serum and lxHAT (hypoxanthine- aminopterin-thymidine). Growing hybridoma clones were screened by ELISA. Positive clones were further assessed for their HDAC11 specificity via western blotting and immunocytochemistry. We tested 264 clones from the fusion of the first mouse and 534 clones from the second mouse. We selected 15 clones and these were cryoprotected. The four out of 15 cryoprotected clones were further subcloned by limited dilution in order to obtain monoclonality. We obtained two clones producing monoclonal anti-HDACl l antibodies: the antibodies are antibodies produced by cell lines deposited at the Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ) under No DSM ACC3170 and DSM ACC3171. Antibody isotyping was assessed by ELISA. The antibodies of IgG isotype were purified by affinity chromatography.

Example 3: Monoclonal antibody purification

Hybridoma cells' supernatant containing antibody was purified using protein A/G sepharose (GE Healthcare) affinity chromatography and eluted into the buffer containing lOOmM citric acid of pH6 for antibody isotype IgGi,3 and pH 3.2 for antibody isotype IgG_2a,2b, respectively.

Example 4: Epitope mapping and permutation analysis

Antibody specificity was addressed by epitope mapping and permutation analysis performed by PEPperPRINT GmbH (Heidelberg, Germany). It was found that (numbers relate to the sequence of SEQ ID NO:4 (Fig. 2a)) Ln (alternatively F or I) and E₂ also contribute to antibody recognition, whereas position M₁₀ is of lower importance and can be exchanged by A, F, G, I, L, N, Q, S, T, V, W and Y. Positions K4 ( replaceable by R, F, M), S₇ (replaceable by T), D₈ (replaceable by E) and Ln (replaceable by F or I) can only be replaced by few selected amino acids predominantly in line of a conservative exchange. E₂, E3, L5, L₆ and S9 cannot be replaced at all and are essential part of the DSM ACC3170 antibody epitope. With linear 13 and 15mer peptides of histone deacetylase 11 isoform 1 , identified the sequence KLLSDSM was identified as minimal consensus motif recognized by the DSM ACC3170 antibody epitope. Mappings of monoclonal DSM ACC3171 antibody against the target protein HDAC11 did not reveal any linear epitope with 10, 12, and 15mer peptides of HDAC11. The results are in consistency with the negative outcome of Western blotting experiments. Therefore, monoclonal DSM ACC3171 antibody apparently recognizes a conformatorial and/or a discontinuous epitope with low affinity of partial epitopes.

Example 5: Immunocytochemistry

For detection and cellular localization studies of the HDAC11 protein, we performed immunocytochemistry (ICC) according to Abeam protocol with minor modifications (Cambridge, UK). Cells were grown on poly-L-lysine coated slides (Thermo Scientific), rinsed shortly with phosphate buffer saline (PBS), fixed with 2% paraformaldehyde for 60 minutes at room temperature, and permeabilized for 15 min in PBS containing 0.1% Triton X-100. Cells were incubated in blocking solution containing 10%> goat serum, 0.25%> bovine serum albumin, 0.1% Triton X-100, PBS for 60 min at room temperature to block unspecific binding of antibodies. Primary antibody incubation was done overnight at 4°C followed by three PBS-washing steps and incubation with secondary anti mouse IgG antibody conjugated to Cy3 fluorophore (Dianova) for 60 min at room temperature in the dark humidified chamber. The slides were washed three times with PBS, incubated with 2.5 μg/ml solution of 4^',6-diamidin-2-phenylindol in PBS to visualize cell nuclei, and then rinsed three times with PBS and mounted with Fluoromount mounting medium (Sigma).

Example 6: Western blot analysis

The whole cell lysates were prepared by cells lysis in urea-containing buffer followed by protein separation by electrophosesis on 15% denaturing polyacrylamid-SDS gels and blotted on PVDF membranes. The membranes were incubated with hybridomas supernatants containing primary antibody overnight at 4°C followed by the incubation with the secondary antibody goat anti mouse IgG horse radish peroxidise conjugated (Sigma) diluted 1 :5000 in 5% non-fat dry milk/PBS. The signals were detected with ECL Plus (Thermo Scientific), according to the manufacturer's instructions. Example 7: Chromatin Immunoprecipitation ChIP was performed according to the standard procedure described in the ChIP Assay Kit (Upstate/ Milipore, Catalog # 17-229). Cells were fixed with 1% formaldehyde for 10 minutes at room temperature and quenched with 125mM glycine for 5 minutes at room temperature, harvested and lysed with lysis buffer containing 1% sodium dodecyl sulfate (SDS). Cell lysates were sonicated using Covaris S2 device (Covaris, Inc. USA). Chromatin complexes containing HDAC11 protein were immunoprecipitated, eluted with buffer containing 1%SDS, 0.1M sodium hydrogen carbonate (NaHC0₃) for two times 15 minutes at room temperature. Chromatin complexes were de-crosslinked with 5M sodium chloride for overnight at 65°C and genomic DNA was isolated. Genomic DNA served as a template for qPCR analysis.

Print Out (Original in Electronic Form)

(This sheet is not part of and does not count as a sheet of the international application)

1 The indications made below relate to

the deposited microorganism(s) or

other biological material referred to in

the description on:

1-1 page 5, 6,23,23,23,25,26,26

1-2 line 21-23,27,3,7,13,13,1,4

1-3 Identification of deposit

1 -3-1 Name of depositary institution DSMZ DSMZ-Deutsche Sammlung von Mikroor- ganismen und Zellkulturen GmbH

1 -3-2 Address of depositary institution Inhoffenstr. 7B, D-38124 Braunschweig,

Germany

1 -3-3 Date of deposit 26 April 2012 (26.04.2012)

1 -3-4 Accession Number DSMZ ACC3171

1-4 Additional Indications HDACll#170/9

1-5 Designated States for Which

Indications are Made All designations

2 The indications made below relate to

the deposited microorganism(s) or

other biological material referred to in

the description on:

2-1 page 5, 6,22,22,22,23,23,25,25,26

2-2 line 21-23,26,16,24,31,6,12,13,31

2-3 Identification of deposit

2-3-1 Name of depositary institution DSMZ DSMZ-Deutsche Sammlung von Mikroor- ganismen und Zellkulturen GmbH

2-3-2 Address of depositary institution Inhoffenstr. 7B, D-38124 Braunschweig,

Germany

2-3-3 Date of deposit 26 April 2012 (26.04.2012)

2-3-4 Accession Number DSMZ ACC3170

2-4 Additional Indications HDACll#35/47

2-5 Designated States for Which

Indications are Made All designations

FOR RECEIVING OFFICE USE ONLY

0-4 This form was received with the

international application: yes

(yes or no)

0-4-1 Authorized officer

Pasche, Constanti nus

Claims

1. An antibody specifically recognizing histone deacetylase 11 (HDACl l), wherein said antibody recognizes native HDAC11 or native and denatured HDAC11.

2. The antibody of claim 1, wherein the antibody is a monoclonal antibody, preferably a monoclonal antibody recognizing an epitope comprising an amino acid sequence as depicted SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9.

3. The antibody of claim 1 or 2, wherein the antibody is an antibody produced by a cell line deposited at the Leibnitz-Institut DSMZ-Deutsche Sammlung fur Mikroorganismen und Zellkulturen GmbH (DSMZ) under No DSM ACC3170 or No DSM ACC3171.

4. A cell line producing the monoclonal antibody of claim 2 or 3.

5. The cell line of claim 4, wherein the cell line is a cell line deposited at the DSMZ under No DSM ACC3170 or No DSM ACC3171.

6. A method for identifying a subject afflicted with cancer as being susceptible to treatment with an HDAC11 inhibitor, comprising

(a) contacting a sample from said subject with the antibody of any one of claims 1 to 3,

(b) determining the amount of HDAC11 /antibody complexes formed in the mixture of (a), and

(c) comparing said amount with a reference amount, thereby identifying a subject afflicted with cancer as being susceptible to treatment with an HDACl l inhibitor.

7. A method for identifying HDAC11 recruitment sites, comprising

(a) contacting a mixture comprising HDACl l and polynucleotides with the antibody of any one of claims 1 to 3, and

(b) detecting polynucleotides specifically bound to HDACl l /antibody complexes formed in the mixture of (a), thereby identifying HDAC11 recruitment sites.

8. A method for identifying a subject afflicted with cancer, comprising

(a) contacting a sample from said subject comprising HDACl l and polynucleotides with the antibody of any one of claims 1 to 3, and

(b) detecting polynucleotides specifically bound to HDAC 11 /antibody complexes formed in the mixture of (a), and

(c) comparing the polynucleotides specifically bound to HDAC 11 /antibody complexes to a reference, thereby identifying a subject afflicted with cancer.

9. A method for identifying an inhibitor of HDAC 11 recruitment, comprising

(a) contacting a mixture comprising HDACl l and polynucleotides with a compound suspected to be an inhibitor of HDAC 11 recruitment,

(b) contacting the mixture of (a) with the antibody of any one of claims 1 to 3, and

(c) determining the polynucleotide content of HDAC 11 /antibody complexes formed in the mixture of (a), thereby identifying an inhibitor of HDACl l recruitment.

10. A method for identifying an HDAC 11 binding compound, comprising

(a) providing a mixture comprising HDACl l and a compound suspected to be an HDAC 11 binding compound,

(c) determining the amount of compound suspected to be an HDACl l binding compound in HDAC 11 /antibody complexes formed in the mixture of (a), thereby identifying an HDAC 11 binding compound.

11. Use of the antibody of any one of claims 1 to 3 for identifying HDAC recruitment sites.

12. Use of the antibody of any one of claims 1 to 3 for identifying an inhibitor of HDAC 11 recruitment.

13. Use of the antibody of any one of claims 1 to 3 for identifying HDACl l binding compounds.

14. A device comprising the antibody of any one of claims 1 to 3.

A kit for carrying out a method of any one of claims 6 to 10, wherein said kit comprises instructions for carrying out said method, an antibody of any one of claims 1 to 3, and standards for a reference.