The present invention relates to antibody, or antibody fragment, or any protein that can be produced by modern technology methods, or any fragment of a protein, or a fusion product containing the protein, or a protein with adduct molecules, or a product with a modified protein, or pharmaceutical composition containing a protein, or an industrial product containing the protein, or a kit containing the protein, and all the above have the property of exhibiting antigenic reactivity against (recognizes specifically) the phosphorylated serine that is located at the C-terminus of a protein that belongs to the H2B histone family, and therefore can bind selectively to said phosphorylated serine. This binding is indicative of damage to the genetic material of the cells, including DNA double-stranded breaks.

Also, the present invention relates to a method and kit for the determination of DNA double-stranded breaks. The method includes processing of a sample comprising proteins of the H2B histone family with an antibody, or antibody fragment, or any protein that can be produced by modern technology methods, or any fragment of a protein, or a fusion product containing the protein, or a fusion of a molecule to a protein, or a product with a modified protein, or pharmaceutical composition containing a protein, or an industrial product containing the protein, or a kit containing the protein, and all the above have the property of exhibiting antigenic reactivity against (recognizes specifically) the phosphorylated serine that is located at the C-terminus of a protein that belongs to the H2B histone family, and therefore can bind selectively to said phosphorylated serine. This binding is indicative of damage to the genetic material of the cells, including DNA double-stranded breaks.

Description of the Invention

The present invention relates to a specific antibody against the phosphorylated serine at position 146 of the B4DR52 protein, as well as to the uses of this antibody intended to detect lesions in the genetic material of human cells. The B4DR52 protein is a histone variant and is a member of the H2B histone family (UniProtKB - B4DR52-1 (B4DR52 HUMAN), Primary number: B4DR52) (Figure 1 : B4DR52 Amino Acid Sequence).

The above histone is also registered in databases containing sequences in the form of nucleotides (DNA and/or RNA sequences) or in the form of amino acids (protein sequences) and they present high degree of homology to the protein "UniProtKB - B4DR52 (B4DR52 HUMAN)" to humans and other non-human organisms. The term "DNA" refers to deoxyribonucleic acid. The term "RNA" refers to ribonucleic acid.

Histones are basic proteins that form a complex with DNA, the nucleosome, which is the structural basis of chromatin and chromosomes in the nucleus of eukaryotic cells. Histones are grouped into five (5) histone families, which are: H2A, H2B, H3, H4 and HI . The members of each family are proteins called histone variants. Histone variants that belong to the same histone families are characterized by an extended homology betwen their sequences, and occupy the same position in the nucleosome, and they have the same topography in the nucleosome. It is now well known that histones and their variants undergo post-translational modifications involving additions of small molecules onto certain amino acids of their sequence by biochemical means (e.g., phosphorylation, acetylation, methylation, etc.). These additions occur as a result of biological processes of the cells. It has been hypothesized that these post-translational modifications constitute structural units for the "Histone Code Hypothesis". It has already been demonstrated by detailed scientific studies that cellular functions are regulated by the "Histone Code", such as transcription of genetic material, repair of genetic material damage, cell signaling etc (1).

The B4DR52 histone variant bears a serine at its carboxy terminus, at position 146, followed by a glutamine, creating a "serine-glutamine" motif (international symbolism: SQ motif). This serine is phosphorylated as a consequence of the creation of DNA double-stranded breaks. This phosphorylated serine participates in the 'DNA repair code' (2).

Histones other than B4DR52 which undergo post-translational modifications and are involved in the "DNA repair Code" have been discovered and are well known. Representative example is the histone variant H2AX that belongs to the H2A histone family and has a phosphorylated serine at position 139, designated γΗ2ΑΧ. The prefix "γ" is indicative of the phosphorylation that occurs as a consequence of DNA double-stranded breaks (3-1 1). In adition, another histone, member of the H2B histone family, has a phosphorylated serine at position 14 in the presence of DNA double-stranded breaks (12, 13).

Given the sever consequenses of DNA double-stranded breaks on cell survival and the risk of cancer in the body, various detection methods have been developed, included: colony survival assays, chromosomal aberration frequency, comet assay, PFGE, prematurely condensed chromosomes-PCC assay, and the detection of DNA double-stranded breaks with anti-yH2AX antibody. γΗ2ΑΧ has been proven to be an excellent marker for detecting DNA double-stranded breaks for three main reasons: 1) unparalleled sensitivity, as it can detect a single double-stranded DNA break in the nucleus of a human cell, 2) specificity of DNA double-stranded breaks (3), and 3) quantification of DNA double- stranded breaks (3, 4, 6).

The B4DR52 histone variant when bears the phosphorylated serine at position 146 is designated herein "yB4DR52", in analogy to γΗ2ΑΧ (3). yB4DR52 signals the existence of DNA double- stranded breaks, with a mechanism similar to that of γΗ2ΑΧ. Specific antibodies recognizing phosphorylated serine 146 of B4DR52 make it possible to label the DNA region around the DNA break.

The present invention relates to a specific antibody, either polyclonal or monoclonal, or a polyclonal antibody fragment or monoclonal antibody fragment that recognizes specifically the phosphorylated serine at position 146 of the B4DR52 protein that belongs to the H2B histone family. The term "antigenic reactivity" refers to protein molecule or fragment of protein molecule, or polypeptide exhibiting strong selective binding with high affinity or avidity to regions of other molecules, according to the biological example of the "antigen-antibody" reaction. The term "antigenically reactive antibody fragment" is a protein segment, specific antibody, selected from a group consisting of Fab, Fab', F(ab')2 and F(v). Also, the present invention relates to any protein that can be produced by modern technological methods, or any portion of a protein, or a fusion product containing the protein or a protein adduct, a protein modified protein product, or a pharmaceutical composition that containing the protein , or an industrial product containing the protein, or a tool kit (KIT) containing the protein, which protein, (including antibodies) exhibits antigenic reactivity against (recognizes specifically) the phosphorylated serine at position 146 of the protein B4DR52, which is a variety that belongs to the H2B histone family.

Also, the present invention relates to an isolated or purified antibody or antibody fragment or portion of a protein having the property of exhibiting antigenic reactivity against (recognizes specifically) the phosphorylated serine at position 146 of the B4DR52 protein. Also, the present invention relates to a fusion protein comprising the isolated or purified antibody or fragment thereof exhibiting antigenic reactivity against (recognizes specifically) the phosphorylated serine at position 146 of the B4DR52 protein.

Also, the present invention relates to all of the above molecules, termed hereinafter "antigenically reactive entity", that exhibit specific recognition and binding against the phosphorylated serine at position 146 of the B4DR52 protein" and are labeled with a detection facilitating agent. The detection facilitating agent for example may be: a secondary antibody, an enzyme, a radioactive isotope, a fluorescent molecule, biotin etc. In addition, the detection facilitating agent may be detected by chemiluminescence. This technology, as well as the methods and tools to be developed on the basis of this technology, serve to detect DNA double-stranded breaks that bear the cells of an organism, to determine the ability of an organism's cells to repair their DNA, as well as the detect and quantify of their apoptosis. In addition, these tools are useful in the study and diagnosis of other cellular conditions and functions mediated by DNA double-stranded breaks, such as genetic recombination, viral DNA incorporation in the host genetic material, etc. yB4DR52 signals the existence of DNA double-stranded breaks, with a mechanism similar to that of γΗ2ΑΧ. Specific antibodies and entities in generial, as defined hereinabove, that selectively recognize the phosphorylated serine 146 of B4DR52, enable the DNA region where the yB4DR52 is located around the double strand break to be probed. The yB4DR52 is created after the generation of DNA double-stranded breaks. Measurements of yB4DR52 amount vs total H2B at regular intervals after the generation of DNA double-stranded breaks show that follows the curve depicted in Figure 2. This curve is characterized by three phases: in the first part of the curve, yB4DR52 increases (phase 1), the second part remains relatively constant (phase 2) (plate, width), and in the third part it is gradually reduced (phase 3). Curve sections vary in shape and length depending on cell type, but the overall shape of the curve remains the same (Figure 2)·

The yB4DR52 is indicative of DNA double-stranded breaks in cells and allows quantitative determination of them. A specific antibody against the phosphorylated serine at position 146 of histone B4DR52, hereafter referred to as anti-yB4DR52, may be used in a number of methods and applications.

A method for detection and quantitative determination of DNA double stranded brakes comprising (i) one or more anti-yB4DR52 antibodies; (ii) a means for labeling the antibodies (e.g., a secondary antibody or a chemical group which binds to said specific antibody and serves to detect the specific antibody, etc.), (iii) a sample containing proteins of the H2B histone family which are complexed with DNA or are derived from a DNA complex. The sample may contain cells, or cell extract. In the case where the sample contains cells, the quantitative determination of DNA double stranded brakes can be done by immunocytochemistry. In the case that the sample contains a cell extract, the quantitative determination of DNA double stranded brakes can be done by immunochemistry techniques. The sample may be derived from sampling of human biological material or members of Hominidae. In the context of this method, detecting the binding of said specific antibody suggests the presence of DNA double-stranded breaks and allows for quantitative determination of DNA double stranded brakes. Based on the above, the above method can be used as a basis in many applications:

In determining the ability of cells to repair DNA. The rate of phosphorylation increase up to the plateau (Figure 2: Phase 1 of the curve) as well as the rate of phosphorylation decrease (Figure 2: Phase 3 of the curve) is indicative of the body's ability to repair its DNA. This method is based on the quantification of the number of DNA double-stranded breaks at different time points, divided by time. The above method is the basis for the assay for determining the sensitivity of an organism to one or more environmental factors, e.g. mutagens, radiation, drugs, chemicals, or in combinations of the above.

In estimation of the degree of exposure of an organism to radiation, and/or the dose of radiation absorbed by the body as a biological radiation dosimeter. This estimation is obtained by quantification of the number of DNA double-stranded breaks in the sample and comparison with a reference curve (one or more standard preparation (s) exposed/exposed to a predetermined radiation dose).

In the detection and quantification of apoptosis. The cells are protected against damage that can be caused to their genetic material by multiple DNA repair mechanisms. In the case that the cell has a large burden of damage to its DNA that cannot be repaired, the mechanism of apoptosis is activated to lead cell to death. The mechanism of apoptosis involves DNA fragmentation through the creation of DNA double-stranded breaks. Therefore, this method can be the basis for detecting apoptosis. In particular: a method of (i) detecting apoptosis in a sample of cells from an individual (healthy or patient); (ii) quantification of apoptosis in said cell population by quantification of DNA double- stranded breaks compared to a standard sample.

The estimation of the percentage of cells in the S phase of the cell cycle in a population of cells through the effect of topoisomerase I inhibitors. A method of determining the percentage of cells in the cell cycle DNA synthesis (international term: S-phase), with reference to the whole of the sample cells, which method includes, among others: collection of a sample of cells exposed to topoisomerase I inhibitors, or alternatively, collection of a sample of cells which are then exposed to topoisomerase I inhibitors,

II. Detection of the specific anti-yB4DR52 binding to the phosphorylated serine 146 of the B4DR52 protein that belongs to the H2B histone family, that is in said sample, where detection of said binding implies the presence of DNA double-stranded breaks under the effect of the topoisomerase inhibitors I on cells in the S phase of the cell cycle in the sample. Additionally, this method can be used for the estimation of the percentage of abnormal cell proliferation in a patient from which said sample is collected.

Also, the present invention relates to a method used to determine homologous recombination in DNA, wherein the detection of DNA double-stranded breaks is indicative of homologous recombination in DNA, and is based on the above mentioned method.

Also, the present invention relates to a tool kit for detection or quantification of DNA double-stranded breaks, or a combination of detection and quantification of DNA double-stranded breaks, wherein said tool kit comprises anti-yB4DR52 and a means for detecting the specific binding of said anti- yB4DR52 in a sample of biological material. Also, the present invention relates to a product modified by modern technology, comprising anti- yB4DR52 and a means for detecting the specific binding of said anti-yB4DR52 to a sample of biological material. Also, the present invention relates to a pharmaceutical composition or product and which comprises anti-yB4DR52 and a means for detecting the specific binding of said anti-yB4DR52 to a sample of biological material.

Also, the present invention relates to an industrial product comprising anti-yB4DR52 and a means for detecting the specific binding of said anti-yB4DR52 to a sample of biological material.

Note: All references to the literature listed in this specification are clearly incorporated by reference.

Bibliography

1. The language of covalent histone modifications. Strahl, B.D., & Allis, CD. Nature 2000 403:41-5.

2. The histone code at DNA breaks: A guide to repair? Van Attikum, H., & Gasser, S. M. Nature Reviews Molecular Cell Biology 2005 6:757-65.

3. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM. J Biol Chem. 1998 273(10):5858-68.

4. Megabase chromatin domains involved in DNA double-strand breaks in vivo. Rogakou EP, Boon C, Redon C, Bonner WM. J Cell Biol. 1999 146(5):905-16.

5. A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Paull TT, Rogakou EP, Yamazaki V, Kirchgessner CU, Gellert M, Bonner WM. Curr Biol. 2000 10(15):886-95.

6. Quantitative detection of (125)IdU-induced DNA double-strand breaks with gamma-H2AX antibody. Sedelnikova OA, Rogakou EP, Panyutin IG, Bonner WM. Radiat Res. 2002 158(4):486- 92.

7. Initiation of DNA fragmentation during apoptosis induces phosphorylation of H2AX histone at serine 139. Rogakou EP, Nieves-Neira W, Boon C, Pommier Y, Bonner WM. J Biol Chem. 2000 Mar 31 275(13):9390-5

8. Phosphorylation of histone H2AX and activation of Mrel 1, Rad50, and Nbsl in response to replication-dependent DNA double-strand breaks induced by mammalian DNA topoisomerase I cleavage complexes. Furuta T, Takemura H, Liao ZY, Aune GJ, Redon C, Sedelnikova OA, Pilch DR, Rogakou EP, Celeste A, Chen HT, Nussenzweig A, Aladjem MI, Bonner WM, Pommier Y. J Biol Chem. 2003 May 30, 278(22):20303-12.

9. Response to RAG-mediated VDJ cleavage by NBS1 and gamma-H2AX. Chen HT, Bhandoola A, Difilippantonio MJ, Zhu J, Brown MJ, Tai X, Rogakou EP, Brotz TM, Bonner WM,

Ried T, Nussenzweig A. Science. 2000 290(5498): 1962-5.

10. Recombinational DNA double-strand breaks in mice precede synapsis. Mahadevaiah SK, Turner JM, Baudat F, Rogakou EP, de Boer P, Bianco-Rodriguez J, Jasin M, Keeney S, Bonner WM, Burgoyne PS. Nat Genet. 2001 Mar 27(3):271-6.

1 1. Histone H2AX is phosphorylated at sites of retroviral DNA integration but is dispensable for postintegration repair. Daniel R, Ramcharan J, Rogakou E, Taganov KD, Greger JG, Bonner W, Nussenzweig A, Katz RA, Skalka AM. J Biol Chem. 2004 279(44):45810-4. 12. Apoptotic phosphorylation of histone H2B is mediated by mammalian sterile twenty kinase. Cheung WL, Ajiro K, Samejima K, Kloc M, Cheung P, Mizzen CA, Beeser A, Etkin LD, Chernoff J, Earnshaw WC, Allis CD. Cell. 2003 1 13(4):507-17.

13. Phosphorylation of histone H2B at DNA double-strand breaks. Fernandez-Capetillo O, Allis CD, Nussenzweig A. J Exp Med. 2004 199(12): 1671-7.

Amino Acid Sequence

B4DR52 Amino Acid Sequence (UniProtKB - B4DR52-1)

10 20 30 40 50

MPDPAKSAPA PKKGSKKAVT KVQKKDGKKR KRSRKESYSV YVYKVLKQVH

60 70 80 90 100

PDTGISSKAM GIMNSFVNDI FERIAGEASR LAHYNKRSTl TSREIQTAVR

110 120 130 140 150

LLLPGELAKH AVSEGTKAVT KYTSSNPRNL SPTKPGGSED RQPPPSQLSA

160

IPPFCLVLRA GIAGQV