Compositions and methods related to K180 dimethylated H1.0 protein

Abstract

To provide compositions and methods related to K180 dimethylated H1.0 protein. [Solution] H1.0K180me2 antibodies, H1.0K180me2 proteins, and H1.0K180me2 peptides, as well as methods for their use in diagnosis and therapy, are provided herein. These antibodies, etc., may also be used for the detection and quantification of histone H1.0 proteins or fragments thereof (H1.0K180me2) containing dimethylated lysine at lysine residue 180. Such compositions and methods are useful for detecting replication aging, DNA damage, genotoxic stress, radiation exposure, and Alzheimer's disease, and are useful for monitoring treatment regimens, patient stratification, and drug screening, and may serve as markers of biological aging in the system.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims priority based on U.S. Provisional Application No. 62 / 325,392, U.S. Provisional Application No. 62 / 325,362, U.S. Provisional Application No. 62 / 325,408, U.S. Provisional Application No. 62 / 355,265, and U.S. Provisional Application No. 62 / 355,277, filed on 20 April 2016, each of these provisional applications is incorporated herein by reference in whole. [Background technology]

[0002] background Cellular chromatin is a dynamic macromolecule that allows for many three-dimensional structures and tends to remodel and reconfigure to receive physiologically relevant inputs. Histone proteins are the major protein components of chromatin, and double-stranded DNA is entangled with histone proteins. When histone proteins change, it becomes possible to differentially alter access to the transcription machinery for certain genes while maintaining intact access to other genes. Differential chromatin condensation achieved by post-translational modifications (PTMs) of histones is fundamental to chromatin packaging (Lunyak and Rosenfeld 92008) Hum. Mol. Genet., Vol. 17: pp. R28-36; Jenuwein and Allis (2001) Science, Vol. 293: pp. 1074-1080). Histone PTMs, such as methylation, can act as epigenetic codes and play a crucial role in many aspects of cellular responses closely related to development, injury, disease, and aging.

[0003] The five main families of histones are H1, H2A, H2B, H3, and H4. Histones H2A, H2B, H3, and H4 are known as core histones, while histones H1 and H5 are known as linker histones. In recent years, numerous H1.0-binding proteins identified by multiple studies have demonstrated the crucial role of H1.0 in protein-protein interactions, suggesting a new paradigm of H1.0 structure and function that extends beyond its effects on chromatin structure. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Lunyak and Rosenfeld (2009) Hum. Mol. Genet., Vol. 17: pp. R28-36 [Non-Patent Document 2] Jenuwein and Allis (2001), Science, Vol. 293: pp. 1074-1080. [Overview of the Initiative] [Means for solving the problem]

[0005] Since H1.0 methylation is involved in disease onset, response to injury, and response to treatment regimens, there is a need to detect H1.0 methylation in various cellular conditions, and a sensitive assay needs to be able to distinguish between different types of cellular conditions. There is also a need to target methylated H1.0-related diseases and conditions for treatment. Methods and compositions for this purpose are provided herein.

[0006] Brief summary H1.0K180me2 antibodies, H1.0K180me2 proteins, and H1.0K180me2 peptides and methods of use therefor in diagnosis and treatment are provided herein. These H1.0K180me2 antibodies, H1.0K180me2 proteins, and H1.0K180me2 peptides may be used in the treatment of methylated H1.0-related diseases or conditions in an individual. These H1.0K180me2 antibodies, H1.0K180me2 proteins, and H1.0K180me2 peptides are also used for the detection and quantification of histone H1.0 proteins or fragments thereof (H1.0K180me2) containing dimethylated lysine at lysine residue 180, and such compositions and methods are useful for detecting replicative senescence, DNA damage, genotoxic stress, radiation exposure, Alzheimer's disease, monitoring treatment regimens, patient stratification, drug screening, and may serve as markers of biological aging within a system.

[0007] In one embodiment, an antibody (H1.0K180me2 antibody) that specifically binds to a dimethylated antigen is provided herein, wherein the dimethylated antigen comprises a dimethylated lysine residue, the lysine residue corresponds to K180 of human histone H1.0, and the dimethylated lysine residue is required for binding. In some embodiments, the dimethylated antigen does not contain any other methylated lysine residue. In some embodiments, if the antigen contains a dimethylated lysine residue at lysine residues corresponding to K166, K172, K174, K175, and / or K177 of the human histone H1.0 protein, the antibody does not bind or binds minimally. In some embodiments, if the antigen contains a monomethylated lysine residue at lysine residues corresponding to K166, K172, K174, K175, K177, and / or K180 of the human histone H1.0 protein, the antibody does not bind or binds minimally. In some embodiments, if the antigen contains a trimethylated lysine residue at lysine residues corresponding to K166, K172, K174, K175, K177, and / or K180 of the human histone H1.0 protein, the antibody will not bind or will bind only minimally. In some embodiments, the antibody is at least twice as specific to the dimethylated antigen than to the monomethylated antigen, where the monomethylated antigen contains a monomethylated lysine residue, and the lysine residue corresponds to K180 of the human histone H1.0 protein. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is labeled. In some embodiments, the antibody is biotinylated. In some embodiments, the antibody is attached to a solid surface. In some embodiments, the antibody is attached to beads, columns, resins, or microplates. In some embodiments, the antibody is a cell-permeable antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a humanized antibody.In some embodiments, the antibody is conjugated to at least one therapeutic agent selected from the group consisting of a radionuclide, a cytotoxin, a chemotherapeutic agent, a drug, a prodrug, a toxin, an enzyme, an immunomodulator, an apoptosis promoter, a cytokine, a hormone, an oligonucleotide, an antisense molecule, siRNA, and a secondary antibody. In some embodiments, the antibody can remove H1.0K180me2 in a sample. In some embodiments, the antibody can remove cells containing H1.0K180me2. In some embodiments, the antibody can remove senescent cells.

[0008] In another aspect, there is provided herein a method of treating a methylated H1.0-related disease or condition in an individual, the method comprising administering to the individual a therapeutically effective amount of any one of the H1.0K180me2 antibodies provided herein. In some embodiments, the methylated H1.0-related disease or condition is selected from the group consisting of Alzheimer's disease, radiation exposure, exposure to genotoxic stress factors, a disease or condition involving the accumulation of senescent cells, and a disease or condition associated with an elevated level of the H1.0K180me2 protein or peptide. In another related aspect, there is provided herein a method of removing H1.0K180me2 in an individual, the method comprising administering to the individual a therapeutically effective amount of any one of the H1.0K180me2 antibodies provided herein for treating an individual suffering from a disease or condition selected from the group consisting of, for example, Alzheimer's disease, radiation exposure, exposure to genotoxic substances, exposure to DNA-damaging agents, and a condition involving the accumulation of senescent cells. In a related aspect, there are provided herein pharmaceutical compositions, kits, and other products comprising any one of the H1.0K180me2 antibodies described herein.

[0009] In another embodiment, histone H1.0 peptides or histone H1.0 proteins containing dimethylated lysine residues are provided herein, wherein the dimethylated lysine residue corresponds to K180 of human histone H1.0 (H1.0K180me2 peptides and H1.0K180me2 proteins). In some embodiments, the peptide includes a sequence selected from the group consisting of SEQ ID NOs: 3 to 35. In some embodiments, the peptide includes a sequence selected from the group consisting of SEQ ID NOs: 3 to 5. In some embodiments, the protein includes the sequence of SEQ ID NO: 2. In some embodiments, the protein includes the sequence of SEQ ID NO: 3. In some embodiments, the peptide or protein does not contain any other methylated lysine residues. In some embodiments, the peptide or protein is labeled. In some embodiments, the peptide or protein is biotinylated. In some embodiments, the peptide or protein is attached to a solid surface. In some embodiments, the peptide or protein is attached to beads, columns, resins, or microplates. In some embodiments, the peptide or protein is conjugated to at least one therapeutic agent selected from the group consisting of radionuclides, cytotoxins, chemotherapeutic agents, drugs, prodrugs, toxins, enzymes, immunomodulators, apoptosis promoters, cytokines, hormones, oligonucleotides, antisense molecules, siRNA, and secondary antibodies. In some embodiments, the peptide or protein can remove or block H1.0K180me2 autoantibodies. In some embodiments, the peptide or protein can remove or block H1.0K180me2 IgG autoantibodies. In some embodiments, the peptide or protein can remove or block H1.0K180me2 IgM autoantibodies.

[0010] In another embodiment, a method for treating a methylated H1.0-related disease or condition in an individual is provided herein, comprising the step of administering to the individual a therapeutically effective amount of one of the H1.0K180me2 protein or H1.0K180me2 peptides described herein. In some embodiments, the methylated H1.0-related disease or condition is selected from the group consisting of Alzheimer's disease, diseases or conditions including radiation exposure, exposure to genotoxic stressors, accumulation of senescent cells, and diseases or conditions accompanied by elevated levels of H1.0K180me2 autoantibodies. In a related embodiment, a method for removing, blocking, or neutralizing an individual's H1.0K180me2 autoantibodies is provided herein, comprising the step of administering to the individual a therapeutically effective amount of one of the H1.0K180me2 protein or H1.0K180me2 peptides described herein. In some embodiments, the individual suffers from a disease or condition selected from the group consisting of Alzheimer's disease, radiation exposure, exposure to genotoxic stressors, accumulation of senescent cells, or a disease or condition accompanied by elevated levels of H1.0K180me2 autoantibodies. In relevant embodiments, pharmaceutical compositions, kits, and other products comprising any one of the H1.0K180me2 peptides or proteins described herein are provided herein.

[0011] In another embodiment, a method is provided herein for determining whether an individual has or is at risk of developing Alzheimer's disease, comprising the steps of (a) contacting a biological sample derived from the individual with one of the H1.0K180me2 antibodies provided herein, and (b) determining the concentration of the H1.0K180me2 antigen in the sample that is bound to the antibody, wherein a decrease in concentration compared to a control indicates that the individual has or is at risk of developing Alzheimer's disease. In one embodiment, if the serum concentration of histone H1.0 protein is less than 5.61 nmol / ml or lower, the individual has or is at risk of developing Alzheimer's disease. In some embodiments, if the PLR(LR+) is 3.6 or higher, the individual has or is at risk of developing Alzheimer's disease. In some embodiments, if the NLR(LR-) is 0.26 or lower, the individual is not at risk of developing Alzheimer's disease. In some embodiments, if an individual is determined to have or be at risk of developing Alzheimer's disease, the method further includes the step of treating the individual with an Alzheimer's disease drug or regimen. Thus, a method for treating an individual with an Alzheimer's disease drug or regimen is provided herein if, as determined by the above method, the serum concentration of histone H1.0 protein is less than or equal to 5.61 nmol / ml and / or the PLR(LR+) measurement is 3.6.

[0012] In another embodiment, a method is provided herein for determining whether an individual has or is at risk of developing Alzheimer's disease, comprising the steps of (a) contacting a biological sample derived from the individual with the H1.0K180me2 protein or H1.0K180me2 peptide provided herein, and (b) determining the concentration of an autoantibody in the sample bound to the protein or peptide, wherein an increase in concentration compared to a control indicates that the individual has or is at risk of developing Alzheimer's disease. In some embodiments, the method includes the step of determining the concentration of an IgM autoantibody in the sample bound to the protein or peptide. In some embodiments, the method includes the step of determining the concentration of an IgG autoantibody in the sample bound to the protein or peptide. In some embodiments, if the serum concentration of IgG autoantibody normalized to the total IgG level is higher than or equal to 9.69 ug / ml, the individual has or is at risk of developing Alzheimer's disease. In some embodiments, if the serum concentration of IgG autoantibodies normalized to serum volume is higher than or equal to 8.23 ​​ug / ml, the individual has or is at risk of developing Alzheimer's disease. In some embodiments, if the serum concentration of IgM autoantibodies normalized to serum volume is higher than or equal to 409 fMol / ml, the individual has or is at risk of developing Alzheimer's disease. In some embodiments, if the ratio of IgM autoantibody concentration to total IgM concentration is 26.6 × 10⁻⁶ -6If the value is greater than the specified value, the individual has or is at risk of developing Alzheimer's disease. In some embodiments, if the PLR(LR+) is 2.4 or greater for IgG autoantibodies, or if the PLR(LR+) is 3.5 or greater for IgM autoantibodies, the individual has or is at risk of developing Alzheimer's disease. In some embodiments, the method includes the steps of determining the concentration of IgM autoantibodies in a sample bound to a protein or peptide, and comparing the measured value to the total IgM concentration in the sample. In some embodiments, if it is determined that the individual has or is at risk of developing Alzheimer's disease, the method includes the step of treating the individual with an Alzheimer's disease drug or regimen.

[0013] In another embodiment, a method is provided herein for determining whether an individual diagnosed with Alzheimer's disease and undergoing treatment for Alzheimer's disease will benefit from or continue to benefit from the treatment, the method comprising: (a) contacting a biological sample derived from the individual with one of the H1.0K180me2 antibodies provided herein; (b) determining the concentration of the H1.0K180me2 antigen in the sample bound to the antibody; and (c) determining, if an increase in concentration relative to a control is present, that the individual will benefit from or continue to benefit from the treatment. In some embodiments, if it is determined that the individual will benefit from or continue to benefit from the treatment, the method further comprises the step of treating the individual with an Alzheimer's disease drug or regimen.

[0014] In another embodiment, a method is provided herein for determining whether an individual diagnosed with Alzheimer's disease and undergoing treatment for Alzheimer's disease will benefit from or continue to benefit from the treatment, the method comprising: (a) contacting a biological sample derived from the individual with the H1.0K180me2 protein or peptide provided herein; (b) determining the concentration of an autoantibody in the sample bound to the protein or peptide; and (c) determining whether the individual will benefit from or continue to benefit from the treatment if a decrease in concentration relative to a control exists. In some embodiments, the method includes the step of determining the concentration of an IgM autoantibody in the sample bound to the protein or peptide. In some embodiments, the method includes the step of determining the concentration of an IgG autoantibody in the sample bound to the protein or peptide.

[0015] In another embodiment, a method is provided herein for determining whether an individual diagnosed with Alzheimer's disease would benefit from a candidate treatment, wherein the individual has not yet initiated the treatment, and the method comprises: (a) administering the candidate treatment to the individual; (b) contacting a biological sample from the individual after administration of the candidate treatment with one of the H1.0K180me2 antibodies provided herein; (c) determining the concentration and / or intracellular localization of the H1.0K180me2 antigen in the sample bound to the antibody; and (d) determining that the individual would benefit from the candidate treatment if there is an increase in concentration or a decrease in intracellular localization to the cytoplasm compared to a control.

[0016] In another embodiment, a method is provided herein for determining whether an individual diagnosed with Alzheimer's disease would benefit from a candidate treatment, wherein the individual has not yet initiated the treatment, and the method comprises: (a) contacting a biological sample derived from the individual with a candidate treatment; (b) contacting the biological sample with one of the H1.0K180me2 antibodies provided herein; (c) determining the concentration and / or intracellular localization of the H1.0K180me2 antigen in the sample bound to the antibody; and (d) determining that the individual would benefit from the candidate treatment if there is an increase in concentration relative to a control or a decrease in intracellular localization in the cytoplasm relative to a control.

[0017] In another embodiment, a method is provided herein for determining whether an individual diagnosed with Alzheimer's disease would benefit from a candidate treatment, wherein the individual has not yet initiated the treatment, and the method comprises: (a) administering the candidate treatment to the individual; (b) contacting a biological sample derived from the individual with the H1.0K180me2 protein or H1.0K180me2 peptide provided herein; (c) determining the concentration of an autoantibody in the sample that binds to the protein or peptide; and (d) determining that the individual would benefit from the candidate treatment if a decrease in concentration relative to a control is present.

[0018] Because the method relates to a method for Alzheimer's disease, in some embodiments, the measured change (increase, decrease, or no change) is relative to a threshold established by patient operating characteristic curve analysis for optimal specificity and sensitivity. In some embodiments, the biological sample is selected from the group consisting of whole blood, plasma, serum, saliva, urine, feces, synovial fluid, cerebrospinal fluid, bronchial lavage, ascites fluid, bone marrow aspirate, pleural fluid, tissue, cells, biopsy material, interstitial fluid, and lymph. In some embodiments, the individual is 60 years of age or older. In some embodiments utilizing antibodies, the antibodies are labeled. In some embodiments, the H1.0K180me2 antibody is attached to beads, columns, resin, or microplates. In some embodiments, the H1.0K180me2 protein or H1.0K180me2 peptide is labeled. In some embodiments, the H1.0K180me2 protein or H1.0K180me2 peptide is biotinylated. In some embodiments, the H1.0K180me2 protein or H1.0K180me2 peptide is attached to beads, resin, column, or microplate. In some embodiments, the H1.0K180me2 protein contains the sequence of SEQ ID NO: 2. In some embodiments, the H1.0K180me2 peptide contains any one of the sequences of SEQ ID NOs: 3 to 35. In some embodiments, the H1.0K180me2 peptide contains the sequence of SEQ ID NO: 3. In some embodiments, the concentration of an autoantibody specific to the H1.0K180me2 antigen is determined. In some embodiments, the concentration of H1.0K180me2 or H1.0K180me2 autoantibody is determined by ELISA. In some embodiments, the concentration of an IgG autoantibody is determined. In some embodiments, the concentration of an IgM autoantibody is determined. In some embodiments, the concentrations of both IgG and IgM autoantibodies are determined.

[0019] In various aspects relating to determining whether an individual diagnosed with Alzheimer's disease will benefit from a candidate treatment, and whether an individual diagnosed with Alzheimer's disease and receiving treatment for Alzheimer's disease will benefit from or continue to benefit from the treatment, the treatment may include APP synthesis inhibitors, beta-secretase inhibitors, gamma-secretase inhibitors and modulators, Aβ aggregation inhibitors, Aβ immunotherapy, cholesterol-lowering drugs, anti-tau drugs, cholinesterase inhibitors, and N-methyl-D-aspartate (NMDA). The group consists of antagonists, atypical antipsychotics, protein S-nitrosylation blockers, glucagon-like peptide-1 receptor agonists, rapamycin, rapalog, endogenous cannabinoids, cannabinoids, neuroprotective substances, molecules that regulate calcium influx, antioxidants, anti-inflammatory drugs, drugs that regulate glutamate homeostasis, autophagy inducers, hormones, hormone regulators, statins, insulin, insulin carriers, multifunctional nanocarriers, vitamins, nutritional supplements, small RNA molecules, peptides, and ultrasound therapy.

[0020] In another embodiment, a method is provided herein for determining whether an individual has been exposed to a DNA damaging agent, comprising the steps of (a) contacting a biological sample derived from the individual with one of the H1.0K180me2 antibodies provided herein, and (b) determining the concentration of the H1.0K180me2 antigen in the sample bound to the antibody, wherein an increase in concentration relative to a control indicates that the individual has been exposed to a DNA damaging agent. In some embodiments, this increase exceeds a threshold established by subject operating characteristic curve analysis for optimal specificity and sensitivity. In some embodiments, the DNA damaging agent is radiation. In some embodiments, the biological sample is selected from the group consisting of whole blood, plasma, serum, saliva, urine, synovial fluid, cerebrospinal fluid, bronchial lavage, ascites fluid, bone marrow aspirate, pleural fluid, tissue, cells, biopsy material, interstitial fluid, and lymph. In some embodiments, the antibody is labeled. In some embodiments, the antibody is attached to beads, resin, column, or microplate. In some embodiments, the concentration is determined by ELISA. In some embodiments, the antibody is at least twice as specific to the dimethylated K180 antigen than to the monomethylated antigen, where the monomethylated antigen contains a monomethylated lysine residue, and the lysine residue corresponds to K180 of the human histone H1.0 protein. In some embodiments, the antibody is at least twice as specific to the dimethylated antigen than to the trimethylated antigen, where the trimethylated antigen contains a trimethylated lysine residue, and the lysine residue corresponds to K180 of the human histone H1.0 protein. In some embodiments, the antibody is an antigen-binding fragment.

[0021] In another embodiment, a method is provided herein for determining whether an individual has been exposed to a DNA damaging agent, comprising the steps of (a) contacting a biological sample derived from the individual with one of the H1.0K180me2 proteins or H1.0K180me2 peptides provided herein, and (b) determining the concentration of an autoantibody in the sample bound to the protein or peptide, wherein an increase in concentration relative to a control indicates that the individual has been exposed to a DNA damaging agent. In some embodiments, this increase exceeds a threshold established by subject operating characteristic curve analysis for optimal specificity and sensitivity. In some embodiments, the DNA damaging agent is radiation. In some embodiments, the biological sample is selected from the group consisting of whole blood, plasma, serum, saliva, urine, synovial fluid, cerebrospinal fluid, bronchial lavage, ascites fluid, bone marrow aspirate, pleural fluid, tissue, cells, biopsy material, interstitial fluid, and lymph. In some embodiments, the protein or peptide includes labeling. In some embodiments, the protein or peptide is attached to beads, resin, column, or microplate. In some embodiments, the concentration is determined by ELISA. In some embodiments, the peptide contains one of the sequences of SEQ ID NOs: 3 to 35. In some embodiments, the protein contains the sequence of SEQ ID NO: 2. In some embodiments, the peptide contains the sequence of SEQ ID NO: 3.

[0022] In another embodiment, a method is provided herein for determining whether an individual being treated with a rapalog is responsive to such treatment, comprising the steps of (a) contacting a biological sample derived from the individual with one of the H1.0K180me2 antibodies provided herein, (b) determining the concentration of the H1.0K180me2 antigen in the sample bound to the antibody, and (c) determining whether the individual is responsive to the treatment, wherein a decrease in concentration relative to a control indicates that the individual is responsive. In some embodiments, the decrease falls below a threshold established by patient operating characteristic curve analysis for optimal specificity and sensitivity. In some embodiments, the rapalog is rapamycin, sirolimus, Rapamune, everolimus, RA 001, Afinitor, Zortress, Temsirolimus, CCI-779, Torisel, Lidaforolimus, AP23573, MK-8669, Deforolimus, Zotarolimus, ABT-578, AZD8055, AZD2014, OSI-027, MLN0128, WYE-132, Torin1, PI-103, P7170, PF The antibodies are selected from the group consisting of -04691502, PF-05212384, PKI-587, GNE477, PKI-180, WJD008, XL765, SAR245409, NVP-BEZ235, BGT226, SF1126, GSK2126458, Ku-0063794, WYE-354, NVP-BEZ235, PF-05212384, XL765, Torin 2, WYE-125132, and OSI-027. In some embodiments, the biological sample is selected from the group consisting of whole blood, plasma, serum, saliva, urine, synovial fluid, cerebrospinal fluid, bronchial lavage, ascites fluid, bone marrow aspirate, pleural fluid, tissue, cells, biopsy material, interstitial fluid, and lymph. In some embodiments, the antibodies are labeled. In some embodiments, the antibody is attached to beads, resin, column, or microplate. In some embodiments, the concentration is determined by ELISA. In some embodiments, the antibody is an antigen-binding fragment.In some embodiments, the antibody is at least twice as specific to the dimethylated K180 antigen than to the monomethylated antigen, where the monomethylated antigen comprises a monomethylated lysine residue, and the lysine residue corresponds to K180 of the human histone H1.0 protein. In some embodiments, the antibody is at least twice as specific to the dimethylated antigen than to the trimethylated antigen, where the trimethylated antigen comprises a trimethylated lysine residue, and the lysine residue corresponds to K180 of the human histone H1.0 protein.

[0023] In another embodiment, a method is provided herein for determining whether an individual being treated with a rapalog is responsive to such treatment, comprising the steps of (a) contacting a biological sample derived from the individual with an H1.0K180me2 peptide or H1.0K180me2 protein, (b) determining the concentration of an autoantibody in the sample bound to the protein or peptide, and (c) determining whether the individual is responsive to the treatment, wherein a change in the concentration of the autoantibody relative to a control indicates that the individual is responsive. In some embodiments, the change is relative to a threshold established by patient operating characteristic curve analysis for optimal specificity and sensitivity. In some embodiments, the rapalog is rapamycin, sirolimus, Rapamune, everolimus, RA 001, Afinitor, Zortress, Temsirolimus, CCI-779, Torisel, Lidaforolimus, AP23573, MK-8669, Deforolimus, Zotarolimus, ABT-578, AZD8055, AZD2014, OSI-027, MLN0128, WYE-132, Torin1, PI-103, P7170, PF The group consists of -04691502, PF-05212384, PKI-587, GNE477, PKI-180, WJD008, XL765, SAR245409, NVP-BEZ235, BGT226, SF1126, GSK2126458, Ku-0063794, WYE-354, NVP-BEZ235, PF-05212384, XL765, Torin 2, WYE-125132, and OSI-027. In some embodiments, the biological sample is selected from the group consisting of whole blood, plasma, serum, saliva, urine, synovial fluid, cerebrospinal fluid, bronchial lavage, ascites fluid, bone marrow aspirate, pleural fluid, tissue, cells, biopsy material, interstitial fluid, and lymph. In some embodiments, the protein or peptide includes labeling. In some embodiments, the protein or peptide is attached to beads, resin, column, or microplate. In some embodiments, the concentration is determined by ELISA. In some embodiments, the protein contains the sequence of SEQ ID NO: 2. In some embodiments, the peptide contains one of the sequences of SEQ ID NOs: 3 to 35.In some embodiments, the peptide includes the sequence of SEQ ID NO: 3.

[0024] In another embodiment, diagnostic kits comprising an H1.0K180me2 peptide or H1.0K180me2 protein are provided herein. In some embodiments, the protein or peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 3 to 35. In some embodiments, the protein or peptide comprises a label. In some embodiments, the protein or peptide is biotinylated. In some embodiments, the protein or peptide is attached to a solid surface. In some embodiments, the protein or peptide is attached to beads, resin, column, or microplate. In some embodiments, the protein or peptide is provided for the detection of H1.0K180me2 autoantibodies in a sample. In some embodiments, the protein or peptide is provided as a reference standard. In some embodiments, the kit further comprises an H1.0K180me2 antibody. In some embodiments, the kit is used for the detection of Alzheimer's disease, radiation exposure, exposure to genotoxic substances, or exposure to DNA damaging agents. In some embodiments, the kit is used for drug screening. In some embodiments, the kit is used for patient stratification. In some embodiments, the kit is used for treatment selection.

[0025] In another embodiment, a transdermal patch for measuring the concentration of a subcutaneous tissue target molecule is provided herein, comprising (a) a substrate comprising (1) an H1.0K180me2 antibody, or (2) an H1.0K180me2 protein or an H1.0K180me2 peptide, and (b) a plurality of microneedles. In some embodiments, the substrate comprises an H1.0K180me2 antibody. In some embodiments, the antibody is labeled. In some embodiments, the antibody is an antigen-binding fragment. In some embodiments, the substrate comprises an H1.0K180me2 protein or an H1.0K180me2 peptide. In some embodiments, the peptide is labeled. In some embodiments, the peptide is biotinylated. In some embodiments, the protein comprises the sequence of SEQ ID NO: 2. In some embodiments, the peptide comprises one of the sequences of SEQ ID NOs: 3 to 35. In some embodiments, the peptide comprises the sequence of SEQ ID NO: 3. In some embodiments, the patch is a transdermal microneedle array patch. In some embodiments, the substrate is elastic and stretchable.

[0026] In another embodiment, a portable device for determining whether an individual has been exposed to radiation or a DNA damaging agent is provided herein, comprising (a) a sample collection device, (b) a reader, (c) an assay module containing either (1) an H1.0K180me2 antibody, or (2) an H1.0K180me2 protein or H1.0K180me2 peptide, and (d) a plurality of microneedles. In some embodiments, the substrate comprises an H1.0K180me2 antibody. In some embodiments, the antibody is labeled. In some embodiments, the antibody is an antigen-binding fragment. In some embodiments, the substrate comprises an H1.0K180me2 protein or an H1.0K180me2 peptide. In some embodiments, the protein or peptide is labeled. In some embodiments, the protein or peptide is biotinylated. In some embodiments, the protein comprises the sequence of SEQ ID NO: 2. In some embodiments, the peptide comprises one of the sequences of SEQ ID NOs: 3 to 35. In some embodiments, the peptide comprises the sequence of SEQ ID NO: 3. In some embodiments, the peptide includes the sequence of SEQ ID NO: 2.

[0027] In another embodiment, a test specimen suitable for a lateral flow assay of an analyte is provided herein, comprising a sample receiving zone, wherein the sample receiving zone comprises either (1) an H1.0K180me2 antibody, or (2) an H1.0K180me2 protein or an H1.0K180me2 peptide. In some embodiments, the receiving zone comprises an H1.0K180me2 antibody. In some embodiments, the antibody is labeled. In some embodiments, the antibody is an antigen-binding fragment. In some embodiments, the receiving zone comprises an H1.0K180me2 protein or an H1.0K180me2 peptide. In some embodiments, the protein or peptide is labeled. In some embodiments, the protein or peptide is biotinylated. In some embodiments, the protein comprises the sequence of SEQ ID NO: 2. In some embodiments, the peptide comprises one of the sequences of SEQ ID NOs: 3 to 35. In some embodiments, the peptide comprises the sequence of SEQ ID NO: 3. In some embodiments, the peptide comprises the sequence of SEQ ID NO: 2.

[0028] In another embodiment, an in vitro method for dimethylating a histone H1.0 peptide or histone H1.0 protein is provided herein, comprising the step of contacting the protein or peptide with a methyltransferase enzyme and a methyl donor under conditions that produce a specifically dimethylated protein or peptide, wherein the protein or peptide is specifically dimethylated at a lysine residue corresponding to K180 of the human histone H1.0 protein, and the methyltransferase enzyme is G9A methyltransferase or GLP methyltransferase. In some embodiments, the peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 42-74. In some embodiments, the protein comprises the sequence of SEQ ID NO: 1. In some embodiments, the protein or peptide is contacted with G9A methyltransferase. In some embodiments, the protein or peptide is contacted with GLP methyltransferase. In some embodiments, the methyl donor is S-adenosyl-L-methionine. In some embodiments, the contacting step is carried out in a methylation buffer. In some embodiments, the product is dimethylated with a lysine residue corresponding to K180 of the human histone H1.0 protein by more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or even more than 99%. In related embodiments, antibodies that specifically bind to the dimethylated protein or peptide produced by the provided in vitro method are provided herein.

[0029] In another embodiment, the following histone H1.0 peptide or histone H1.0 protein is provided herein, which is specifically dimethylated at a lysine residue corresponding to K180 of a human histone H1.0 protein, produced by a method comprising contacting an H1.0 protein or H1.0 peptide with a methyltransferase enzyme and a methyl donor under conditions that enable specific dimethylation, wherein the methyltransferase enzyme is G9A methyltransferase or GLP methyltransferase. In some embodiments, the H1.0 peptide includes a sequence selected from the group consisting of SEQ ID NOs: 42-74. In some embodiments, the H1.0 protein includes the sequence of SEQ ID NO: 1. In some embodiments, the produced specifically dimethylated H1.0 protein includes the sequence of SEQ ID NO: 2. In some embodiments, the produced specifically dimethylated H1.0 peptide includes the sequences of SEQ ID NOs: 3-35. In related embodiments, antibodies that specifically bind to the dimethylated protein or peptide produced by the provided in vitro method are provided herein.

[0030] In another embodiment, a kit for in vitro methylation of an H1.0 protein or H1.0 peptide is provided herein, comprising (a) an H1.0 protein or H1.0 peptide, (b) a G9A methyltransferase or GLP methyltransferase enzyme, and a methyl donor. In some embodiments, the kit comprises an H1.0 protein, the H1.0 protein comprising the sequence of SEQ ID NO: 1. In some embodiments, the kit comprises an H1.0 peptide. In some embodiments, the H1.0 peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 42 to 74. In some embodiments, the kit comprises a G9A methyltransferase enzyme. In some embodiments, the kit comprises a GLP methyltransferase enzyme. In some embodiments, the methyl donor is S-adenosyl-L-methionine. In some embodiments, the kit further comprises a methylation buffer.

[0031] In another embodiment, a complex comprising a histone H1.0 peptide and a methyltransferase enzyme, which is in vitro, is provided herein. In some embodiments, the H1.0 peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 42 to 74. In some embodiments, the methyltransferase enzyme is a G9A methyltransferase enzyme. In some embodiments, the methyltransferase enzyme is a GLP methyltransferase enzyme. In certain embodiments, for example, the following items are provided: (Item 1) An antibody that specifically binds to a dimethylated antigen, wherein the dimethylated antigen contains a dimethylated lysine residue, the lysine residue corresponds to K180 of human histone H1.0, and the dimethylated lysine residue is required for binding. (Item 2) The antibody described in item 1, wherein the dimethylated antigen does not contain any other methylated lysine residues. (Item 3) The antibody described in item 1, which does not bind or binds minimally if the antigen contains a dimethylated lysine residue at the lysine residues corresponding to K166, K172, K174, K175, and / or K177 of the human histone H1.0 protein. (Item 4) The antibody described in item 1, which does not bind or binds minimally if the antigen contains a monomethylated lysine residue in the lysine residues corresponding to K166, K172, K174, K175, K177, and / or K180 of the human histone H1.0 protein. (Item 5) The antibody described in item 1, which does not bind or binds minimally if the antigen contains a trimethylated lysine residue in the lysine residues corresponding to K166, K172, K174, K175, K177, and / or K180 of the human histone H1.0 protein. (Item 6) The antibody according to item 1, wherein the antibody is at least twice as specific to the dimethylated antigen as to the monomethylated antigen, the monomethylated antigen comprises a monomethylated lysine residue, and the lysine residue corresponds to K180 of the human histone H1.0 protein. (Item 7) The antibody according to item 1, wherein the antibody is at least twice as specific to the dimethylated antigen as to the trimethylated antigen, the trimethylated antigen comprises a trimethylated lysine residue, and the lysine residue corresponds to K180 of the human histone H1.0 protein. (Item 8) The antibody described in item 1, including the label. (Item 9) The biotinylated antibody described in item 8. (Item 10) Antibodies described in item 1, attached to a solid surface. (Item 11) Antibodies as described in item 10, attached to beads, columns, resin, or microplates. (Item 12) An antibody as described in item 1, conjugated to at least one therapeutic agent selected from the group consisting of radionuclides, cytotoxins, chemotherapeutic agents, drugs, prodrugs, toxins, enzymes, immunomodulators, apoptosis promoters, cytokines, hormones, oligonucleotides, antisense molecules, siRNAs, and secondary antibodies. (Item 13) The antibody described in item 1, which can remove H1.0K180me2 from the sample. (Item 14) The antibody described in item 1, which can remove cells containing H1.0K180me2. (Item 15) The antibody described in item 1, which can remove senescent cells. (Item 16) A synthetic histone H1.0 peptide or synthetic histone H1.0 protein containing a dimethylated lysine residue, wherein the dimethylated lysine residue corresponds to K180 of human histone H1.0. (Item 17) The peptide according to item 16, wherein the protein or peptide includes a label. (Item 18) The peptide according to item 16, wherein the protein or peptide is biotinylated. (Item 19) The peptide according to item 16, wherein the protein or peptide does not contain any other dimethylated lysine residues. (Item 20) The peptide described in item 16, wherein the protein or peptide does not contain any other methylated lysine residues. (Item 21) The peptide according to item 16, wherein the protein or peptide is attached to beads, microplates, columns, or resin. (Item 22) A peptide listed in item 16, containing one of the sequences of sequence numbers 3 to 35. (Item 23) The peptide described in item 22, containing the sequence of sequence number 3. (Item 24) The peptide described in item 16, wherein the protein contains the sequence of SEQ ID NO: 2. (Item 25) The peptide according to item 16, wherein the protein or peptide is conjugated to at least one therapeutic agent selected from the group consisting of radionuclides, cytotoxins, chemotherapeutic agents, drugs, prodrugs, toxins, enzymes, immunomodulators, apoptosis promoters, cytokines, hormones, oligonucleotides, antisense molecules, siRNA, and secondary antibodies. (Item 26) The peptide described in item 16, wherein the protein or peptide can remove or block the H1.0K180me2 autoantibody. (Item 27) The peptide described in item 26, wherein the protein or peptide can remove or block the H1.0K180me2 IgG autoantibody. (Item 28) The peptide described in item 26, wherein the protein or peptide can remove or block the H1.0K180me2 IgM autoantibody. (Item 29) A method for determining whether an individual has or is at risk of developing Alzheimer's disease, (a) The step of contacting the biological sample derived from the individual with the antibody described in item 1, (b) A step of determining the concentration of the dimethylated antigen in the sample that is bound to the antibody, wherein a decrease in the concentration compared to a control indicates that the individual has or is at risk of developing Alzheimer's disease. A method that includes this. (Item 30) The method according to item 29, wherein if the serum concentration of histone H1.0 protein is less than 5.61 nmol / ml or lower, the individual has or is at risk of developing Alzheimer's disease. (Item 31) The method according to item 29, wherein the aforementioned decrease falls below a threshold established by patient operating characteristic curve analysis for optimal specificity and sensitivity. (Item 32) The method according to item 29, wherein if the PLR(LR+) is 3.6 or greater, the individual has or is at risk of developing Alzheimer's disease. (Item 33) The method according to item 44, wherein if the NLR(LR-) is 0.26 or less, the individual is not at risk of developing Alzheimer's disease. (Item 34) The method according to item 29, further comprising the step of treating the individual with an Alzheimer's disease drug or regimen if the individual is determined to have or be at risk of developing Alzheimer's disease. (Item 35) A method for determining whether an individual diagnosed with Alzheimer's disease and receiving treatment for Alzheimer's disease benefits from or continues to benefit from said treatment, (a) The step of contacting the biological sample derived from the individual with the antibody described in item 1, (b) A step of determining the concentration of the dimethylated antigen in the sample that binds to the antibody, (c) If an increase in concentration relative to the control exists, the step of determining whether the individual benefits from or continues to benefit from the treatment. A method that includes this. (Item 36) The method according to item 35, wherein the increase exceeds a threshold established by patient operating characteristic curve analysis for optimal specificity and sensitivity. (Item 37) The method of item 35, further comprising the step of treating the individual with an Alzheimer's disease drug or regimen if it is determined that the individual benefits from or continues to benefit from the treatment. (Item 38) A method for determining whether an individual diagnosed with Alzheimer's disease would benefit from a candidate treatment, wherein the individual has not yet initiated the treatment, and the method (a) A step of administering the candidate treatment to the individual, (b) The step of contacting the biological sample derived from the individual after administration of the candidate treatment with the antibody described in item 1, (c) A step of determining the concentration and / or intracellular localization of the dimethylated antigen in the sample that binds to the antibody, (d) If there is an increase in the concentration or a decrease in intracellular localization to the cytoplasm compared to the control, the step of determining that the individual will benefit from the candidate treatment. Methods that include... (Item 39) The method according to item 38, wherein the increase in the concentration exceeds a threshold established by patient operating characteristic curve analysis for optimal specificity and sensitivity. (Item 40) The method according to item 38, further comprising the step of treating the individual with an Alzheimer's disease drug or regimen if it is determined that the individual would benefit from the treatment. (Item 41) A method for determining whether an individual diagnosed with Alzheimer's disease would benefit from a candidate treatment, wherein the individual has not yet initiated the treatment, and the method (a) A step of bringing a biological sample derived from the individual into contact with a candidate treatment, (b) The step of contacting the biological sample with the antibody described in item 1, (c) A step of determining the concentration and / or intracellular localization of the dimethylated antigen in the sample that binds to the antibody, (d) If there is an increase in the concentration compared to the control or a decrease in intracellular localization in the cytoplasm compared to the control, the step of determining that the individual will benefit from the candidate treatment. Methods that include... (Item 42) The method according to item 41, wherein the increase is relative to a threshold established by patient operating characteristic curve analysis for optimal specificity and sensitivity. (Item 43) The method according to item 41, further comprising the step of treating the individual with an Alzheimer's disease drug or regimen if it is determined that the individual would benefit from the treatment. (Item 44) The method according to any one of items 38 to 42, wherein the treatment is selected from the group consisting of APP synthesis inhibitors, beta-secretase inhibitors, gamma-secretase inhibitors and modulators, Aβ aggregation inhibitors, Aβ immunotherapy, cholesterol-lowering agents, antitau agents, cholinesterase inhibitors, N-methyl-D-aspartate (NMDA) antagonists, atypical antipsychotics, protein S-nitrosylation blockers, glucagon-like peptide-1 receptor agonists, rapamycin, rapalog, endogenous cannabinoids, cannabinoids, neuroprotective substances, molecules that regulate calcium influx, antioxidants, anti-inflammatory agents, drugs that regulate glutamate homeostasis, autophagy inducers, hormones, hormone regulators, statins, insulin, insulin carriers, multifunctional nanocarriers, vitamins, nutritional supplements, small RNA molecules, peptides, and ultrasound therapy. (Item 45) The method according to any one of items 29 to 44, wherein the biological sample is selected from the group consisting of whole blood, plasma, serum, saliva, urine, feces, synovial fluid, cerebrospinal fluid, bronchial lavage, ascites fluid, bone marrow aspirate, pleural fluid, tissue, cells, biopsy material, interstitial fluid, and lymph fluid. (Item 46) The method according to any one of items 29 to 45, wherein the individual is 60 years of age or older. (Item 47) The method according to any one of items 29 to 46, wherein the antibody is labeled. (Item 48) The method according to any one of items 29 to 47, wherein the antibody is attached to beads, microplates, columns, or resin. (Item 49) The method according to any one of items 29 to 48, wherein the concentration is determined by ELISA. (Item 50) The method according to any one of items 29 to 49, wherein the antibody is at least twice as specific to the dimethylated antigen than to the monomethylated antigen, the monomethylated antigen comprises a monomethylated lysine residue, and the lysine residue corresponds to K180 of the human histone H1.0 protein. (Item 51) The method according to any one of items 29 to 49, wherein the antibody is at least twice as specific to the dimethylated antigen than to the trimethylated antigen, the trimethylated antigen comprises a trimethylated lysine residue, and the lysine residue corresponds to K180 of the human histone H1.0 protein. (Item 52) A method for determining whether an individual has or is at risk of developing Alzheimer's disease, (a) The step of contacting the biological sample derived from the individual with the protein or peptide described in item 16, (b) A step of determining the concentration of an autoantibody in the sample that is bound to the protein or peptide, wherein an increase in the concentration compared to a control indicates that the individual has or is at risk of developing Alzheimer's disease. A method that includes this. (Item 53) The method according to item 52, comprising the step of determining the concentration of an IgM autoantibody in the sample that binds to the protein or peptide. (Item 54) The method according to item 52, comprising the step of determining the concentration of an IgG autoantibody in the sample that is bound to the protein or peptide. (Item 55) The method according to item 52, wherein if the serum concentration of IgG autoantibodies normalized to total IgG levels is higher than or equal to 9.69 ug / ml, the individual has or is at risk of developing Alzheimer's disease. (Item 56) The method according to item 52, wherein if the serum concentration of IgG autoantibodies normalized to serum volume is higher than or equal to 8.23 ​​ug / ml, the individual has or is at risk of developing Alzheimer's disease. (Item 57) The method according to item 52, wherein if the serum concentration of IgM autoantibodies normalized to serum volume is higher than or equal to 409 fMol / ml, the individual has or is at risk of developing Alzheimer's disease. (Item 58) The ratio of the concentration of IgM autoantibodies to the total IgM concentration is 26.6 × 10⁻⁶. -6 The method according to item 52, wherein, if greater than, the individual has or is at risk of developing Alzheimer's disease. (Item 59) The method according to item 52, wherein the increase exceeds a threshold established by patient operating characteristic curve analysis for optimal specificity and sensitivity. (Item 60) The method according to item 52, wherein the individual has or is at risk of developing Alzheimer's disease if PLR(LR+) is 2.4 or greater for IgG autoantibodies, or if PLR(LR+) is 3.5 or greater for IgM autoantibodies. (Item 61) The method according to item 52, comprising the steps of determining the concentration of an IgM autoantibody in the sample that is bound to the protein or peptide, and comparing the measured value with the total IgM concentration in the sample. (Item 62) The method according to item 52, further comprising the step of treating the individual with an Alzheimer's disease drug or regimen if the individual is determined to have or be at risk of developing Alzheimer's disease. (Item 63) A method for determining whether an individual diagnosed with Alzheimer's disease and receiving treatment for Alzheimer's disease benefits from or continues to benefit from said treatment, (a) The step of contacting the biological sample derived from the individual with the protein or peptide described in item 16, (b) A step of determining the concentration of the autoantibody in the sample that is bound to the protein or peptide, (c) If a decrease in the concentration relative to the control exists, the step of determining that the individual will benefit from or continue to benefit from the treatment. A method that includes this. (Item 64) The method according to item 63, wherein the reduction falls below a threshold established by patient operating characteristic curve analysis for optimal specificity and sensitivity. (Item 65) The method according to item 63, comprising the step of determining the concentration of an IgM autoantibody in the sample that binds to the protein or peptide. (Item 66) The method according to item 63, comprising the step of determining the concentration of an IgG autoantibody in the sample that is bound to the protein or peptide. (Item 67) A method for determining whether an individual diagnosed with Alzheimer's disease would benefit from a candidate treatment, wherein the individual has not yet initiated the treatment, and the method (a) A step of administering the candidate treatment to the individual, (b) The step of contacting the biological sample derived from the individual with the protein or peptide described in item 16, (c) A step of determining the concentration of the autoantibody in the sample that is bound to the protein or peptide, (d) If a decrease in concentration relative to the control exists, the step of determining that the individual will benefit from the candidate treatment. Methods that include... (Item 68) The method of item 67, wherein the reduction falls below a threshold established by patient operating characteristic curve analysis for optimal specificity and sensitivity. (Item 69) The method according to item 67, comprising the step of determining the concentration of an IgM autoantibody in the sample that is bound to the protein or peptide. (Item 70) The method according to item 67, comprising the step of determining the concentration of an IgG autoantibody in the sample that is bound to the protein or peptide. (Item 71) The method according to any one of items 63 to 70, wherein the treatment is selected from the group consisting of APP synthesis inhibitors, beta-secretase inhibitors, gamma-secretase inhibitors and modulators, Aβ aggregation inhibitors, Aβ immunotherapy, cholesterol-lowering agents, antitau agents, cholinesterase inhibitors, N-methyl-D-aspartate (NMDA) antagonists, atypical antipsychotics, S-nitrosylation blockers of proteins, glucagon-like peptide-1 receptor agonists, rapamycin, rapalog, endogenous cannabinoids, cannabinoids, neuroprotective substances, molecules that regulate calcium influx, antioxidants, anti-inflammatory drugs, drugs that regulate glutamate homeostasis, autophagy inducers, hormones, hormone regulators, statins, insulin, insulin carriers, multifunctional nanocarriers, vitamins, nutritional supplements, small RNA molecules, peptides, and ultrasound therapy. (Item 72) The method according to any one of items 52 to 71, wherein the biological sample is selected from the group consisting of whole blood, plasma, serum, saliva, urine, feces, synovial fluid, cerebrospinal fluid, bronchial lavage, ascites fluid, bone marrow aspirate, pleural fluid, tissue, cells, biopsy material, interstitial fluid, and lymph fluid. (Item 73) The method according to any one of items 52 to 72, wherein the individual is 60 years of age or older. (Item 74) The method according to any one of items 52 to 73, wherein the protein or peptide is labeled. (Item 75) The method according to any one of items 52 to 74, wherein the protein or peptide is biotinylated. (Item 76) The method according to any one of items 52 to 75, wherein the protein or peptide is attached to beads, microplates, columns, or resin. (Item 77) The method according to any one of items 52 to 76, wherein the peptide comprises one of the sequences of sequence numbers 3 to 35. (Item 78) The method according to any one of items 52 to 76, wherein the peptide comprises the sequence of SEQ ID NO: 3. (Item 79) The method according to any one of items 52 to 76, wherein the protein comprises the sequence of Sequence ID No. 2. (Item 80) The method according to any one of items 52 to 79, wherein the concentration of an autoantibody specific to H1.0K180me2 is determined. (Item 81) The method described in any one of items 52 to 79, which determines the concentration of IgG autoantibodies. (Item 82) The method described in any one of items 52 to 79, which determines the concentration of IgM autoantibodies. (Item 83) The method according to any one of items 52 to 79, wherein the concentrations of IgG and IgM autoantibodies are determined. (Item 84) The method according to any one of items 52 to 79, wherein the total IgG and / or IgM concentrations are determined. (Item 85) The method according to any one of items 52 to 79, wherein the ratio of the concentration of IgG autoantibodies to the concentration of total IgG is determined. (Item 86) The method according to any one of items 52 to 79, wherein the concentration of IgM autoantibodies relative to the total IgM concentration is determined. (Item 87) A method for determining whether an individual has been exposed to a DNA damaging agent, (a) The step of contacting the biological sample derived from the individual with the antibody described in item 1, (b) A step of determining the concentration of the dimethylated antigen in the sample that is bound to the antibody, wherein an increase in the concentration relative to a control indicates that the individual has been exposed to a DNA damaging agent. A method that includes this. (Item 88) The method according to item 87, wherein the increase exceeds a threshold established by patient operating characteristic curve analysis for optimal specificity and sensitivity. (Item 89) The method according to item 87, wherein the DNA damaging agent is radiation. (Item 90) The method according to item 87, wherein the biological sample is selected from the group consisting of whole blood, plasma, serum, saliva, urine, synovial fluid, cerebrospinal fluid, bronchial lavage, ascites fluid, bone marrow aspirate, pleural fluid, tissue, cells, biopsy material, interstitial fluid, and lymph fluid. (Item 91) The method according to item 87, wherein the antibody includes a label. (Item 92) The method according to item 87, wherein the antibody is attached to beads, microplates, columns, or resin. (Item 93) The method according to item 87, wherein the concentration is determined by ELISA. (Item 94) The method according to item 87, wherein the antibody is at least twice as specific to the dimethylated antigen than to the monomethylated antigen, the monomethylated antigen comprises a monomethylated lysine residue, and the lysine residue corresponds to K180 of the human histone H1.0 protein. (Item 95) The method according to item 87, wherein the antibody is at least twice as specific to the dimethylated antigen than to the trimethylated antigen, the trimethylated antigen comprises a trimethylated lysine residue, and the lysine residue corresponds to K180 of the human histone H1.0 protein. (Item 96) A method for determining whether an individual has been exposed to a DNA damaging agent, (a) The step of contacting the biological sample derived from the individual with the protein or peptide described in item 16, (b) A step of determining the concentration of an autoantibody in the sample that is bound to the protein or peptide, wherein an increase in the concentration relative to a control indicates that the individual has been exposed to a DNA damaging agent. A method that includes this. (Item 97) The method of item 96, wherein the increase exceeds a threshold established by patient operating characteristic curve analysis for optimal specificity and sensitivity. (Item 98) The method according to item 96, comprising the step of determining the concentration of an IgM autoantibody in the sample that is bound to the protein or peptide. (Item 99) The method according to item 96, comprising the step of determining the concentration of an IgG autoantibody in the sample that is bound to the protein or peptide. (Item 100) The method according to item 96, wherein the DNA damaging agent is radiation. (Item 101) The method according to item 96, wherein the biological sample is selected from the group consisting of whole blood, plasma, serum, saliva, urine, synovial fluid, cerebrospinal fluid, bronchial lavage, ascites fluid, bone marrow aspirate, pleural fluid, tissue, cells, biopsy material, interstitial fluid, and lymph fluid. (Item 102) The method according to item 96, wherein the protein or peptide is labeled. (Item 103) The method according to item 96, wherein the protein or peptide is attached to beads, microplates, columns, or resin. (Item 104) The method according to item 96, wherein the concentration is determined by ELISA. (Item 105) The method according to item 96, wherein the peptide comprises one of the sequences of sequence numbers 3 to 35. (Item 106) The method according to item 105, wherein the peptide comprises the sequence of SEQ ID NO: 3. (Item 107) The method according to item 96, wherein the protein comprises the sequence of SEQ ID NO: 2. (Item 108) A method for determining whether an individual receiving treatment with a laparog is responsive to such treatment, (a) The step of contacting the biological sample derived from the individual with the antibody described in item 1, (b) A step of determining the concentration of the dimethylated antigen in the sample that binds to the antibody, (c) A step of determining whether the individual is responsive to the treatment, wherein a decrease in the concentration relative to the control indicates that the individual is responsive. A method that includes this. (Item 109) The method according to item 108, wherein the reduction falls below a threshold established by patient operating characteristic curve analysis for optimal specificity and sensitivity. (Item 110) The aforementioned rapagnoses include rapamycin, sirolimus, Rapamune, everolimus, and RA. 001, Afinitor, Zortress, Temsirolimus, CCI-779, Torisel, Lidaforolimus, AP23573, MK-8669, Deforolimus, Zotarolimus, ABT-578, AZD8055, AZD2014, OSI-027, MLN0128, WYE-132, Torin1, PI-103, P7170, PF The method described in item 108, selected from the group consisting of -04691502, PF-05212384, PKI-587, GNE477, PKI-180, WJD008, XL765, SAR245409, NVP-BEZ235, BGT226, SF1126, GSK2126458, Ku-0063794, WYE-354, NVP-BEZ235, PF-05212384, XL765, Torin 2, WYE-125132, and OSI-027. (Item 111) The method according to item 108, wherein the biological sample is selected from the group consisting of whole blood, plasma, serum, saliva, urine, synovial fluid, cerebrospinal fluid, bronchial lavage, ascites fluid, bone marrow aspirate, pleural fluid, tissue, cells, biopsy material, interstitial fluid, and lymph fluid. (Item 112) The method according to item 108, wherein the antibody is labeled. (Item 113) The method according to item 108, wherein the antibody is attached to beads, microplates, columns, or resin. (Item 114) The method according to item 108, wherein the concentration is determined by ELISA. (Item 115) The method according to item 108, wherein the antibody is at least twice as specific to the dimethylated antigen than to the monomethylated antigen, the monomethylated antigen comprises a monomethylated lysine residue, and the lysine residue corresponds to K180 of the human histone H1.0 protein. (Item 116) The method according to item 108, wherein the antibody is at least twice as specific to the dimethylated antigen than to the trimethylated antigen, the trimethylated antigen comprises a trimethylated lysine residue, and the lysine residue corresponds to K180 of the human histone H1.0 protein. (Item 117) A method for determining whether an individual receiving treatment with a laparog is responsive to such treatment, (a) The step of contacting the biological sample derived from the individual with the protein or peptide described in item 16, (b) A step of determining the concentration of the autoantibody in the sample that is bound to the protein or peptide, (c) A step of determining whether the individual is responsive to treatment, wherein a change in the concentration of autoantibodies against a control indicates that the individual is responsive. A method that includes this. (Item 118) The method according to item 117, wherein the change is relative to a threshold established by patient operating characteristic curve analysis for optimal specificity and sensitivity. (Item 119) The method according to item 117, comprising the step of determining the concentration of an IgM autoantibody in the sample that binds to the protein or peptide. (Item 120) The method according to item 117, comprising the step of determining the concentration of an IgG autoantibody in the sample that is bound to the protein or peptide. (Item 121) The aforementioned rapagnoses include rapamycin, sirolimus, Rapamune, everolimus, and RA. 001, Afinitor, Zortress, Temsirolimus, CCI-779, Torisel, Lidaforolimus, AP23573, MK-8669, Deforolimus, Zotarolimus, ABT-578, AZD8055, AZD2014, OSI-027, MLN0128, WYE-132, Torin1, PI-103, P7170, PF The method described in item 117, selected from the group consisting of -04691502, PF-05212384, PKI-587, GNE477, PKI-180, WJD008, XL765, SAR245409, NVP-BEZ235, BGT226, SF1126, GSK2126458, Ku-0063794, WYE-354, NVP-BEZ235, PF-05212384, XL765, Torin 2, WYE-125132, and OSI-027. (Item 122) The method according to item 117, wherein the biological sample is selected from the group consisting of whole blood, plasma, serum, saliva, urine, synovial fluid, cerebrospinal fluid, bronchial lavage, ascites fluid, bone marrow aspirate, pleural fluid, tissue, cells, biopsy material, interstitial fluid, and lymph fluid. (Item 123) The method according to item 117, wherein the protein or peptide is labeled. (Item 124) The method according to item 117, wherein the protein or peptide is attached to beads, microplates, columns, or resin. (Item 125) The method according to item 117, wherein the aforementioned concentration is determined by ELISA. (Item 126) The method according to item 117, wherein the peptide comprises one of the sequences of sequence numbers 3 to 35. (Item 127) The method according to item 126, wherein the peptide comprises the sequence of SEQ ID NO: 3. (Item 128) The method according to item 117, wherein the protein comprises the sequence of sequence number 2. (Item 129) A diagnostic kit containing the protein or peptide described in item 16. (Item 130) The kit according to item 129, wherein the peptide comprises a sequence selected from the group consisting of SEQ ID NOs. 3 to 35. (Item 131) The kit according to item 130, wherein the protein or peptide is labeled. (Item 132) The kit according to item 131, wherein the protein or peptide is biotinylated. (Item 133) The kit according to item 129, wherein the protein or peptide is attached to a solid surface. (Item 134) The kit described in item 133, wherein the protein or peptide is attached to beads, microplates, columns, or resin. (Item 135) The kit described in item 129 provides the aforementioned protein or peptide for the detection of H1.0K180me2 autoantibodies in a sample. (Item 136) The aforementioned protein or peptide is provided as a reference standard in the kit described in item 130. (Item 137) The kit described in item 129 further contains the H1.0K180me2 antibody. (Item 138) The kit described in item 129, used for the detection of Alzheimer's disease, radiation exposure, exposure to genotoxic substances, or exposure to DNA damaging agents. (Item 139) A kit described in item 129, used for drug screening. (Item 140) The kit described in item 129, used for patient stratification. (Item 141) The kit described in item 129, used for selecting a treatment. (Item 142) A transdermal patch for measuring the concentration of subcutaneous tissue target molecules, (a) A substrate comprising either the antibody described in item 1 (1) or the protein or peptide described in item 16 (2), and (b) Multiple microneedles A transdermal patch containing [the specified ingredient]. (Item 143) The patch according to item 142, wherein the substrate comprises the antibody described in item 1. (Item 144) The aforementioned antibody is a patch as described in item 143, which includes a label. (Item 145) The patch according to item 142, wherein the substrate comprises the protein or peptide described in item 16. (Item 146) The aforementioned peptide is a patch according to item 145, which includes a label. (Item 147) The patch described in item 146, wherein the peptide is biotinylated. (Item 148) The patch described in item 145, wherein the peptide contains one of the sequences of sequence numbers 3 to 35. (Item 149) The patch described in item 148, wherein the peptide contains the sequence of SEQ ID NO: 3. (Item 150) The patch described in item 142, wherein the protein contains the sequence of sequence number 2. (Item 151) The patch described in item 142 is a transdermal microneedle array patch. (Item 152) The patch according to item 142, wherein the substrate is elastic and stretchable. (Item 153) A portable device for determining whether an individual has been exposed to radiation or a DNA damaging agent, (a) Sample collection device, (b) Reader, (c)(1) an assay module comprising either the antibody described in item 1, or (2) the protein or peptide described in item 16, and (d) Multiple microneedles A device that includes this. (Item 154) The apparatus described in item 153, wherein the substrate contains the antibody described in item 1. (Item 155) The apparatus according to item 154, wherein the antibody includes a label. (Item 156) The apparatus according to item 153, wherein the substrate contains the protein or peptide described in item 16. (Item 157) The apparatus according to item 153, wherein the protein or peptide is labeled. (Item 158) The apparatus according to item 157, wherein the protein or peptide is biotinylated. (Item 159) The apparatus according to item 153, wherein the peptide comprises one of the sequences of sequence numbers 3 to 35. (Item 160) The apparatus according to item 153, wherein the peptide comprises the sequence of Sequence ID No. 3. (Item 161) The apparatus according to item 153, wherein the protein comprises the sequence of Sequence ID No. 2. (Item 162) A test specimen suitable for a lateral flow assay of an analyte, comprising a sample-receiving zone, wherein the sample-receiving zone contains either (1) the antibody described in item 1, or (2) the protein or peptide described in item 16. (Item 163) The test specimen according to item 162, wherein the receptor zone contains the antibody described in item 1. (Item 164) The antibody is a test specimen according to item 163, including a label. (Item 165) The test specimen according to item 162, wherein the receptor zone contains the protein or peptide described in item 16. (Item 166) The test specimen according to item 162, wherein the protein or peptide is labeled. (Item 167) The test specimen according to item 162, wherein the protein or peptide is biotinylated. (Item 168) The test specimen according to item 162, wherein the peptide contains one of the sequences of sequence numbers 3 to 35. (Item 169) The test specimen according to item 168, wherein the peptide contains the sequence of SEQ ID NO: 3. (Item 170) The test specimen according to item 162, wherein the protein contains the sequence of sequence number 2. (Item 171) A method for treating a methylation H1.0-related disease or condition in an individual, comprising the step of administering to the individual a therapeutically effective amount of an antibody described in any one of items 1 to 15. (Item 172) The method according to item 171, wherein the disease or condition is selected from the group consisting of Alzheimer's disease, diseases or conditions involving radiation exposure, exposure to genotoxic stressors, accumulation of senescent cells, and diseases or conditions accompanied by elevated levels of H1.0K180me2 protein or peptide. (Item 173) A method for treating a methylation H1.0-related disease or condition in an individual, comprising the step of administering to the individual a therapeutically effective amount of one of the proteins or peptides listed in items 16 to 28. (Item 174) The method according to item 173, wherein the disease or condition is selected from the group consisting of Alzheimer's disease, radiation exposure, exposure to genotoxic stressors, disease or condition involving the accumulation of senescent cells, or disease or condition accompanied by elevated levels of H1.0K180me2 autoantibodies. (Item 175) A method for removing H1.0K180me2 from an individual, comprising the step of administering to the individual a therapeutically effective amount of an antibody described in any one of items 1 to 15. (Item 176) The method according to item 175, wherein the individual suffers from a disease or condition selected from the group consisting of Alzheimer's disease, radiation exposure, exposure to genotoxic substances, exposure to DNA damaging agents, and accumulation of senescent cells. (Item 177) A method for removing, blocking, or neutralizing an H1.0K180me2 autoantibody from an individual, comprising the step of administering to the individual a therapeutically effective amount of one of the proteins or peptides listed in items 16 to 28. (Item 178) The method according to item 177, wherein the individual suffers from a disease or condition selected from the group consisting of Alzheimer's disease, radiation exposure, exposure to genotoxic stressors, accumulation of senescent cells, and diseases and conditions accompanied by elevated levels of H1.0K180me2 autoantibodies. (Item 179) A pharmaceutical composition comprising one of the antibodies, proteins, or peptides listed in items 1 to 28, and a pharmaceutically acceptable excipient. (Item 180) A sterile composition as described in item 179. (Item 181) A kit comprising one of the antibodies, proteins, peptides, or compositions described in items 1 to 28 or 179 to 180. (Item 182) A product comprising any one of the compositions described in items 1 to 28 or 179 to 181. [Brief explanation of the drawing]

[0032] [Figure 1A] Figures 1A and 1B show the identification of a histone H1.0 protein (H1.0K180me2) containing dimethylated lysine at residue 180 as a post-translational modification in cellular senescence of hADSCs using discovery-based mass spectrometry. Figure 1A shows the discovery pipeline by mass spectrometry. Figure 1B shows the entire mass spectrometry spectrum for the unmodified dimethylated peptide. [Figure 1B]Figures 1A and 1B show the identification of a histone H1.0 protein (H1.0K180me2) containing dimethylated lysine at residue 180 as a post-translational modification in cellular senescence of hADSCs using discovery-based mass spectrometry. Figure 1A shows the discovery pipeline by mass spectrometry. Figure 1B shows the entire mass spectrometry spectrum for the unmodified dimethylated peptide.

[0033] [Figure 2A] Figures 2A-2B show the expression levels of six distinct variants of histone H1 (Figure 2A) and RNA-seq reads (Figure 2B) from H1.0 in SR (self-renewal) and REP-SEN (replica-senescence) hADSCs. [Figure 2B] Figures 2A-2B show the expression levels of six distinct variants of histone H1 (Figure 2A) and RNA-seq reads (Figure 2B) from H1.0 in SR (self-renewal) and REP-SEN (replica-senescence) hADSCs.

[0034] [Figure 3A-C] Figures 3A–D show the specificity of the H1.0K180me2 antibody provided herein. Figure 3A shows amino acid-adjacent histone H1.0K180 compared to different methylation sites of other lysine residues in the H1.0K180me2 and H3.0 proteins. Figures 3B and 3C show the results of a slot blot assay demonstrating the specificity of the antibody against H1.0K180me2. Figure 3D demonstrates the specificity of the antibody against H1.0K180me2. Figure 3E shows a ClustalW2 alignment of the histone H1 variant protein sequence, showing the embedding of H1.0K180 in a unique sequence of amino acids not present in other H1 variants. [Figure 3D]Figures 3A–D show the specificity of the H1.0K180me2 antibody provided herein. Figure 3A shows amino acid-adjacent histone H1.0K180 compared to different methylation sites of other lysine residues in the H1.0K180me2 and H3.0 proteins. Figures 3B and 3C show the results of a slot blot assay demonstrating the specificity of the antibody against H1.0K180me2. Figure 3D demonstrates the specificity of the antibody against H1.0K180me2. Figure 3E shows a ClustalW2 alignment of the histone H1 variant protein sequence, showing the embedding of H1.0K180 in a unique sequence of amino acids not present in other H1 variants. [Figure 3E] Figures 3A–D show the specificity of the H1.0K180me2 antibody provided herein. Figure 3A shows amino acid-adjacent histone H1.0K180 compared to different methylation sites of other lysine residues in the H1.0K180me2 and H3.0 proteins. Figures 3B and 3C show the results of a slot blot assay demonstrating the specificity of the antibody against H1.0K180me2. Figure 3D demonstrates the specificity of the antibody against H1.0K180me2. Figure 3E shows a ClustalW2 alignment of the histone H1 variant protein sequence, showing the embedding of H1.0K180 in a unique sequence of amino acids not present in other H1 variants.

[0035] [Figure 4] Figure 4 shows the use of indirect ELISA with labeled H1.0K180me2 peptide for the detection of H1.0K180me2 autoantibodies in body fluid samples.

[0036] [Figure 5A] Figure 5A shows the use of sandwich ELISA for the direct detection of the H1.0K180me2 antigen using antibodies against the H1.0K180me2 epitope in a body fluid sample. Figure 5B shows the standard curve for the sandwich ELISA test for the H1.0K180me2 antigen. [Figure 5B]Figure 5A shows the use of sandwich ELISA for the direct detection of the H1.0K180me2 antigen using antibodies against the H1.0K180me2 epitope in a body fluid sample. Figure 5B shows the standard curve for the sandwich ELISA test for the H1.0K180me2 antigen.

[0037] [Figure 6] Figure 6 shows that H1.0 mRNA expression increases after aging induced by acute DNA damage and genotoxic stress.

[0038] [Figure 7A] Figure 7A shows the dimethylation of H1.0K180 (H1.0K180me2) in chromatin immediately after DNA damage, as determined by Western blot analysis. Figure 7B shows the secretion of H1.0K180me2 after DNA damage. [Figure 7B] Figure 7A shows the dimethylation of H1.0K180 (H1.0K180me2) in chromatin immediately after DNA damage, as determined by Western blot analysis. Figure 7B shows the secretion of H1.0K180me2 after DNA damage.

[0039] [Figure 8] Figures 8A and 8B show that dimethylation of H1.0K180 occurs upstream of PARP-1 activity. Figure 8A shows a Western blot analysis using the H1.0K180me2 antibody, revealing that methylation of H1.0K180 occurs upstream of PARP-1 functional activity in the DNA damage repair pathway. Figure 8B evaluates the inhibition of PARP activity by the PARP-1 inhibitor AG14361, supporting the idea that methylation of H1.0K180 occurs upstream of PARP-1 functional activity in the DNA damage repair pathway.

[0040] [Figure 9]Figures 9A and 9B show LC-MS / MS analysis (Figure 9A) and slot blot analysis (Figure 9B) of hADSCs, acute DNA damage, and genotoxic stress-induced aging in SR cells, revealing that dimethylated H1.0K180 (H1.0K180me2) is released from chromatin after genotoxic stress-induced aging (Figure 9A) and secreted from cells into the extracellular matrix / cell culture medium (Figure 9B).

[0041] [Figure 10A] Figures 10A–10C show the effect of ionizing radiation on H1.0K180me2 levels. Figure 10A shows that exposure to ionizing radiation induces an increase in circulating H1.0K180me2 levels in mouse serum. Figures 10A and 10B show slot blot analysis of the presence (Figure 10A) and quantification (Figure 10B) of H1.0K180me2 using antibodies specific to the H1.0K180me2 epitope in serum. Figure 10C shows Western blot analysis of the presence of the H1.0K180me2 epitope level in mouse serum after X-ray irradiation (7 Gy), using antibodies specific to the H1.0K180me2 epitope. [Figure 10B-C] Figures 10A–10C show the effect of ionizing radiation on H1.0K180me2 levels. Figure 10A shows that exposure to ionizing radiation induces an increase in circulating H1.0K180me2 levels in mouse serum. Figures 10A and 10B show slot blot analysis of the presence (Figure 10A) and quantification (Figure 10B) of H1.0K180me2 using antibodies specific to the H1.0K180me2 epitope in serum. Figure 10C shows Western blot analysis of the presence of the H1.0K180me2 epitope level in mouse serum after X-ray irradiation (7 Gy), using antibodies specific to the H1.0K180me2 epitope.

[0042] [Figure 11A-B]Figures 11A–11C show age-related accumulation of circulating H1.0K180me2 in brain tissue and serum. Western blot analysis of H1.0K180me2 in mouse (Figure 11A) and human (Figure 11B) brain tissue is shown, revealing that its abundance increases with biological age. Figure 11C shows H1.0K180me2 levels in human serum samples normalized by total IgG serum levels. [Figure 11C] Figures 11A–11C show age-related accumulation of circulating H1.0K180me2 in brain tissue and serum. Western blot analysis of H1.0K180me2 in mouse (Figure 11A) and human (Figure 11B) brain tissue is shown, revealing that its abundance increases with biological age. Figure 11C shows H1.0K180me2 levels in human serum samples normalized by total IgG serum levels.

[0043] [Figure 12A] Figures 12A-12C demonstrate the usefulness of measuring serum H1.0K180me2 as a biomarker for Alzheimer's disease. Figure 12A shows that serum H1.0K180me2 is decreased in individuals with Alzheimer's disease compared to age-matched controls (normalized by serum volume). Figure 12B shows that serum H1.0K180me2 is decreased in individuals with Alzheimer's disease compared to age-matched controls (normalized by total serum IgG levels). Figure 12C shows that serum H1.0K180me2 is decreased in individuals with Alzheimer's disease compared to age-matched controls (normalized by total serum protein concentration). [Figure 12B]Figures 12A-12C demonstrate the usefulness of measuring serum H1.0K180me2 as a biomarker for Alzheimer's disease. Figure 12A shows that serum H1.0K180me2 is decreased in individuals with Alzheimer's disease compared to age-matched controls (normalized by serum volume). Figure 12B shows that serum H1.0K180me2 is decreased in individuals with Alzheimer's disease compared to age-matched controls (normalized by total serum IgG levels). Figure 12C shows that serum H1.0K180me2 is decreased in individuals with Alzheimer's disease compared to age-matched controls (normalized by total serum protein concentration). [Figure 12C] Figures 12A-12C demonstrate the usefulness of measuring serum H1.0K180me2 as a biomarker for Alzheimer's disease. Figure 12A shows that serum H1.0K180me2 is decreased in individuals with Alzheimer's disease compared to age-matched controls (normalized by serum volume). Figure 12B shows that serum H1.0K180me2 is decreased in individuals with Alzheimer's disease compared to age-matched controls (normalized by total serum IgG levels). Figure 12C shows that serum H1.0K180me2 is decreased in individuals with Alzheimer's disease compared to age-matched controls (normalized by total serum protein concentration).

[0044] [Figure 13A] Figure 13A shows that human IgG autoantibodies against H1.0K180me2 can be detected in various human biological fluids (plasma, urine, saliva) using indirect ELISA. Figure 13B demonstrates the usefulness of measuring IgG autoantibodies against H1.0K180me2 as a biomarker for Alzheimer's disease. [Figure 13B]Figure 13A shows that human IgG autoantibodies against H1.0K180me2 can be detected in various human biological fluids (plasma, urine, saliva) using indirect ELISA. Figure 13B demonstrates the usefulness of measuring IgG autoantibodies against H1.0K180me2 as a biomarker for Alzheimer's disease.

[0045] [Figure 14A] Figure 14A shows a schematic diagram of measuring IgM antibodies against H1.0K180me2 under test and normalized conditions.

[0046] [Figure 14B] Figure 14B shows the measurement results for total IgM and a standard curve of anti-IgM molar concentration against optical density (OD) at 450 nm. IgM concentration is inferred from this curve. The box plot shows total IgM concentration, demonstrating that total IgM levels do not distinguish between individuals with and without Alzheimer's disease (AD).

[0047] [Figure 14C] Figure 14C is a schematic diagram of the use of an indirect ELISA assay for the measurement and quantification of H1.0K180me2 IgM autoantibodies.

[0048] [Figure 14D] Figure 14D shows the standard curve of anti-IgG molar concentration against optical density (OD) at 450 nm. IgM concentration can be inferred from this curve.

[0049] [Figure 14E] Figure 14E demonstrates the usefulness of measuring IgM autoantibodies targeting H1.0K180me2 as a biomarker for Alzheimer's disease (the figure shows unnormalized raw data).

[0050] [Figure 15A]Figure 15A demonstrates the usefulness of measuring IgM autoantibodies against H1.0K180me2 as a biomarker for Alzheimer's disease (the figure shows data normalized to total IgM levels).

[0051] [Figure 15B] Figure 15B demonstrates the stability and reproducibility of the diagnostic characteristics of serological indirect ELISA for the measurement and quantification of H1.0K180me2 IgM autoantibodies between different operators and different laboratory settings.

[0052] [Figure 16A] Figure 16A demonstrates that the correlation between total IgM and H1.0K180me2 IgM autoantibodies is not significant (relative to R2 value) in both Alzheimer's disease patients and neurological control patients.

[0053] [Figure 16B] Figure 16B demonstrates that total unmodified H1.0 protein levels (left graph) and total H1.0 IgM autoantibody levels (right graph) do not distinguish between individuals with and without Alzheimer's disease.

[0054] [Figure 17] Figure 17 demonstrates that patients with Alzheimer's disease can be stratified into distinct subpopulations by measuring H1.0K180me2 IgG and H1.0K180me2 IgM autoantibodies and examining the correlation between the two measurements.

[0055] [Figure 18] Figure 18 shows the Western blot analysis of H1.0K180me2 in SR hADSCs after bleomycin treatment, demonstrating that everolimus, a derivative of rapamycin, can block the appearance of H1.0K180me2 during DNA damage.

[0056] [Figure 19]Figure 19 shows Western blot analysis of H1.0K180me2 in SR hADSCs after bleomycin or temozolomide treatment, revealing that temozolomide, a chemotherapeutic agent capable of inducing the base excision repair pathway, also leads to H1.0K180 methylation.

[0057] [Figure 20-1] Figure 20 shows the effects of mTOR and PI3K inhibitors on H1.0K180me2 kinetics. [Figure 20-2] Figure 20 shows the effects of mTOR and PI3K inhibitors on H1.0K180me2 kinetics.

[0058] [Figure 21] Figures 21A and 21B show the results of an in vitro methylation assay using G9A methyltransferase. G9A methyltransferase is capable of methylating H1.0 peptides (Figure 21A) and full-length H1.0 proteins (Figure 21B).

[0059] [Figure 22A] Figure 22A shows that in the presence of unmethylated H1.0 peptides, G9A methyltransferase specifically and extensively dimethylates H1.0K180 (99.9% of the total peptide). [Figure 22B] Figure 22B shows a tabular representation of the methylation efficiencies of G9 and GLP methyltransferases.

[0060] [Figure 23] Figure 23 shows that minimal further methylation occurs in the presence of the dimethylated H1.0 peptide (only 2.64% of the peptide is further methylated).

[0061] [Figure 24]Figure 24 shows that in the presence of recombinant full-length H1.0, G9A methyltransferase methylates the C-terminal lysine residue containing H1.0K180me2.

[0062] [Figure 25] Figure 25 shows that in the presence of the unmethylated H1.0 peptide, GLP methyltransferase specifically dimethylates H1.0K180 (96.6% of the total peptide) to produce H1.0K180me2.

[0063] [Figure 26] Figure 26 shows that in the presence of the K180 dimethylated H1.0 peptide, GLP methyltransferase further methylates only H1.0K180 and H1.0K174 with very low efficiency (1.02% of the peptide is further methylated).

[0064] [Figure 27] Figure 27 shows the methylation of recombinant full-length H1.0 protein by GLP methyltransferase.

[0065] [Figure 28A] Figures 28A-28B show that siRNA knockdown of G9A in human adipocyte-derived stem cells (hADSCs) led to a reduction in H1.0K180me2 levels (Figure 28B) following bleomycin treatment (Figure 28A). [Figure 28B] Figures 28A-28B show that siRNA knockdown of G9A in human adipocyte-derived stem cells (hADSCs) led to a reduction in H1.0K180me2 levels (Figure 28B) following bleomycin treatment (Figure 28A). [Modes for carrying out the invention]

[0066] Detailed explanation This specification provides an antibody (H1.0K180me2 antibody) that specifically binds to a dimethylated antigen, wherein the dimethylated antigen is a histone H1.0 peptide or histone H1.0 protein containing a dimethylated lysine residue, the lysine residue corresponds to K180 of the human histone H1.0 protein, and the antibody is required for the binding of the dimethylated lysine residue.

[0067] Histone H1.0 peptides or histone H1.0 proteins containing dimethylated lysine residues, wherein the dimethylated lysine residue corresponds to K180 of human histone H1.0 protein (H1.0K180me2 peptides and H1.0K180me2 proteins), are also provided herein.

[0068] These H1.0K180me2 antibodies, H1.0K180me2 peptides, and H1.0K180me2 proteins are provided herein for therapeutic and diagnostic applications. These H1.0K180me2 antibodies, H1.0K180me2 proteins, and H1.0K180me2 peptides may be used in the treatment of methylation H1.0-related diseases or conditions in individuals. These H1.0K180me2 antibodies, H1.0K180me2 proteins, and H1.0K180me2 peptides may be used to detect replication aging, DNA damage, genotoxic stress, radiation exposure, and Alzheimer's disease, and to monitor treatment regimens, patient stratification, and drug screening, and may serve as markers of biological aging within a system.

[0069] These and related compositions and methods are described herein. I. Dimethylated proteins and peptides A. Dimethylated H1.0K180me2 proteins and peptides

[0070] The terms “peptide,” “protein,” and “polypeptide,” as used herein, refer to macromolecules of amino acids, and unless otherwise specified, include abnormal amino acids that function in a manner similar to naturally occurring amino acids.

[0071] Histone H1.0 proteins and histone H1.0 peptides containing a dimethylated lysine residue, wherein the lysine residue corresponds to K180 of human histone H1.0 protein, are provided herein. Histone H1.0 proteins containing dimethylated lysine at the residue corresponding to K180 of human H1.0 protein are referred herein to as "H1.0K180me2 proteins." Histone H1.0 peptides containing dimethylated lysine at the residue corresponding to K180 of human H1.0 protein are referred herein to as "H1.0K180me2 peptides." These may also be collectively referred to simply as "H1.0K180me2."

[0072] The residue locations of the H1.0 protein discussed herein are identified with respect to a reference amino acid sequence. The human H1.0 histone protein used to identify the residue locations is the NCBI reference sequence: NP_005309.1, which can be accessed at http: / / www.ncbi.nlm.nih.gov / protein / NP_005309.1. In this case, "H1.0 residue lysine" References to "180" or "H1.0K180" or "K180" identify the residue in human histone H1.0 where methionine is the first residue and the 180th amino acid from the N-terminus. The 180th residue is lysine (K) in human H1.0. Those skilled in the art will recognize that the K180 residue may occupy different positions in H1 proteins or different isoforms from different species.

[0073] In this specification, the term "K180" refers to the residue corresponding to K180 in human H1.0 proteins. Similarly, the term "K172" refers to the residue corresponding to K172 in human H1.0 proteins, etc.

[0074] Table 1 provides the 194-amino acid sequence of the full-length human H1.0 protein, numbered consecutively from 1 to 194 (NCBI reference sequence: NP_005309.1). The K180 residue is K * It is shown as follows. [Table 1]

[0075] Table 2 provides the 194-amino acid sequences of full-length human H1.0 protein dimethylated with K180, numbered consecutively from 1 to 194. K180me2 residues are indicated as K(me2). [Table 2]

[0076] In certain embodiments, the dimethylated H1.0K180me2 peptide (containing a K180me2 epitope recognized by the H1.0K180me2 antibody provided herein) comprises an amino acid sequence selected from the sequences presented in Table 3A. [Table 3A-1] [Table 3A-2]

[0077] The H1.0K180me2 proteins and peptides described herein may be further conjugated for use in a variety of applications, including, but not limited to, therapeutic, detection, diagnostic, visualization, quantification, and screening, and for use in biological assays relating to these therapeutic applications.

[0078] In some embodiments, the H1.0K180me2 protein and peptide include labels, e.g., detectable labels, spin labels, colorimetric labels, radioactive labels, enzymatic labels, fluorescent labels, or magnetic labels (e.g., conjugated to the label). In exemplary embodiments, the H1.0K180me2 protein / peptide is biotinylated. In some embodiments, the H1.0K180me2 protein / peptide is conjugated to or attached to a solid surface, e.g., beads (e.g., magnetic, glass, or plastic beads), column, resin, or microplate. In some embodiments, the H1.0K180me2 protein / peptide is coated onto a microplate. In some embodiments, the H1.0K180me2 protein / peptide is conjugated to or includes effector molecules, e.g., radionuclides, cytotoxins, chemotherapeutic agents, drugs, prodrugs, toxins, enzymes, immunomodulators, apoptosis promoters, cytokines, hormones, oligonucleotides, antisense molecules, siRNA, and secondary antibodies, although these are not limited to these.

[0079] In some embodiments, the amino acid sequence further includes additional terminal residues, for example, for conjugation purposes. In some embodiments, the amino acid sequence further includes a C-terminal residue. In such embodiments, the H1.0K180me2 peptide has the amino acid sequences CAKPVKASKPKKAKPVKPK (SEQ ID NO: 36), CAKPVKASKPKKAKPVKPKC (SEQ ID NO: 37), AKPVKASKPKKAKPVKPKC (SEQ ID NO: 38), CAKPVKASKPKKAKPVK (me2) PK (SEQ ID NO: 39), CAKPVKASKPKKAKPVK (me2) PKC (SEQ ID NO: 40), or AKPVKASKPKKAKPVK (me2)PKC (SEQ ID NO: 41) may be included. In some embodiments, the H1.0K180me2 peptide comprises any one fragment of the H1.0K180me2 peptides provided herein. In some embodiments, the H1.0K180me2 peptide is conjugated to KLH (keyhole limpet hemocyanin), OVA (ovalbumin), BC (bacterial cellulose), or BSA (bovine serum albumin).

[0080] The H1.0K180me2 protein and peptides provided herein are useful for inclusion as reference standards in the detection of pathophysiology and in methods that incorporate the quantitative assessment of the presence of H1.0K180me2.

[0081] In vitro methylation of proteins or peptides has traditionally presented challenges in terms of specificity (e.g., specificity for the substrate to be methylated and its control). A method for selective dimethylation of the K180 residue of an H1.0 protein (or its peptide fragment) using G9A methyltransferase or GLP methyltransferase is described herein.

[0082] Compositions and methods relating to the specific dimethylation of histone H1.0 protein with lysine residue 180 (K180) (H1.0 having K180me2) and the specific dimethylation of peptide fragments of histone H1.0 protein with lysine corresponding to K180 are provided herein. B.H1.0K180me2 peptide production

[0083] The H1.0K180me2 peptide is also useful as a reference standard for the indirect detection of pathophysiology, and not limited to, (1) at least DNA damage, genotoxic stress (e.g., associated with environmental exposure), radiation exposure, chemotherapy, and immunotherapy, radiotherapy, and molecular diagnostics of Alzheimer's disease using antibodies with DNA damage payloads, (2) monitoring of treatment regimens and patient stratification, (3) drug screening, and (4) quantitative analysis of data in methods including therapeutic applications.

[0084] The H1.0K180me2 peptides provided herein are in the range of 5 to 193 amino acid (aa) lengths. In various embodiments, the lengths of the H1.0K180me2 peptides are 5aa, 6aa, 7aa, 8aa, 9aa, 10aa, 11aa, 12aa, 13aa, 14aa, 15aa, 16aa, 17aa, 18aa, 19aa, 20aa, 21aa, 22aa, 23aa, 24aa, 25aa, 26aa, 27aa, 28aa, 29aa, or 30aa. In a particular exemplary embodiment, the length of the H1.0K180me2 peptide is 15aa, 16aa, 17aa, 18aa, 19aa, or 20aa.

[0085] Table 3A lists exemplary H1.0K180me2 peptides that can be produced by synthesis using the methods and compositions described herein. In some embodiments, the H1.0K180me2 peptide of the present invention comprises one of the sequences selected from those presented in Table 3A. In some embodiments, the H1.0K180me2 peptide consists of one of the sequences selected from those presented in Table 3A. In exemplary embodiments, the H1.0K180me2 peptide comprises the sequence of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In exemplary embodiments, the H1.0K180me2 peptide consists of the sequence of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. The peptides provided in Table 3A may further include labels, such as biotin.

[0086] Methods for the in vitro generation of H1.0K180me2 peptide (H1.0K180me2 peptide produced by synthesis) are provided herein. Generally, the method involves contacting a substrate peptide with a G9A methyltransferase enzyme or a G9A-like protein (GLP) methyltransferase enzyme and a methyl donor under conditions that allow for specific dimethylation (in vitro methylation) of the lysine residue corresponding to K180 of the histone H1.0 protein. In various embodiments, the substrate H1.0 peptide includes a sequence selected from the group of sequences presented in Table 3B. In relevant embodiments, the peptide is a sequence selected from the group of sequences presented in Table 3B.

[0087] The peptides used for methylation may be produced by synthesis or by methods familiar to those skilled in the art. [Table 3B-1] [Table 3B-2]

[0088] Generally, a method for dimethylating a histone H1.0 peptide includes the steps of contacting the peptide with a methyltransferase enzyme and a methyl donor under conditions that produce a specifically dimethylated peptide, wherein the peptide is specifically dimethylated at the lysine residue corresponding to K180 of the human histone H1.0 protein, and the methyltransferase enzyme is G9A methyltransferase or GLP methyltransferase.

[0089] In embodiments of the method for in vitro generation of the H1.0K180me2 peptide, the G9A methyltransferase may be a recombinant G9A methyltransferase, a purified G9A mammalian methyltransferase, a human G9A methyltransferase, a mouse G9A methyltransferase, etc. The G9A methyltransferase includes artificially or naturally generated isoforms containing enzymatically active orthologs, chimeras, and enzyme domains.

[0090] In embodiments of the method for in vitro generation of the H1.0K180me2 peptide, the GLP methyltransferase may be a recombinant GLP methyltransferase, a purified GLP mammalian methyltransferase, a human GLP methyltransferase, a mouse GLP methyltransferase, etc. The GLP methyltransferase includes artificially or naturally generated isoforms containing enzymatically active orthologs, chimeras, and enzyme domains.

[0091] In embodiments of the method for in vitro generation of the H1.0K180me2 peptide, the methyl donor may be S-adenosyl-L-methionine (SAM).

[0092] In embodiments of the method for in vitro generation of the H1.0K180me2 peptide, the contacting step may be performed in a methylation buffer.

[0093] In embodiments of the method for in vitro generation of the H1.0K180me2 peptide, the peptide may contain a label (e.g., biotin) prior to methylation.

[0094] In embodiments of the method for in vitro generation of the H1.0K180me2 peptide, the peptide may be conjugated to a label (e.g., biotin) after methylation.

[0095] In embodiments of methods for in vitro methylation of the H1.0K180me2 peptide substrate, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or even more than 99.9% of the product contains K180me2. Similarly, in embodiments of methods for in vitro production of the H1.0K180me2 peptide, in various embodiments, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, less than 0.5%, or less than 0.35% of the product also contains other methylated residues (e.g., K172me1, K172me2, K172me3, K174me1, K174me2, K174me3, K175me1, K175me2, K175me3, K177me1, K177me2, K177me3, K166me1, K166me2, K166me3, K180me1, and / or K180me3). In an exemplary embodiment, less than 0.35% of the product having K180me2 also contains K174me3, K175me3, and K177me1, less than 1.25% of the product having K180me2 also contains K166me1, and less than 1.04% of the product having K180me2 contains K174me1 and K180me3.

[0096] In some embodiments, the H1.0K180me2 peptide binds to an antibody specific for the H1.0K180me2 antigen (H1.0K180me2 antibody). Antibodies that specifically bind to a dimethylated antigen, wherein the dimethylated antigen is a histone H1.0 peptide containing a dimethylated lysine residue and the lysine residue corresponds to K180 of the human histone H1.0 protein, are provided herein.

[0097] In certain embodiments, the H1.0K180me2 peptide binds to the H1.0K180me2 antibody with a dissociation constant (Kd) of about 0.0001 nM to about 1 μM. For example, the Kd of the peptide may be about 1 μM, about 100 nM, about 50 nM, about 10 nM, about 5 nM, about 1 nM, about 0.5 nM, about 0.1 nM, about 0.05 nM, about 0.01 nM, about 0.005 nM, about 0.001 nM, about 0.0005 nM, or even about 0.0001 nM.

[0098] In some embodiments, the H1.0K180me2 peptide is specific for an anti-human H1.0K180me2 antibody. In some embodiments, the H1.0K180me2 peptide is cross-reactive with H1.0K180me2 antibodies from other species.

[0099] In some embodiments, the H1.0K180me2 peptide is selective for H1.0K180me2 antibodies and shows little or no binding to H1.0K180me1 or H1.0K180me3 antibodies.

[0100] In some embodiments, the binding preference (e.g., affinity) of the H1.0K180me2 peptide for H1.0K180me2 antibodies is generally at least about 2-fold, about 5-fold, or at least about 10, 20, 50, 10 2 、10 3 、10 4 、10 5 、or 10 6 -fold higher than for non-specific target antibodies (e.g., randomly generated antibodies).

[0101] The H1.0K180me2 peptide can be equally specific / selective for antibodies having a payload (e.g., including, but not limited to, radionuclides, cytotoxins, chemotherapeutic agents, drugs, prodrugs, toxins, enzymes, immunomodulators, apoptosis promoters, cytokines, hormones, antagonists, agonists or receptor decoys).

[0102] The H1.0K180me2 peptides provided herein may be further conjugated for various purposes, such as, but not limited to, detection, diagnosis, visualization, quantification, sorting, treatment, and for use in biological assays.

[0103] In some embodiments, the H1.0 peptide or H1.0K180me2 peptide is labeled (e.g., conjugated either before or after methylation), such as a detectable label, spin label, colorimetric label, radioactive label, enzymatic label, fluorescent label, magnetic label, etc.

[0104] In some embodiments, a complex comprising a histone H1.0 peptide and a methyltransferase enzyme is provided herein, which is present in vitro (e.g., in a test tube, tube, reaction chamber, reaction vessel, etc.). In some embodiments, the H1.0 peptide comprises a sequence selected from the group consisting of SEQ ID NOs. 42 to 74. In some embodiments, the methyltransferase enzyme is a G9A methyltransferase enzyme. In some embodiments, the methyltransferase enzyme is a GLP methyltransferase enzyme. Production of C.H1.0K180me2 protein

[0105] The full-length H1.0K180me2 protein described herein is also useful as a reference standard in methods including, but not limited to, (1) at least DNA damage, genotoxic stress (e.g., associated with environmental exposure), radiation exposure, chemotherapy, and immunotherapy, radiotherapy, and molecular diagnostics of Alzheimer's disease using antibodies with DNA damage payloads, (2) monitoring of treatment regimens and patient stratification, (3) drug screening, and (4) use as a therapeutic agent.

[0106] In some embodiments, an in vitro method for dimethylating a histone H1.0 protein is provided herein, comprising the step of contacting the protein with a methyltransferase enzyme and a methyl donor under conditions that produce a specifically dimethylated protein, wherein the protein is specifically dimethylated at a lysine residue corresponding to K180 of the human histone H1.0 protein, and the methyltransferase enzyme is G9A methyltransferase or GLP methyltransferase.

[0107] In some embodiments, methods for the in vitro production of the H1.0K180me2 protein are provided herein. In one embodiment, the method comprises the step of contacting a full-length H1.0 protein with a G9A methyltransferase enzyme and a methyl donor under conditions that allow for specific dimethylation of the K180 residue. The G9A methyltransferase includes artificially or naturally produced isoforms containing orthologues, chimeric and enzyme domains that are enzymatically active.

[0108] In some embodiments, methods for the in vitro production of the H1.0K180me2 protein are provided herein. In one embodiment, the method comprises contacting a full-length H1.0 protein with a GLP methyltransferase enzyme and a methyl donor under conditions that allow for specific dimethylation of the K180 residue. The GLP methyltransferase includes artificially or naturally produced isoforms containing orthologues, chimeric and enzyme domains that are enzymatically active.

[0109] Full-length H1.0 unmethylated protein substrates used for in vitro methylation may be produced by isolation, synthesis, or recombination using methods familiar to those skilled in the art. Nucleic acids encoding the H1.0K180me2 protein are provided herein. Vectors containing any of the nucleic acids encoding the H1.0K180me2 protein provided herein are also provided herein.

[0110] In some embodiments of the methods for in vitro methylation or in vitro production of the H1.0K180me2 protein, the G9A methyltransferase may be recombinant G9A methyltransferase, purified mammalian G9A methyltransferase, human G9A methyltransferase, mouse G9A methyltransferase, etc.

[0111] In some embodiments of the methods for in vitro methylation or in vitro production of the H1.0K180me2 protein, the GLP methyltransferase may be recombinant GLP methyltransferase, purified GLP mammalian methyltransferase, human GLP methyltransferase, or mouse GLP methyltransferase.

[0112] In some embodiments of the method for the in vitro production of the H1.0K180me2 protein, the protein may contain a label (e.g., biotin) prior to methylation.

[0113] In some embodiments of the method for the in vitro generation of the H1.0K180me2 protein, the protein may be conjugated with a label (e.g., biotin) after methylation.

[0114] In some embodiments of methods for in vitro methylation or in vitro generation of the H1.0K180me2 protein, the methyl donor is S-adenosyl-L-methionine.

[0115] In some embodiments of the method for the in vitro generation of the H1.0K180me2 protein, the contact step is performed in a methylation buffer.

[0116] In embodiments of methods for in vitro methylation of the H1.0K180me2 protein substrate, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or even more than 99.9% of the product may contain K180me2. Similarly, in some embodiments of methods for in vitro production of the H1.0K180me2 protein, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, less than 0.5%, or less than 0.35% of the product also contains other methylated residues (e.g., K172me1, K172me2, K172me3, K174me1, K174me2, K174me3, K175me1, K175me2, K175me3, K177me1, K177me2, K177me3, K166me1, K166me2, K166me3, K180me1, and / or K180me3). In an exemplary embodiment, less than 0.35% of the product having K180me2 also contains K174me3, K175me3, and K177me1, less than 1.25% of the product having K180me2 also contains K166me1, and / or less than 1.04% of the product having K180me2 contains K174me1 and K180me3.

[0117] Antibodies that specifically bind to a dimethylated antigen, wherein the dimethylated antigen is found in a histone H1.0 protein containing a dimethylated lysine residue and the lysine residue corresponds to K180 of the human histone H1.0 protein are provided herein.

[0118] In some embodiments, the H1.0K180me2 protein is selective for an antibody specific for H1.0K180me2 (H1.0K180me2 antibody). In some embodiments, the peptide binds to an antibody that is non-specific for H1.0K180me2.

[0119] In certain embodiments, the H1.0K180me2 protein binds to the H1.0K180me2 antibody with a dissociation constant (Kd) of approximately 0.0001 nM to approximately 1 μM. For example, Kd may be approximately 1 μM, approximately 100 nM, approximately 50 nM, approximately 10 nM, approximately 5 nM, approximately 1 nM, approximately 0.5 nM, approximately 0.1 nM, approximately 0.05 nM, approximately 0.01 nM, approximately 0.005 nM, approximately 0.001 nM, approximately 0.0005 nM, or even approximately 0.0001 nM.

[0120] In some embodiments, the H1.0K180me2 protein is specific to anti-human H1.0K180me2 antibodies. In some embodiments, the H1.0K180me2 protein is cross-reactive with H1.0K180me2 antibodies from other species.

[0121] In some embodiments, the H1.0K180me2 protein is selective for the H1.0K180me2 antibody and shows little to no binding affinity to the H1.0K180me1 or H1.0K180me3 antibody.

[0122] In some embodiments, the binding preference (e.g., affinity) of the H1.0K180me2 protein to the H1.0K180me2 antibody is generally at least about 2 times, about 5 times, or at least about 10, 20, 50, 10 times compared to nonspecific target antibodies (e.g., randomly generated antibodies). 2 , 10 3 , 10 4 , 10 5 , or 10 6 It is double.

[0123] The H1.0K180me2 protein provided herein may be further conjugated for use in various applications, including, but not limited to, detection, diagnosis, visualization, quantification, screening, and therapeutic use, and in biological assays.

[0124] In some embodiments, the H1.0 protein (H1.0 substrate) or H1.0K180me2 protein is conjugated with a label (either before or after methylation), such as a detectable label, spin label, colorimetric label, radioactive label, enzymatic label, fluorescent label, or magnetic label. II. Antibodies that bind to dimethylated histone H1.0 proteins and peptides A.H1.0K180me2 antibody

[0125] This specification provides antibodies that specifically bind to a dimethylated antigen, wherein the dimethylated antigen is a histone H1.0 peptide or histone H1.0 protein containing a dimethylated lysine residue, and the lysine residue corresponds to K180 of the human histone H1.0 protein. This dimethylated antigen is the H1.0K180me2 antigen, also referred to as the H1.0K180me2 epitope. These antibodies specifically bind to the H1.0K180me2 epitope of the H1.0K180me2 protein and H1.0K180me2 peptide. These antibodies specifically bind to the dimethylated antigen (specifically bind to the H1.0K180me2 epitope) and require the presence of a dimethyl group at K180 for binding. The terms “anti-H1.0K180me2” or “H1.0K180me2 antibody” or “anti-H1.0K180me2 antibody” are interchangeable to refer to these antibodies.

[0126] The term “antibody,” as used throughout this specification, is in its broadest sense and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, non-human antibodies, chimeric antibodies, bispecific antibodies, multispecific antibodies, multifunctional antigen-binding fragments (e.g., Fab fragments, Fab'2 fragments, CDRs, or ScFvs), antibody-drug conjugates, and other antibody fragments that retain specificity for the H1.0K180me2 antigen. In some embodiments, the antibody is a single-chain antibody that retains specificity for the H1.0K180me2 antigen.

[0127] The schematic binding of the H1.0K180me2 antibody is shown in Figure 5A.

[0128] In some embodiments, the H1.0K180me2 antibody provided herein is a diagnostic antibody.

[0129] In some embodiments, the H1.0K180me2 antibody provided herein is a therapeutic antibody.

[0130] In some embodiments, the H1.0K180me2 antibody is present at a high titer.

[0131] In some embodiments, the H1.0K180me2 antibody is an affinity-purified antibody.

[0132] The H1.0K180me2 antibodies provided herein may be further conjugated for use in various applications, including, but not limited to, detection, diagnosis, visualization, quantification, screening, and therapeutic use, and in biological assays.

[0133] In some embodiments, the H1.0K180me2 antibody includes a label, such as a detectable label, spin label, colorimetric label, radioactive label, enzyme label, fluorescent label, or magnetic label (e.g., conjugated to the label).

[0134] In some embodiments, the antibody is conjugated or attached to a solid surface, such as beads (e.g., magnetic, glass, or plastic beads), a column, a resin, or a microplate. In some embodiments, the antibody is coated onto a microplate.

[0135] In some embodiments, the antibody is conjugated to or contains effector molecules, such as, but not limited to, radionuclides, cytotoxins, chemotherapeutic agents, drugs, prodrugs, toxins, enzymes, immunomodulators, apoptosis promoters, cytokines, hormones, oligonucleotides, antisense molecules, siRNA, and secondary antibodies.

[0136] It is recognized that the H1.0K180me2 biomarker may be conjugated to chromatin or released from chromatin into the nucleus, cytoplasm, or extracellular space. The antibodies provided herein can bind to extracellular H1.0K180me2 and / or intracellular H1.0K180me2. If intracellular, the H1.0K180me2 antigen may be further conjugated to chromatin, released in the nucleus, released from the nucleus into cytoplasmic space, or further localized into the substructures of the cytoplasm.

[0137] In some embodiments, the antibody (e.g., a therapeutic antibody) is a neutralizing antibody that neutralizes one or more biological activities of H1.0K180me2. For example, the antibody binds to extracellular H1.0K180me2 and neutralizes any binding or signaling activity that the antibody may possess.

[0138] The antibodies provided herein may be of any immunoglobulin type, such as IgG, IgA, IgE, IgD, or IgM. In some embodiments, the antibodies are of the IgG subtype and may be IgG1, IgG2, IgG3, or IgG4 antibodies.

[0139] Antibodies specific to H1.0K180me2 derived from any species are provided herein. In some embodiments, the H1.0K180me2 antibody is specific to human H1.0K180me2. In some embodiments, the H1.0K180me2 antibody is cross-reactive with H1.0K180me2 derived from other species.

[0140] The antibodies provided herein bind specifically to H1.0K180me2. In some embodiments, these antibodies bind specifically and selectively to H1.0K180me2.

[0141] The antibodies provided herein specifically bind to dimethylated antigens, the dimethylated H1.0 antigens comprising dimethylated lysine residues, the lysine residues corresponding to K180 of the human histone H1.0 protein (H1.0K180me2 antigen), and in some embodiments, the dimethylated antigens do not contain any other methylated lysine residues.

[0142] In some embodiments, if the antigen contains dimethylated lysine residues, the H1.0K180me2 antibody will not bind, or will bind only minimally, with the lysine residues corresponding to K166, K172, K174, K175, and / or K177 of the human histone H1.0 protein.

[0143] In some embodiments, the H1.0K180me2 antibody does not bind to, or binds minimally to, an antigen containing one or more of the following residues: K172me1, K172me2, K172me3, K174me1, K174me2, K174me3, K175me1, K175me2, K175me3, K177me1, K177me2, K177me3, K166me1, K166me2, K166me3, K180me1, and / or K180me3.

[0144] In some embodiments, the H1.0K180me2 antibody contains a dimethylated K180 residue, but does not bind to, or binds minimally to, an antigen that also contains one or more of the following residues: K172me1, K172me2, K172me3, K174me1, K174me2, K174me3, K175me1, K175me2, K175me3, K177me1, K177me2, K177me3, K166me1, K166me2, K166me3, K180me1, and / or K180me3.

[0145] In some embodiments, if the antigen contains a monomethylated lysine residue at the lysine residues corresponding to K166, K172, K174, K175, K177, and / or K180 of the human histone H1.0 protein, the H1.0K180me2 antibody will not bind, or will bind only minimally.

[0146] In some embodiments, if the antigen contains trimethylated lysine residues in the lysine residues corresponding to K166, K172, K174, K175, K177, and / or K180 of the human histone H1.0 protein, the H1.0K180me2 antibody will not bind, or will bind only minimally.

[0147] In some embodiments, the H1.0K180me2 antibody is selective for dimethylation at residue K180 and shows little to no binding affinity to the H1.0K180me1 or H1.0K180me3 epitope.

[0148] The H1.0K180me2 antibody binds to the H1.0K180me2 epitope in any culture medium.

[0149] In some embodiments, the H1.0K180me2 antibody exhibits at least 1.5, 2, 2.5, 2.7, 5, or even 10 times higher specificity (binding preference, affinity) to the dimethylated antigen of K180 (H1.0K180me2 antigen) than to the monomethylated antigen of K180 (H1.0K180me1 antigen). (Figure 3D) In ​​some embodiments, the specificity to the H1.0K180me2 antigen is generally at least about 2, about 5, or at least about 10, 20, 50, 10 times higher specificity to nonspecific target molecules (e.g., randomly generated molecules lacking a specifically recognized site), to monomethylated K180 residues, to trimethylated K180 residues, or to H1.0 proteins methylated at any other residue. 2 , 10 3 , 10 4 , 10 5 , or 10 6 It is double.

[0150] In some embodiments, the binding efficiency of the H1.0K180me2 antibody is monitored by an ELISA assay. In some embodiments, the antibody binds to the H1.0K180me2 peptide at least 2.7 times more efficiently than the H1.0K180me1 peptide. In some embodiments, one antibody molecule recognizes one of 117 molecules of the H1.0K180me2 peptide, but only one of 316 molecules of the H1.0K180me1 peptide.

[0151] In certain embodiments, the antibodies provided herein have a dissociation constant (Kd) in the range of 0.0001 nM to 1 μM. For example, the Kd of an antibody may be about 1 μM, about 100 nM, about 50 nM, about 10 nM, about 5 nM, about 1 nM, about 0.5 nM, about 0.1 nM, about 0.05 nM, about 0.01 nM, about 0.005 nM, about 0.001 nM, about 0.0005 nM, or even about 0.0001 nM. Generation of B.H1.0K180me2 antibody

[0152] Antibodies that are specifically immunoreactive to H1.0K180me2 may be selected using various immunoassay formats. For example, a monoclonal antibody specific to H1.0K180me2 may be selected using a solid-phase ELISA immunoassay (see, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that may be used to determine specific immunoreactivity).

[0153] The production of antibodies provided herein may be by any method known to those skilled in the art. For example, in some embodiments, antibodies are produced by recombinant cells engineered to express desired VH, VL, and constant domains of the desired antibody. In some embodiments, antibodies are produced by hybridomas.

[0154] In some embodiments, any peptide containing the H1.0K180me2 antigen, optionally ligated to an immunogenic carrier, is used for immunization using a standard protocol. In some embodiments, a peptide containing a sequence derived from those shown in Table 3A, optionally ligated to an immunogenic carrier, is used for immunization using a standard protocol. In exemplary embodiments, AKPVKASKPKKAKPVK, optionally ligated to an immunogenic carrier, is used. me2 The peptide containing PK (SEQ ID NO: 3) is used for immunization using a standard protocol. The quality and titer of the resulting antibody may be evaluated using techniques known to those skilled in the art.

[0155] The compositions of the present invention described herein also include nucleic acids encoding antibodies, vectors comprising any of the nucleic acids encoding antibodies, and host cells comprising any of such vectors.

[0156] As those skilled in the art will readily recognize, antibodies can also be prepared by any of several commercial services. Diagnosis Direct and indirect detection of A.H1.0K180me2

[0157] The H1.0K180me2 antibody, H1.0K180me2 protein, and H1.0K180me2 peptide provided herein are useful for various diagnostic purposes.

[0158] Assays for both direct and indirect detection and quantification of H1.0K180me2 concentrations are provided herein. Such quantification may be useful for detecting replication senescence, DNA damage, genotoxic stress, radiation exposure, Alzheimer's disease, and biological aging. Quantification may also be useful for therapeutic regimens, drug screening, and monitoring patient stratification as responders or non-responders to drug treatments aimed at restoring cell viability, preventing DNA damage, increasing cellular metabolism and autophagy, inhibiting cellular senescence, and blocking the accumulation of insoluble protein waste in the cytoplasm of cells. Depending on the application, H1.0K180me2 may be detected and quantified in vivo, in vitro, ex vivo, in situ, or cell-free systems.

[0159] Direct detection of H1.0K180me2 involves detecting H1.0K180me2 using an H1.0K180me2 antibody.

[0160] Indirect detection of H1.0K180me2 involves using an H1.0K180me2 protein or H1.0K180me2 peptide that binds to an autoantibody produced in response to the presence of the H1.0K180me2 antigen. In this context, the H1.0K180me2 protein can be referred to as an H1.0K180me2 autoantibody-binding protein, and the H1.0K180me2 peptide can be referred to as an H1.0K180me2 autoantibody-binding peptide.

[0161] H1.0K180me2 may be detected by any number of methods well known to those skilled in the art. The H1.0K180me2 antibody, H1.0K180me2 protein, and H1.0K180me2 peptide provided herein are readily usable in a variety of immunoassays. These immunoassays include, but are not limited to, enzyme-coupled immunosorbent assays (ELISA), Western blotting, radioimmunoassays (RIA), flow cytometry, lateral flow immunoassays, slot blotting, magnetic immunoassays, radioimmunoassays, indirect immunofluorescence assays, direct immunofluorescence assays, ambient fiber optic immunoassays (SOFIA), spectrophotography, radiography, electrophoresis, immunoelectrophoresis, capillary electrophoresis, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), high-diffusion chromatography, fluid or gel precipitation reactions, immunodiffusion, spectroscopy, mass spectrometry, quantitative mass spectrometry, all types of multiplex assays, and all types of microfluidic assays.

[0162] The H1.0K180me2 antibodies and H1.0K180me2 proteins or peptides provided herein may include labels, such as detectable labels, spin labels, colorimetric labels, radioactive labels, enzymatic labels, fluorescent labels, or magnetic labels (for example, they may be conjugated to the labels).

[0163] The H1.0K180me2 antibodies and H1.0K180me2 proteins and peptides provided herein may include detectable labels. The detectable group may be any material having detectable physical or chemical properties, for example, detectable by spectroscopic, photochemical, biochemical, immunochemical, fluorescent, electrical, optical or chemical methods. Useful labels in the present invention include, but are not limited to, magnetic beads (e.g., DYNABEADS®), fluorescent dyes (e.g., fluorescein isothiocyanate, red, rhodamine, etc.), and radiolabels (e.g., 3 H, 125 I, 35 S, 14C, or 32 Examples of colorimetric labels include enzymes (e.g., LacZ, CAT, horseradish peroxidase, alkaline phosphatase, etc., which are typically used as detectable enzymes, either as marker gene products or in ELISA), biotin, avidin, or streptavidin and colloidal gold-colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and nanoparticles. In exemplary embodiments, biotin is the label.

[0164] The labels provided herein may be directly or indirectly coupled to desired components of assays following methods well known in the art. As indicated above, a wide range of labels may be used, selected according to the required sensitivity, ease of compound conjugation, stability requirements, available instruments, and disposable provision. Labels are often attached indirectly. Generally, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand then binds to an antiligand molecule (e.g., streptavidin), which is either inherently detectable or covalently bound to a signaling system such as a detectable enzyme, fluorescent compound, or chemiluminescent compound. Several ligands and antiligands may be used. If the ligand has a natural antiligand, e.g., biotin, the ligand may be used in combination with a labeled, naturally occurring antiligand. Alternatively, any hapten or antigenic compound may be used in combination with an antibody. Components may also be directly conjugated to signaling compounds, for example, by conjugation with an enzyme or fluorophore. Examples of enzymes suitable for labeling include, but are not limited to, hydrolases, phosphatases, esterases, glycosidases, or oxitranscription factoreductases, and peroxidases. Examples of fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, and umbelliferone. Examples of chemiluminescent compounds include luciferin and 2,3-dihydrophthalazinedione, such as luminol.

[0165] Methods for detecting labels are well known to those skilled in the art. Therefore, for example, if the label is radioactive, the detection method includes a scintillation counter or photographic film, such as in autoradiography. If the label is fluorescent, it may be detected by exciting a fluorescent dye with a suitable wavelength of light and detecting the resulting fluorescence, for example, by microscopic examination, visual inspection, photographic film, or by the use of an electron detector such as a charge-coupled device (CCD) or photomultiplier tube. Similarly, enzyme labels may be detected by providing a suitable substrate to an enzyme and detecting the resulting reaction product. Finally, simple colorimetric labels may be detected simply by observing the color associated with the label. Thus, in various dipstick assays, various conjugated beads appear as the color of the bead, while conjugated gold often appears pink.

[0166] In immunoassays that detect autoantibodies specific to H1.0K180me2, specific secondary antibodies can be used to differentiate between IgG, IgM, IgA, IgE, and IgD autoantibody types.

[0167] During detection, the concentration of H1.0K180me2 in a specific fraction may be quantified, for example, in the intracellular fraction, in the soluble and chromatin-bound fraction, or in the cytoplasmic fraction. For example, an increase in the concentration of H1.0K180me2 in the cytoplasmic fraction indicates cellular senescence. In some embodiments, the localization of H1.0K180me2 is visualized by imaging intact cells, cultured cells, or cells in section cultures. For example, increased intracellular localization of H1.0K180me2 outside the nucleus indicates senescence. In some embodiments, the release of H1.0K180me2 into the cytosol or extracellular matrix indicates senescence.

[0168] Detection may be performed on any biological sample. Biological samples include, but are not limited to, whole blood, plasma, serum, saliva, urine, feces, synovial fluid, cerebrospinal fluid, bronchial lavage, ascites fluid, bone marrow aspirate, pleural fluid, tissue, cells, biopsy material, interstitial fluid, lymph, or fractions thereof, all derived from an individual. In some embodiments, the biological sample includes cells, which are in culture, in suspension, on a slide, in intact tissue, or in a preparation prepared for FAC analysis.

[0169] Biological specimens are obtained from individuals. As used herein, individuals refer to humans, domesticated animals and livestock, as well as any animal classified as a mammal, including zoo animals, sporting animals, or pets, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, and cats. Individuals may be male or female. In one embodiment, the individual is female. In one embodiment, the individual is male.

[0170] In some embodiments, the individual is over 50 years old. In some embodiments of the methods described herein, the individual is under 50 years old. In some embodiments of the methods described herein, the individual is at least 50 years old, at least 55 years old, at least 60 years old, at least 65 years old, at least 70 years old, at least 75 years old, or at least 80 years old. In exemplary embodiments, the individual is at least 60 years old.

[0171] Biological samples are obtained by standard methods well known to those skilled in the art. The samples are optionally pre-treated, if desired, by dilution or concentration in a suitable buffer solution. At physiological pH, one of several standard buffer solutions using various buffers such as phosphate or Tris may be used.

[0172] The concentration of H1.0K180me2 in a biological sample may be quantified using the direct detection methods described herein. In some embodiments, the H1.0K180me2 protein or peptide may be used cooperatively, for example, as a positive control or as a competitor in a competitive immunoassay, and may be labeled or unlabeled depending on the format of the assay being performed.

[0173] The concentration of H1.0K180me2 autoantibodies in biological samples may be quantified using the indirect detection methods described herein. In some embodiments, H1.0K180me2 antibodies may be used cooperatively, for example, as a positive control or as a competitor in a competitive immunoassay, and may be labeled or unlabeled depending on the format of the assay being performed.

[0174] Those skilled in the art will recognize that in some embodiments, it may be necessary to compare a determined concentration of the H1.0K180me2 antigen or autoantibody with a control (i.e., a reference control). Relative comparison may allow for the determination, for example, whether an individual has or is at risk of developing a disease (e.g., Alzheimer's disease), or whether an individual is responsive to or may be responsive to a particular treatment (e.g., Alzheimer's disease treatment or treatment with a laparog). The control may be an age-matched control, a sex-matched control, an age and sex-matched control, a healthy control, an unmanipulated control, or a reference standard equivalent to a collection of reference standards. For example, it may be compared prior to treatment (e.g., Alzheimer's disease treatment or treatment with a laparog) or prior to exposure to a genotoxic substance, DNA damaging agent, or radiation. It may also be compared with a sample from the same individual derived from an unaffected area, e.g., unaffected tissue.

[0175] Those skilled in the art recognize that it is often desirable to reduce nonspecific binding in immunoassays and during analyte detection. When the assay involves H1.0K180me2 antibodies or H1.0K180me2 proteins and peptides immobilized on a solid substrate, it may be desirable to minimize the amount of nonspecific binding to the substrate. Methods for reducing such nonspecific binding are known to those skilled in the art. Typically, this involves the step of coating the substrate with a proteinaceous composition. In some embodiments, proteinaceous compositions such as bovine serum albumin (BSA), skim milk powder, and gelatin may be used.

[0176] Sensitivity, specificity, positive and negative predictive values ​​(PPV and NPV), and positive and negative likelihood ratios (PLR and NLR) can be calculated for each diagnostic trial design. Statistical methods help predict the presence or absence of disease in patients.

[0177] Sensitivity is generally defined as the probability that a test result will be positive when a disease is present (prevalence accuracy).

[0178] Specificity generally refers to the probability that a test result will be negative when the disease is not present (the accuracy of disease detection).

[0179] The positive predictive value (PPV) is generally the probability that a disease is present when the test is positive, and it explains the pre-test prevalence of the disease (for example, the pre-test prevalence for Alzheimer's disease is 10%).

[0180] The negative predictive value (NPV) is generally the probability that the disease is not present when the test is negative, and it explains the pre-test prevalence of AD being 10% (Prince, MJ, Am J Epidemiol, 1996).

[0181] The positive likelihood ratio (LR+ or PLR) is generally the probability of a person having a disease test positive divided by the probability of a person not having a disease test positive. The positive likelihood ratio (PLR) generally indicates how much the probability of having the disease increases if the test is positive. A PLR greater than 1 indicates an increased probability of having the target disorder, a PLR less than 1 indicates a decreased probability of having the target disorder, and a PLR of 1 means that the test does not change the probability of having the disease.

[0182] The negative likelihood ratio (LR- or NLR) is generally the probability of a person having a negative disease test divided by the probability of a person not having a negative disease test. The negative likelihood ratio (NLR) generally indicates how much the probability of having the disease decreases when the test is negative.

[0183] In some embodiments, a comparison is made to threshold levels established by patient operating characteristic curve analysis for optimal specificity and sensitivity. The ROC curve, threshold, and area under the curve (AUC) are shown for each of the test designs provided herein.

[0184] In some embodiments, the diagnostic methods provided herein may be used, for example, as confirmatory tests to definitively confirm that an individual has a disease, such as Alzheimer's disease.

[0185] In other embodiments, the diagnostic methods provided herein may be used (for testing, screening, etc.) as predictors to determine, for example, the likelihood that an individual will develop a disease, such as Alzheimer's disease. In such embodiments, the diagnosis may include the calculation of a likelihood ratio.

[0186] In other embodiments, the diagnostic methods provided herein may be used as companion diagnostics. In such embodiments, the diagnostics may include the calculation of positive predictive value (PPV) and negative predictive value (NPV).

[0187] In some embodiments, a diagnostic odds ratio (OR) may be established using the diagnostic method provided herein. In such embodiments, the diagnosis may include the calculation of sensitivity and specificity, which are measures of the effectiveness of the diagnostic test. B. H1.0K180me2 antibody for diagnostic purposes - Direct detection and quantification of H1.0K180me2

[0188] Antibodies that specifically bind to the H1.0K180me2 antigen, useful for diagnosis, are provided herein. The H1.0K180me2 antibodies provided herein require dimethylation of the K180 residue to bind.

[0189] In some embodiments, the H1.0K180me2 antibody includes a label, such as a detectable label, spin label, colorimetric label, radioactive label, enzyme label, fluorescent label, or magnetic label (e.g., conjugated to the label).

[0190] In some embodiments, the antibody is conjugated or attached to a solid surface, such as beads (e.g., magnetic, glass, or plastic beads), a column, a resin, or a microplate. In some embodiments, the antibody is coated onto a microplate.

[0191] The H1.0K180me2 antibody is discussed in more detail in the preceding Section II.

[0192] Direct detection is schematically shown in Figure 5A. C. H1.0K180me2 protein and peptide for diagnostic purposes - Indirect detection and quantification of H1.0K180me2

[0193] H1.0k180me2 protein and H1.0k180me2 peptide are provided herein for the detection of naturally occurring H1.0k180me2 autoantibodies in samples.

[0194] It is recognized that the production of the H1.0K180me2 antigen in organisms responding to certain stimuli may result in the generation of antigen-specific, naturally occurring autoantibodies. Therefore, in some embodiments of the present invention, assays for these naturally occurring autoantibodies against H1.0K180me2 may be desirable. Detection and measurement of autoantibodies are proposed as alternative measures for H1.0K180me2 production.

[0195] Methods and compositions for the detection and measurement of naturally occurring autoantibodies specific to the H1.0K180me2 antigen are provided herein. For use herein, autoantibodies specific to the H1.0K180me2 protein or fragments thereof may interchangeably be referred to as H1.0K180me2 autoantibodies.

[0196] The autoantibodies measured may be any immunoglobulin type, such as IgG, IgM, IgE, IgD, or IgA. In some embodiments, IgG autoantibodies against H1.0K180me2 are measured. In some embodiments, IgM autoantibodies against H1.0K180me2 are measured. In some embodiments, more than one type of autoantibody against H1.0K180me2 is measured; for example, in some embodiments, IgG and IgM autoantibodies against H1.0K180me2 are measured. In some embodiments, more than one type of autoantibody against H1.0K180me2 is measured and a ratio is calculated; for example, in some embodiments, IgG and IgM autoantibodies against H1.0K180me2 are measured and the ratio of IgG autoantibody against H1.0K180me2 to IgM autoantibody against H1.0K180me2 is calculated. In other embodiments, the ratio of H1.0K180me2 IgM autoantibody to total IgM (e.g., serum IgM) is measured. In other embodiments, the ratio of H1.0K180me2 IgM autoantibody to total IgM (e.g., serum IgM) is measured and related as a function to the ratio of H1.0K180me2 IgG autoantibody to total IgG (e.g., serum IgG). In some embodiments, more than one autoantibody against H1.0K180me2 is measured and compared to transferrin, ferritin, or serum albumin content. In other embodiments, more than one autoantibody against H1.0K180me2 is measured and normalized to the total amount of protein in the sample or to the volume of the sample.

[0197] In an exemplary embodiment, a screening test for Alzheimer's disease includes measuring the ratio of H1.0K180me2 IgM autoantibody to total IgM (Figure 17). In another exemplary embodiment, a screening test for Alzheimer's disease includes measuring the ratio of H1.0K180me2 IgG autoantibody to total IgG (Figure 17). In some embodiments, these are compared to each other to stratify the patient population (Figure 17).

[0198] Looking at the schematic diagrams, Figures 4 and 14C illustrate a method for indirectly measuring H1.0K180me2 levels. As illustrated in Figures 4 and 14C, a labeled H1.0K180me2 peptide is brought into contact with the sample, autoantibodies in the sample produced in response to H1.0K180me2 are bound to the labeled peptide, a secondarily labeled antibody is added to bind to the autoantibodies in the sample, followed by detection and quantification. In Figure 4, the autoantibodies in the sample can be of any type (IgG, IgM, IgE, IgD, or IgA). In this assay, the secondary antibody used to bind to the autoantibodies can be distinguished among IgG, IgM, IgE, IgD, and IgA antibodies, and as a result, each type can be quantified independently, if so desired. In some embodiments, the secondary antibody is an anti-IgG antibody, and the assay is used to quantify IgG autoantibodies against H1.0K180me2. In some embodiments, the secondary antibody is an anti-IgM antibody, and the assay is used to quantify IgM autoantibodies against H1.0K180me2 (Figure 14C). In some embodiments, two secondary antibodies are used to quantify more than one autoantibody; for example, in some embodiments, both anti-IgG and anti-IgM secondary antibodies are used, and the assay is used to quantify IgG and IgM autoantibodies against H1.0K180me2.

[0199] In some embodiments, the H1.0K180me2 protein contains the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the H1.0K180me2 peptide contains an amino acid sequence selected from those provided in Table 3A (SEQ ID NOs: 3-35). In some embodiments, the H1.0K180me2 peptide contains the amino acid sequence AKPVKASKPKKAKPVK (me2)Includes PK (SEQ ID NO: 3). In some embodiments, the amino acid sequence further includes additional terminal residues, for example, for conjugation purposes. In some embodiments, the amino acid sequence further includes a C-terminal residue. In such embodiments, the H1.0K180me2 peptide has the amino acid sequences CAKPVKASKPKKAKPVKPK (SEQ ID NO: 36), CAKPVKASKPKKAKPVKPKC (SEQ ID NO: 37), AKPVKASKPKKAKPVKPKC (SEQ ID NO: 38), CAKPVKASKPKKAKPVK (me2) PK (SEQ ID NO: 39), CAKPVKASKPKKAKPVK (me2) PKC (SEQ ID NO: 40), or AKPVKASKPKKAKPVK (me2) PKC (SEQ ID NO: 41) may be included. In some embodiments, the H1.0K180me2 peptide comprises any one fragment of the H1.0K180me2 peptides provided herein. In some embodiments, the H1.0K180me2 peptide is conjugated to KLH (keyhole limpet hemocyanin), OVA (ovalbumin), BC (bacterial cellulose), or BSA (bovine serum albumin).

[0200] In some embodiments, the H1.0K180me2 protein or peptide is synthetic, for example, a product of in vitro methylation using a G9A methyltransferase enzyme or a G9A-like protein (GLP) methyltransferase enzyme under conditions that allow for specific dimethylation of the K180 residue (as described herein).

[0201] The H1.0K180me2 proteins and peptides provided herein may be further conjugated for use in a variety of applications, including, but not limited to, detection, diagnosis, visualization, quantification, sorting, therapy, and biological assays. In some embodiments, the H1.0K180me2 protein or peptide is labeled, e.g., with a detectable label, spin label, colorimetric label, radioactive label, enzymatic label, fluorescent label, or magnetic label (e.g., conjugated to a label). In exemplary embodiments, the H1.0K180me2 protein or peptide is biotinylated. In some embodiments, the H1.0K180me2 protein or peptide is conjugated to or attached to a solid surface, e.g., beads (e.g., magnetic, glass, or plastic beads), column, or microplate. In some embodiments, the H1.0K180me2 protein or peptide is coated onto a microplate. In some embodiments, the H1.0K180me2 protein or peptide is conjugated to, or includes, effector molecules, including, but not limited to, radionuclides, cytotoxins, chemotherapeutic agents, drugs, prodrugs, toxins, enzymes, immunomodulators, apoptosis promoters, cytokines, hormones, oligonucleotides, antisense molecules, siRNA, and secondary antibodies.

[0202] In some embodiments, the H1.0K180me2 protein or peptide is selective for autoantibodies that are specific to H1.0K180me2. In some embodiments, the protein or peptide binds to autoantibodies that are nonspecific to H1.0K180me2.

[0203] In certain embodiments, the H1.0K180me2 protein or peptide provided herein binds to a target autoantibody and has a dissociation constant (Kd) in the range of 0.0001 nM to 1 μM. For example, the Kd of the H1.0K180me2 protein or peptide may be about 1 μM, about 100 nM, about 50 nM, about 10 nM, about 5 nM, about 1 nM, about 0.5 nM, about 0.1 nM, about 0.05 nM, about 0.01 nM, about 0.005 nM, about 0.001 nM, about 0.0005 nM, or even about 0.0001 nM.

[0204] In some embodiments, the H1.0K180me2 protein or peptide is specific to human H1.0K180me2 autoantibodies. In some embodiments, the H1.0K180me2 protein or peptide is cross-reactive with H1.0K180me2 autoantibodies from other species.

[0205] In some embodiments, the H1.0K180me2 protein or peptide is selective for H1.0K180me2 autoantibodies and shows little to no binding affinity to H1.0K180me1 or H1.0K180me3 autoantibodies. In some embodiments, the H1.0K180me2 protein or peptide binds to H1.0K180me2 autoantibodies but also shows binding affinity to H1.0K180me1 and / or anti-H1.0K180me3 autoantibodies.

[0206] In some embodiments, the binding preference (e.g., affinity) of the H1.0K180me2 protein or peptide to the H1.0K180me2 autoantibody is generally at least about 2 times, about 5 times, or at least about 10, 20, 50, 10 times compared to nonspecific autoantibodies (e.g., autoantibodies targeting a different target). 2 , 10 3 , 10 4 , 10 5 , or 10 6 It is double.

[0207] It is also possible to evaluate the H1.0K180me2 autoantibody protein or peptide using methods familiar to those skilled in the art to determine whether the H1.0K180me2 autoantibody protein or peptide has specificity and / or selectivity for the H1.0K180me2 antibody.

[0208] Nucleic acids encoding the H1.0K180me2 protein and peptide described herein are provided herein. Vectors containing any of the nucleic acids encoding the H1.0K180me2 protein and peptide described herein are also provided herein. D. Detection of Alzheimer's disease

[0209] H1.0K180me2 antibodies (for determining H1.0K180me2 levels) and H1.0K180me2 proteins and peptides (for determining H1.0K180me2 autoantibody levels) are provided herein for use in screening individuals for Alzheimer's disease, for use in identifying whether an individual is at risk of developing Alzheimer's disease, for use in estimating the likelihood of an individual developing Alzheimer's disease, for use in diagnosing Alzheimer's disease, for use in early detection of Alzheimer's disease, for use in prognosis of Alzheimer's disease, for use in selecting individuals who may respond to Alzheimer's disease treatment with Alzheimer's disease drugs or regimens, for use in selecting / determining treatment options for those diagnosed with Alzheimer's disease, for use in monitoring treatment for those diagnosed with Alzheimer's disease and undergoing ongoing treatment with Alzheimer's disease drugs or regimens, or for use in screening for Alzheimer's disease drugs and regimens.

[0210] H1.0K180me2 proteins and peptides for use in stratifying patients into distinct populations are also provided herein (for determining H1.0K180me2 IgG and H1.0K180me2 IgM autoantibody levels, also referred to as anti- or autoanti-H1.0K180me2 IgG and anti-autoanti-H1.0K180me2 IgM). In one embodiment, these distinct populations may be those that are likely to respond to immunotherapy for Alzheimer's disease, compared to those that are unlikely to respond to or not at all to immunotherapy for Alzheimer's disease.

[0211] Figure 17 demonstrates that by simultaneously measuring anti-H1.0K180me2 IgG and anti-H1.0K180me2 IgM autoantibody levels, it is possible to stratify individuals with Alzheimer's disease into distinct subpopulations based on the correlation between anti-H1.0K180me2 IgM normalized by total IgM and anti-H1.0K180me2 IgG normalized by total IgG in patient serum.

[0212] These uses are based on the observation that levels of H1.0K180me2 IgG autoantibodies and H1.0K180me2 IgM autoantibodies against H1.0K180me2 are altered in patients with Alzheimer's disease (Figures 12A-12C, 13B, 14A-14E, and 15A-15B).

[0213] Alzheimer's disease patients showed lower H1.0K180me2 concentrations than healthy, age-matched healthy controls, demonstrating that H1.0K180me2 concentration can effectively isolate patients with Alzheimer's disease from healthy individuals (Figure 12A). While serum H1.0K180me2 concentrations were sufficient to identify Alzheimer's disease patients, normalization of serum samples by total IgG (Figure 12B) or total protein (Figure 12C) allowed for direct inter-individual comparisons regardless of variables that may alter overall serum concentrations, such as the protocol used to obtain serum, changes in operators, patient hydration status, and patient activity level. For example, H1.0K180me2 serum levels were observed to be elevated in healthy older individuals compared to healthy younger individuals, while patients with Alzheimer's disease showed significantly lower, normalized H1.0K180me2 serum levels compared to healthy older individuals (over 60 years of age) (Figures 12B, 12C).

[0214] Alzheimer's disease patients also showed altered levels of H1.0K180me2 autoantibodies compared to age-matched healthy controls, demonstrating that patients with Alzheimer's disease can be effectively isolated from healthy individuals by serum concentrations of anti-H1.0K180me2 IgG and / or IgM (concentrations of H1.0K180me2 IgG and / or IgM autoantibodies). Alzheimer's disease patients showed higher serum concentrations of anti-H1.0K180me2 IgG than healthy, age-matched controls (Figures 13, 14A-E, 15A, 15B). Anti-H1.0K180me2 IgG and IgM levels in human serum were quantified by indirect ELISA analysis using biotinylated H1.0K180me2 capture peptide (H1.0K180me2 peptide) followed by a suitable secondary antibody (targeting either IgG or IgM antibodies). Equal volumes of serum were analyzed from healthy individuals aged 30–40 years (n=7) or over 60 years (n=9), and individuals over 60 years with clinically diagnosed Alzheimer's disease (n=10).

[0215] Figure 13B shows the quantification of anti-H1.0K180me2 IgG levels determined by indirect ELISA in Alzheimer's disease patients and age-matched controls. The concentration of anti-H1.0K180me2 IgG in each serum sample was calculated using a standard curve created from serial dilutions of H1.0K180me2-specific antibodies included in the ELISA experiment.

[0216] Figure 14E shows the quantification of raw, unnormalized anti-H1.0K180me2 IgM levels determined by indirect ELISA in patients with Alzheimer's disease (AD) and age-matched controls (AD-free, neurological controls).

[0217] Figures 15A and 15B show the quantification of normalized anti-H1.0K180me2 IgM levels determined by indirect serological ELISA in Alzheimer's disease patients (with AD) and age-matched controls (without AD, neurological controls). Figure 15B demonstrates that the diagnostic characteristics remained consistent regardless of different operators and laboratory settings.

[0218] Exemplary Method, H1.0K180me2 Level: More specifically, in one embodiment, a method for determining the H1.0K180me2 level for use in screening an individual for Alzheimer's disease, identifying an individual at risk of developing Alzheimer's disease, estimating the likelihood of an individual developing Alzheimer's disease, determining whether an individual has Alzheimer's disease, detecting early signs of Alzheimer's disease in an individual, and for use in assessing the prognosis of Alzheimer's disease in an individual, wherein (a) a biological sample derived from an individual is H1 A method is provided herein that includes the steps of (b) contacting the sample with a .0K180me2 antibody and (b) determining the concentration of H1.0K180me2 in the sample bound to the antibody, wherein a decrease in concentration relative to a control may indicate that the individual has Alzheimer's disease, is at risk of developing Alzheimer's disease, or has a greater likelihood of developing Alzheimer's disease, and an increase in concentration relative to a control or no change in concentration may indicate that the individual does not have Alzheimer's disease, is not at risk of developing Alzheimer's disease, or does not have a greater likelihood of developing Alzheimer's disease. Controls may include, but are not limited to, healthy controls (e.g., age-matched, sex-matched), a reference standard equivalent to a collection of healthy controls, or control samples from the same individual isolated early. In some embodiments, the concentration of circulating H1.0K180me2 is determined. In some embodiments, the change in concentration is relative to a threshold established by patient operating characteristic curve analysis for optimal specificity and sensitivity. In some embodiments, the method further includes the step of treating an individual with an Alzheimer's disease drug or regimen if it is determined that the individual has or is at risk of developing Alzheimer's disease.

[0219] In one embodiment of the method, a serum concentration of less than or equal to 5.61 nmol / ml of H1.0K180me2 indicates that an individual has or is likely to develop Alzheimer's disease. In one embodiment, a serum concentration of less than or equal to 5.61 nmol / ml of H1.0K180me2 indicates that an individual has or is likely to develop Alzheimer's disease with 78% specificity, 80% sensitivity, and a positive likelihood ratio of 3.6. In one embodiment, this represents a 24% increase in the probability of having Alzheimer's disease (post-test probability) compared to the pre-test probability. The post-test probability is calculated based on the following formula: pre-test odds × LR / (1 + pre-test odds × LR) (wherein pre-test odds is the clinical suspicion of the presence of the disease before the test). Post-test probabilities are typically calculated using the Likelihood Ratio Nomogram or Fagan Nomogram (NEJM 1975; Vol. 293: p. 257).

[0220] In another embodiment of the method, 4.76 × 10⁶ of H1.0K180me2 relative to total protein in serum is used. -4 A ratio less than or equal to this indicates that the individual has or is likely to develop Alzheimer's disease. In one embodiment, the ratio of H1.0K180me2 to total protein in serum is 4.76 × 10⁻⁶. -4 Concentrations below or equal to this indicate that the individual has Alzheimer's disease or is likely to develop Alzheimer's disease with 70% specificity, 90% sensitivity, and a positive likelihood ratio of 3.00. In one embodiment, this represents a 14.8% increase in the probability of having Alzheimer's disease compared to the probability before the test.

[0221] Exemplary Method, H1.0K180me2 Autoantibody Level: In another embodiment, the H1.0K180me2 protein or peptide is used to screen an individual for Alzheimer's disease, to identify an individual at risk of developing Alzheimer's disease, to estimate the likelihood of an individual developing Alzheimer's disease, to determine whether an individual has Alzheimer's disease, to detect early signs of Alzheimer's disease in an individual, and for use in assessing the prognosis of Alzheimer's disease in an individual (H1.0K180me2 autoantibody level, example). This specification provides a method for determining IgG autoantibody levels or IgM autoantibody levels, comprising the steps of (a) contacting a biological sample from an individual with the H1.0K180me2 protein or peptide, and (b) determining the concentration of an autoantibody in the sample that binds to the peptide, wherein an increase in concentration relative to a control may indicate that the individual has or is at risk of developing Alzheimer's disease, and a decrease in concentration relative to a control or no change in concentration may indicate that the individual does not have or is not at risk of developing Alzheimer's disease. Examples of controls, but not limited to these, include healthy controls (e.g., age-matched, sex-matched), a reference standard equivalent to a collection of healthy controls, or control samples from the same individual that were isolated early. In some embodiments, the change in concentration is relative to a threshold established by patient operating characteristic curve analysis for optimal specificity and sensitivity. In some embodiments, the method further includes the step of treating the individual with an Alzheimer's disease drug or regimen if it is determined that the individual has or is at risk of developing Alzheimer's disease.

[0222] In one embodiment, a serum concentration of IgG autoantibody against H1.0K180me2 higher than or equal to 9.69 ug / ml (normalized to total IgG levels) indicates that the individual has or is likely to develop Alzheimer's disease. In one embodiment, a serum concentration of IgG autoantibody against H1.0K180me2 higher than or equal to 9.69 ug / ml indicates that the individual has or is likely to develop Alzheimer's disease with 89% specificity, 60% sensitivity, and a positive likelihood ratio of 5.4. In one embodiment, this represents a 30% increase in the probability of having Alzheimer's disease compared to the probability before the test.

[0223] In some embodiments, if the serum concentration of IgG autoantibodies is higher than or equal to 8.23 ​​ug / ml normalized to serum volume, the individual has or is at risk of developing Alzheimer's disease. In some embodiments, if the serum concentration of IgM autoantibodies is higher than or equal to 409 fMol / ml normalized to serum volume, the individual has or is at risk of developing Alzheimer's disease. In some embodiments, if the ratio of the concentration of IgM autoantibodies to the total IgM concentration is 26.6 × 10⁻⁶ -6 If the value is greater than this, the individual has or is at risk of developing Alzheimer's disease. In one embodiment, if PLR(LR+) is 2.4 or greater than this relative to IgG autoantibodies, or if PLR(LR+) is 3.5 or greater than this relative to IgM autoantibodies, the individual has or is at risk of developing Alzheimer's disease.

[0224] In relevant embodiments, the methods provided herein are used in observational studies. Examples of observational studies, but not limited to, include: (a) cross-sectional studies (single time-point design or delayed cross-sectional) - testing one or more specimens / samples per patient collected at a single time point; (b) longitudinal studies - testing multiple specimens / samples per patient collected over a long period (e.g., weeks, months, years); (c) retrospective studies - testing previously collected specimens (characterized specimens) where the status of the analyte and the clinical status of the patient are known prior to the start of the study; (d) prospective studies - testing specimens collected before or during the study, except that both the status of the analyte and the clinical status of the patient are established during the study; and (e) prospective-retrospective studies - testing previously collected specimens where the clinical status is known, but the status of the analyte is unknown and will be established during the study.

[0225] In relevant embodiments, the methods provided herein are intended for the diagnosis of Alzheimer's disease and may be used to determine, verify, or confirm a patient's clinical condition as the sole determinant. In these embodiments, this type of test also includes a sole confirmatory assay (to verify previous test results) and a sole exclusion assay (to exclude specific conditions).

[0226] In the relevant embodiments, the methods provided herein are intended to provide “aid-to-diagnostic” assistance for Alzheimer’s disease and for assessing the clinical status of a patient. It may be used to provide additional information to aid in decision-making or verification. This test may be used to assess the patient's current condition, although it is not necessarily the sole determinant.

[0227] In relevant embodiments, the methods provided herein are intended for screening for Alzheimer's disease and may be used to determine the status of the disease, impairment, or other physiological condition in asymptomatic individuals. Depending on the condition and the nature of the target patient population, the screening methods may be used routinely or limited to patients at "risk." In this context, the methods described herein are used to assess the current condition of a patient.

[0228] In relevant embodiments, the methods provided herein are intended to determine predisposition to Alzheimer's disease, and the methods described herein may be used to determine the likelihood of a patient developing the disease before symptoms appear (e.g., to assess the risk of developing the disease in the future), and for patients at high risk (as determined by test results), preventive interventions may be taken.

[0229] In relevant embodiments, the methods provided herein are intended for the prognosis of Alzheimer's disease, and the methods described herein may be used to measure factors related to clinical outcomes independently of treatment. The methods described herein may be used to estimate the natural progression of the disease (e.g., outcomes without treatment), or the methods described herein are designed to assess the future condition of a patient.

[0230] In relevant embodiments, the methods provided herein are intended for determining the physiological status ("aging clock") of an aging population, and the methods described herein may be used to assess the physiological state of an individual for the purpose of identifying a person's state or characteristics regarding aging, or the risk of age-related Alzheimer's disease.

[0231] In related embodiments, quantification of the H1.0K180me2 antigen and / or quantification of the H1.0K180me2 autoantibody may be used to increase the confidence of screening, diagnosing, or detecting Alzheimer's disease.

[0232] In related embodiments, the quantification of H1.0K180me2 antigen and / or H1.0K180me2 autoantibody is performed, but is not limited to these, Aβ 42 , T-tau, p-tau, Aβ 42 It may be used in conjunction with other cerebrospinal fluid (CSF) tests, including the measurement of the / T-tau ratio and Aβ42 / p-tau.

[0233] In related embodiments, the quantification of the H1.0K180me2 antigen and / or the quantification of the H1.0K180me2 autoantibody may be used in conjunction with assessments of cognitive status tests, such as the MMSE (Mini-Mental State Examination), GDS (Global Deterioration Rate), and CDR (Clinical Dementia Scale) tests.

[0234] In related embodiments, quantification of the H1.0K180me2 antigen and / or quantification of the H1.0K180me2 autoantibody may be used in conjunction with neuroimaging.

[0235] In some embodiments of the methods described herein, the level of H1.0K180me2 may be normalized to total IgG in the biological sample or to total protein in the biological sample. In some embodiments of the methods described herein, the concentration of H1.0K180me2 may be determined as a relative ratio to an unmethylated, labeled, synthetic H1.0 peptide.

[0236] In embodiments of the methods described herein, the individual is over 50 years old. In some embodiments of the methods described herein, the individual is under 50 years old. In some embodiments of the methods described herein, the individual is at least 50 years old, at least 55 years old, at least 60 years old, at least 65 years old, at least 70 years old, at least 75 years old, or at least 80 years old. In exemplary embodiments, the individual is at least 60 years old. E. Companion diagnostics for Alzheimer's disease

[0237] H1.0K180me2 antibodies (for determining H1.0K180me2 levels) and H1.0K180me2 proteins and peptides (for determining H1.0K180me2 autoantibody levels, e.g., IgG autoantibody levels or IgM autoantibody levels) are also provided herein for use in methods for selecting individuals who may respond to Alzheimer's disease treatment with Alzheimer's disease drugs or regimens, for use in selecting / determining treatment options for those diagnosed with Alzheimer's disease, for use in monitoring treatment for those diagnosed with Alzheimer's disease and receiving ongoing treatment with Alzheimer's disease drugs or regimens, or for use in screening for Alzheimer's disease drugs and regimens.

[0238] In these embodiments, the Alzheimer's disease drugs and regimens / treatments include, but are not limited to, APP synthesis inhibitors, beta-secretase inhibitors, gamma-secretase inhibitors and modulators, Aβ aggregation inhibitors, Aβ immunotherapy, cholesterol-lowering drugs, anti-tau drugs, cholinesterase inhibitors, N-methyl-D-aspartate (NMDA) antagonists, atypical antipsychotics, protein S-nitrosylation blockers, glucagon-like peptide-1 receptor agonists, rapamycin, rapalog, endogenous cannabinoids, cannabinoids, neuroprotective substances, molecules that regulate calcium influx, antioxidants, anti-inflammatory drugs, drugs that regulate glutamate homeostasis, autophagy inducers, hormones, hormone regulators, statins, insulin, insulin carriers, multifunctional nanocarriers, vitamins, nutritional supplements, small RNA molecules, peptides, or ultrasound therapy.More specifically, APP synthesis inhibitors (+fencerin), beta-secretase inhibitors (MK-8931, E2609, LY2811376, LY2886721, PF-05297909), gamma-secretase inhibitors and modulators (semagacestat LY450139, abagacestat BMS-708163, PF-3084014, ELND006, tarenflurvir, CHF5074), Aβ aggregation inhibitors (tramiprosate (3APS), cryoquinol (PBT1), PBT2, ELND005 (scyllo- Nositol), PQ912), Aβ immunotherapy (GSK933776, AN1802+QS21, ACC-001, Alzheimer's Disease-106, bapineozumab, solanezumab, gantenerumab (RO4909832), ponezumab (PF-04360365), MABT5102A (crenezumab), BAN2401, intravenous immunoglobulin, gantenerumab (R1450 or RO4909832)), antitau drugs (lithium, tidogliusib (NP031112), LMTX (methylene blue)), cholinesterase inhibitors (Raza dyne® (galantamine), Exelon® (rivastigmine), and Aricept® (donepezil), N-methyl D-aspartate (NMDA) antagonists (Aricept® and Namzaric®, Namenda® and donepezil combinations), atypical antipsychotics (olanzapine, quetiapine, risperidone), protein S-nitrosylation blockers, glucagon-like peptide 1 receptor agonists, rapamycin and rapalog, endogenous canna Vinoids and cannabinoids, neuroprotective substances, molecules that regulate calcium influx, antioxidants (vitamin E, vitamin C, alpha-lipoic acid, coenzyme Q), anti-inflammatory molecules and drugs, drugs that regulate glutamate homeostasis, autophagy inducers, hormones and hormone regulators, statins, insulin and intranasal insulin, insulin carriers including long-acting insulin and thalidomide, ramipril, resveratrol, multifunctional nanocarriers, vitamins and nutritional supplements, small RNA molecules, peptides, and ultrasound therapy.In some embodiments, Alzheimer's disease drugs and regimens are selected from a list of FDA-registered clinical trials for drug approval, which are pending FDA approval and available at the U.S. Food and Drug Administration's worldwide web address.

[0239] Exemplary Method, H1.0K180me2 Level: In one embodiment, a method is provided herein for using an H1.0K180me2 antibody to determine whether an individual diagnosed with Alzheimer's disease and undergoing ongoing treatment will benefit from or continue to benefit from the ongoing treatment, comprising the steps of (a) preparing a biological sample from an individual undergoing ongoing treatment; (b) contacting the biological sample with an H1.0K180me2 antibody; (c) determining the concentration of H1.0K180me2 in the sample bound to the antibody; and (d) selecting an individual that will benefit from or continue to benefit from the treatment, wherein an increase in concentration relative to a control may indicate that the individual will benefit from or continue to benefit from the treatment, and a decrease in concentration relative to a control or no change in concentration may indicate that the individual does not tend to benefit from or continue to benefit from the treatment, or is not responsive to the treatment. Examples of controls include, but are not limited to, samples from an individual with Alzheimer's disease who is not undergoing treatment, or control samples from the same individual isolated early prior to the initiation of treatment. In some embodiments, the concentration of circulating H1.0K180me2 is determined. In some embodiments, the change in concentration is relative to a threshold established by patient operating characteristic curve analysis for optimal specificity and sensitivity.

[0240] Methods using the H1.0K180me2 antibody are also provided herein for use in selecting treatments for individuals diagnosed with Alzheimer's disease, for determining treatment options for individuals diagnosed with Alzheimer's disease, and for determining which individuals may benefit from a particular treatment. In one embodiment, the method includes (a) preparing a biological sample derived from an individual before the individual is treated for Alzheimer's disease; (b) contacting the biological sample with a candidate treatment; (c) contacting the biological sample with the H1.0K180me2 antibody; (d) determining the concentration and / or intracellular localization of H1.0K180me2 in the sample bound to the antibody; and (e) selecting individuals who may benefit from the treatment, wherein an increase in concentration compared to a control, or a decrease in intracellular localization in the cytoplasm, may indicate that the individual may benefit from the treatment, while a decrease in concentration compared to a control, no change in concentration, or an increase in intracellular localization in the cytoplasm, or no change in intracellular localization, may indicate that the individual does not benefit from the treatment. In another embodiment, the method includes (a) administering a candidate treatment to an individual; (b) preparing a biological sample derived from the individual after administration of the treatment; (c) contacting the biological sample with an H1.0K180me2 antibody; (d) determining the concentration and / or intracellular localization of H1.0K180me2 in the sample bound to the antibody; and (e) selecting individuals that may benefit from the treatment. An increase in concentration or a decrease in intracellular localization in the cytoplasm compared to a control may indicate that the individual may benefit from the treatment, while a decrease in concentration or no change in concentration compared to a control, or an increase or no change in intracellular localization in the cytoplasm, may indicate that the individual does not benefit from the treatment. Controls may include, but are not limited to, samples derived from healthy individuals or biological samples being contacted with a placebo treatment. In some embodiments, the concentration of circulating H1.0K180me2 is determined. In some embodiments, the change in concentration is relative to a threshold established by patient operating characteristic curve analysis for optimal specificity and sensitivity.

[0241] Exemplary Method, H1.0K180me2 Autoantibody Levels: In another embodiment, a method is provided herein for using the H1.0K180me2 protein or peptide to determine whether an individual diagnosed with Alzheimer's disease and undergoing ongoing treatment will benefit from or continue to benefit from the ongoing treatment, comprising the steps of (a) preparing a biological sample derived from the individual; (b) contacting the biological sample with the H1.0K180me2 protein or peptide; (c) determining the concentration of autoantibodies in the sample bound to the protein or peptide; and (d) selecting an individual that will benefit from or continue to benefit from the treatment, wherein a decrease in the concentration of autoantibodies relative to a control may indicate that the individual will benefit from or continue to benefit from the treatment, and no change or increase in the concentration of autoantibodies relative to a control may indicate that the individual will not benefit from or no longer benefit from the treatment. Controls may include, but are not limited to, samples derived from individuals with Alzheimer's disease who are not receiving treatment, or control samples from the same individual isolated early prior to the initiation of treatment. In some embodiments, the change in concentration is relative to a threshold established by patient operating characteristic curve analysis for optimal specificity and sensitivity. In some embodiments, the concentration of anti-H1.0K180me2 IgG autoantibody is determined. In some embodiments, the concentration of anti-H1.0K180me2 IgM autoantibody is determined. In some embodiments, the concentrations of anti-H1.0K180me2 IgG and IgM autoantibodies are determined.

[0242] Methods using the H1.0K180me2 protein or peptide are also provided herein for use in selecting treatments for individuals diagnosed with Alzheimer's disease, for determining treatment options for individuals diagnosed with Alzheimer's disease, and for determining whether an individual may benefit from a particular treatment. In one embodiment, the method may include the steps of (a) administering a candidate treatment to an individual, (b) preparing a biological sample derived from the individual after administration, (c) contacting the sample with the H1.0K180me2 protein or peptide, (d) determining the concentration of autoantibodies in the sample bound to the protein or peptide, and (e) selecting individuals who may benefit from the treatment, where a decrease in the concentration of autoantibodies relative to a control may indicate that the individual benefits from the particular treatment, and no change or increase in the concentration of autoantibodies relative to a control may indicate that the individual does not benefit from the particular treatment. Reference controls may include, but are not limited to, samples derived from healthy individuals or biological samples being contacted with a placebo treatment. In some embodiments, the change in concentration is relative to a threshold established by patient operating characteristic curve analysis for optimal specificity and sensitivity. In some embodiments, the concentration of anti-H1.0K180me2 IgG autoantibody is determined. In some embodiments, the concentration of anti-H1.0K180me2 IgM autoantibody is determined. In some embodiments, the concentrations of anti-H1.0K180me2 IgG and IgM autoantibodies are determined.

[0243] Methods using the H1.0K180me2 protein or peptide are also provided herein for stratifying Alzheimer's disease patients into distinct groups: one group prone to responding to any type of Alzheimer's disease treatment and another group (possibly more restrictive) prone to not responding to any type of Alzheimer's disease treatment. In one embodiment, the group prone to responding to any type of Alzheimer's disease treatment may respond to both immunotherapy and non-immunotherapy-based treatments for Alzheimer's disease. In one embodiment, the group prone to not responding to any type of Alzheimer's disease treatment may respond only to non-immunotherapy-based treatments for Alzheimer's disease. In some embodiments, as provided herein, such stratification may be performed by calculating the ratio of the concentration of anti-H1.0K180me2 IgG antibody to the concentration of anti-H1.0K180me2 IgM antibody and / or relating these to each other and fitting them to a statistical model. Such methods can provide a range of viable treatment options for individuals diagnosed with Alzheimer's disease and for determining whether an individual may benefit from a particular treatment. In one embodiment, the method may include the steps of (a) preparing a biological sample derived from an individual; (b) contacting the sample with the H1.0K180me2 protein or peptide; (c) determining the concentrations of IgG and IgM autoantibodies in the sample that bind to the protein or peptide; and (d) selecting individuals who may benefit from immunotherapy-based treatment for Alzheimer's disease.

[0244] In the relevant embodiments, the methods provided herein are useful in ensuring that H1.0K180me2 or H1.0K180me2 autoantibody levels are within physiological levels or within the range of established therapeutic agents.

[0245] In relevant embodiments, the methods provided herein are useful for monitoring Alzheimer's disease and may be used to measure H1.0K180me2 or H1.0K180me2 autoantibody levels for the purpose of adjusting treatment / intervention as needed. In relevant embodiments, the methods provided herein are useful for monitoring the effects of Alzheimer's disease drugs or nutritional regimens or lifestyle adjustments in individuals receiving such treatment.

[0246] In relevant embodiments, the methods provided herein are useful for monitoring clinical outcomes in any observational or interventional clinical study for Alzheimer's disease, where (a) an observational study is a study in which the results obtained during the study are not used in the management of patients and do not influence treatment decisions, and (b) an interventional study is a study in which the results obtained during the study may influence decisions regarding the management of patients and may be used to guide treatment.

[0247] In relevant embodiments, the methods provided herein are useful for a series of measurements, thereby obtaining numerous decisions over time. These types of monitoring methods may be used for detecting / assessing disease progression / regression, disease recurrence, minimal residual disease, response / resistance to treatment, and / or adverse effects of treatment. These types of monitoring methods may be designed to assess changes in an individual's condition.

[0248] In relevant embodiments, the methods provided herein are useful for predicting responses or reactions to Alzheimer's disease treatments, and the methods described herein may be used to measure factors that determine the likelihood of a patient's response or adverse reaction to a particular therapy. Predictive methods specifically designed for use as companion diagnostics are described herein.

[0249] In some embodiments of the methods described herein, the level of H1.0K180me2 may be normalized to total IgG in the biological sample or to total protein in the biological sample. In some embodiments of the methods described herein, the concentration of H1.0K180me2 may be determined as a relative ratio to an unmethylated, labeled, synthetic H1.0 peptide.

[0250] In embodiments of the methods described herein, the individual is over 50 years old. In some embodiments of the methods described herein, the individual is under 50 years old. In some embodiments of the methods described herein, the individual is at least 50 years old, at least 55 years old, at least 60 years old, at least 65 years old, at least 70 years old, at least 75 years old, or at least 80 years old. In exemplary embodiments, the individual is at least 60 years old.

[0251] In related embodiments, the method may be used for screening for new Alzheimer's disease drugs and regimens. F. Detection of aging

[0252] H1.0K180me2 antibodies and H1.0K180me2 proteins and peptides for use in detecting aging are provided herein. Where provided herein, aging is related to replication aging (REP-SEN), genotoxic stress-induced aging, and radiation-induced aging.

[0253] Generally, methods for detecting aging include direct or indirect detection of H1.0K180me2. For direct detection of H1.0K180me2, the method generally includes (a) contacting an organism-derived biological sample with an H1.0K180me2 antibody, and (b) determining the concentration of the H1.0K180me2 antigen in the sample bound to the antibody, where an increase in concentration relative to a control indicates the presence of senescent cells in the biological sample. For indirect detection of H1.0K180me2, the method generally includes (a) contacting an organism-derived biological sample with an H1.0K180me2 protein or peptide, and (b) determining the concentration of the H1.0K180me2 antigen in the sample bound to the peptide, where an increase in concentration relative to a control indicates the presence of senescent cells in the biological sample. In some embodiments, the change in concentration is relative to a threshold established by patient operating characteristic curve analysis for optimal specificity and sensitivity.

[0254] In some embodiments, the method may be used to identify individuals that have experienced genotoxic aging. In some embodiments, genotoxic stress-induced aging is the result of an individual's exposure to DNA damaging agents, drugs or toxins, such as radiation, UV light, bleomycin and, but not limited to, trazodone, etotifen, cephalexin, nisoldipine (Nisoldipme), CGS 15943, clotrimazole, 5-nonyltryptamine, doxepin, pergolide, paroxetine, resveratrol, quercetin, honokiol, 7-nitroindazole, megestrol, fluvoxamine, etoposide, veliparib, rucaparib, olaparib, camptothecin, or any other genotoxic agents and common chemotherapeutic agents, including terbinafine.

[0255] In some embodiments, the method is useful for identifying aging in individuals undergoing chemotherapy to ensure the effectiveness of the chemotherapy. The chemotherapeutic agents in these embodiments include alemtuzumab (Campath), alitretinoin (Panretin), allopurinol (Zyloprim), altretamine (Hexalen), amifostin (Ethyol), anastrozole (Arimidex), arsenite (Trisenox), asparaginase (Elspar), and BCG Live (TICE). The following may be selected from the group consisting of BCG, bexarotene (Targretin), bleomycin (Blenoxane), intravenous busulfan (Busulfex), oral busulfan (Myleran), carsterone (Methosarb), capecitabine (Xeloda), streptozocin (Zanosar), tel (Sclerosol), tamoxifen (Nolvadex), temozolomide (Temodar), teniposide, VM-26 (Vumon), testolactone (Teslac), thioguanine, 6-TG (Thioguanine), thiotepa (Thioplex), and topotecan (Hycamtin). G. Detection of DNA damage

[0256] H1.0K180me2 antibodies, as well as H1.0K180me2 proteins and peptides, are provided herein for use in detecting DNA damage, such as acute DNA damage.

[0257] Generally, methods for detecting DNA damage include direct or indirect detection of H1.0K180me2. For direct detection of H1.0K180me2, the method generally includes (a) contacting an organism-derived biological sample with an H1.0K180me2 antibody, and (b) determining the concentration of the H1.0K180me2 antigen in the sample bound to the antibody, where an increase in concentration relative to a control indicates that the biological sample has undergone DNA damage. For indirect detection of H1.0K180me2, the method generally includes (a) contacting an organism-derived biological sample with an H1.0K180me2 protein or peptide, and (b) determining the concentration of the H1.0K180me2 antigen in the sample bound to the peptide, where an increase in concentration relative to a control indicates that the biological sample has undergone DNA damage. In some embodiments, the change in concentration is relative to a threshold established by patient operating characteristic curve analysis for optimal specificity and sensitivity.

[0258] In some embodiments, DNA damage is the result of exposure of cells or organisms to DNA damaging agents, drugs, or toxins, such as radiation, bleomycin, or other DNA damaging agents (e.g., the chemotherapeutic agents mentioned above).

[0259] In some embodiments, the method is useful for determining DNA damage within 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 24 hours, 48 ​​hours, 3 days, 4 days, or up to 5 days from such exposure to a genotoxic substance or DNA damaging agent.

[0260] In some embodiments, portable devices for detecting DNA damage are provided herein. The device may include a sample collection device, a reader, and an assay module containing an H1.0K180me2 antibody. The device may also include a sample collection device, a reader, and an assay module containing an H1.0K180me2 protein or peptide. H. Detection of radiation exposure

[0261] H1.0K180me2 antibodies, as well as H1.0K180me2 proteins and peptides, are provided herein for use in detecting radiation exposure.

[0262] Generally, methods for detecting radiation exposure include direct or indirect detection of H1.0K180me2. For direct detection of H1.0K180me2, the method generally includes (a) contacting an organism-derived biological sample with an H1.0K180me2 antibody, and (b) determining the concentration of the H1.0K180me2 antigen in the sample bound to the antibody, where an increase in concentration relative to a control indicates radiation exposure. For indirect detection of H1.0K180me2, the method generally includes (a) contacting an organism-derived biological sample with an H1.0K180me2 protein or peptide, and (b) determining the concentration of the H1.0K180me2 antigen in the sample bound to the peptide, where an increase in concentration relative to a control indicates radiation exposure. In some embodiments, the change in concentration is relative to a threshold established by patient operating characteristic curve analysis for optimal specificity and sensitivity. In one exemplary embodiment, the change in the concentration of H1.0K170me2 antigen 2 hours after 7 Gy of X-ray exposure is from 12 umol / L to 21 umol / L, and 48 hours later it is from 26 umol / L to 35 umol / L.

[0263] In some embodiments, the method is useful for determining radiation exposure within 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 24 hours, 48 ​​hours, 3 days, 4 days, or up to 5 days from such exposure.

[0264] In some embodiments, the method is useful for military personnel to determine such exposure in field conditions, for example, in a combat zone.

[0265] In some embodiments, portable devices for detecting radiation damage are provided herein. The device may include a sample collection device, a reader, and an assay module containing an H1.0K180me2 antibody. The device may also include a sample collection device, a reader, and an assay module containing an H1.0K180me2 protein or peptide. I.H1.0K180me2 and Rapalog

[0266] Mammalian targets (mTORs) of rapamycin have emerged as promising therapeutic targets. Rapamycin and several rapamycin derivatives, rapamycin analogs, and other mTOR inhibitors are drugs approved by the FDA for the treatment of certain disease conditions.

[0267] The inventors have found that the detection of H1.0K180me2 may be useful for screening individual responsiveness to rapamycin, rapamycin derivatives, rapamycin analogs, and other mTOR inhibitors collectively referred to as "rapalogs," and may be useful for monitoring rapalog-based therapeutic regimens. The inventors have also found that the detection of H1.0K180me2 may be useful for screening additional rapalogs for drug screening purposes. Specifically, it is shown here that rapamycin derivatives and the immunosuppressant everolimus block the appearance of H1.0K180me2 in response to DNA damage (Figure 18). It has also been observed that treatment with rapalogs prior to or concurrent with exposure to genotoxic stress may reduce the effects of genotoxic stressors, as evidenced by changes in the concentration and / or intracellular localization of H1.0K180me2.

[0268] As used herein, rapalogs include FDA-approved rapalogs and rapalogs currently undergoing clinical trials. FDA-approved rapalogs include rapamycin, sirolimus, rapamycin, everolimus, RAD001, Afinitor, Zortress, temsirolimus, CCI-779, Torisel, ridafololimus, AP23573, MK-8669, defololimus, zotarolimus, and ABT-578. Other RPAlogs include AZD8055, AZD2014, OSI-027, MLN0128, WYE-132, Torin1, PI-103, P7170, PF-04691502, PF-05212384, PKI-587, GNE477, PKI-180, WJD008, XL765, SAR245409, NVP-BEZ235, BGT226, SF1126, GSK2126458, Ku-0063794, WYE-354, NVP-BEZ235, PF-05212384, XL765, Torin 2, WYE-125132, and OSI-027.

[0269] Generally, methods for monitoring the effects of ongoing rapalog-based treatments in individuals diagnosed with cancer, immunodeficiency, diabetes, arthritis, Alzheimer's disease and other neurodegenerative diseases, cardiovascular diseases, autoimmune diseases, and other age-related pathologies are provided herein.

[0270] Accordingly, in one embodiment, a method is provided herein for determining whether an individual receiving treatment with a rapalog is responsive to such treatment, comprising the steps of (a) contacting a biological sample derived from the individual with an H1.0K180me2 antibody, (b) determining the concentration and / or localization of H1.0K180me2 in the sample bound to the antibody, and (c) determining whether the individual is responsive to the treatment, wherein a decrease in established concentration or a change in localization compared to a control indicates that the individual is responsive to the rapalog. In some embodiments, if there is an increase in the extracellular concentration of H1.0K180me2 compared to a control, it is determined that the treatment with the rapalog is ineffective or needs to be modified. In some embodiments, if there is an increase in the cytoplasmic localization of H1.0K180me2 compared to a control, it is determined that the treatment with the rapalog is ineffective or needs to be modified. In some embodiments, the decrease is compared to an age-matched control that has not been diagnosed with the associated disease to which the rapalog is being administered. In some embodiments, if a decrease in the extracellular or cytoplasmic concentration of H1.0K180me2 protein is observed compared to the control, it is determined that the treatment is effective and should be continued. In some embodiments, the method further includes a step of using information to modify the type, process, duration, and / or dosage of the treatment. In some embodiments, the concentration of circulating H1.0K180me2 is determined. In some embodiments, the change in concentration is relative to a threshold established by patient operating characteristic curve analysis for optimal specificity and sensitivity.

[0271] Similarly, when autoantibodies against H1.0K180me2 are used for measurement, the method includes (a) contacting an individual-derived biological sample with the H1.0K180me2 protein or peptide, (b) determining the concentration of the autoantibody bound to the protein or peptide, and (c) determining whether the individual is responsive to the treatment, where a change in the concentration of the autoantibody relative to a control indicates that the individual is responsive to the rapalog. In some embodiments, if there is no change in the concentration of H1.0K180me2 autoantibodies relative to a control, it is determined that the treatment with the rapalog is ineffective or needs to be modified. In some embodiments, the decrease is relative to an age-matched control that has not been diagnosed with the associated disease to which the rapalog is being administered. In some embodiments, if there is a change in the concentration of H1.0K180me2 autoantibodies relative to a control, it is determined that the treatment is effective and should be continued. In some embodiments, the change in concentration is relative to a threshold established by patient operating characteristic curve analysis for optimal specificity and sensitivity.

[0272] In some embodiments, methods are provided herein for selecting individuals who have been diagnosed with or are suspected of having cancer, immunodeficiency, diabetes, Alzheimer's disease or other neurodegenerative diseases, cardiovascular diseases, autoimmune diseases, arthritis, and other age-related pathologies and who may benefit from treatment with a laparog, i.e., for determining whether an individual may respond to treatment with a laparog. In some embodiments, the method comprises the steps of preparing a biological sample derived from an individual, treating the sample with a laparog in vitro, ex vivo, in section culture, or in tissue culture, and determining the concentration and / or intracellular localization of H1.0K180me2 in the sample. In some embodiments, the concentration of H1.0K180me2 is determined by measuring the concentration of H1.0K180me2 in the sample using an H1.0K180me2 antibody. In some embodiments, the concentration of H1.0K180me2 is determined by measuring the concentration of an anti-H1.0K180me2 autoantibody in the sample. In some embodiments, it is determined that treatment with rapalog may not be effective if there is an increase in the intracellular or extracellular concentration of H1.0K180me2 compared to the control. In some embodiments, it is determined that treatment with rapalog may not be effective if there is an increase in the intracellular localization of H1.0K180me2 compared to the control. In some embodiments, the decrease is in age-matched controls who have not been diagnosed with a disease that may warrant treatment with rapalog. In some embodiments, it is determined that treatment may be effective if there is a decrease in the intracellular or extracellular concentration of the H1.0K180me2 protein compared to the control. In some embodiments, the change in concentration is in relation to a threshold established by patient operating characteristic curve analysis for optimal specificity and sensitivity.

[0273] In some embodiments, the methods provided herein further include a step of using information to modify the type, process, duration, and / or dosage of treatment.

[0274] In other embodiments, the use of H1.0K180me2 detection to monitor the capabilities of a novel rapalog identified (for drug screening applications) is provided herein. J. Detection of biological aging

[0275] H1.0K180me2 antibodies, as well as H1.0K180me2 proteins and peptides, are provided herein for use in detecting biological aging. Since aging is a fundamental property of most organisms, biological aging markers or biomarkers of aging are expected to find many applications in biological investigations.

[0276] A difference can exist between chronological age and biological age. Some individuals age more rapidly, while others age more slowly and remain healthier and "youthful" longer due to good habits, genetics, and / or the absence of environmental stressors. Being able to track that biological age may help in modifying lifestyles (similar to tracking body mass index) or in implementing anti-aging procedures.

[0277] Accurate measurement of biological age using aging markers is useful for testing age-related disease theories about biological aging, such as (i) diagnosing various age-related diseases and defining cancer subtypes, (ii) predicting / foreseeing the onset of various diseases, and (iii) serving as surrogate markers for evaluating therapeutic interventions, including rejuvenation approaches.

[0278] Generally, methods for detecting biological aging include direct or indirect detection of H1.0K180me2. For direct detection of H1.0K180me2, the method generally includes (a) contacting an individual-derived biological sample with an H1.0K180me2 antibody, and (b) determining the concentration of the H1.0K180me2 antigen in the sample bound to the antibody, where an increase in concentration compared to a control indicates that the biological sample originates from an individual that has experienced biological aging. For indirect detection of H1.0K180me2, the method generally includes (a) contacting an individual-derived biological sample with an H1.0K180me2 protein or peptide, and (b) determining the concentration of the H1.0K180me2 antigen in the sample bound to the peptide, where an increase in concentration compared to a control indicates that the biological sample originates from an individual that has experienced biological aging. In some embodiments, the change in concentration is relative to a threshold established by patient operating characteristic curve analysis for optimal specificity and sensitivity. K. Diagnostic kits and products

[0279] Kits useful for detecting H1.0K180me2 are provided herein. In some embodiments, the kit comprises one or more H1.0K180me2 antibodies or H1.0K180me2 peptides described herein. In certain embodiments, the antibody or peptide is labeled. Furthermore, the kit may optionally include explanatory materials for performing any of the methods described herein. Explanatory materials typically include, but are not limited to, written or printed materials. Any medium in which such instructions can be stored and communicated to end users is contemplated herein. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD-ROMs), etc. Such media may include addresses to internet sites providing such explanatory materials.

[0280] The kit may also contain additional components to facilitate the specific application for which the kit is designed. For example, if the kit contains labeled anti-H1.0K180me2, the kit may further contain reagents for detecting the label (e.g., enzyme substrates for enzyme labeling, filter sets for detecting fluorescent labeling, appropriate secondary labels, etc.). The kit may also further contain buffers and other reagents routinely used for the practice of a particular method.

[0281] In addition to the H1.0K180me2 antibody, exemplary kits useful in immunoassays for detecting H1.0K180me2 may include the H1.0K180me2 protein or peptide. This peptide may be used, for example, as a positive control or as a competitor in a competitive immunoassay, and may be labeled or unlabeled depending on the format of the assay performed.

[0282] In addition to the anti-H1.0K180me2 peptide, another exemplary kit useful in immunoassays for detecting H1.0K180me2 would include an H1.0K180me2 antibody. This antibody may be used, for example, as a positive control or as a competitor in a competitive immunoassay, and may be labeled or unlabeled depending on the format of the assay performed.

[0283] A transdermal patch for measuring the concentration of a target H1.0K180me2 protein and peptide in subcutaneous tissue is provided herein, comprising a substrate containing an H1.0K180me2 antibody and a plurality of microneedles. In another embodiment, a transdermal patch for measuring the concentration of an H1.0K180me2 autoantibody is provided herein, comprising a substrate and an H1.0K180me2-binding protein or peptide specific for the detection of the H1.0K180me2 autoantibody. In some embodiments, the patch is a transdermal microneedle array patch. In some embodiments, the substrate of the patch is elastically stretchable. In some embodiments, a kit comprising a patch containing the antibody or peptide provided herein, and optionally instructions for use, is provided herein. In some embodiments, the patch is useful for detecting and measuring the concentration of H1.0K180me2, or for detecting and measuring the concentration of H1.0K180me2 antibodies in biological samples, for the purpose of detecting replication senescence, DNA damage, genotoxic stress, radiation exposure, and Alzheimer's disease, and is useful for monitoring treatment regimens and for drug screening.

[0284] Portable devices for detection, for example, for the detection of DNA damage or radiation exposure, are also provided herein. The device may include a sample collection device, a reader, and an assay module containing an H1.0K180me2 antibody. The device may also include a sample collection device, a reader, and an assay module containing an H1.0K180me2 protein or peptide.

[0285] Lateral flow pieces or test pieces suitable for lateral flow assays of analytes are also provided herein, comprising a sample receiving zone, wherein the sample receiving zone contains either an H1.0K180me2 antibody or an anti-H1.0K180me2 conjugated peptide. In some embodiments, the antibody or peptide includes a label. IV. Therapeutic Drugs A. Treatment of Methylated H1.0-Related Diseases and Conditions

[0286] Therapeutic H1.0K180me2 antibodies, therapeutic H1.0K180me2 proteins, and therapeutic H1.0K180me2 peptides are provided herein for the treatment of methylated H1.0-related diseases or conditions.

[0287] As used herein, “methylated H1.0-related disease or condition” is characterized by elevated levels of H1.0K180me2, increased endogenous dimethylation of K180 H1.0 protein / peptide substrates, increased release of H1.0K180me2 from chromatin, increased release of H1.0K180me2 from the nucleus into the cytoplasm, increased intracytoplasmic deposition of H1.0K180me2, elevated levels of H1.0K180me2 in extracellular space, elevated circulating levels of H1.0K180me2 in body fluids (e.g., serum, urine, saliva, cerebrospinal fluid, etc.), and / or elevated levels of autoantibodies specific to H1.0K180me2.

[0288] Methylated H1.0-related diseases and conditions include, but are not limited to, age-related pathologies, Alzheimer's disease, conditions involving increased senescent cells, radiation exposure, exposure to genotoxic stressors, accumulation of senescent cells associated with external and internal stressors, and autoimmune diseases and conditions associated with high levels of autoantibodies against H1.0K180me2.

[0289] A method for treating a methylation H1.0-related disease or condition in an individual by binding to and removing H1.0K180me2 is provided herein, comprising the step of administering to the individual a therapeutically effective amount of a therapeutic H1.0K180me2 antibody.

[0290] A method for treating a methylation H1.0-related disease or condition in an individual for binding to and removing an H1.0K180me2 autoantibody is provided herein, comprising the step of administering to the individual a therapeutically effective amount of therapeutic H1.0K180me2 protein or therapeutic H1.0K180me2 peptide.

[0291] As used herein, "individual" refers to any animal classified as a mammal, including humans, domesticated and livestock animals, as well as zoo animals, sporting animals, or pets, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, and cats. An individual may be male or female.

[0292] In some embodiments of the methods described herein, the individual is over 50 years old. In some embodiments of the methods described herein, the individual is under 50 years old. In some embodiments of the methods described herein, the individual is at least 50 years old, at least 55 years old, at least 60 years old, at least 65 years old, at least 70 years old, at least 75 years old, or at least 80 years old. In exemplary embodiments, the individual is at least 60 years old.

[0293] Looking more specifically at methylation H1.0-related diseases and conditions during aging, there is an increase in the accumulation of intracytoplasmic H1.0K180me2 in human brain tissue (Figure 11B). These observations correlate with age-related increases in circulating H1.0K180me2 antigen observed in serological tests (Figure 11C).

[0294] However, when comparing individuals diagnosed with Alzheimer's disease with a control group (those without Alzheimer's disease pathology) to a reference control (diagnosed Alzheimer's disease patients) (Figures 12A-12C), a decrease in the circulating level of H1.0K180me2 antigen observed in serological ELISA tests was observed (Figure 11C).

[0295] Compared to age-matched individuals without pathology, this decrease in circulating levels of H1.0K180me2 antigen in the Alzheimer's disease group is accompanied by elevated levels of IgG and IgM autoantibodies against the H1.0K180me2 epitope (Figures 13B, 14B, 14D, 14E, 15A, 16A, 16B).

[0296] Therefore, in the context of Alzheimer's disease, treatment with a therapeutically effective dose of H1.0K180me2 antibody (e.g., a cell-permeable antibody or a cell-clearing antibody) is provided.

[0297] Furthermore, compared to age-matched controls, naturally occurring serum IgG and IgM autoantibodies produced in response to H1.0K180me2 are increased in individuals with Alzheimer's disease (Figures 13B, 14B, 14D, 14E, 15A, 16A, 16B) and may be harmful in the sense that they attack and destroy cells expressing H1.0K180me2. Therefore, in this context, treatment with a therapeutically effective dose of H1.0K180me2 antibody that binds to (neutralizes) the protein or peptide is provided.

[0298] In the context of DNA damaging agents, dimethylation of H1.0K180 (H1.0K180me2) is observed in chromatin after acute DNA damage induced by the chemomichian bleomycin (Figure 7A). Therefore, in this context, treatment with a therapeutically effective dose of H1.0K180me2 antibody (e.g., a cell-permeable antibody or a cell-removal antibody) is offered. Furthermore, naturally occurring autoantibodies arising in response to the increase in H1.0K180me2 in the dimethylation of H1.0 proteins are targets for treatment with a therapeutically effective dose of H1.0K180me2 antibody that binds to (neutralizes) the protein or peptide.

[0299] In the context of genotoxic stress, H1.0K180me2 is released from chromatin during genotoxic stress-induced aging (a few days after treatment with a DNA damaging agent) (Figure 9A) and secreted from cells into the extracellular space (Figure 9B). Therefore, in this context, treatment with a therapeutically effective dose of H1.0K180me2 antibody is offered. Furthermore, naturally occurring autoantibodies that arise in response to an increase in H1.0K180me2 are targets for treatment with a therapeutically effective dose of H1.0K180me2 antibody that binds to (neutralizes) proteins or peptides.

[0300] In the context of radiation exposure, exposure to ionizing radiation induces an increase in the levels of circulating H1.0K180me2 in the serum (Figures 10A and 10B). Therefore, in this context, treatment with a therapeutically effective dose of H1.0K180me2 antibody is offered. Furthermore, naturally occurring autoantibodies that arise in response to the increase in H1.0K180me2 are targets for treatment with a therapeutically effective dose of H1.0K180me2 antibody that binds to (neutralizes) proteins or peptides.

[0301] In some embodiments, a diagnostic method is performed to determine whether the individual being treated is actually suffering from a methylated H1.0-related disease or condition. Assays testing for the presence, localization, chromatin fraction, intracellular accumulation, serum levels, and autoantibody levels of H1.0K180me2 or H1.0K180me2 antibodies indicating a methylated H1.0-related disease or condition may be performed prior to treatment. B. Therapeutic H1.0K180me2 antibody

[0302] As discussed in Section (II)(A) above, antibodies that recognize and specifically bind to the H1.0K180me2 epitope and may be used for therapeutic purposes are provided herein.

[0303] In addition to the H1.0K180me2 antibody that recognizes the dimethylated K180 antigen, in some embodiments, the therapeutic antibody is an anti-idiotype antibody. Such an idiotype antibody recognizes disease-related IgG autoantibodies or disease-related IgM autoantibodies, or other methylated H1.0-related diseases or conditions, in patients with Alzheimer's disease. In some embodiments, in vitro IgM (IVIgM) may be derived from the plasma of an Alzheimer's disease patient donor and used as a prophylactic treatment or vaccine for Alzheimer's disease. Such IVIgM may contain an anti-idiotype antibody that recognizes the patient's disease-related autoantibodies. Therefore, in some embodiments, IVIgM may have an idiotype antibody that can inactivate IgG and / or IgM autoantibodies against H1.0K180me2.

[0304] The therapeutic antibodies provided herein may be of any immunoglobulin type, such as IgG, IgA, IgE, IgD, or IgM, or they may be anti-idiotype antibodies against IgG, IgA, IgE, IgD, or IgM. In some embodiments, the antibody is of the IgG subtype and may be an IgG1 antibody, IgG2 antibody, IgG3 antibody, or IgG4 antibody. In some embodiments, the antibody is of the IgM subtype.

[0305] The antibodies provided herein may be further conjugated for use in a variety of applications, including, but not limited to, detection, visualization, quantification, screening, and therapeutic use, and in biological assays related to such therapeutic applications.

[0306] In some embodiments, the therapeutic antibody is a neutralizing antibody that neutralizes one or more biological activities of H1.0K180me2. For example, the antibody can bind to extracellular H1.0K180me2 and neutralize any binding or signaling activity it may possess. In some embodiments, the antibody may remove or block H1.0K180me2 in cells or samples. In some embodiments, the antibody may remove cells containing H1.0K180me2.

[0307] In some embodiments, the therapeutic antibody may remove senescent cells. In some embodiments, the antibody may remove cells / tissues or protein products that cause symptoms of Alzheimer's disease. In some embodiments, the antibody may remove cells damaged by radiation, DNA damaging agents, and other genotoxic substances. In some embodiments, the antibody is a cell-permeable antibody. In other embodiments, the membrane of affected cells is included, allowing entry of the therapeutic H1.0K180me2 antibody provided herein.

[0308] In some embodiments, the therapeutic antibodies provided herein have antibody-dependent cell-mediated cytotoxicity (ADCC) activity. Effector cells, including cytotoxic T cells, natural killer (NK) cells, macrophages, neutrophils, eosinophils, dendritic cells, or monocytes, which have Fc gamma receptors (FcγR or FCGR) on their surface, recognize and bind to the Fc region of antibodies bound to target cells. Such binding may induce activation of intracellular signaling pathways that lead to cell death.

[0309] In some embodiments, therapeutic antibodies possess complement-dependent cell-mediated cytotoxicity (CDC) activity. Antibody-induced CDC is mediated via proteins of the classical complement cascade and is induced by binding the complement protein Clq to the antibody. The antibody Fc region that binds to Clq may induce activation of the complement cascade.

[0310] In some embodiments, therapeutic antibodies possess antibody-dependent cell phagocytic (ADCP) activity. Phagocytic cells, including monocytes and macrophages, which have Fc receptors on their cell surface, recognize and bind to the Fc region of antibodies that bind to target cells. The binding of the Fc receptor to the antibody-bound target cell may initiate phagocytosis in the target cell.

[0311] In some embodiments, therapeutic antibodies may form immune complexes. For example, the immune complexes may be cells that express or extrude the H1.0K180me2 antigen, which is coated by the antibody. C. Therapeutic H1.0K180me2 protein and H1.0K180me2 peptide

[0312] The production of the H1.0K180me2 antigen in individuals suffering from methylated H1.0-related diseases or conditions leads to the generation of naturally occurring autoantibodies specific to the H1.0K180me2 antigen. Therefore, therapeutic approaches that bind to and eliminate these naturally occurring autoantibodies are provided herein. For this purpose, in some embodiments, therapeutic H1.0K180me2 proteins and therapeutic H1.0K180me2 peptides that hold dimethylated lysine corresponding to K180 and may bind to and further neutralize naturally occurring autoantibodies that arise in response to H1.0K180me2 epitopes in cells or in circulation are provided herein.

[0313] In some embodiments, therapeutic H1.0K180me2 proteins / peptides may block the biological activity of H1.0K180me2 autoantibodies in an individual's serum. Such blocking of autoantibody levels may reduce the immune response and alleviate adverse symptoms of methylated H1.0 diseases or conditions.

[0314] In one embodiment, the therapeutic H1.0K180me2 protein is the full-length H1.0K180me2 protein (SEQ ID NO: 1). The therapeutic H1.0K180me2 peptide provided herein may be any fragment of the full-length H1.0K180me2 protein that retains a dimethylated K180 residue. In some embodiments, the therapeutic H1.0K180me2 peptide is in the range of 5 to 193 amino acids (aa) in length. In some embodiments, the length of the therapeutic H1.0K180me2 peptide is 5aa, 6aa, 7aa, 8aa, 9aa, 10aa, 11aa, 12aa, 13aa, 14aa, 15aa, 16aa, 17aa, 18aa, 19aa, 20aa, 21aa, 22aa, 23aa, 24aa, 25aa, 26aa, 27aa, 28aa, 29aa, or even 30aa. In a particular exemplary embodiment, the length of the therapeutic H1.0K180me2 peptide is 15aa, 16aa, 17aa, 18aa, 19aa, or 20aa. Table 3A provides exemplary sequences of the therapeutic H1.0K180me2 peptide provided herein. Therefore, in some embodiments, the therapeutic H1.0K180me2 peptide comprises one of the sequences selected from those presented in Table 3A. In related embodiments, the therapeutic H1.0K180me2 peptide consists of one of the sequences selected from those presented in Table 3A. In exemplary embodiments, the therapeutic H1.0K180me2 peptide comprises the sequence of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In exemplary embodiments, the H1.0K180me2 antibody-conjugated peptide consists of the sequence of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0315] In some embodiments, the therapeutic H1.0K180me2 protein or peptide is synthesized. In some embodiments, the therapeutic H1.0K180me2 protein or peptide is the product of an in vitro methylation reaction using a G9A methyltransferase enzyme or a G9A-like protein (GLP) methyltransferase enzyme under conditions that allow for specific dimethylation of the K180 residue, for example, as described in more detail herein.

[0316] As provided in Section I, the therapeutic H1.0K180me2 protein / peptide provided herein may be further conjugated for a variety of purposes, for example, but not limited to, use in therapy and use in biological assays related to its therapeutic applications.

[0317] In some embodiments, the therapeutic H1.0K180me2 protein / peptide includes labeling, such as detectable labeling, spin labeling, colorimetric labeling, radioactive labeling, enzymatic labeling, fluorescent labeling, or magnetic labeling (e.g., conjugated to the label). In exemplary embodiments, the therapeutic H1.0K180me2 protein / peptide is biotinylated. In some embodiments, the therapeutic H1.0K180me2 protein / peptide is conjugated to or attached to a solid surface, such as beads (e.g., magnetic, glass, or plastic beads), a column, or a microplate. In some embodiments, the therapeutic H1.0K180me2 protein / peptide is coated onto a microplate. In some embodiments, the therapeutic H1.0K180me2 protein / peptide is conjugated to or includes effector molecules, including, but not limited to, radionuclides, cytotoxins, chemotherapeutic agents, drugs, prodrugs, toxins, enzymes, immunomodulators, apoptosis promoters, cytokines, hormones, oligonucleotides, antisense molecules, siRNA, and secondary antibodies.

[0318] In some embodiments, the therapeutic H1.0K180me2 protein / peptide is selective for autoantibodies that are specific to H1.0K180me2. In some embodiments, the therapeutic H1.0K180me2 protein / peptide binds to autoantibodies that are nonspecific to H1.0K180me2.

[0319] In certain embodiments, the therapeutic H1.0K180me2 protein / peptide provided herein has a dissociation constant (Kd) in the range of 0.0001 nM to 1 μM. For example, the Kd of an anti-H1.0K180me2 binding peptide may be about 1 μM, about 100 nM, about 50 nM, about 10 nM, about 5 nM, about 1 nM, about 0.5 nM, about 0.1 nM, about 0.05 nM, about 0.01 nM, about 0.005 nM, about 0.001 nM, about 0.0005 nM, or even about 0.0001 nM.

[0320] In some embodiments, the therapeutic H1.0K180me2 protein / peptide is specific to human H1.0K180me2 autoantibodies. In some embodiments, the therapeutic H1.0K180me2 protein / peptide is cross-reactive with H1.0K180me2 autoantibodies from other species.

[0321] In some embodiments, the therapeutic H1.0K180me2 protein / peptide is selective for H1.0K180me2 autoantibodies and shows little to no binding affinity to autoantibodies that bind to H1.0K180me1 or H1.0K180me3. In some embodiments, the therapeutic H1.0K180me2 protein / peptide binds to H1.0K180me2 autoantibodies but also shows binding affinity to H1.0K180me1 and / or H1.0K180me3 autoantibodies.

[0322] In some embodiments, the binding preference (e.g., affinity) of the therapeutic H1.0K180me2 protein / peptide to the H1.0K180me2 antibody is generally at least about 2 times, about 5 times, or at least about 10, 20, 50, 10 times compared to a nonspecific target antibody (e.g., a randomly generated antibody). 2 , 10 3 , 10 4 , 10 5 , or 10 6 It is double.

[0323] It is also possible to evaluate the therapeutic H1.0K180me2 protein / peptide using methods familiar to those skilled in the art to determine whether the therapeutic H1.0K180me2 protein / peptide has specificity and / or selectivity for the H1.0K180me2 antibody.

[0324] Nucleic acids encoding the therapeutic H1.0K180me2 peptide described herein are provided herein. Vectors containing any of the nucleic acids encoding the therapeutic H1.0K180me2 peptide described herein are also provided herein. D. Combination therapy

[0325] Administration of either the therapeutic H1.0K180me2 antibody or the therapeutic H1.0K180me2 protein / peptide provided herein may be administered in combination with other known drugs / treatments for diseases manifesting as H1.0 methylation.

[0326] In the context of Alzheimer's disease prevention, any of the therapeutic H1.0K180me2 antibodies may be administered in combination with the administration of Alzheimer's disease prophylactic agents or regimens, including, but not limited to, APP synthesis inhibitors, beta-secretase inhibitors, gamma-secretase inhibitors and modulators, Aβ aggregation inhibitors, Aβ immunotherapy, cholesterol-lowering agents, antitau agents, cholinesterase inhibitors, N-methyl-D-aspartate (NMDA) antagonists, atypical antipsychotics, protein S-nitrosylation blockers, glucagon-like peptide-1 receptor agonists, rapamycin, rapalog, endogenous cannabinoids, cannabinoids, neuroprotective substances, molecules that regulate calcium influx, antioxidants, anti-inflammatory agents, drugs that regulate glutamate homeostasis, autophagy inducers, hormones, hormone modulators, statins, insulin, insulin carriers, multifunctional nanocarriers, vitamins, nutritional supplements, small RNA molecules, peptides, and ultrasound therapy.

[0327] In the context of Alzheimer's disease treatment, any of the therapeutic H1.0K180me2 proteins / peptides, but not limited to these, may be administered in combination with the administration of Alzheimer's disease drugs or regimens including, but not limited to, APP synthesis inhibitors, beta-secretase inhibitors, gamma-secretase inhibitors and modulators, Aβ aggregation inhibitors, Aβ immunotherapy, cholesterol-lowering agents, antitau agents, cholinesterase inhibitors, N-methyl-D-aspartate (NMDA) antagonists, atypical antipsychotics, protein S-nitrosylation blockers, glucagon-like peptide-1 receptor agonists, rapamycin, rapalog, endogenous cannabinoids, cannabinoids, neuroprotective substances, molecules that regulate calcium influx, antioxidants, anti-inflammatory agents, drugs that regulate glutamate homeostasis, autophagy inducers, hormones, hormone modulators, statins, insulin, insulin carriers, multifunctional nanocarriers, vitamins, nutritional supplements, small RNA molecules, peptides, and ultrasound therapy. E. Administration of therapeutic H1.0K180me2 antibody and therapeutic anti-H1.0K180me2 binding protein / peptide

[0328] In vivo administration of the therapeutic H1.0K180me2 antibodies and therapeutic H1.0K180me2 proteins / peptides described herein may be performed intravenously, intramuscularly, subcutaneously, topically, orally, percutaneously, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebrally, or intranasally. An effective dose of the treatment may be administered to treat diseases or conditions that manifest in H1.0 methylation or diseases or conditions that manifest elevated levels of H1.0K180me2 autoantibodies. The appropriate dose of the treatment may be determined based on the type of disease or condition being treated, the type of therapeutic antibody, protein, or peptide, the severity and progression of the disease or condition, the individual's clinical status, the individual's clinical history and response to the treatment, and the discretion of the attending physician.

[0329] For in vivo administration of the therapeutic H1.0K180me2 antibody and therapeutic H1.0K180me2 protein / peptide described herein, the usual dose may vary depending on the route of administration, from approximately 1 ng / kg to approximately 1000 mg / kg or more of the individual's body weight per day. For repeated administration over several days or longer, the treatment may be continued until the desired suppression of symptoms is achieved, depending on the severity of the methylated H1.0-related disease or condition being treated. Depending on the pattern of pharmacokinetic breakdown that the physician wishes to achieve, a dosage regimen may be useful. For example, administration to an individual 1 to 21 times per week is provided herein. In certain embodiments, the frequency of administration is three times daily, twice daily, once daily, every other day, once weekly, every two weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every nine weeks, every ten weeks, or once monthly, every two months, every three months, or at longer intervals. The progression of treatment may be monitored by conventional techniques and assays. The administration regimen may be changed over time, independently of the dose used. F. Pharmaceutical Compositions

[0330] This application provides compositions comprising a therapeutic H1.0K180me2 antibody and a therapeutic H1.0K180me2 protein / peptide, comprising a pharmaceutical composition comprising any one or more of the therapeutic antibodies, proteins, or peptides described herein and one or more pharmaceutically acceptable excipients. In some embodiments, the composition is sterile. The pharmaceutical composition generally contains an effective amount of the therapeutic antibody, protein, or peptide. G. Kits and Products

[0331] This application provides a kit comprising the therapeutic H1.0K180me2 antibody, therapeutic H1.0K180me2 protein, and therapeutic H1.0K180me2 peptide composition described herein. In some embodiments, the kit further comprises components selected from a secondary antibody, reagents for immunohistochemical analysis, pharmaceutically acceptable excipients and instructions for use, and any combination thereof. In one embodiment, the kit comprises any one or more of the therapeutic compositions described herein and one or more pharmaceutically acceptable excipients.

[0332] This application also provides a product comprising any one of the therapeutic compositions or kits described herein. An example of a product is a vial (including a sealed vial).

[0333] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. [Examples]

[0334] (Example 1) material and method Materials and methods used in subsequent embodiments are provided herein. In vitro methylation of H1.0 proteins and peptides - Radiolabeled in vitro methylation assay

[0335] To visualize the methylation of H1.0 peptide by G9A methyltransferase in vitro, a methylation assay was performed by incorporating a radiolabeled methyl donor on a gel after autoradiography exposure, enabling visualization of the methylated peptide. The following methylation reaction was set up in a 1.5 ml tube: 1× HMT reaction buffer (50 mM Tris-HCl, 5 mM MgCl2, 4 mM dithiothreitol, pH 9.0), 10 U G9A methyltransferase (NEB, M0235S, lot number 0031201), 3.2 mM adenosyl-L-methionine, S-[methyl-3H] (Perkin Elmer, NET155H, lot number 1664720), 10 μg H1.0 peptide (ThermoFisher Scientific, biotin-AKPVKASKPKKAKPVKPK (SEQ ID NO: 75)), final volume 10 μl. A control reaction solution identical to the one described above was also prepared, except that it did not contain the H1.0 peptide. The reaction solution was incubated in a thermocycler at 37°C for 1 hour. The reaction was stopped by incubation on ice for 5 minutes, and then separated on a 16.5% tricine gel (BioRad, 4563063). The gel was immersed in 30% methanol and 5% glycerol for 30 minutes, and then vacuum-dried at room temperature for 24 hours. Subsequently, autoradiography analysis of the dried gel was performed using a phosphoimager (Molecular Dynamics) to evaluate the effective methylation of the H1.0 peptide with a methyl-3H group by G9A methyltransferase. Liquid chromatography and high-resolution mass spectrometry (LC-MS)

[0336] To pinpoint the exact sites of methylation of histone H1.0 by G9A and GLP methyltransferases, in vitro methylation assays were performed and subsequently analyzed by liquid chromatography and high-resolution mass (LC-MS) analysis. Methylation reactions were set up using either G9A methyltransferase (NEB, M0235S, lot number 0031201) or GLP methyltransferase (Cayman Chemical, 10755). The methyl donor for each reaction was unlabeled S-adenosyl-L-methionine (NEB, B9003S). The H1.0 substrates for each reaction were biotin (ThermoFisher Scientific, biotin-AKPVKASKPKKAKPVKPK (SEQ ID NO: 75)) and dimethylated H1.0 peptide (ThermoFisher Scientific, biotin-AKPVKASKPKKAKPVK). (me2) The peptide was either PK (SEQ ID NO: 76) or an unmodified H1.0 peptide labeled with full-length recombinant human H1.0 (NEB, M2501S). The methylation reaction was set up in triplicate for each condition as follows: 1× HMT reaction buffer (50 mM Tris-HCl, 5 mM MgCl2, 4 mM dithiothreitol, pH 9.0); 10 U G9A or GLP methyltransferase; 3.2 mM S-adenosyl-L-methionine; 500 ng unmodified or modified H1.0 peptide, or 1 μg full-length protein; final volume 10 μl. As a control, a reaction mixture was set up as described above but without any methyltransferase. All reaction mixtures were incubated in a thermocycler at 37°C for 1 hour, then stopped by incubation on ice for 5 minutes. Samples were frozen at -80°C prior to LC-MS analysis.

[0337] For analysis by liquid chromatography and high-resolution mass spectrometry (LC-MS), the sample was prepared as described above, and approximately 1 μg of the product was injected into a Thermo Scientific Easy nLC system consisting of a 10 cm × 100 μm trap column and a 25 cm × 100 μm ID decomposition column. Buffer A consisted of 98% water, 2% methanol, and 0.2% formic acid. Buffer B consisted of 10% water, 10% isopropanol, 80% acetonitrile, and 0.2% formic acid. The sample was loaded at 4 μL / min for 10 minutes, and a gradient of 0–45% of buffer B was run at 375 nL / min for 130 minutes, for a total run time of 150 minutes (including regeneration and sample loading). A Thermo Scientific LTQ Orbitrap Velos mass spectrometer was run on a standard Top-10 data-dependent structure, except that a higher switching limit (20K) was used to ensure that MS2 did not interfere with the full scan duty cycle. This ensured optimal full scan data for quantification. Fragmentation and analysis of MS2 were performed on the ion trap mass spectrometer. Samples were run in triplicate. LC-MS data analysis

[0338] Protein identification was performed using the RefSeqHuman sequence database and Thermo Scientific Proteome Discoverer version 1.4 (including the Sequest and Percolator algorithms). These searches were performed against a control reaction mixture lacking any methyltransferase enzymes. The peptide confidence filter in Percolator was set to "High". Protein quantification was performed using Pinpoint version 1.4 software. The Pinpoint quantification workflow included importing the Proteome Discoverer.msf file as a spectral library. The identified peptides were then quantified in MS.raw files using Pinpoint peak detection, chromatogram alignment, and area calculation algorithms.

[0339] To pinpoint the exact location of G9A methylation in the unmodified H1.0 peptide (AKPVKASKPKKAKPVKPK (SEQ ID NO: 42)), a methylation reaction was set up and the product was identified by LC-MS. The methylation reaction involved recombinant G9A, an unlabeled methyl donor (S-adenosyl-L-methionine), and the unmodified H1.0 peptide. The methylation reaction was then analyzed by LC-MS to identify each spectral peak (corresponding to the peptide species in the final reaction) and quantified using spectral counting. The number of circles labeled "me" in Figure 22A represents the methylation status of the lysine residue (mono-, di-, or tri-methylation). Figure 22A shows that G9A specifically and abundantly dimethylates H1.0K180 (99.9% of all peptides) in the presence of the unmethylated H1.0 peptide. hADSC culture

[0340] Human adipocyte-derived mesenchymal stem cells (hADSCs) were commercially obtained from Life Technologies (R7788-115) and the American Type Culture Collection, ATCC (PCS-500-011). All cell lines were isolated from human adipose tissue obtained from three healthy adult female Caucasian donors aged 38, 45, and 49 years who routinely underwent liposuction. Cells confirmed by flow cytometry and immunohistochemical staining were positive for CD29, CD44, CD73, CD90, CD105, and CD166, and negative for CD14, CD31, CD34, and CD45. The cell lines were confirmed to be capable of adipogenesis, chondrogenesis, and osteogenic differentiation under in vitro conditions.

[0341] Isolated adipose-derived stem cell lines were grown in DMEM / F12 medium (Life Technologies, 11330-057) supplemented with 10% (v / v) fetal bovine serum (FBS) and 50 U / ml penicillin / streptomycin at 37°C / 5% CO2. Cumulative population doubling (PD) was calculated as a function of growth days in culture over multiple passages using the formula PD = log(N / N0) × 3.33 (where N0 is the number of cells plated in the flask and N is the number of cells recovered in this passage). PD4-10 of hADSCs for the regenerating population (SR) and PD41-46 for the replicating senescent population (REP-SEN) were used in all experiments. Induction and evaluation of aging

[0342] As a replication senescence, hADSCs were grown in culture until replication depletion was reached (PD41-46). As an acute DNA damage condition, SR hADSCs (PD4-10) were treated with 50 μg / ml bleomycin sulfate in growth medium for 2 hours (Cayman Chemical, 13877). As a genotoxic stress-induced senescence, SR hADSCs (PD4-10) were treated with 50 μg / ml bleomycin sulfate (Cayman Chemical, 13877) in growth medium for 2 hours, then washed with PBS and transferred to fresh growth medium without bleomycin. The cells were then grown for 3 days before harvesting.

[0343] To assess cellular senescence, cells were scored against senescence markers, including growth arrest, SA-β-gal activity, and the presence of persistent DNA damage foci. Assays to monitor pH-dependent senescence-associated β-galactosidase activity (SA-β-Gal) expression were performed as described in the manufacturer's kit (BioVision). Cultured hADSCs were fixed in fixative solution at room temperature for 15 minutes, washed twice with PBS, and stained overnight at 37°C with X-Gal containing staining supplements. Cells were washed twice with PBS and images were acquired using light microscopy (Leica, DMiL). DNA damage foci were evaluated by immunostaining against γH2A.X foci, as described below. The appearance of γH2A.X foci indicates DNA double-strand breaks or DNA damage. γH2A.X foci are a widely accepted molecular marker for double-strand breaks. antibody

[0344] Primary antibodies used: Anti-H1.0K180me2, 1:100 dilution, rabbit polyclonal, Aviva Systems Biology. Anti-H1.0 whole, 1:500 dilution, EMD Millipore MABE446. Anti-γH2A.X (Ser139Ph), 1:500-1000 dilution, EMD Millipore 05-636. Anti-beta-actin, 1:2000, Abcam ab6276. Anti-poly-(ADP)-ribose, 1:500, Enzo Life Sciences ALX-804-220-R100. Anti-G9A, ​​1:500, Bethyl Laboratories A300-933A. Anti-GLP, 1:500, Bethyl Laboratories A301-643A. Anti-H3K9me2, 1:1000, Abcam A301-643A.

[0345] Secondary antibodies: Goat-anti-mouse-HRP, 1:4000, Biorad 1706516. Goat-anti-rabbit-HRP, 1:4000, Biorad 1706515. Goat-anti-human-HRP, 1:4000, Biorad 1721050; AlexaFluor-488-donkey-anti-mouse, 1:5000, Life Technologies A-21202; AlexaFluor-488-donkey-anti-rabbit, 1:5000, Life Technologies A-21206; AlexaFluor-555-donkey-anti-mouse, 1:5000, Life Technologies A-31570; AlexaFluor-555-donkey-anti-rabbit, 1:5000, Life Technologies A-31572; and anti-IgM HRP (rabbit anti-human IgM (Mu chain) (HRP-Conjugate) (Abcam, ab97210, lot number GR169227-10)). ELISA

[0346] The following protocol describes a generalized enzyme-coupled immunosorbent assay (ELISA) for the direct or indirect detection of serum H1.0K180me2 IgG levels.

[0347] Figure 4 illustrates the use of a sandwich ELISA for the indirect detection of serum H1.0K180me2 levels (e.g., for the indirect detection of IgG or IgM levels). ELISA is used for the detection of antibodies against the H1.0K180me2 peptide epitope in body fluid samples. The following protocol describes a generalized enzyme-coupled immunosorbent assay (ELISA) (for the indirect detection of serum H1.0K180me2 IgG or IgM levels) that uses a biotinylated peptide to capture specific antibodies in a serum sample, followed by detection with a secondary antibody conjugated to HRP, and incubation with tetramethylbenzidine (TMB). Wells of a streptavidin-coated microplate (ThermoFisher, 15501) were washed three times with washing buffer (Tris-buffered saline (25 mM Tris, 150 mM NaCl; pH 7.6), 0.1% BSA, and 0.05% Tween-20). 50 pmol of custom biotinylated H1.0K180me2 peptide (ThermoFisher) was diluted in washing buffer and allowed to bind to each well at room temperature for 2 hours with gentle shaking. Unbound peptides were washed three times with washing buffer to remove them. Serial dilutions of H1.0K180me2 antibody in washing buffer were prepared as standard solutions (1:25000, 1:50000, 1:100000, 1:200000, 1:400000, storage concentration 0.54 mg / ml). Serum samples were diluted in wash buffer (1:1000). 100 μL of standard solution and sample were added to two wells in a double well. The wells were incubated at room temperature for 1 hour with gentle shaking. Unbound standard solution or sample was washed three times with wash buffer to remove any residue. Secondary antibodies conjugated to HRP (either anti-IgG or anti-IgM secondary antibodies) were diluted in wash buffer (1:1000) (serum sample: goat-anti-human-HRP, BioRad 1721050; standard solution: goat-anti-rabbit-HRP, BioRad 1706515). 100 μL of the secondary antibody dilution was added to the well. The wells were incubated at room temperature for 1 hour with gentle shaking. Unbound secondary antibody was washed three times with wash buffer to remove any residue.100 μL of TMB solution (PeproTech) was added to each well and incubated at room temperature for 15 minutes with gentle shaking. 100 μL of stop solution (0.18 M H2SO4) was added to each well and incubated at room temperature for 5 minutes with gentle shaking. Upon oxidation, TMB forms a water-soluble blue reaction product that can be spectrophotometrically measured at 650 nm. Upon acidification with the stop solution, the reaction product turns yellow with an absorbance peak at 450 nm. The absorbance of each well was measured at 450 nm using a plate reader (Molecular Devices). The concentrations of H1.0K180me2 IgG or IgM in each sample were determined by extrapolation from the standard curve and are further described below. Figure 14C shows the use of ELISA for indirect detection of autoanti-H1.0K180me2 IgM levels in body fluids.

[0348] Figure 5A shows the use of sandwich ELISA for the direct detection of the H1.0K180me2 peptide epitope in a body fluid sample. Typically, an antibody specific to the H1.0K180me2 epitope is provided and immobilized (coated) on a microplate. A clinical sample containing the H1.0K180me2 epitope is provided for quantification. The sample is added to the microplate, and the H1.0K180me2 epitope binds to the immobilized antibody. Unbound material is washed away. Next, a detection antibody, e.g., HRP or any other labeled antibody, is added. These detection antibodies bind to the captured epitope. Unbound detection antibodies are washed away. A detection substrate solution is added, and the fluorophore or color change is measured. This is then quantified against a standard curve, and the level of the H1.0K180me2 epitope in the clinical sample is reported.

[0349] Figure 5B shows the standard curve for a sandwich ELISA assay of the H1.0K180me2 antigen. In vitro methylated full-length H1.0K180me2 was used as the standard for a 2-fold dilution series from 50 ng / ml to 3.125 ng / ml. The standard concentrations are plotted against the OD measured at 450 nm. A linear trend line was plotted between points exceeding the detection limit, and the equation of the line was calculated (shown in the plot). Inserts show the raw data. ELISAs were performed in double series, and the mean OD value was used for curve construction. Western blot

[0350] Materials for Western blot analysis (cultured cells or tissues) were dissolved in ice-cold RIPA lysis buffer (Thermo Scientific 89900) and sonicated using a Covaris S2 sonicator (10% duty cycle, intensity 5, bursts of 100 per minute, 120 seconds). The total protein concentration in each sample was quantified using Quick Start Bradford 1x staining reagent (BioRad, 5000205) according to the manufacturer's protocol. The samples were then mixed with NuPAGE LDS sample loading buffer (ThermoFisher, NP0007) and NuPAGE sample reduction buffer (ThermoFisher, NP0004) and denatured by heating at 70°C for 10 minutes. Proteins were separated by electrophoresis on 4–12% precast polyacrylamide gel (ThermoFisher, NP0321) and transferred to a 0.45 μm nitrocellulose membrane. The membranes were blocked with 5% nonfat milk in PBS-T for 30 minutes at room temperature, and then immunoblotted overnight at 4°C with the primary antibody listed above. Proteins were detected with the HRP secondary antibody listed above for 1 hour at room temperature, followed by detection on ECL Western Blotting Substrate (ThermoFisher, 32106) using the manufacturer's instructions. All washing between steps was done with PBS-T. The membranes were imaged using the Omega LUM-C imaging system (Gel Company). Immunofluorescence

[0351] For immunofluorescence analysis, cells were cultured, processed on chamber slides, fixed with neutral 10% formalin, and permeabilized with PBS containing 0.5% Triton X-100. After washing, the slides were blocked with PBS containing 1% BSA and 4% donkey serum. After washing, the slides were incubated with the primary antibodies listed above, washed again, incubated with the AlexaFluor secondary antibodies listed above, and mounted on slow-fade gold (Molecular Probes) containing DAPI (to visualize the nuclei). Cells were observed by fluorescence microscopy, and images were acquired for analysis using Spotfire software (Diagnostics Instruments). Slot blot analysis

[0352] 0.5 μl of serum sample or a known amount of synthetic peptide was diluted in 200 μl of TBS. The sample was then thermally denatured at 70°C for 10 minutes and transferred to a nitrocellulose membrane using a vacuum manifold slot blotting apparatus (BioRad, 1706542). The membrane was blocked with 5% nonfat milk in PBS-T at room temperature for 30 minutes, and then immunoblotted with the primary antibody listed above at 4°C overnight. Proteins were detected with the HRP secondary antibody listed above at room temperature for 1 hour, followed by detection with ECL Western Blotting Substrate (ThermoFisher, 32106) using the manufacturer's instructions. All washing between steps was performed using PBS-T. The membrane was then bled into Omega LUM-C Images were generated using an imaging system (Gel Company). Subsequently, the slot blot bands for each sample were quantified using ImageJ. RNASeq analysis

[0353] Total RNA was isolated from self-replicating and replicating senescent cell culture samples using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Samples from two different hADSC cell lines were combined under relevant conditions, and RNA concentrations were measured using a Qubit 2.0 fluorometer with an RNA HS assay kit (Invitrogen, Life Technologies). ERCC RNA Spike-In Control mix (Ambion, Life Technologies) was added to the total RNA for quality control analysis. Next, rRNA depletion was performed using a Low Input Ribominus Eukaryote System v2 (Ambion, Life Technologies). The cDNA library was analyzed using Ion. The libraries were constructed using the Total RNA-Seq Kit V2 (Ambion, Life Technologies) and barcoded using the Ion Xpress RNA-Seq Barcode (Ambion, Life Technologies). Library size distribution and quantification were performed using the Bioanalyzer 2100 (Agilent Technologies) with the DNA HS Bioanalyzer Kit. Library sequencing was performed using the Ion Proton System with the P1 chip (Life Technologies), and each library was sequenced three times.

[0354] RNAseq reads from individual Ion Proton System sequencing runs were combined for each library. Sequence reads were mapped to the reference human genome assembly hg19 (GRCh37) using the Torrent Mapping Alignment Program (TMAP, Life Technologies). The quality of RNAseq runs for each condition was assessed by comparing the predicted count of ERCC spike-in RNA sequences, obtained from the manufacturer's website, to the measured count of RNAseq tags mapping to the same sequences. Initial gene expression levels were obtained as the sum of reads mapped to exons for individual NCBI RefSeq gene models (c), and genes with low expression (read count <1 per million) were removed from subsequent analyses. For each library, individual gene expression levels were normalized using the beta-actin (ACTB) expression level (cACTB) and the sum of exon lengths l per gene. For library j, the beta-actin normalization factor sj was:

number

[0355] The final normalized expression value for gene i in library j is:

number

[0356] Bleomycin treatment: Cell growth medium was supplemented with 50 μg / ml bleomycin (Cayman Chemical 13877) for 2 hours to induce DNA double-strand breaks. Cells were either harvested immediately after bleomycin treatment (acute DNA damage) or grown for 3 days after bleomycin treatment (genotoxic stress-induced senescence). PARP-1 inhibitor: Cell growth medium was supplemented with 1 μM of the effective PARP-1 inhibitor AG14361 (Selleckchem S2178) for 24 hours prior to downstream analysis. Rapamycin treatment: Cell growth medium was supplemented with 500 nM rapamycin (Cayman Chemical 11346) for 24 hours prior to downstream analysis or treatment. Everolimus treatment: Cell growth medium was supplemented with 500 nM everolimus (Cayman Chemical 11597) for 24 hours prior to downstream analysis or treatment. Temozolomide treatment: Cell growth medium was supplemented with 50 μg / ml temozolomide (Cayman Chemical 14163) for 2 hours prior to downstream analysis. Analysis of H1.0K180me2 in mice after irradiation.

[0357] Blood was collected from two 13-month-old mice by cheek puncture, and serum was separated using Microtainer Tubes® with an SST Serum Separator (BD, 365956). The same mice were then exposed to 7 Gy of ionizing radiation (in the form of X-rays). Blood was again collected from one mouse 2 hours after irradiation and from the other mouse 48 hours after irradiation. Serum was again separated from the blood using Microtainer Tubes®. The serum from each mouse before and after irradiation was then analyzed by slot blotting and Western blotting. Isolation of adipose-derived stem cells (ADSCs) from mice

[0358] Mouse adipose-derived stem cells were isolated from wild-type mice. Subcutaneous or perirenal white adipose tissue was collected and suspended in Hanks buffer solution (HBSS), 3.5% bovine serum albumin (BSA), and 1% collagenase type II (Sigma) in a ratio of 1:3 w / v, and shaken at 37°C for 50 minutes. Cells were filtered through a 70 μm mesh cell strainer (BD Falcon No. 352350), treated with erythrocyte lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.3), expanded ex vivo in DMEM / F12 complete medium (DMEM / F12, 10% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin, 2.5 μg / ml amphotericin B; Invitrogen) at 37°C with 10% CO2, and subcultured at 80% confluence, changing the medium every 72–96 hours. The cells were then used for Western blotting analysis as described elsewhere. (Example 2) Discovery of the H1.0K180me2 peptide in aging

[0359] Self-regenerating (SR) hADSCs and replicated aging (REP-SEN) hADSCs were obtained according to the method described in Example 1. To identify post-translational modifications (PTMs) associated with novel histone aging, lysates from SR and REP-SEN hADSCs were evaluated by M / Z Pair Tag LC-MS according to the discovery pipeline in Figure 1A. Five technical iterative injections of self-regenerating and replicated aging hADSC lysates were processed with a full-scan optimized setting. It was determined that chromatographic and instrumental methods for optimal full-scan quantitative measurement competed with methods for optimal fragmentation scanning. Therefore, the accurate mass and wide dynamic range capabilities of the mass spectrometer were utilized by incorporating two separate paths of data measurement into the analysis. The first path focused on obtaining uncompromising, optimized full-scan (MS) data for reproducible quantification. This first full-scan quantitative path was used to create an inclusion list of potentially interesting features. Next, the inclusion list was used as a subset of the data sample to acquire targeted fragmentation scans during the second pass. As shown in Figure 1B, the mass spectra revealed the unmodified (AKPVKASKPKKAKPVKPK (SEQ ID NO: 42)) peptide of SR hADSC and the dimethylated (AKPVKASKPKKAKPVK) peptide of REP-SEN hADSC. me2 The PK (SEQ ID NO: 3) peptide has been identified.

[0360] RNA-seq experiments comparing the expression profiles of SR and REP-SEN hADSCs were performed according to the method described in Example 1. As shown in Figure 2A, both SR (black bars) and REP-SEN (gray bars) hADSCs contain six distinct variants of histone H1: H1.0, H1.1, H1.2, H1.3, H1.4, and H1.5. The values ​​on the y-axis represent the sum of mapped reads of exons for each gene, normalized by relative beta-actin expression in each sample and further normalized by the full length of the exon for each gene. Histone H1.0 was found to be the fourth most abundant variant of H1 in terms of gene expression levels. H1.0 mRNA accounted for 8% of total H1 expression in SR hADSCs and 14% of total H1 expression in REP-SEN hADSCs. However, unlike the other variants, its expression remained constant during replication senescence. A genome browser diagram (Figure 2B) of the unique mapping of all reads to the histone H1.0 gene identified by RNA-seq analysis showed higher H1.0 expression in SR hADSCs (upper track) than in REP-SEN hADSCs (lower track). Read counts were normalized by the relative beta-actin expression of each sample.

[0361] Alignment of various peptide sequences centered on known methylated lysine residues revealed that the amino acid sequence around H1.0K180 is unique and not similar to that of other methylated lysine residues (Figure 3A). (Example 3) H1.0K180me2 antibody

[0362] Polyclonal antibodies specific to the H1.0K180me2 antigen were produced as follows: Two New Zealand rabbits were immunized for antibody production. A keyhole limpet hemocyanin (KLH) emulsion conjugated with the H1.0K180me2 peptide (peptide: CAKPVKASKPKKAKPVK(me2)PK (SEQ ID NO: 39)) was administered subcutaneously (SQ) by injection; the conjugated peptide was (KLH-CAKPVKASKPKKAKPVK(me2)PK (SEQ ID NO: 77)), where the C-terminal C was artificially added to the peptide sequence in complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) to create a covalent linkage to KLH. Initial immunization was performed at 10SQ sites using 0.5 mg of antigen in CFA. Six subsequent booster immunizations were performed at routine intervals over a period of 7-8 weeks. The booster consisted of 0.25 mg of antigen in IFA at the 4SQ site. Rabbits were collected at week 5 (approximately 25 ml of blood per rabbit) and week 8 (approximately 50 ml of blood per rabbit). Immunoassay titer was evaluated using ELISA by comparing blood samples before and after immunization. Antibodies were then purified using antigen affinity chromatography. The antigens used for purification were either biotin or bovine serum albumin (BSA) conjugated H1.0K180me2 peptides (biotin-CAKPVKASKPKKAKPVK(me2)PK (SEQ ID NO: 78)) or BSA-CAKPVKASKPKKAKPVK(me2)PK (SEQ ID NO: 79)). The peptides were dimethylated using the in vitro methylation method provided herein.

[0363] The H1.0K180me2 antibody was tested as follows: To check the specificity of the H1.0K180me2 antibody, histone H1.0 peptides methylated with K172, K174, K175, K177, or K180 were transferred to the membrane in a dilution series using vacuum manifold slot blotting. The membrane was then immunoblotted with the H1.0K180me2 antibody to identify potential cross-reactivity with other methyl-lysine groups. The H1.0K180me2 antibody was highly specific to H1.0K180me2 even at low concentrations (Figure 3B). To further check the specificity of the H1.0K180me2 antibody, histone H3 peptides methylated with K4, K9, K27, K36, or K79 were transferred to the membrane in a dilution series using vacuum manifold slot blotting. Next, the membrane was immunoblotted using the H1.0K180me2 antibody to identify potential cross-reactivity with other methyl-lysine groups. This antibody did not show cross-reactivity with any of the methylated H3 peptides analyzed (Figure 3C). The slot blot analysis protocol was performed according to the method described in Example 1.

[0364] The specificity of the rabbit anti-H1.0K180me2 IgG antibody is that of anti-H1.0K180me2 The determination was made using IgG ELISA. Wells were coated with either H1.0K180me2 peptide, H1.0K180me1 peptide, or unmodified peptide in a 2-fold dilution series from 6.25 pmol to 781 fmol. 6.67 fmol of rabbit anti-H1.0K180me2 IgG antibody was added to each well in 100 μl of buffer, and antibody binding efficiency was monitored by ELISA. Curves for each peptide were created by plotting the amount of peptide against OD measured at 450 nm. Raw ELISA data for two repeated experiments are shown in tables and figures (Figure 3D).

[0365] Rabbit anti-H1.0K180me2 IgG antibody binds to H1.0K180me2 peptide 2.7 times more efficiently than H1.0K180me1 peptide. Within the optimal linear range, one molecule of rabbit anti-H1.0K180me2 IgG antibody recognizes one out of 117 H1.0K180me2 peptide molecules, but only one out of 316 H1.0K180me1 peptide molecules. Unmodified peptides are not recognized within this range.

[0366] ClustalW2 alignment of histone H1 variant protein sequences revealed that H1.0K180 is embedded in a unique amino acid sequence not present in other H1 variants (Figure 3E). The peptide identified in the initial discovery experiment is highlighted in gray. The globular domain region containing three α-helices is also shown. (Example 4) Indirect detection of serum H1.0K180me2 IgG and H1.0K180me2 IgM autoantibody levels using H1.0K180me2 peptide in ELISA. Indirect detection of serum H1.0K180me2 IgG autoantibodies

[0367] The following example describes an enzyme-coupled immunosorbent assay (ELISA) using biotinylated therapeutic H1.0K180me2 peptide to capture specific autoantibodies in serum samples, followed by detection with a secondary antibody conjugated to HRP, and incubation with tetramethylbenzidine (TMB). Wells of a streptavidin-coated microplate (ThermoFisher, 15501) were washed three times with washing buffer (Tris-buffered saline (25 mM Tris, 150 mM NaCl; pH 7.6) with 0.1% BSA and 0.05% Tween-20). 50 pmol of custom biotinylated H1.0K180me2 peptide (ThermoFisher) was diluted in washing buffer and allowed to bind to each well at room temperature for 2 hours with gentle shaking. Unbound peptides were washed three times with washing buffer. Serial dilutions of H1.0K180me2 antibody in wash buffer were prepared as standard solutions (1:25000, 1:50000, 1:100000, 1:200000, 1:400000, storage concentration 0.54 mg / ml). Serum samples were diluted in wash buffer (1:1000). 100 μL of standard solution and sample were added to two wells in a row. The wells were incubated at room temperature for 1 hour with gentle shaking. Unbound standard solution or sample was washed three times with wash buffer to remove any residue. Secondary antibodies conjugated to HRP (secondary labeled antibodies shown in step 3 of Figure 4) were diluted in wash buffer (1:1000) (serum sample: goat-anti-human-HRP, Biorad 1721050; standard solution: goat-anti-rabbit-HRP, Biorad 1706515). 100 μL of diluted secondary antibody was added to each well. The wells were incubated at room temperature for 1 hour with gentle shaking. Unbound secondary antibody was washed away three times with washing buffer. 100 μL of TMB solution (PeproTech) was added to each well and incubated at room temperature for 15 minutes with gentle shaking. 100 μL of stop solution (0.18 M H2SO4) was added to each well and incubated at room temperature for 5 minutes with gentle shaking. Upon oxidation, TMB forms a water-soluble blue reaction product that may be spectrophotometrically measurable at 650 nm.Upon acidification with stop solution, the reaction product turns yellow with an absorbance peak at 450 nm. The absorbance of each well was measured at 450 nm using a plate reader (molecular devices). The concentration of H1.0K180me2 IgG in each sample was determined by extrapolation from the standard curve.

[0368] Indirect detection of serum H1.0K180me2 IgG levels using ELISA is generally shown in Figure 4. Indirect detection of serum H1.0K180me2 IgM autoantibodies

[0369] The detection of serum H1.0K180me2 IgM autoantibodies involved three steps. The test included measuring total IgM in the patient's serum using a commercially available sandwich ELISA kit (Step 1), followed by measuring anti-H1.0K180me2 IgM in the same sample using an indirect ELISA assay (Step 2). As a final step, the anti-H1.0K180me2 IgM levels were normalized by the total IgM in the sample (Step 3).

[0370] Step 1: Total human IgM in the patient's serum was measured using a commercially available sandwich ELISA kit (Human IgM ELISA Ready-SET-Go!, part number 88-50620, lot number 123620111) purchased from Affymetrix eBioscience.

[0371] Step 2: Measurement of anti-H1.0K180me2 IgM in patient serum using a customized indirect ELISA assay. Biotin-labeled H1.0K180me2 capture peptides were coated onto a 96-well streptavidin-coated microplate (ThermoFisher, 15501, lot number QH218700). Excess free peptides were washed away with a washing buffer (Tris-buffered saline (25 mM Tris, 150 mM NaCl; pH 7.6) with 0.1% BSA and 0.05% Tween-20). Nonspecific binding sites on the microplate were blocked using a blocking buffer (Thermo Scientific, 37536, lot number QJ222191) containing d-biotin (VWR, 97061-444, lot number 1405C080; to block unbound streptavidin on the plate surface) to reduce background noise. Diluted patient serum was added to microplate wells in triplicate. Anti-H1.0K180me2 autoantibodies in the patient samples recognized and bound to the capture peptide on the plate surface. In parallel, a dilution series of anti-H1.0K180me2 IgG antibody standard solutions was processed on the same plate. The wells were then washed with wash buffer to remove any unbound samples or standards. The bound anti-H1.0K180me2 IgM autoantibodies were then detected using anti-IgM HRP detection antibody (secondarily labeled antibody shown in Figure 4, step 3; rabbit anti-human IgM (Mu chain) (HRP conjugate) (Abcam, ab97210, lot number GR169227-10)) added to each sample well. Standard solutions were detected using anti-IgG HRP detection antibody (goat anti-rabbit IgG(H+L)(HRP conjugate) (BioRad, 1706515, lot number 350003080)) added to each standard solution well. Excess, unbound detection antibody was then washed away with wash buffer. The activity of bound HRP was then determined by adding tetramethylbenzidine (TMB) substrate (KPL, 52-00-01, lot number 10164336) to the wells.This reaction was stopped by adding a stop solution ((0.18 M H2SO4) (KPL, 50-85-04, lot number 10164328)), and the absorbance of the well contents was read at 450 nm. The absorbance is directly proportional to the concentration of anti-H1.0K180me2 IgM in the sample. The molar concentration of anti-H1.0K180me2 IgM in each sample was then determined by extrapolation from the standard curve and multiplication by the initial dilution factor.

[0372] The standard curve utilized an anti-H1.0K180me2 rabbit polyclonal IgG antibody. This antibody was confirmed to be highly specific to H1.0K180me2. It does not recognize methylation of any other lysine of H1.0 and is specific to the degree of methylation, for example, it can distinguish between mono(me1), di(me2), and tri(me3) lysine K180 (Figure 3A). This antibody does not cross-react with methylation sites of other common histone proteins (Figure 3A). The standard curve was obtained using a fixed-concentration biotinylated epitope (synthetic peptide AKPVKASKPKKAKPVK). me2 Dilution series of anti-H1.0K180me2 IgG (72, 36, 18, 12, 9, 4.5 fmol / ml) or total IgM antibody (556, 444, 222, 111, 56, 28, 18, or 0 fmol / ml) were prepared using either PK (SEQ ID NO: 3) or a fixed amount of secondary anti-IgM antibody, respectively. The molar concentration of anti-H1.0K180me2 IgM in each sample was estimated from the molar concentration curve of anti-H1.0K180me2 IgG.

[0373] Step 3: Normalization of anti-H1.0K180me2 IgM serum concentration by total IgM serum concentration. Total IgM in the patient's serum was measured in three consecutive sets. The average total IgM concentration in each sample was then calculated from the three sets of measurements. Anti-H1.0K180me2 IgM in the patient's serum was measured in three consecutive sets. The average anti-H1.0K180me2 IgM concentration in each sample was then calculated from the three sets of measurements. To obtain the final test value, the average anti-H1.0K180me2 IgM concentration was divided by the average total IgM concentration for each patient's sample.

number

[0374] Indirect detection of serum H1.0K180me2 IgM levels using ELISA is generally shown in Figure 4. Measurement of anti-H1.0 (unmodified H1.0) IgM levels in the patient's serum.

[0375] The procedure was identical to that of indirect ELISA for anti-H1.0K180me2 IgM, but the captured peptide was different. In this case, the peptide was an unmodified peptide lacking K180me2 (unmodified H1.0). The standard substance used was rabbit polyclonal IgG produced in response to the unmodified peptide (Figure 16B, right panel). (Example 5) The use of the H1.0K180me2 antibody indicates that H1.0K180me2 is associated with acute DNA damage.

[0376] Figure 6 shows that H1.0 mRNA expression increases after DNA damage and genotoxic stress-induced aging, according to the method described in Example 1. RT-PCR analysis of histone H1.0 mRNA expression was performed on total RNA from self-replicating hADSCs, hADSCs treated with bleomycin for 2 hours (acute DNA damage), and hADSCs treated with bleomycin and aged for 3 days (genotoxic stress-induced aging). H1.0 mRNA expression increased more than twofold after acute DNA damage and remained high after genotoxic stress-induced aging (1.5 times relative to SR).

[0377] To evaluate the relationship between H1.0K180 methylation and DNA damage, SR hADSCs and hADSCs treated with bleomycin, a DNA damaging agent, for 2 hours (according to the method described in Example 1) were lysed, fractionated, and a chromatin-bound fraction was obtained. Western blot analysis using α-H1.0K180me2 antibody (performed according to the method described in Example 1) showed chromatin H1.0K180 methylation during DNA damage (Figure 7A). H1.0K180me2 was localized in the cytoplasm during treatment of SR hADSCs with bleomycin, which induces DNA double-strand breaks (according to the method described in Example 1).

[0378] Slot blot immunoassay revealed that H1.0K180me2 was detected in the conditioned medium of hADSCs in a time-dependent manner after bleomycin-induced genotoxic injury, with the maximum secretion of H1.0K180me2 detected within 48 hours (Figure 7B). This indicates secretion from cells. Increased secretion of DNA fragmentation factors related to GSI-SEN (DFFB / DFF40 / CAD) was observed in a similar manner to H1.0K180me2, starting 48 hours after treatment (Figure 7B). The protein is not secreted in measurable amounts upon the development of acute DNA damage (ADD). (Example 6) Methylation of H1.0K180 is unrelated to the activity of the DNA damage repair protein PARP-1 and occurs upstream of it.

[0379] To further evaluate whether H1.0K180 methylation is dependent on the activity of the DNA damage repair pathway protein PARP-1, Western blot analysis was performed according to the method described in Example 1 to investigate chromatin-bound H1.0K180me2 and γH2A.X upon treatment with bleomycin. PARP-1, an early player in the DNA damage response pathway, was inhibited using the effective inhibitor AG14361 according to the method described in Example 1. As shown in Figure 8A, H1.0K180me2 appeared in chromatin upon DNA damage, and its appearance was independent of PARP-1 activity. Indeed, inhibition of PARP-1 resulted in increased accumulation of H1.0K180me2 and γH2A.X (Figure 8A). Western blot analysis of PARP-1 inhibitory activity in the entire cell lysate revealed that bleomycin treatment led to an increase in intracellular poly-(ADP)-ribose (PAR) levels, which were abolished by the PARP-1 inhibitor, suggesting efficient PARP-1 inhibition (Figure 8B). Therefore, it was determined that methylation of H1.0K180 is unrelated to the activity of PARP-1, a protein in the DNA damage repair pathway. (Example 7) The use of the H1.0K180me2 antibody indicates that during genotoxic stress-induced aging, H1.0K180me2 is released from chromatin and secreted into the extracellular matrix.

[0380] SR hADSCs, hADSCs treated with bleomycin for 2 hours (acute DNA damage), and hADSCs treated with bleomycin and aged for 3 days (genotoxic stress-induced aging) were lysed and fractionated into soluble and chromatin-bound fractions. These fractions were then subjected to LC-MS / MS analysis. Peptide expression levels were obtained as the total area under the relative intensity curve in LC-MS / MS, and individual peptides were clearly assigned to proteins using Pinpoint software, version 1.4 (Thermo Scientific). The areas of all peptides assigned to individual proteins were summed to obtain protein expression levels, which were normalized to the total protein library size for each sample. For each cell fraction, the abundance of H1.0 peptides is expressed as a percentage of the total H1.0 peptides observed under each condition. The normalized absolute values ​​of H1.0 peptide levels in each fraction are also shown. As shown in Figure 9A, chromatin-bound H1.0 decreased from approximately 60% total in SR and acute DNA damage to approximately 30% total in genotoxic stress-induced aging. Culture media derived from bleomycin-treated SR hADSCs were collected for slot blot analysis using α-H1.0 and α-H1.0K180me2 antibodies to evaluate the secretion of H1.0 into the extracellular matrix (ECM). The experiment was performed according to the method described in Example 1. Secreted H1.0 was detectable in the cell culture medium, and secreted H1.0K180me2 was also readily detectable 24 hours after bleomycin treatment (Figure 9B). These results confirm that methylated H1.0K180 is released from chromatin and secreted into the ECM during genotoxic stress-induced aging. (Example 8) The use of H1.0K180me2 antibodies indicates that exposure to ionizing radiation induces an increase in serum levels of H1.0K180me2.

[0381] The effect of ionizing radiation on serum H1.0K180me2 levels was investigated. Serum was collected from wild-type mice before exposure to 7 Gy of ionizing radiation and either 2 hours or 48 hours after exposure, according to the method described in Example 1. Serum levels of H1.0K180me2 2 hours (mouse 1) or 48 hours (mouse 2) after irradiation were compared to initial levels before treatment using slot blotting and immunoblotting with α-H1.0K180me2 antibody (Figure 10A). The concentration of H1.0K180me2 in each serum sample was calculated using the standard curve of the H1.0K180me2 peptide included in each analysis. Mouse serum albumin was used as a loading control. The dot blot band for H1.0K180me2 was quantified and normalized by serum albumin. The relative increase in H1.0K180me2 after irradiation is shown (Figure 10B). Western blot analysis of equal volumes of mouse serum containing α-H1.0K180me2 antibody also showed an increase in H1.0K180me2 after irradiation (Figure 10C). (Example 9) The use of H1.0K180me2 antibodies and labeled H1.0K180me2 peptides indicates that H1.0K180me2 levels in the brain and serum are indicators of Alzheimer's disease.

[0382] Whole cell lysates from mouse brain samples derived from 1-month-old (young) and 24-month-old (aged) mice were compared by Western blotting analysis using α-H1.0K180me2, α-H1.0, α-γH2A.X, and α-β-actin antibodies according to the method described in Example 1. H1.0K180me2 levels were elevated in 24-month-old mice and correlated with elevated γH2A.X levels (Figure 11A).

[0383] Further research was conducted using human clinical samples.

[0384] Human serum was obtained from the Cooperative Human Tissue Network (CHTN) and Nuclea Biotechnologies (NCB). The serum came from three groups: 1) healthy middle-aged donors with no significant medical conditions (n=7, age range=32-38 years); 2) healthy elderly donors with no significant medical conditions (n=9, age range=63-76 years); and 3) elderly donors with clinically diagnosed Alzheimer's disease (n=10, age range=75-103 years). Further details for each donor are shown in Table 4. [Table 4]

[0385] Whole cell lysates from human brain samples from healthy individuals aged 23 years (young) and / or over 60 years (elderly) were also analyzed by Western blotting using the antibodies and methods described above. Table 4 provides characteristics of the brain tissue donors. As shown in Figure 11B, older individuals show increased expression of H1.0K180me2. These results indicate that H1.0K180me2 increases with biological age.

[0386] The objective of this study was to demonstrate the usefulness of serum H1.0K180me2 measurements and / or serum antibody measurements against H1.0K180me2 as biomarkers of predictive performance for Alzheimer's disease. To ensure the correct predictive ability of the novel diagnostic biomarkers, a patient stratification method was developed and tested for its diagnostic accuracy. The performance of the tests was evaluated by comparison with registered reference standards (e.g., a combination of several tests recommended by the National Institute of Aging-Alzheimer's Association (NIA-AA) for the definitive diagnosis of AD). This study was designed and performed according to the Standards for Reporting of Diagnostic Accuracy (STARD). Statistical analysis included optimization of diagnostic accuracy parameters, cross-testing, and the Youden index. According to the patient stratification strategy and using the predictive power of the analysis, patients can be stratified as a category of patients with a high probability of developing Alzheimer's disease. Sensitivity, specificity, positive and negative predictive values ​​(PPV and NPV), and positive and negative likelihood ratios (PLR and NLR) are shown for each trial design. In Figures 12A-12C, 13B, 14B, 14E, 15A, and 15B, the centerline of the box plots represents the median; the ends of the boxes represent the 25th and 75th percentiles determined by R software; the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots; and data points are plotted as white circles. The dashed lines represent thresholds calculated by ROC curve (Treatment Operating Characteristic Curve) analysis. Sensitivity, specificity, positive and negative predictive values ​​(PPV and NPV), and positive and negative likelihood ratios (PLR and NLR) are shown for each trial design. Statistical methods help predict the presence or absence of disease in patients. The extent to which the trial results modify the pre-trial probability of disease is expressed by the "likelihood ratio" based on Bayes' theorem. The positive likelihood ratio (PLR) represents how much the probability of having the disease increases if the test is positive. The negative likelihood ratio (NLR) represents how much the probability of having the disease decreases if the test is negative.A PLR greater than 1 indicates an increased probability of the target disorder being present, a PLR less than 1 indicates a decreased probability of the target disorder being present, and a PLR of 1 means that the probability of the disorder does not change due to the trial. The ROC curve, threshold, and area under the curve (AUC) are shown for each trial design. A value of 0 in the 2x2 matrix (TP, FN, FP, TN) may make it impossible to calculate the ratio. To control this, a "pseudo-count" of 0.5 was added. The "pseudo-count" is added to all values ​​and therefore does not affect the relative values. In Figures 12A-12C, 13B, 14B, 14E, 15A, and 15B, the centerlines of the box plots indicate the median; the ends of the boxes indicate the 25th and 75th percentiles as determined by R software; the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots; and data points are plotted as white circles.

[0387] To test whether H1.0K180me2 levels can serve as a diagnostic indicator for Alzheimer's disease, H1.0K180me2 levels in human serum were quantified by slot blot analysis using α-H1.0K180me2 antibody according to the method described in Example 1. Equal volumes of serum were analyzed from healthy individuals aged 30–40 years (n=7) or over 60 years (n=9), and individuals over 60 years with clinically diagnosed Alzheimer's disease (n=10) (Table 4 provides serum donor characteristics). H1.0K 180me2 level

[0388] Figures 12A-C show that the detection of H1.0K180me2 antigen in human serum (Figures 12A, 12B, 12C), the detection of naturally occurring H1.0K180me2 IgG autoantibodies (Figure 13B), and the detection of naturally occurring H1.0K180me2 IgM autoantibodies (Figures 14A-E, 15A-B) may serve as tools for the early diagnosis of Alzheimer's disease.

[0389] Figure 12A shows the quantification of H1.0K180me2 levels determined by slot blot analysis in Alzheimer's disease patients and age-matched controls. The quantification of H1.0K180me2 levels was determined by slot blot analysis in Alzheimer's disease patients and age-matched controls. The concentration of H1.0K180me2 in each serum sample was calculated using the standard curve for the H1.0K180me2 peptide included in each analysis. As shown in Figure 12A, Alzheimer's disease patients showed lower serum H1.0K180me2 concentrations than healthy, age-matched controls, suggesting that serum H1.0K180me2 concentrations may be effective in separating patients with Alzheimer's disease from healthy individuals and can act as a diagnostic tool for detecting Alzheimer's disease. As shown in Table 5, a serum concentration of H1.0K180me2 antigen of 5.61 nmol / ml or less indicates the presence of the disease with a 24% probability compared to the pre-test probability, and the positive likelihood ratio (PLR or LR+) is 3.6. The post-test probability is calculated based on the following formula: pre-test odds × LR / (1 + pre-test odds × LR) (where pre-test odds is the clinical suspicion of the presence of the disease before the test). The post-test probability is usually calculated from the Likelihood Ratio Nomogram or Fagan Nomogram (NEJM 1975; vol. 293: p. 257). [Table 5]

[0390] Figure 12B shows the H1.0K180me2 levels in human serum normalized by total IgG serum levels. To normalize the H1.0K180me2 levels in human serum by total IgG serum levels, total IgG levels were determined for each serum sample by slot blot analysis using a goat anti-human IgG secondary antibody. The concentration of H1.0K180me2 was normalized by the measured IgG levels in each sample. As shown in Figure 12B, H1.0K180me2 levels were elevated in healthy individuals over 60 years of age compared to healthy young individuals (30-40 years of age), but patients with Alzheimer's disease showed significantly lower normalized levels of H1.0K180me2 compared to healthy elderly individuals (over 60 years of age). The left portion of the box graph represents patients without a diagnosis of prodromal Alzheimer's disease or mild cognitive impairment (MCI). The right portion of the box graph represents patients with post-mortem confirmation of Alzheimer's disease pathology. Table 6 shows the values ​​derived from ROC analysis. [Table 6]

[0391] Figure 12C shows the H1.0K180me2 levels in human serum normalized by total protein levels. To normalize the H1.0K180me2 levels in human serum by total protein levels, total serum protein was measured for each sample using Qubit (Invitrogen). The H1.0K180me2 concentration was then normalized by the measured protein concentration in each sample. As shown in Figure 12C, H1.0K180me2 levels were elevated in healthy elderly individuals over 60 years of age, but patients with Alzheimer's disease showed significantly lower normalized H1.0K180me2 levels compared to healthy elderly individuals (over 60 years of age). Figure 12C shows the serological quantification of the H1.0K180me2 antigen relative to the total protein composition of serum. Table 7 shows 4.76 × 10⁶ -4 A ratio of H1.0K180me2 antigen to total serum protein less than 3.00 indicates a 24.8% probability of disease presence compared to the pre-test probability, and the PLR ​​is 3.00. [Table 7]

[0392] While serum concentrations of H1.0K180me2 are sufficient to identify Alzheimer's disease patients, the use of normalization of serum samples by total IgG (Figure 12B) or total protein (Figure 12C) allows for direct comparisons between individuals regardless of variables that may alter overall serum concentrations, such as the protocol used to obtain the serum, changes in operators, patient hydration status, and patient activity level. IgG is very abundant in human serum, accounting for approximately 11% of serum proteins. Therefore, IgG can act as an indicator of serum concentration and may be used to normalize between patients. Total serum protein provides a direct indicator of serum concentration and may be used to normalize H1.0K180me2 concentrations between patients. In each example, normalized H1.0K180me2 levels were observed to increase with healthy aging (above 60 years). Serum H1.0K180me2 levels in Alzheimer's disease patients were significantly lower than those of age-matched controls. While the observed trend remained unchanged using standard normalization procedures, it was shown that direct comparisons of H1.0K180me2 levels between patients were possible regardless of serum collection and processing procedures or differences in patients' baseline serum concentration levels. Anti-H1.0K180me2 IgG (H1.0K180me2 IgG autoantibody) level

[0393] Figure 13A shows that human anti-H1.0K180me2 IgG can be detected in various human bodily fluids using indirect ELISA. Human plasma, urine, and saliva were tested for H1.0K180me2 IgG autoantibodies using indirect ELISA and labeled H1.0K180me2 peptide, respectively. A linear relationship between plasma concentration and OD measured at 450 nm was detected when plasma was diluted 500-fold, 1000-fold, and 2000-fold in loading buffer. Similarly, a linear relationship between urine and saliva concentrations and OD measured at 450 nm was detected when these fluids were diluted 80-fold and 160-fold in loading buffer. This suggests that indirect ELISA testing for anti-H1.0K180me2 IgG has utility in detecting anti-H1.0K180me2 IgG in a variety of human bodily fluids.

[0394] In related experiments, anti-H1.0K180me2 IgG levels in human serum were quantified by indirect ELISA analysis using biotinylated H1.0K180me2 capture peptide (H1.0K180me2 autoantibody-binding peptide), followed by a secondary antibody specific to the IgG autoantibody. Equal volumes of serum were analyzed from healthy individuals aged 30–40 years (n=7) or over 60 years (n=9), and individuals over 60 years with clinically diagnosed Alzheimer's disease (n=10) (Figure 13B).

[0395] Figure 13B shows the quantification of autoanti-H1.0K180me2 IgG levels determined by indirect ELISA in Alzheimer's disease patients and age-matched controls. The concentration of anti-H1.0K180me2 IgG in each serum sample (autoantibody IgG level determined by indirect ELISA) was calculated using a standard curve created by serial dilutions of H1.0K180me2-specific antibodies included in the ELISA experiment shown in Figure 12E. Alzheimer's disease patients show higher concentrations of serum anti-H1.0K180me2 IgG than healthy, age-matched controls. Serum concentrations of anti-H1.0K180me2 IgG may effectively isolate patients with Alzheimer's disease from healthy individuals and can act as a diagnostic tool for detecting Alzheimer's disease. As shown in Table 8, measured concentrations of autoanti-H1.0K180me2 antibody equal to or higher than 8.23 ​​ug / ml indicate the presence of the disease with a 30% probability compared to the pre-test probability, and the PLR ​​is 5.4. Figure 13B shows a standard curve for quantifying H1.0K180me2 autoantibodies in human serum. Total serum protein was measured for each sample using the Bradford assay. Anti-H1.0K180me2 IgG concentrations were normalized by the measured protein concentrations in each sample. Anti-H1.0K180me2 IgG levels are lower in healthy individuals over 60 years of age compared to healthy young individuals (30-40 years of age). Patients with Alzheimer's disease show elevated normalized levels of anti-H1.0K180me2 IgG compared to healthy elderly individuals (over 60 years of age). [Table 8] Anti-H1.0K180me2 IgM (H1.0K180me2 IgM autoantibody) level

[0396] In another related experiment, anti-H1.0K180me2 IgM levels in human serum were quantified by indirect ELISA analysis using biotinylated H1.0K180me2 capture peptide (H1.0K180me2 autoantibody-binding peptide), followed by a secondary antibody specific to IgM antibodies (normalized by volume). Equal volumes of serum were analyzed from healthy individuals over 60 years of age (n=9) and individuals over 60 years of age with clinically diagnosed Alzheimer's disease (n=10) (raw data in Figure 14E; data normalized to total IgM levels in Figure 15A).

[0397] Table 9 below shows the raw data derived from indirect ELISA analysis and further analyzed in Figures 14D-14E. ELISA was performed in triplicate for each patient sample. Anti-H1.0K180me2 IgM concentrations were calculated for each replication from the standard curve shown in Figure 14D. The average concentration of anti-H1.0K180me2 IgM for each sample was calculated from the three technical replications. The standard deviation of the samples between the three technical replications was also calculated. The coefficient of embodiment between replications was calculated as CV% = standard deviation / mean × 100%.

[0398] Since a standard curve for anti-H1.0K180me2 was not available, a standard curve was created using anti-H1.0K180me2 IgG antibody (Figure 14D). The molar concentration of anti-H1.0K180me2 IgG was plotted against OD at 450 nm. The curve was then used to estimate the molar concentration of anti-H1.0K180me2 IgM in each sample. [Table 9]

[0399] Figure 14E demonstrates the usefulness of measuring IgG autoantibodies against H1.0K180me2 as a biomarker for Alzheimer's disease (figure shows raw data). The left panel shows ROC curve analysis used to evaluate overall predictive performance and select the optimal threshold cutoff value to distinguish between positive and negative test results. An empirical ROC curve (solid line) is created by plotting to show the relationship between percent specificity and sensitivity at each possible threshold. The optimal threshold cutoff value, depicted in the right panel, was calculated using the empirical ROC curve. The optimal threshold is shown as a gray dot on the empirical ROC curve. The right panel of Figure 14E shows the box plot distribution of anti-H1.0K180me2 IgM concentrations in patients with and without Alzheimer's disease (neurological controls). Individual measurements for all samples are shown as dots, and box plots show the distribution for each sample, along with the upper and lower quartiles, and the distances from the upper and lower quartiles to the maximum and minimum non-outlier points. Samples above the threshold cutoff (dashed line) are considered positive for the test. Samples below the threshold cutoff are considered negative for the test. The 2x2 matrix below Figure 14E shows that the distribution may be divided into four groups: true positive (TP; index is positive and AD is present); false positive (FP; index is positive but AD is absent); true negative (TN; index is negative and AD is absent); and false negative (FN; index is negative but AD is present). The number of samples in each group is plotted in the 2x2 matrix.

[0400] Table 10 below shows the raw data for evaluating the test performance of anti-H1.0K180me2 IgM. [Table 10]

[0401] Table 11 below shows the normalized data (anti-H1.0K180me2 IgM normalized by total IgM level) derived from indirect ELISA analysis and further analyzed in Figures 15A and 15B. ELISA was performed in triplicate on each patient's sample. The concentration of anti-H1.0K180me2 IgM was calculated for each replication from the standard curve shown in Figure 14D and normalized by the mean of the total IgM concentration measured in the sample (Figure 14B). The mean normalized concentration of anti-H1.0K180me2 IgM for each sample was calculated from the three technical replications. The standard deviation of the samples between the three technical replications was also calculated. The coefficient of embodiment between replications was calculated as CV% = standard deviation / mean × 100%. [Table 11]

[0402] Figure 15A demonstrates the usefulness of measured IgM autoantibodies against H1.0K180me2 as a biomarker for Alzheimer's disease (the figure shows normalized data). The left panel shows ROC curve analysis used to evaluate overall predictive performance and select optimal threshold cutoff values ​​to distinguish between positive and negative test results. An empirical ROC curve (solid line) is created by plotting to show the relationship between percent specificity and sensitivity at each possible threshold. The optimal threshold cutoff values, depicted in the right panel, were calculated using the empirical ROC curve. The optimal thresholds are shown as gray dots on the empirical ROC curve. The right panel of Figure 15A shows the box plot distribution of normalized anti-H1.0K180me2 IgM concentrations in patients with and without Alzheimer's disease (neurological controls). Individual measurements for all samples are shown as dots, and box plots are shown for each distribution. The box plot shows the upper and lower quartiles, as well as the median, along with the distance from the upper and lower quartiles to the maximum and minimum non-outlier points. Samples above the threshold cutoff (dashed line) are considered positive for the test. Samples below the threshold cutoff are considered negative for the test. The 2x2 matrix below Figure 15A shows that the distribution may be divided into four groups: true positive (TP; index is positive and AD is present); false positive (FP; index is positive but AD is absent); true negative (TN; index is negative and AD is absent); and false negative (FN; index is negative but AD is present). The number of samples in each group is plotted in the 2x2 matrix. Figure 15B demonstrates that the test characteristics do not vary by laboratory settings and different operators.

[0403] Table 12 below shows the evaluation of the test performance of anti-H1.0K180me2 IgM / total IgM. [Table 12]

[0404] The results of this trial adjust the probability of disease from 10% before the trial to 63% after the trial. The positive likelihood ratio (LR+) is 15.0 (95% CI: 0.98, 229), and the positive predictive value (PPV) is 94% (95% CI: 53, 100). The negative likelihood ratio (LR-) is 0.26 (95% CI: 0.09, 0.78), and the negative predictive value is 79% (95% CI: 47, 95). contrast

[0405] Figure 14B shows the standard curve: the molar concentrations of the human IgG dilution series were plotted against an OD of 450 nm. The total molar concentration of IgM in each sample was then estimated by extrapolation using the curve.

[0406] Table 13 below shows the data from ELISA for total IgM. ELISA was performed in triplicate on each patient's sample. The molar concentration of total IgM was calculated for each replication from the standard curve shown in Figure 14B. The average molar concentration of total IgM for each sample was calculated from the three technical replications. The standard deviation of the samples between the three technical replications was also calculated. The coefficient of embodiment between replications was calculated as CV% = standard deviation / mean × 100%. [Table 13]

[0407] Figure 14B demonstrates that total IgM levels do not distinguish between individuals with and without Alzheimer's disease. Individual measurements for all samples are shown as dots, and box plots are shown for each distribution. The box plots show the median, along with the upper and lower quartiles, and the distances from the upper and lower quartiles to the maximum and minimum points that are not outliers.

[0408] Figure 16A demonstrates no correlation between total IgM levels and anti-H1.0K180me2 IgM levels in patient samples. All patient samples were plotted on a scatter plot based on measured anti-H1.0K180me2 IgM concentrations against measured total IgM concentrations. Anti-H1.0K180me2 IgM levels were associated with low R2 As demonstrated by the values, it is not affected by the total IgM level in the patient's serum.

[0409] Figure 16B demonstrates that total unmodified H1.0 protein levels (left graph) and total anti-H1.0 IgM levels do not distinguish between individuals with and without Alzheimer's disease. In the left panel, total H1.0 (unmodified) levels were measured in serum samples from Alzheimer's disease and control patients using a custom chemiluminescent slot blot immunoassay. In the right panel, IgM autoantibodies against the unmodified H1.0 peptide were measured in the patients' serum using indirect ELISA. There was no statistically significant difference in the levels of IgM autoantibodies against the unmodified H1.0 peptide (p=0.72), indicating that the diagnostic usefulness for Alzheimer's disease is specific to anti-H1.0K180me2 IgM in the patients' serum. For all samples, relative measurements for both panels are shown as dots, and box plots show each distribution. The box plot shows the upper and lower quartiles, as well as the median, along with the distances from the upper and lower quartiles to the maximum and minimum non-outlier points. Correlation of anti-H1.0K180me2 IgG and IgM levels

[0410] Figure 17 demonstrates that patients with Alzheimer's disease can be stratified into distinct populations by measuring H1.0K180me2 IgG and IgM autoantibodies. Anti-H1.0K180me2 IgG levels were measured in samples from Alzheimer's disease patients and normalized by total IgG levels. The correlation between normalized anti-H1.0K180me2 IgM levels in Alzheimer's disease patient samples and these normalized anti-H1.0K180me2 IgG levels was then examined using a scatter plot. This analysis allows for the stratification of Alzheimer's disease patients into distinct populations. Patients marked 75, 77, 94, and 78 in Figure 17 tend to respond to any Alzheimer's disease treatment. Patients marked 90, 91, 93, 83, and 103 in Figure 17 tend to respond only to specific Alzheimer's disease treatments, e.g., only to non-immunomodulatory Alzheimer's disease treatments. (Example 10) Rapamycin and its derivatives block the accumulation of H1.0K180me2 in the cytoplasm after DNA damage.

[0411] To test whether rapamycin derivatives may block the emergence of H1.0K180me2 during DNA damage, SR hADSCs were treated with bleomycin for 2 hours with or without 24-hour pretreatment with rapamycin or everolimus (a derivative of rapamycin). The cells were then lysed according to the method described in Example 1 and analyzed by Western blotting for H1.0K180me2, γH2A.X, and β-actin. As shown in Figure 18, both rapamycin and everolimus reduced the emergence of H1.0K180me2 during bleomycin treatment, suggesting that everolimus may also block the emergence of H1.0K180me2 during DNA damage.

[0412] SR hADSCs were treated with bleomycin or temozolomide, compounds that induce the base excision repair pathway, for 2 hours. The cells were then lysed and analyzed by Western blotting for H1.0K180me2, γH2A.X, and β-actin. As shown in Figure 19, both bleomycin and temozolomide were able to induce methylation of H1.0K180me2. (Example 11) mTOR and PI3K inhibitors block the accumulation of H1.0K180me2 after DNA damage.

[0413] Figure 20 shows the effects of mTOR and PI3K inhibitors on H1.0K180me2 kinetics. SR hADSCs were treated with bleomycin for 2 hours with or without 24-hour pretreatment with the chemoinhibitors mTOR1, mTOR2, and / or PI3K. Cells were lysed and chromatin extracted for Western blotting analysis of H1.0K180me2, whole H1.0, γH2A.X, and whole histone H4. The drug concentration used and the specific inhibitory target for each drug are given after the drug name. All inhibitors tested were able to reduce the appearance of H1.0K180me2 upon bleomycin treatment. (Example 12) In vitro methylation by G9A and analysis of the product

[0414] Figures 21A and 21B show the results of this in vitro G9A methylation assay.

[0415] G9A methyltransferase (Figure 21A) is capable of methylating the H1.0 peptide. Next, the methylation reaction was degraded on a gel and visualized by autoradiography. The appearance of a band at 4 kDa indicates that the tritium-labeled methyl group is transferred to the peptide, allowing G9A methyltransferase to methylate the H1.0 peptide, which enables visualization. A control reaction lacking the H1.0 peptide did not produce a tritium-labeled product.

[0416] G9A methyltransferase (Figure 21B) can methylate full-length recombinant H1.0. Figure 21B shows an in vitro methylation assay of recombinant full-length histone H1.0 with increasing amounts of G9A. G9A can dimethylate full-length H1.0 at K180.

[0417] To pinpoint the exact location of G9A methylation in the unmodified H1.0 peptide (AKPVKASKPKKAKPVKPK (SEQ ID NO: 36)), a methylation reaction was set up and the product was identified by LC-MS. The methylation reaction involved recombinant G9A, an unlabeled methyl donor (S-adenosyl-L-methionine), and the unmodified H1.0 peptide. The methylation reaction was then analyzed by LC-MS, and each spectral peak (corresponding to the peptide species in the final reaction) was identified and quantified using spectral counting. In Figure 22A, the number of circles labeled "me" represents the methylation status of the lysine residue (mono-, di-, or tri-methylation). Figure 22A shows that in the presence of the unmethylated H1.0 peptide, G9A specifically and abundantly dimethylates H1.0K180 (99.9% of all peptides).

[0418] More specifically, the data demonstrated that G9A dimethylates lysine K180 but not other lysines (K166, K174, K175, or K177) present in the same peptide fragment, with an estimated methylation efficiency of approximately 99% (Figures 22A and 22B). To further investigate the sensitivity and specificity of G9A methylation of H1.0 peptides to lysine 180, K180me2 peptides (H1.0 AA165-182) were used as substrates for similar in vitro methylation experiments. As shown in Figures 22B and 23, only very small amounts of further methylated peptides were detected: H1.0K166me1K180me2 (1.27% of the total peptides in the reaction), H1.0K174me1K180me3 (1.06% of the total peptides in the reaction), and H1.0K174me3K175me3K177me1K180me2 (0.35% of the total peptides in the reaction).

[0419] To pinpoint the exact location of G9A methylation in full-length H1.0 protein, methylation reactions were set up and the products were identified by LC-MS. The methylation reactions involved recombinant G9A, an unlabeled methyl donor (S-adenosyl-L-methionine), and recombinant human H1.0 protein. The methylation reactions were then analyzed by LC-MS to identify the methylation sites. In Figure 24, the number of circles labeled "me" represents the methylation status of the lysine residue (mono-, di-, or tri-methylation). Figure 24 shows that in the presence of recombinant full-length H1.0, G9A methylates the C-terminal lysine residue containing H1.0K180me2. (Example 13) In vitro GLP-mediated methylation and product analysis

[0420] To pinpoint the exact location of GLP methylation in the unmodified H1.0 peptide (AKPVKASKPKKAKPVKPK (SEQ ID NO: 36)), a methylation reaction was set up and the product was identified by LC-MS. The methylation reaction involved recombinant GLP, an unlabeled methyl donor (S-adenosyl-L-methionine), and the unmodified H1.0 peptide. The methylation reaction was then analyzed by LC-MS to identify each spectral peak (corresponding to the peptide species in the final reaction) and quantified using spectral counting. The number of circles labeled "me" in Figure 25 represents the methylation status of the lysine residue (mono-, di-, or tri-methylation). Figure 25 shows that in the presence of the unmethylated H1.0 peptide, GLP specifically dimethylates H1.0K180 (96.6% of all peptides).

[0421] K180 Dimethylated H1.0 Peptide (AKPVKASKPKKAKPVK (me2)To pinpoint the exact location of GLP methylation in PK (SEQ ID NO: 3), a methylation reaction was set up and the product was identified by LC-MS. The methylation reaction involved recombinant GLP, an unlabeled methyl donor (S-adenosyl-L-methionine), and the H1.0K180me2 peptide. The methylation reaction was then analyzed by LC-MS, and each spectral peak (corresponding to the peptide species in the final reaction) was identified and quantified using spectral counting. The number of circles labeled "me" in Figure 26 represents the methylation state of the lysine residue (mono-, di-, or tri-methylation). Figure 26 shows that in the presence of the K180 dimethylated H1.0 peptide, GLP only further methylates H1.0K180 and H1.0K174, and its efficiency is very low (1.02% of the peptide is further methylated).

[0422] To pinpoint the exact location of GLP methylation in full-length H1.0 protein, methylation reactions were set up and the products were identified by LC-MS. The methylation reactions involved recombinant GLP, an unlabeled methyl donor (S-adenosyl-L-methionine), and recombinant human H1.0 protein. The methylation reactions were then analyzed by LC-MS to identify the methylation sites. The number of circles labeled "me" in Figure 27 represents the methylation status (mono-, di-, or tri-methylation) of the lysine residues. Figure 27 shows that GLP does not methylate full-length H1.0 in the presence of recombinant full-length H1.0 under the conditions described herein. Under the conditions provided herein, GLP methylates numerous lysine residues in t...

Claims

[Claim 1] An antibody that specifically binds to a dimethylated antigen, wherein the dimethylated antigen comprises a dimethylated lysine residue, the lysine residue corresponds to K180 of human histone H1.0, and the dimethylated lysine residue is required for binding.

Images