Antibody compositions and methods for targeting extracellular nucleosome proteins

Abstract

Provided herein are methods and compositions of internalizing anti-nucleosome protein antibodies into a cell. The methods and compositions can be used for disease diagnosis and therapeutic treatments.

Description

ANTIBODY COMPOSITIONS AND METHODS FOR TARGETINGEXTRACELLULAR NUCLEOSOME PROTEINSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 566,085, filed March 15, 2024, the disclosure of which is herein incorporated by reference in its entirety for all purposes.SEQUENCE LISTING SUBMITTED VIA ELECTRONIC FILING SYSTEM

[0002] The instant application contains a Sequence Listing which has been filed electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on March 11, 2025, is named 110618-0885WO-1494030_SL.xml and is 6.3 KB in size.BACKGROUND

[0003] In eukaryotic cells, genomic DNA is packed as chromatin, of which nucleosomes are the basic structural repeating units. A nucleosome is composed of a double-stranded (dsDNA) fragment wrapped around a histone octamer (each with two H2A, H2B, H3 and H4 histones). Under certain pathological conditions, especially during apoptosis, chromatin fragments leading to the release of nucleosomes into the blood circulation system. Free nucleosomes then can enter the cytoplasm of mammalian cells by clathrin- and caveolin-dependent endocytosis and trigger immune responses by activating cGMP-AMP synthase (cGAS)

[0004] Oligonucleotides and dsDNA fragments, due to their negative charge and hydrophilic features, cannot easily enter cells. In contrast, Lys- and Arg-rich histones are positively charged small proteins that can be efficiently taken up by mammalian cells. Nucleosomes themselves are DNA-histone complexes and form zwitterionic nanoparticles. Such physicochemical features may allow nucleosomes to be directly taken up by mammalian cells. It has been demonstrated that extracellular nucleosomes can enter the cytoplasm of mammalian cells by clathrin- and caveolin-dependent endocytic pathways, and after cellular entry, these nucleosomes trigger innate immune responses via cGAS activation in the cytosol (see. NucleicAcids Res. 2021 Dec 2;49(21): 12306-12319). However, such internalization of extracellular nucleosome has not been applied in diagnostic, prognostic and / or theranostic fields.SUMMARY

[0005] In one aspect, the present disclosure provides a method of internalizing one or more antibodies into a mammalian cell. In some embodiments, the method comprises contacting the cell with the one or more antibodies, wherein the one or more antibodies bind to a nucleosome protein on the surface of the cell and the one or more antibodies are internalized into the cell. In some embodiments, the nucleosome protein comprises a nuclear protein, a protein bound to the nuclear protein, a DNA binding protein, or a protein bound to the DNA binding protein. In some embodiments, the nucleosome protein comprises histone Hl, histone H2A, histone H2B, histone H3, histone H4, a modified histone protein, a histone modifying enzyme, a histone acetyltranferase, a histone deacetylase, a histone lysine methyl-transferase, a histone lysine demethylase, a transcription factor, a DNA ligase, nucleophosmin, nucleolin, Ku70, Ku80, centromere protein A (CENPA), high mobility group protein 14 (HMG14), high mobility group protein 17 (HMG17 or HMGN2), retinoblastoma-binding protein 5 (RBBP5), Holliday junction recognition protein (HJURP), a DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a transcription factor co-activator or a transcription factor co-repressor.

[0006] In some embodiments, the antibodies are bound by a secondary antibody. In some embodiments, the antibodies are coated on a gold nanoparticle. In some embodiments, at least two antibodies are linked together as a polymer. In some embodiments, the polymer is a dimer, a trimer, a tetramer, or a pentamer. In some embodiments, at least two antibodies are crosslinked together. In some embodiments, further comprising contacting the cell with one or more DNA nucleases.

[0007] In some embodiments, the one or more DNA nucleases comprises an endonuclease, an exonuclease, or a combination thereof. In some embodiments, the endonuclease is a deoxyribonuclease (DNase), a serratia marcescens nuclease (Benzonase), a micrococcal nuclease (MNase), a restriction enzyme, nuclease SI, nuclease Pl, a sequence specific endonuclease, or a sequence non-specific endonuclease. In some embodiments, the one or more DNA nucleases comprise a single stranded DNA (ssDNA) nuclease, a double-stranded DNA (dsDNA) nuclease, or a combination thereof. In some instances, the DNA nucleases and the antibodies are contacted to the cell at the same time, optionally wherein the DNA nucleases areattached to the antibodies. In other instances, the DNA nucleases are contacted to the cell before the antibodies.

[0008] In some embodiments, the antibodies are covalently or non-covalently linked to at least one payload. In some embodiments, the antibodies are bound by a secondary antibody, and wherein the secondary antibody is covalently or non-covalently linked to at least one payload. In some embodiments, the antibodies and / or the secondary antibody are linked to the payload via a linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the non-cleavable linker is a maleimide alkane linker, or a maleimide cyclohexane linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is cleaved following contacting the cell with the one or more antibodies, thereby releasing the payload on or in the cell. In some embodiments, the cleavable linker is a hydrazone linker, a cathepsin B-responsive linker, a disulfide linker, or a pyrophosphate diester linker. In some embodiments, the cleavable linker is a protease-sensitive linker, a pH-sensitive linker, a radiation-sensitive linker, a disulfide linker, or a glutathionesensitive linker. In some embodiments, the cleavable linker is a Valine-Citrulline (Val-Cit) linker.

[0009] In some embodiments, the antibody and / or the secondary antibody are attached to a biotin moiety, and the at least one payload is attached to a streptavidin. In some embodiments, the at least one payload comprises a small molecule, peptide, protein, nucleic acid, toxin, therapeutic agent, drug, chemotherapeutic agent, liposome, nanoparticle, dendrimer, detectable label, or any derivative, fragment, or combination thereof.

[0010] In some embodiments, the therapeutic agent is selected from the group consisting of an antitumor agent, antineoplastic agent, prodrug, lysosome destabilizing agent (e.g., chloroquine), alkylating agent, alkaloid, allosteric inhibitor, anti-folic, anti-inflammatory agent, antibiotics anti-bacterial, antifungal, antifibrotic agent, anti-infective agent, anti- parasitic agent, antiviral agent, antimycobacterial agent, antineoplastic agent, antiprotozoal agent, antiviral agent, bioactive peptide, steroid hormone, photosensitizer substance, radiopharmaceutical, anti-prion agent, and any combination thereof.

[0011] In some embodiments, the antitumor agent is selected from the group consisting of an aromatase inhibitor; an anti-estrogen; an anti-androgen; a gonadorelin agonist; a topoisomerase I inhibitor; a topoisomerase II inhibitor; a microtubule inhibitor; an alkylating agent; a retinoid; a carotenoid; a tocopherol; a cyclooxygenase inhibitor; an MMP inhibitor; amTOR inhibitor; an antimetabolite; a platin compound; a methionine aminopeptidase inhibitor; a bisphosphonate; an antiproliferative antibody; a heparinase inhibitor; an inhibitor of Ras oncogenic isoforms; a telomerase inhibitor; a proteasome inhibitor; a Flt-3 inhibitor; an Hsp90 inhibitor; a kinesin spindle protein inhibitor; a MEK inhibitor; a PARP inhibitor; a Tyrosine kinase inhibitor; a PI3K inhibitor; an AKT inhibitor; an EGFR inhibitor; an antitumor antibiotic; a nitrosourea; a compound targeting / decreasing protein or lipid kinase activity; a compound targeting / decreasing protein or lipid phosphatase activity; any further anti- angiogenic compound; and any combination thereof.

[0012] In some embodiments, the antitumor agent is selected from the group consisting of azacitidine, axathioprine, bleomycin, capecitabine, carboplatin, chlorabucil, cisplatin, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, etoposide, fenretinide, fluorouracil, gemcitabine, herceptin, idarubicin, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, tafluposide, teniposide, tioguanine, retinoic acid, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, receptor tyrosine kinase inhibitor, and any combination thereof. In some embodiments, the antitumor agent is a tubule inhibitor, a DNA inhibitor, or an RNA inhibitor.

[0013] In some embodiments, the tubule inhibitor is selected from the group consisting of monomethyl auri statin F (MMAF), monomethyl auri statin E (MMAE), maytansine, maytansinoid, mertansine (emtansine, DM1), ravtansine (soravtansine, DM4), tubulysin, halichondrin (eribulin), cryptophycin, EG5 inhibitor, and any derivative thereof. In some embodiments, the DNA inhibitor is selected from the group consisting of alkylator, duocarmycin, duocarmycin DM, calicheamicin, pyrrolobenzodiazepine (PDB), enediyne, uncialamycin, topoisomerase inhibitor, topotecan, camptothecin (CPT), exatecan, and any derivative thereof. In some embodiments, the RNA inhibitor is selected from the group consisting of RNA splicing inhibitor, RNA polymerase II inhibitor, thailanstatin, amatoxin, and any derivative thereof.

[0014] In some embodiments, the detectable label is selected from the group consisting of magnetic label, fluorescent moiety, enzyme, light emitting particle, chemiluminescent probe, metal particle, non-metal colloidal particle, polymeric dye particle, pigment molecule, electrochemically active species, semiconductor nanocrystal, nanoparticle, quantum dot, gold particles, fluorophore, or radioactive label.

[0015] In some embodiments, the cell is a human cell. In some embodiments, the cell is in vitro. In some embodiments, the cell is in vivo. In some embodiments, the cell has an aneuploidy and / or DNA repair deficiency. In some embodiments, the cell comprises a functionally impaired transcription factor or regulator selected from the group consisting of p53, NF-Kb, AP-1, E2F1, and BRCA1. In some embodiments, the functional impairment of the transcription factor or regulator is caused by a gene mutation. In some embodiments, the cell comprises a functionally impaired transcription factor p53.

[0016] In another aspect, the present disclosure provides a method of delivering one or more payloads inside of a cell. In some embodiments, the method comprises contacting the cell with one or more payloads covalently or non-covalently linked to an antibody, wherein the antibody binds to a nucleosome protein on the surface of the cell and delivers the one or more payloads inside of the cell.

[0017] In some embodiments, the nucleosome protein comprises histone Hl, histone H2A, histone H2B, histone H3, histone H4, centromere protein A (CENPA), high mobility group protein 14 (HMG14), high mobility group protein 17 (HMG17 or HMGN2), retinoblastomabinding protein 5 (RBBP5), or Holliday junction recognition protein (HJURP).

[0018] In some embodiments, the one or more payloads comprise a small molecule, peptide, protein, nucleic acid, toxin, therapeutic agent, drug, chemotherapeutic agent, liposome, nanoparticle, dendrimer, detectable label, or any derivative, fragment, or combination thereof.

[0019] In some embodiments, the therapeutic agent is selected from the group consisting of an antitumor agent, antineoplastic agent, prodrug, lysosome destabilizing agent (e.g., chloroquine), alkylating agent, alkaloid, allosteric inhibitor, anti-folic, anti-inflammatory agent, antibiotics anti-bacterial, antifungal, antifibrotic agent, anti-infective agent, anti- parasitic agent, antiviral agent, antimycobacterial agent, antineoplastic agent, antiprotozoal agent, antiviral agent, bioactive peptide, steroid hormone, photosensitizer substance, radiopharmaceutical, anti-prion agent, and any combination thereof.

[0020] In some embodiments, the antitumor agent is selected from the group consisting of an aromatase inhibitor; an anti-estrogen; an anti-androgen; a gonadorelin agonist; a topoisomerase I inhibitor; a topoisomerase II inhibitor; a microtubule inhibitor; an alkylating agent; a retinoid; a carotenoid; a tocopherol; a cyclooxygenase inhibitor; an MMP inhibitor; a mTOR inhibitor; an antimetabolite; a platin compound; a methionine aminopeptidase inhibitor; a bisphosphonate; an antiproliferative antibody; a heparinase inhibitor; an inhibitor of Rasoncogenic isoforms; a telomerase inhibitor; a proteasome inhibitor; a Flt-3 inhibitor; an Hsp90 inhibitor; a kinesin spindle protein inhibitor; a MEK inhibitor; a PARP inhibitor; a Tyrosine kinase inhibitor; a PI3K inhibitor; an AKT inhibitor; an EGFR inhibitor; an antitumor antibiotic; a nitrosourea; a compound targeting / decreasing protein or lipid kinase activity; a compound targeting / decreasing protein or lipid phosphatase activity; any further anti- angiogenic compound; and any combination thereof.

[0021] In some embodiments, the antitumor agent is selected from the group consisting of azacitidine, axathioprine, bleomycin, capecitabine, carboplatin, chlorabucil, cisplatin, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, etoposide, fenretinide, fluorouracil, gemcitabine, herceptin, idarubicin, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, tafluposide, teniposide, tioguanine, retinoic acid, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, receptor tyrosine kinase inhibitor, and any combination thereof.

[0022] In some embodiments, the antitumor agent is a tubule inhibitor, a DNA inhibitor, or an RNA inhibitor. In some embodiments, the tubule inhibitor is selected from the group consisting of monomethyl auri statin F (MMAF), monomethyl auri statin E (MMAE), maytansine, maytansinoid, mertansine (emtansine, DM1), ravtansine (soravtansine, DM4), tubulysin, halichondrin (eribulin), cryptophycin, EG5 inhibitor, and any derivative thereof. In some embodiments, the DNA inhibitor is selected from the group consisting of alkylator, duocarmycin, duocarmycin DM, calicheamicin, pyrrolobenzodiazepine (PDB), enediyne, uncialamycin, topoisomerase inhibitor, topotecan, camptothecin (CPT), exatecan, and any derivative thereof. In some embodiments, the RNA inhibitor is selected from the group consisting of RNA splicing inhibitor, RNA polymerase II inhibitor, thailanstatin, amatoxin, and any derivative thereof.

[0023] In some embodiments, the detectable label is selected from the group consisting of magnetic label, fluorescent moiety, enzyme, light emitting particle, chemiluminescent probe, metal particle, non-metal colloidal particle, polymeric dye particle, pigment molecule, electrochemically active species, semiconductor nanocrystal, nanoparticle, quantum dot, gold particles, fluorophore, or radioactive label.

[0024] In some instances, the one or more payloads are covalently linked to the antibody. In other instances, the one or more payloads are non-covalently linked to the antibody. In someembodiments, the antibody is bound by a secondary antibody, and wherein the one or more payloads are conjugated to the second antibody.

[0025] In some embodiments, the method further comprises contacting the cell with one or more nucleases, wherein the one or more nucleases comprise a single stranded DNA (ssDNA) nuclease, a double-stranded DNA (dsDNA) nuclease, or a combination thereof. In some embodiments, the cell is in vitro. In some embodiments, the cell is in vivo. In some embodiments, the cell has an aneuploidy and / or DNA repair deficiency. In some embodiments, the cell comprises a functionally impaired transcription factor or regulator selected from the group consisting of p53, NF-Kb, AP-1, E2F1, and BRCA1. In some embodiments, the functional impairment of the transcription factor or regulator is caused by a gene mutation. In some embodiments, the cell comprises a functionally impaired transcription factor p53.

[0026] In some embodiments, the cell is a cancer cell and wherein the payloads such as therapeutic drugs kill or inhibit growth or division of the cancer cell. In some embodiments, the cancer comprises an acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical carcinoma; AIDS-related cancer; AIDS-related lymphoma; anal cancer; appendix cancer; astrocytomas; atypical teratoid / rhabdoid tumor; basal cell carcinoma; bladder cancer; brain stem glioma; brain tumor; brain stem glioma; central nervous system atypical teratoid / rhabdoid tumor; central nervous system embryonal tumors; astrocytomas; craniopharyngioma; ependymoblastoma; ependymoma; medulloblastoma; medulloepithelioma; pineal parenchymal tumors of intermediate differentiation; supratentorial primitive neuroectodermal tumors and pineoblastoma; breast cancer; bronchial tumors; Burkitt lymphoma; cancer of unknown primary site (CUP); carcinoid tumor; carcinoma of unknown primary site; central nervous system atypical teratoid / rhabdoid tumor; central nervous system embryonal tumors; cervical cancer; childhood cancers; chordoma; chronic lymphocytic leukemia; chronic myelogenous leukemia; chronic myeloproliferative disorders; colon cancer; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; endocrine pancreas islet cell tumors; endometrial cancer; ependymoblastoma; ependymoma; esophageal cancer; esthesioneuroblastoma; Ewing sarcoma; extracranial germ cell tumor; extragonadal germ cell tumor; extrahepatic bile duct cancer; gallbladder cancer; gastric (stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal cell tumor; gastrointestinal stromal tumor (GIST); gestational trophoblastic tumor; glioma; hairy cell leukemia; head and neck cancer; heart cancer; Hodgkin lymphoma; hypopharyngeal cancer; intraocular melanoma; islet cell tumors; Kaposi sarcoma; kidney cancer; Langerhans cell histiocytosis; laryngeal cancer;lip cancer; liver cancer; lung cancer; malignant fibrous histiocytoma bone cancer; medulloblastoma; medulloepithelioma; melanoma; Merkel cell carcinoma; Merkel cell skin carcinoma; mesothelioma; metastatic squamous neck cancer with occult primary; mouth cancer; multiple endocrine neoplasia syndromes; multiple myeloma; multiple myeloma / plasma cell neoplasm; mycosis fungoides; myelodysplastic syndromes; myeloproliferative neoplasms; nasal cavity cancer; nasopharyngeal cancer; neuroblastoma; Non-Hodgkin lymphoma; nonmelanoma skin cancer; non-small cell lung cancer; oral cancer; oral cavity cancer; oropharyngeal cancer; osteosarcoma; other brain and spinal cord tumors; ovarian cancer; ovarian epithelial cancer; ovarian germ cell tumor; ovarian low malignant potential tumor; pancreatic cancer; papillomatosis; paranasal sinus cancer; parathyroid cancer; pelvic cancer; penile cancer; pharyngeal cancer; pineal parenchymal tumors of intermediate differentiation; pineoblastoma; pituitary tumor; plasma cell neoplasm / multiple myeloma; pleuropulmonary blastoma; primary central nervous system (CNS) lymphoma; primary hepatocellular liver cancer; prostate cancer; rectal cancer; renal cancer; renal cell (kidney) cancer; renal cell cancer; respiratory tract cancer; retinoblastoma; rhabdomyosarcoma; salivary gland cancer; Sezary syndrome; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma; squamous neck cancer; stomach (gastric) cancer; supratentorial primitive neuroectodermal tumors; T-cell lymphoma; testicular cancer; throat cancer; thymic carcinoma; thymoma; thyroid cancer; transitional cell cancer; transitional cell cancer of the renal pelvis and ureter; trophoblastic tumor; ureter cancer; urethral cancer; uterine cancer; uterine sarcoma; vaginal cancer; vulvar cancer; Waldenstrom macroglobulinemia; or Wilm’s tumor.

[0027] In some embodiments, the cancer comprises an acute myeloid leukemia (AML), breast carcinoma, cholangiocarcinoma, colorectal adenocarcinoma, extrahepatic bile duct adenocarcinoma, female genital tract malignancy, gastric adenocarcinoma, gastroesophageal adenocarcinoma, gastrointestinal stromal tumor (GIST), glioblastoma, head and neck squamous carcinoma, leukemia, liver hepatocellular carcinoma, low grade glioma, lung bronchioloalveolar carcinoma (BAC), non-small cell lung cancer (NSCLC), lung small cell cancer (SCLC), lymphoma, male genital tract malignancy, malignant solitary fibrous tumor of the pleura (MSFT), melanoma, multiple myeloma, neuroendocrine tumor, nodal diffuse large B-cell lymphoma, non-epithelial ovarian cancer (non-EOC), ovarian surface epithelial carcinoma, pancreatic adenocarcinoma, pituitary carcinomas, oligodendroglioma, prostatic adenocarcinoma, retroperitoneal or peritoneal carcinoma, retroperitoneal or peritonealsarcoma, small intestinal malignancy, soft tissue tumor, thymic carcinoma, thyroid carcinoma, or uveal melanoma.

[0028] In some embodiments, the cell is in a human subject having a disease, and wherein the delivery of the therapeutic drug is effective to treat the disease.

[0029] In some embodiments, the method disclosed herein further comprises administering one or more DNA nucleases to the human subject. In some embodiments, the human subject has a cancer and wherein the cell is a cancer cell.

[0030] In some embodiments, the method disclosed herein further comprises determining, prior to the contacting, whether the cell has aneuploidy, a DNA repair deficiency, and / or a functionally impaired transcription factor or regulator selected from the group consisting of p53, NF-Kb, AP-1, E2F1, and BRCA1. In some embodiments, the functional impairment of the transcription factor or regulator is caused by a gene mutation. In some embodiments, the cell comprises a functionally impaired transcription factor p53.

[0031] In another aspect, the present disclosure provides a composition comprising an antibody that binds to a nucleosome protein. In some embodiments, the nucleosome protein comprises a nuclear protein, a protein bound to the nuclear protein, a DNA binding protein, or a protein bound to the DNA binding protein. In some embodiments, the nucleosome protein comprises histone Hl, histone H2A, histone H2B, histone H3, histone H4, a modified histone protein, a histone modifying enzyme, a histone acetyltranferase, a histone deacetylase, a histone lysine methyl-transferase, a histone lysine demethylase, a transcription factor, a DNA ligase, nucleophosmin, nucleolin, Ku70, Ku80, centromere protein A (CENPA), high mobility group protein 14 (HMG14), high mobility group protein 17 (HMG17 or HMGN2), retinoblastoma-binding protein 5 (RBBP5), Holliday junction recognition protein (HJURP), a DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a transcription factor coactivator or a transcription factor co-repressor.

[0032] In some embodiments, the composition comprises a secondary antibody that binds the antibody that binds to the nucleosome protein. In some embodiments, the antibody is coated on a gold nanoparticle. In some embodiments, at least two antibodies that bind to the nucleosome protein are linked together as a polymer. In some embodiments, the polymer is a dimer, a trimer, a tetramer, or a pentamer. In some embodiments, at least two antibodies that bind to the nucleosome protein are crosslinked together.

[0033] In some embodiments, the composition further comprises one or more payloads covalently or non-covalently linked to the antibody and / or the secondary antibody. In some embodiments, the one or more payloads comprise a small molecule, peptide, protein, nucleic acid, toxin, therapeutic agent, drug, chemotherapeutic agent, liposome, nanoparticle, dendrimer, detectable label, or any derivative, fragment, or combination thereof. In some embodiments, the therapeutic agent is selected from the group consisting of an antitumor agent, antineoplastic agent, prodrug, lysosome destabilizing agent (e.g., chloroquine), alkylating agent, alkaloid, allosteric inhibitor, anti-folic, anti-inflammatory agent, antibiotics antibacterial, antifungal, antifibrotic agent, anti-infective agent, anti-parasitic agent, antiviral agent, antimycobacterial agent, antineoplastic agent, antiprotozoal agent, antiviral agent, bioactive peptide, steroid hormone, photosensitizer substance, radio-pharmaceutical, anti-prion agent, and any combination thereof. In some embodiments, the antitumor agent is selected from the group consisting of an aromatase inhibitor; an anti-estrogen; an anti-androgen; a gonadorelin agonist; a topoisomerase I inhibitor; a topoisomerase II inhibitor; a microtubule inhibitor; an alkylating agent; a retinoid, a carotenoid, or a tocopherol; a cyclooxygenase inhibitor; an MMP inhibitor; a mTOR inhibitor; an antimetabolite; a platin compound; a methionine aminopeptidase inhibitor; a bisphosphonate; an antiproliferative antibody; a heparinase inhibitor; an inhibitor of Ras oncogenic isoforms; a telomerase inhibitor; a proteasome inhibitor; a Fit- 3 inhibitor; an Hsp90 inhibitor; a kinesin spindle protein inhibitor; a MEK inhibitor; a PARP inhibitor; a Tyrosine kinase inhibitor; a PI3K inhibitor; an AKT inhibitor; an EGFR inhibitor; an antitumor antibiotic; a nitrosourea, a compound targeting / decreasing protein or lipid kinase activity, a compound targeting / decreasing protein or lipid phosphatase activity, any further anti-angiogenic compound, and any combination thereof.

[0034] In some embodiments, the antitumor agent is selected from the group consisting of azacitidine, axathioprine, bleomycin, capecitabine, carboplatin, chlorabucil, cisplatin, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, etoposide, fenretinide, fluorouracil, gemcitabine, herceptin, idarubicin, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, tafluposide, teniposide, tioguanine, retinoic acid, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, receptor tyrosine kinase inhibitor, and any combination thereof.

[0035] In some embodiments, the antitumor agent is a tubule inhibitor, a DNA inhibitor, or an RNA inhibitor. In some embodiments, the tubule inhibitor is selected from the groupconsisting of monomethyl auri statin F (MMAF), monomethyl auri statin E (MMAE), maytansine, maytansinoid, mertansine (emtansine, DM1), ravtansine (soravtansine, DM4), tubulysin, halichondrin (eribulin), cryptophycin, EG5 inhibitor, and any derivative thereof. In some embodiments, the DNA inhibitor is selected from the group consisting of alkylator, duocarmycin, duocarmycin DM, calicheamicin, pyrrolobenzodiazepine (PDB), enediyne, uncialamycin, topoisomerase inhibitor, topotecan, camptothecin (CPT), exatecan, and any derivative thereof. In some embodiments, the RNA inhibitor is selected from the group consisting of RNA splicing inhibitor, RNA polymerase II inhibitor, thailanstatin, amatoxin, and any derivative thereof.

[0036] In some embodiments, the detectable label is selected from the group consisting of magnetic label, fluorescent moiety, enzyme, light emitting particle, chemiluminescent probe, metal particle, non-metal colloidal particle, polymeric dye particle, pigment molecule, electrochemically active species, semiconductor nanocrystal, nanoparticle, quantum dot, gold particles, fluorophore, or radioactive label.

[0037] In some embodiments, the composition disclosed herein further comprises one or more DNA nucleases. In some embodiments, the one or more DNA nucleases comprise an endonuclease, an exonuclease, or a combination thereof. In some embodiments, the endonuclease is a deoxyribonuclease (DNase), a serratia marcescens nuclease (Benzonase), a micrococcal nuclease (MNase), a restriction enzyme, nuclease SI, nuclease Pl, a sequence specific endonuclease, or a sequence non-specific endonuclease. In some embodiments, the one or more DNA nucleases comprise a single stranded DNA (ssDNA) nuclease, a doublestranded DNA (dsDNA) nuclease, or a combination thereof.

[0038] In another aspect, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of the composition described herein, and a pharmaceutically acceptable excipient, carrier, and / or diluent.

[0039] In yet another aspect, the present disclosure provides a method of treating or ameliorating a disease or disorder in a human subject in need thereof, comprising administering the pharmaceutical composition described herein to the subject, optionally wherein the disease or disorder comprises a cancer. In some embodiments, the method described herein further comprises determining, prior to the administering, whether the cancer has an aneuploidy, a DNA repair deficiency, and / or a functionally impaired transcription factor or regulator selected from the group consisting of p53, NF-Kb, AP-1, E2F1, and BRCA1. In some embodiments,the functional impairment of the transcription factor or regulator is caused by a gene mutation. In some embodiments, the cancer comprises a functionally impaired transcription factor p53 resulting from a mutation of the TP53 gene. In some embodiments, the administering comprises at least one of intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intracerebral, intravaginal, transdermal, rectal, by inhalation, topical administration, or any combination thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0040] FIGS. 1A-1D depict a schematic drawing of binding and internalization of antinucleosome antibody to a cell. FIG. 1A shows an anti-nucleosome antibody binding to extracellular nucleosome on a cell surface, and the anti-nucleosome antibody is also recognized and linked to a 2ndantibody (115-005-071, Jackson ImmunoResearch) conjugated with a desired payload, such as a therapeutic drug or fluorescent label. Internalization of the complex of extracellular nucleosome with the anti-nucleosome antibody and the 2ndADC results in intracellular payload delivery, such as cell killing (when the conjugate is a therapeutic drug) or cell imaging (when the conjugate is a fluorescent label). FIG. IB shows a similar schematic as FIG. 1A except that the payloads are directly attached to an anti-nucleosome antibody. FIG. 1C shows the payloads are conjugated to both an anti-nucleosome antibody and a 2ndantibody. FIG. ID shows an anti-nucleosome antibody with drug conjugates binds to extracellular nucleosome on a cell surface and enter the cell without a 2ndantibody. FIG. IE depicts a schematic drawing of various antibodies that bind to either a DNA nuclear protein (e.g., a histone), or a nuclear protein-binding protein (e.g., a hi stone-modifying enzyme), or a DNA- binding protein (e.g., a transcription factor), or a DNA binding protein-binding protein (e.g., a transcription factor activator and repressor). FIG. IF depicts a schematic drawing of possible formats of anti-nucleosome ADCs / ADC complexes.

[0041] FIGS. 2A-2D depict fluorescent imaging of the internalization of the anti-histone H3 antibody, which can be enhanced by nuclease treatment. The anti-histone H3 antibody (ab309551, Abeam) or an isotype antibody was pre-mixed with an Alexa Fluor 647-labeled anti-mouse IgG 2ndantibody (115-605-071, Jackson ImmunoResearch) before being applied to HER2+ human breast cancer cells in an 18-well glass-bottom chambered coverslips plate. Half of the wells were additionally treated with 20 units / ml of DNase I (M0303S, New England Biolabs). FIG. 2A indicates cells incubated with the isotype antibody and the labeled secondary antibody; FIG. 2B indicates cells incubated with the anti-histone H3 antibody and the labeled secondary antibody; FIG. 2C indicates cells incubated with the isotype antibody,the labeled secondary antibody, and DNase I; and FIG. 2D indicates cells incubated with the anti-histone H3 antibody, the labeled secondary antibody, and DNase I. Arrows in FIGs. 2B and 2D indicate the internalization of the anti-histone H3 antibody into the cells.

[0042] FIGS. 3A-3B depict the exclusive binding specificity of the anti-DNA and anti- histone antibodies through Western blot assay (FIG. 3A) and dot blot DNA binding assay (FIG. 3B)

[0043] FIGS. 4A-4D depict cytotoxicity of anti-nucleosome antibody drug conjugates (ADCs) can be increased by nuclease treatment. Human HER2+ breast cancer cells AU565 were treated with anti -nucleosome antibodies (anti-nucleosome antibody Abeam 3519 or anti- histone antibody H3) + 2ndantibody-drug conjugates with drug conjugates for 3 days (FIG. 4A) or 6 days (FIG. 4B). Human breast cancer cells BT474 were treated with anti-nucleosome antibodies (anti-nucleosome antibody Abeam 3519 or anti-histone antibody H3) + 2ndantibody-drug conjugates with drug conjugates for 3 days (FIG. 4C) or 6 days (FIG. 4D). Cells treated with anti-nucleosome ADCs and DNase I showed a significant stronger cytotoxicity response, in comparison to the cells treated with anti-nucleosome ADCs without DNase I.

[0044] FIGS. 5A-5B depict cytotoxicity of anti-DNA ADCs (FIG. 5A) and anti-histone ADCs (FIG. 5B) with different concentrations / durations of nuclease treatments, indicating that excessive nuclease activity can reduce cytotoxicity of anti-nucleosome ADCs.

[0045] FIGS. 6A-6B depict cytotoxicity of anti-DNA ADCs and anti-histone ADCs with or without DNase I treatments in human HER2+ breast cancer cells (AU565) for 3 days (FIG. 6A) or 6 days (FIG. 6B). AU565 cells comprise a TP53 R175H mutation.

[0046] FIGS. 7A-7B depict cytotoxicity of anti-DNA ADCs and anti-histone ADCs with or without DNase I treatments in human non-small cell lung carcinoma cells (A549) for 3 days (FIG. 7A) or 6 days (FIG. 7B). A549 cells comprise wild type TP53.DETAILED DESCRIPTIONI. Introduction

[0047] The present disclosure provides compositions and methods of internalizing antinucleosome antibodies into a mammalian cell. In particular, the antibodies recognize and bind to nucleosome proteins. The present disclosure also provides that contacting the cell with one or more DNA nucleases can facilitate this internalization. As shown herein, antibodyinternalization can be greatly increased when the cell is further contacted with one or more DNA nucleases. The present disclosure further provides that two or more anti-nucleosome antibodies combining as a complex or cluster can also facilitate the internalization. When the anti-nucleosome antibodies are conjugated with one or more payloads, including without limitation therapeutic agents and / or detectable labels, the anti-nucleosome antibodies can be used to deliver the desired payloads to or into the cells. Therefore, the anti-nucleosome antibodies with drug conjugates or detectable labels are particularly useful for use in diagnostic and therapeutic applications.II. Definitions

[0048] Throughout this disclosure, various quantities, such as amounts, sizes, dimensions, proportions and the like, are presented in a range format. The description of a quantity in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any embodiment. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as all individual numerical values within that range unless the context clearly dictates otherwise. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual values within that range, for example, 1.1, 2, 2.3, 4.62, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, unless the context clearly dictates otherwise.

[0049] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of any embodiment. As used herein, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “includes”, “comprises”, “including” and / or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0050] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items. Additionally, it should be appreciated that items included in a list in the form of “at least one of A, B, and C” can mean (A); (B); (C); (A and B); (B and C); (A and C); or (A, B, and C). Similarly, items listed in the form of “at least one of A, B, or C” can mean (A); (B); (C); (A and B); (B and C); (A and C); or (A, B, and C).

[0051] The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms encompass to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymer.

[0052] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y- carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, z.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0053] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0054] “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refer to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA,GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences.

[0055] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

[0056] The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0057] In the present application, the term “antibody” is used in the broadest sense (unless explicitly noted otherwise), and specifically encompasses, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies containing two light chains and two heavy chains), polyclonal antibodies, multi-specific antibodies (for example, bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single-domain antibodies. The term “monoclonal antibody” generally refers to an antibody obtained from a group of substantially homogeneous antibodies, that is, a cluster in which several antibodies are the same, except for a few natural mutants that may exist. The monoclonal antibody is generally highly specific for a single antigen site. Moreover, unlikeconventional polyclonal antibody preparations (which generally comprise different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. The term “chimeric antibody” generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Generally, the variable region is derived from an antibody (“parent antibody”) in an experimental animal such as a rodent, and the constant region is derived from a human antibody, such that the possibility of causing an adverse immune response in an individual human by the resulting chimeric antibody is reduced as compared with the parental (for example, mouse-derived) antibody. The term “humanized antibody” generally refers to an antibody in which some or all of the amino acids outside the CDR of a non-human antibody (such as a mouse antibody) have been replaced by corresponding amino acids derived from human immunoglobulins. In the CDR, small additions, deletions, insertions, substitutions, or modifications to the amino acids may also be allowed, as long as they still retain the capability of the antibody to bind to a specific antigen. The humanized antibody may optionally comprise at least a portion of a constant region of a human immunoglobulin. The “humanized antibody” reserves the antigen specificity similar to that of the original antibody. The “humanized” form of a non-human (for example, mouse antibody) antibody may minimally comprise a chimeric antibody derived from a non-human immunoglobulin sequence. In some cases, CDR residues in a human immunoglobulin (receptor antibody) may be replaced with CDR residues from a non-human species (donor antibody) (such as a mouse, a rat, a rabbit, or a non-human primate) with the desired properties, affinity, and / or capability. In some cases, FR residues of the human immunoglobulin may be replaced with corresponding non-human residues. In addition, the humanized antibody may comprise an amino acid modification that is not present in the receptor antibody or in the donor antibody. These modifications may be made to further improve the properties such as binding affinity of the antibody. The term “fully human antibody” generally refers to an antibody that is obtained by transferring a human antibodyencoding gene into a genetically engineered antibody gene-deficient animal to allow the animal to express it. All portions of the antibody (comprising the variable and constant regions of the antibody) are encoded by genes originating from humans. The fully human antibody can greatly reduce the side immune effects caused by heterologous antibodies on the human body. Methods for obtaining the fully human antibody in the art may include the phage display technology, the transgenic mouse technology, the ribosome display technology, the RNA-polypeptide technology, etc.

[0058] An antibody can consist of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. An "antibody" functions as a binding protein and is structurally defined as comprising an amino acid sequence from or derived from the framework region of an immunoglobulin encoding gene of an animal producing antibodies.

[0059] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.

[0060] The term "antibody" as used herein includes antibody fragments that retain binding specificity. For example, there are a number of well characterized antibody fragments. Thus, for example, pepsin digests an antibody C-terminal to the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab')2 dimer into an Fab' monomer. The Fab' monomer is essentially an Fab with part of the hinge region (see, Fundamental Immunology, W.E. Paul, ed., Raven Press, N.Y. (1993), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that fragments can be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized using recombinant DNA methodologies.

[0061] Antibodies can include VH-VL dimers, including single chain antibodies (antibodies that exist as a single polypeptide chain), such as single chain Fv antibodies (sFv or scFv) in which a variable heavy and a variable light region are joined together (directly or through a peptide linker) to form a continuous polypeptide. The single chain Fv antibody is a covalentlylinked VH-VL which may be expressed from a nucleic acid including VH- and VL- encoding sequences either joined directly or joined by a peptide-encoding linker (e.g., Huston, et al. Proc. Nat. Acad. Set. USA, 85:5879-5883, 1988). While the VH and VL are connected to each as a single polypeptide chain, the VH and VL domains associate non-covalently. Alternatively, the antibody can be another fragment. Other fragments can also be generated, e.g., using recombinant techniques, as soluble proteins or as fragments obtained from display methods. Antibodies can also include diantibodies and miniantibodies. Antibodies also include heavy chain dimers, such as antibodies from camelids, or antibodies such as nanobodies.

[0062] As disclosed herein, the term “antibody” also includes multi-paratopic antibody (e.g., biparatopic antibody, tri-paratopic antibody) that binds to two or more distinct epitopes of the same antigen. By binding of one antibody, additional epitopes on the target remain exposed allowing for the binding of additional antibodies, leading to crosslinking of the antigen and antibody clustering. In such cases, a plurality of same kind multi-paratopic antibodies (e.g., biparatopic antibodies, tri-paratopic antibodies, etc.) can cluster together through binding to the same antigen. Alternatively, two, three, or more different antibodies (i.e., specific for different binding sites / epitopes) for the same antigen, mixed as an antibody cocktail, also can form a large antibody complex through binding to the same antigen.

[0063] The term “antigen” refers a molecule, moiety, foreign particulate matter, or an allergen that can bind to a specific antibody or T-cell receptor. Antigens can be proteins, peptides (amino acid chains), polysaccharides (chains of simple sugars), lipids, or nucleic acids (e.g., DNA). Antigens exist on normal cells, cancer cells, parasites, viruses, fungi, and bacteria. In some embodiments, the antigen is a nucleosome. In particular embodiments, the antigen is a nucleosome protein, such as histone Hl, histone H2A, histone H2B, histone H3, histone H4, centromere protein A (CENPA), high mobility group protein 14 (HMG14), high mobility group protein 17 (HMG17 or HMGN2), retinoblastoma-binding protein 5 (RBBP5), Holliday junction recognition protein (HJURP), or any derivative, fragment or fusion thereof. In some cases, antibodies are antigen-specific, meaning that an antibody can only react to and bind one specific antigen. In other instances, antibodies may cross-react to bind more than one antigen. The reaction between an antigen and an antibody is called the antigen-antibody reaction.

[0064] Unless specifically stated or obvious from context, as used herein, the term “about” in reference to a number or range of numbers is understood to mean the stated number andnumbers + / - 10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.III. Detailed Description of the Embodiments

[0065] The present disclosure provides a method of internalizing one or more antinucleosome antibodies (which is understood to include nucleosome-binding antibody fragments herein) into a mammalian cell. In some embodiments, the anti-nucleosome antibodies can be antibodies recognizing and binding to a nucleosome protein (such as a nuclear protein, a nuclear protein-binding protein, a DNA binding protein, or a DNA binding proteinbinding protein). In some embodiments, the anti-nucleosome antibodies can also be antibodies recognizing and binding to nucleosome dsDNA. In some embodiments, the anti-nucleosome antibodies can also be a combination of anti-nucleosome protein antibodies and antinucleosome DNA antibodies. In some embodiments, the anti-nucleosome antibodies form an antibody polymer, complex, or cluster, instead of a single antibody, when entering the cell. In some embodiments, the anti-nucleosome antibodies can covalently or non-covalently link to at least one payload.

[0066] The method comprises contacting the cell with the one or more anti-nucleosome antibodies that bind to extracellular nucleosome on the surface of the cell and are internalized into the cell. Alternatively, the method can further comprise contacting the cell with one or more DNA nucleases before, after or at the same time with the one or more anti-nucleosome antibodies disclosed herein. Furthermore, the anti-nucleosome antibodies can be covalently or non-covalently attached to one or more payloads, including but not limited to therapeutic agents and detectable labels. Therefore, the methods disclosed herein have broad applications, such as for diagnostic and therapeutic uses.NUCLEOSOME PROTEIN

[0067] As disclosed herein, the nucleosome protein can be a nuclear protein, a nuclear protein-binding protein, a DNA binding protein, or a DNA binding protein-binding protein. Examples of nucleosome proteins that bind to nucleosome directly or indirectly, include but not limited to, as those described in Squatrito M, Gorrini C, Amati B. Tip60 in DNA damage response and growth control: many tricks in one HAT. Trends Cell Biol. 2006;16(9):433-442, the entire disclosure of which is herein incorporated by reference.

[0068] In some embodiments, the nucleosome protein is a nuclear protein such as histone Hl, histone H2A, histone H2B, histone H3, histone H4, a modified histone protein, and any histone variant. The nuclear proteins form the basic unit of chromatin structure and consists of a protein complex of eight highly conserved core histones (comprising of a pair of each of the histones H2A, H2B, H3, and H4). Around this complex is wrapped approximately 146 base pairs of DNA. Another histone, Hl, acts as a linker and is involved in chromatin compaction. The DNA is wound around consecutive nucleosomes in a structure often said to resemble “beads on a string” and this forms the basic structure of open or euchromatin. In compacted or heterochromatin this string is coiled and super coiled into a closed and complex structure.

[0069] The terms “histones” or “histone proteins” are recognized by the skilled artisan as referring to the predominant class of proteins found in the chromatin of eukaryotic cells. Histones comprise 6 major proteins termed: Hl, H2A, H2B, H3 and H4, which form a structure around which DNA is wound. Two each of the histone classes, H2A, H2B, H3 and H4, the so- called core histones, assemble to form one octameric nucleosome core particle by wrapping 146 base pairs of DNA around the protein spool in 1.65 left-handed super-helical turn. The linker histone, Hl, binds the nucleosome, as well as the entry and exit sites of the DNA, thus locking the DNA into place and allowing the formation of higher order structure. The most basic overall structure is the 10 nm fiber or beads on a string conformation. This structure entails the wrapping of DNA around nucleosomes with approximately 50 base pairs of DNA spaced between each nucleosome. See, e.g., Chromatin: Structure and Function, A. Wolffe (1998), Academic Press, London, for a review. Histones are highly conserved proteins in eukaryotes, and the sequences of histones from many species are known in the art and are available from publicly accessible databases such as GenBank. Examples of representative GenBank accession numbers for histone sequences include: AAC61625, CAA47464, AAA63187, P62807, P62805, among many others that may be obtained through GenBank.

[0070] Histone variants are proteins that substitute for the core canonical histones (H3, H4, H2A, H2B) and the linker histone (Hl) in nucleosomes in eukaryotes and often confer specific structural and functional features. Histone variants include, but not limited to, centromere protein A (CENPA), H3.3, H3.1, H3.2, TS H3.4, H3.5, H3.Y, H4.V, H2A.X, H2A.Z, H2A.B, H2A, L, H2A.P, H2A.W, H2B.1, H2B.Z, H2B.E. and any derivative thereof.

[0071] In some embodiments, the nucleosome protein is a nuclear protein-binding protein or a protein that binds to a nuclear protein. The nuclear protein-binding protein can be any proteinthat is bound to a nuclear protein. Unlimited examples of nuclear protein-binding proteins include a histone modifying enzyme, a histone acetyltranferase, a histone deacetylase, a histone lysine methyl-transferase, a histone lysine demethylase, a histone acetyltransferase (HAT) (such as Gcn5-related N-acetyltransferases (GNATs), MYST HATs, p300 / CBP, nuclear receptor coactivators (e.g., ACTR / SRC-1), TAFII250, TFIIIC, Rttl09, and CLOCK), and any derivative thereof. Examples of histone modifying enzymes, include but not limited to, as those described in Loscalzo J, Handy DE. Epigenetic modifications: basic mechanisms and role in cardiovascular disease. Pulm Circ. 2014;4(2): 169-174, the entire disclosure of which is herein incorporated by reference.

[0072] In some embodiments, the nucleosome protein is a DNA binding protein. The DNA binding protein includes, but not limited to, a transcription factor (such as achaete-scute complex homolog 2 (ASCL2)), a DNA ligase, a DNA methyltransferase (such as myeloid / lymphoid or mixed-lineage leukemia 1 (MLL1), MLL3), nucleophosmin, nucleolin, Ku70, Ku80, centromere protein A (CENPA), high mobility group protein 14 (HMG14), high mobility group protein 17 (HMG17 or HMGN2), retinoblastoma-binding protein 5 (RBBP5), Holliday junction recognition protein (HJURP). and any derivative thereof.

[0073] In some embodiments, the nucleosome protein is a protein that binds to a DNA binding protein or a nuclear protein binding protein. The DNA / nuclear protein binding proteinbinding protein includes but not limited to, a transcription factor activator (such as nuclear receptor co-activator 6 (NCOA6)), a transcription factor repressor, a DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku70, Ku80, and any derivative thereof. Examples of transcription activators and suppressors which indirectly bind to chromatin, include but not limited to, as those described in Henley, M.J., Koehler, A.N. Advances in targeting ‘undruggable’ transcription factors with small molecules. Nat Rev Drug Discov 20, 669-688 (2021), the entire disclosure of which is herein incorporated by reference.ANTI-NUCLEOSOME ANTIBODY

[0074] As used herein, an “anti-nucleosome antibody” or “nucleosome-binding antibody” refers to an antibody or antibody fragment that can recognize and bind to a nucleosome protein, a polypeptide / epitope / site / fragment of a nucleosome protein, or a nucleosome DNA. In some instances, the anti-nucleosome antibodies recognize and bind to nucleosome DNA. In other instances, the anti-nucleosome antibodies recognize and bind to a nucleosome protein. As disclosed above, the nucleosome protein can be a nuclear protein, a nuclear protein-bindingprotein, a DNA binding protein, or a DNA binding protein-binding protein. In yet other instances, the anti-nucleosome antibodies recognize and bind to both nucleosome DNA and proteins. In some embodiments, the anti-nucleosome antibodies recognize and bind to extracellular nucleosome on the surface of a cell. In some embodiments, the anti -nucleosome antibodies recognize and bind to free nucleosome in the vicinity around a cell.

[0075] The anti-nucleosome antibody disclosed herein can recognize and bind to a nucleosome protein. In some embodiments, the antibody recognizes and binds to a nuclear protein, such as histone Hl, histone H2A, histone H2B, histone H3, histone H4, or a histone variant. In some embodiments, the antibody recognizes and binds to a nuclear protein-binding protein, such as RBBP5, GNAT, MYST, HAT, p300 / CBP, ACTR / SRC-1, TAFII250, TFIIIC, Rttl09, CLOCK, HJURP, or any derivative thereof. In some embodiments, the antibody recognizes and binds to a DNA binding protein, such as a transcription factor, ASCL2, a DNA ligase, a DNA methyltransferase, MLL1, MLL3, HMG14, HMG17, or any derivative thereof. In some embodiments, the antibody recognizes and binds to a DNA binding protein-binding protein, such as a transcription factor activator, NCOA6, a transcription factor repressor, a DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku70, Ku80, or any derivative thereof.

[0076] As disclosed herein, to facilitate internalization, a plurality of anti-nucleosome antibodies can form an antibody polymer, complex, or cluster, instead of a single antibody, when entering the cell. Such antibody polymer, complex, or cluster can be made by various ways, such as engineering antibodies to form a polymer such as a dimer, a trimer, a tetramer, a pentamer, or a polymer with any desired number of antibodies; coating antibodies on a carrier such as a gold nanoparticle, using multi-paratopic antibodies which bind to distinct epitopes of the same DNA, and / or forming antibody cocktails.

[0077] As disclosed herein, the anti-nucleosome antibody can covalently or non-covalently link to at least one payload. The payload can be a small molecule, peptide, protein, nucleic acid, toxin, therapeutic agent, diagnostic agent, drug, chemotherapeutic agent, liposome, nanoparticle, dendrimer, detectable label, or any derivative, fragment, or combination thereof. In some embodiments, the payload is directly conjugated to the anti-nucleosome antibody. In some embodiments, the anti-nucleosome antibody is bound by a secondary antibody, and the payload is conjugated to the secondary antibody. In some embodiments, the one or morepayloads are bound to both the anti-nucleosome antibodies and the secondary antibody. Some exemplary anti-nucleosome ADCs / ADC complexes are shown in FIG. IF.

[0078] In some embodiments, multiple anti-nucleosome antibodies can bind on one carrier such as a nanoparticle to form an antibody complex / cluster facilitating internalization (FIG. IF, anti-nucleosome ADC conjugated on nanoparticle). As disclosed herein, the nanoparticle can be a polymer-based, non-polymeric, or lipid-based nanoparticle. Polymer-based nanoparticles include dendrimers, nanoparticles, micelles, nanogels, protein nanoparticles, and drug conjugates. Non-polymeric nanoparticles include carbon nanotubes, nanodiamonds, metallic nanoparticles, quantum dots, and silica-based nanoparticles. Lipid-based nanoparticles can be divided into liposomes and solid lipid nanoparticles. (Yetisgin AA, Cetinel S, Zuvin M, Kosar A, Kutlu O. Therapeutic Nanoparticles and Their Targeted Delivery Applications. Molecules. 2020;25(9):2193.) In some embodiments, the nanoparticle is a gold nanoparticle. In some embodiments, 2, 3, 4, 5, 6, 7, 8, or more anti-nucleosome antibodies are coated on a gold nanoparticle. The synthesis of spherical gold nanoparticles surface- functionalized with an antibody drug conjugate (ADC) can be referenced from Cruz E, Kayser V. Synthesis and Enhanced Cellular Uptake In Vitro of Anti-HER2 Multifunctional Gold Nanoparticles. Cancers (Basel). 2019; 11(6):870. In some embodiments, same kind of antinucleosome antibodies can be coated on one carrier. In some embodiments, different antinucleosome antibodies can be coated on the same carrier.

[0079] In some embodiments, the anti-nucleosome antibody is a multi-paratopic antibody binding to two or more distinct sites within one nucleosome fragment (either nucleosome DNA or nucleosome protein). In such cases, the multi-paratopic antibodies bind to same or different nucleosome fragments (either nucleosome DNA or nucleosome proteins) and form an antibody cluster promoting internalization. In some cases, the anti-nucleosome antibody is a biparatopic antibody. In some cases, the anti-nucleosome antibody is a tri-paratopic antibody. Some exemplary bi-paratopic anti-nucleosome ADCs or ADC complexes are demonstrated in FIG. IF

[0080] The compositions and methods disclosed herein can also be used to internalize 2, 3, 4, 5, 6, 7, 8, or more anti-nucleosome antibodies. In some instances, the multiple antinucleosome antibodies entering the cell are same kind of anti-nucleosome antibody recognizing / binding same nucleosome epitope / site / fragment. In other instances, the multiple anti-nucleosome antibodies entering the cell are different anti-nucleosome antibodies. Theterm “different anti-nucleosome antibodies” refers to a group of anti-nucleosome antibodies that bind to different nucleosome epitopes / sites / fragments. In some instances, multiple different anti-nucleosome antibodies bind to different nucleosome DNA epitopes / sites / fragments. In other instances, multiple different anti-nucleosome antibodies bind to different nucleosome proteins or different nucleosome protein epitopes / sites / fragments. In yet other instances, some different anti-nucleosome antibodies bind to different DNA and protein epitopes / sites / fragments when entering the cell. In some cases, the multiple antinucleosome antibodies entering the cell are a combination of same and different antinucleosome antibodies.

[0081] As disclosed herein, multiple anti-nucleosome antibodies can form any format of a cluster / complex when binding to extracellular nucleosomes on the cell surface and entering the cell. In some instances, the multiple anti-nucleosome antibodies can form a polymer, such as a dimer, a trimer, a tetramer, a pentamer, or a polymer with any desired number of antibodies, when entering the cell. In other instances, the multiple anti-nucleosome antibodies can bind to 2ndantibodies when entering the cell. In yet other instances, the multiple anti -nucleosome antibodies can be coated on a carrier when entering the cell. In yet other instances, the multiple anti-nucleosome antibodies can crosslink each other when entering the cell. In yet other instances, the multiple anti-nucleosome antibodies can be mixed as an antibody cocktail when entering the cell.

[0082] A variety of anti-nucleosome antibodies are known and in some embodiments are used in the compositions and methods described herein. Examples of commercially-available anti-nucleosome DNA antibodies include, but are not limited to antibody 121-3 (Abeam), antibody 3519 DNA (Abeam), antibody SPM603 (Abeam), antibody DSD958 (Abeam), antibody BV16-13 (Millipore), antibody AE-2 (Millipore), antibody 4565 (NeoBio), antibody TNT-3 (Millipore), antibody 16-19 (Millipore), and antibody F7-26 (Millipore). Examples of commercially-available anti-nucleosome protein antibodies include, but are not limited to antihistone H2A antibody (CL5039, Abeam), anti-histone H2AX antibody (JBW301, SigmaAldrich), anti-histone H2B antibody (52484, Abeam), anti-histone H3 antibody (1B1B2, Abeam), and anti-histone H4 antibody (17036, Abeam).NUCLEASE

[0083] As disclosed herein, the method of anti-nucleosome antibody internalization can further include contacting the target cell with one or more agents that break extracellularnucleosome into smaller pieces. Without being bound by theory, smaller DNA fragments may promote internalization of the anti-nucleosome antibody or fragment into the cell. In some embodiments, such agents comprise one or more nucleases. The terms “nuclease”, “nucleodepolymerase” and “polynucleotidase” are used interchangeably herein to refer to an enzyme capable of cleaving the phosphodiester bonds between nucleotides of nucleic acids. In some embodiments, the nuclease is a mammalian nuclease. In some embodiments, the nuclease is a human nuclease. In some embodiments, the nuclease is a recombinant human nuclease. In some embodiments, the nuclease is a DNA nuclease. In some instances, the DNA nuclease is an exonuclease. In other instances, the DNA nuclease is an endonuclease. In yet other instances, the DNA nuclease is an exo-endonuclease which displays both endo- and exonuclease functions. As disclosed herein, the DNA nuclease has DNA cleaving activity but may also be able to cleave RNA.

[0084] In some embodiments, the nuclease is an endonuclease selected from the group consisting of a deoxyribonuclease (DNase), a serratia marcescens nuclease (Benzonase), a micrococcal nuclease (MNase), a Type I restriction enzyme, a Type II restriction enzyme, a Type III restriction enzyme, a Type IV restriction enzyme, a Type V restriction enzyme, Nuclease SI, Nuclease Pl, a sequence specific endonuclease, a sequence non-specific endonuclease, and any derivative, fragment or fusion thereof.

[0085] In some embodiments, the nuclease is a DNase I. DNase I is an endonuclease of the DNase family coded by the gene DNASE1. DNase I cleaves DNA preferentially at phosphodiester linkages adjacent to a pyrimidine nucleotide, yielding 5'-phosphate-terminated polynucleotides with a free hydroxyl group on position 3', on average producing tetranucleotides. It acts on ssDNA, ds DNA, and chromatin. In some embodiments, the DNase l is a recombinant human DNase I. In some embodiments, the DNase l is a Domase alfa (brand name Pulmozyme), an FDA approved drug for cystic fibrosis treatment.

[0086] In some embodiments, the nuclease is a serratia marcescens nuclease (Benzonase). Serratia marcescens nuclease (or serratia nuclease) is a DNA / RNA non-specific endonuclease that hydrolyzes both double- and single-stranded substrate DNA or RNA to 5'- phosphomononucleotide and 5'-phosphooligonucleotide end-products. Commercially available serratia marcescens nucleases include, but not limited to, Benzonase, Basemuncher, Benzo Nuclease, Benz-Neburase, Decontaminase, Denarase, Dr. Nuclease, GENIUS Nuclease, Pierce Universal Nuclease, and TurboNuclease.

[0087] In some embodiments, the nuclease is a micrococcal nuclease (MNase). Micrococcal nuclease is an endo-exonuclease that preferentially digests single-stranded nucleic acids. The rate of cleavage is 30 times greater at the 5' side of A or T than at G or C and results in the production of mononucleotides and oligonucleotides with terminal 3 '-phosphates. The enzyme is also active against double-stranded DNA and RNA and all sequences will be ultimately cleaved.

[0088] In some embodiments, the nuclease is a restriction enzyme (restriction endonuclease, REase). Restriction enzyme is an endonuclease that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. As disclosed herein, the restriction enzyme can be a Type I restriction enzyme, a Type II restriction enzyme, a Type III restriction enzyme, a Type IV restriction enzyme, or a Type V restriction enzyme. In some instances, the restriction enzyme cuts their DNA substrate at their recognition site. In other instances, the recognition and cleavage sites of the restriction enzyme are separate from one another.

[0089] In some embodiments, the nuclease is a nuclease SI or nuclease Pl. Nuclease SI derived from Aspergillus oryzae, and nuclease Pl derived from Penicillium cilrimim. are nonsequence specific endonuclease enzyme that splits single-stranded DNA (ssDNA) and RNA into oligo- or mononucleotides. In some embodiments, nuclease SI or nuclease Pl also can introduce single-stranded breaks in double-stranded DNA or RNA, or DNA-RNA hybrids. In some embodiments, nuclease SI or nuclease Pl hydrolyses single stranded region in duplex DNA such as loops or gaps. In some embodiments, nuclease SI or nuclease Pl cleaves a strand opposite a nick on the complementary strand.

[0090] In some embodiments, the nuclease is a sequence specific endonuclease. In some embodiments, the nuclease is a sequence non-specific endonuclease. In some embodiments, the nuclease is any derivative, fragment, or fusion of the enzyme disclosed herein.

[0091] In some instances, the nuclease is a ribonuclease targeting on RNA. In other instances, the nuclease is a deoxyribonuclease targeting on DNA. In yet other instances, the nuclease targets on both RNA and DNA, such as Benzonase. In some instances, the nuclease is a single stranded DNA nuclease, such as MNase and nuclease Sl / Pl preferentially targeting ssDNA. In other instances, the nuclease is a double stranded DNA nuclease such as most restriction enzymes preferentially targeting dsDNA. In yet other instances, the nuclease targets both ssDNA and dsDNA.

[0092] In some embodiments, the method disclosed herein comprises contacting the cell with one nuclease (e.g., a DNA nuclease). In some embodiments, the method disclosed herein comprises contacting the cell with two, three, four, five, six, seven, eight or more different nucleases. In some instances, the one or more nucleases (e.g., DNA nucleases) and the antinucleosome antibodies are contacted to the cell at the same time. In other instances, the one or more nucleases (e.g., DNA nucleases) are contacted to the cell before the anti-nucleosome antibodies. In yet other instances, the one or more nucleases (e.g., DNA nucleases) are contacted to the cell after the anti-nucleosome antibodies.

[0093] In some embodiments, the one or more DNA nucleases include, but not limited to, DNase I, or Benzonase, or restriction enzymes. In some instances, the nuclease is DNase I. In other instances, the nuclease is Benzonase.PAYLOAD

[0094] As disclosed herein, the method of internalizing anti-nucleosome antibodies can be used to deliver desired payload, linked to the antibody, to the target cells, including without limitation therapeutic agents and / or detectable labels. In some embodiments, the antinucleosome antibody is attached to at least one payload. In some embodiments, the antinucleosome antibody is bound by a secondary antibody, wherein the secondary antibody is attached to at least one payload. Such attachments can be covalent, non-covalent, or both. In some instances, the anti-nucleosome antibody or the secondary antibody is covalently linked to the payload. In some instances, the anti-nucleosome antibody or the secondary antibody is non-covalently linked to the payload. For example, in some embodiments, the anti-nucleosome antibody or the secondary antibody is attached to a biotin moiety, and the at least one payload is attached to a streptavidin, thereby the antibody and the payload is non-covalently linked. The anti-nucleosome antibody, covalently or non-covalently linked to at least one payload, can specifically target a cell of interest and deliver one or more payloads to the cell. In some embodiments, the payload and the anti-nucleosome antibody are produced as a translational fusion. In some embodiments, the anti-nucleosome antibody is prepared and then attached to the payload via a chemical linker.

[0095] As noted, the method provided herein can be used to deliver one or more payloads to the target cell. In some embodiments, one anti-nucleosome antibody can deliver one, two, three, four, five, six, seven, eight or more identical or different payloads into the cell. In some instances, one anti-nucleosome antibody delivers multiple copies of a payload molecule intothe cell. In other instances, one anti-nucleosome antibody delivers multiple structurally different payloads into the cell. As used herein, a payload can be any desired molecule, complex, or other entity that can be attached to the anti-nucleosome antibody. The binding of one or more payloads to the anti-nucleosome antibody will not impact the binding affinity of the antibody to the target DNA.

[0096] Any useful and desired payload can be delivered using the method provided herein. Such flexibility allows the anti-nucleosome antibodies to be used in multiple applications, such as diagnostics, prognostics, or theranostics. The term “theranostics” refers to therapy-related diagnostics, including without limitation using diagnostic information to predict or monitor drug response.

[0097] In some embodiments, the at least one payload comprises a small molecule, peptide, protein, nucleic acid, toxin, chemotherapeutic agent, liposome, nanoparticle, dendrimer, detectable label, or any useful combination thereof. In some embodiments, the at least one payload can be carried by drug carrying particles such as (A) Lipid-based nanocarriers; (B) Inorganic nanoparticles; (C) Polymeric nanoparticles for examples but not limited to as in Lobo, G.C.N.B.et al. Pharmaceutics 2021, 13, 1167. As a non-limiting example, the small molecule could be a therapeutic agent such as a drug that is specifically delivered to a cell harboring a certain mutation using the anti-nucleosome antibody, such a tumor cell. Such an application may be intended to provide a therapeutic effect.

[0098] Examples of therapeutic agents (also refer as therapeutic drugs) that may be attached as payload to the anti-nucleosome antibodies provided herein include, but are not limited to, antitumor agents, antineoplastic agents, prodrugs, lysosome destabilizing agents (e.g., chloroquine), alkylating agents, alkaloids, allosteric inhibitors, antifolics, anti-inflammatory agents, antibiotics, antibacterials, antifungals, antifibrotic agents, anti-infective agents, anti- parasitic agents, antiviral agents, antimycobacterial agents, antineoplastic agents, antiprotozoal agents, antiviral agents, drugs, bioactive peptides, steroid hormones, nucleic acids, photosensitizer substances, radio-pharmaceuticals, antiprion agents, and combinations thereof.

[0099] For example, the therapeutic agent (or therapeutic drug) may be an antitumor agent selected from the group consisting of an aromatase inhibitor; an anti-estrogen; an antiandrogen; a gonadorelin agonist; a topoisomerase I inhibitor; a topoisomerase II inhibitor; a microtubule active agent (e.g., a microtubule inhibitor); an alkylating agent; a retinoid, a carotenoid, or a tocopherol; a cyclooxygenase inhibitor; an MMP inhibitor; a mTOR inhibitor;an antimetabolite; a platin compound; a methionine aminopeptidase inhibitor; a bisphosphonate; an antiproliferative antibody; a heparanase inhibitor; an inhibitor of Ras oncogenic isoforms; a telomerase inhibitor; a proteasome inhibitor; a Flt-3 inhibitor; an Hsp90 inhibitor; a kinesin spindle protein inhibitor; a MEK inhibitor; a PARP inhibitor, a Tyrosine kinase inhibitor, a PI3K inhibitor, an AKT inhibitor, an EGFR inhibitor, an antitumor antibiotic; a nitrosourea, a compound targeting / decreasing protein or lipid kinase activity, a compound targeting / decreasing protein or lipid phosphatase activity, any further anti- angiogenic compound, and combinations thereof.

[0100] Specific examples of antitumor agents include, but are not limited to, azacitidine, axathioprine, bevacizumab, bleomycin, capecitabine, carboplatin, chlorabucil, cisplatin, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, etoposide, fenretinide, fluorouracil, gemcitabine, herceptin, idarubicin, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, tafluposide, teniposide, tioguanine, retinoic acid, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, receptor tyrosine kinase inhibitors, and combinations thereof. Additional examples of antitumor and other therapeutic agents are known in the art.

[0101] In some embodiments, the antitumor agent is a tubulin inhibitor. The terms “tubulin inhibitor”, “microtubule inhibitor” and “mitotic inhibitor” are used interchangeably herein to refer to a drug that inhibits mitosis, or cell division, and is used in treating cancer and other diseases. Specific examples of tubule inhibitors include, but are not limited to, monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), maytansine, maytansinoid, mertansine (emtansine, DM1), ravtansine (soravtansine, DM4), tubulysin, halichondrin (eribulin), cryptophycin, EG5 inhibitor, and any derivative thereof. MMAE and MMAF, both derived from Dolastatin 10, are antimitotic agents which inhibit cell division by blocking the polymerisation of tubulin. MMAE is more hydrophobic than MMAF. MMAF with a charged C-terminal phenylalanine that attenuates its cytotoxic activity compared to its uncharged counterpart, MMAE. Maytansine such as DM1 and DM4, blocks the polymerization of tubulin dimers by inhibiting the formation of mature microtubules. These tubulin inhibitors are the common payloads used in clinical ADC drugs. For examples, MMAF is part of the approved drug belantamab mafodotin in multiple myeloma and some experimental anti-cancer antibodydrug conjugates (ADCs) such as vorsetuzumab mafodotin and SGN-CD19A. MMAE is another antimitotic auristatin that often conjugates to a monoclonal antibody (MAb), such as Brentuximab (cAClO), Glembatumumab (CR011, CDX-011), AGS67E, Sofituzumab,Polatuzumab, Enfortumab, Pinatuzumab, Lifastuzumab, Brentuximab, Glembatumumab, Tisotumab, Indusatumab. Any tubule inhibitor and other ADC payload listed in Zhijia Wang, Hanxuan Li, Lantu Gou, Wei Li, Yuxi Wang, Antibody-drug conjugates: Recent advances in payloads, Acta Pharmaceutica Sinica B, Volume 13, Issue 10, 2023 Pages 4025-4059, can be linked to an anti-nucleosome antibody as disclosed herein.

[0102] In some embodiments, the antitumor agent is a DNA inhibitor. DNA inhibitors act on the whole cell cycle by destroying DNA through double-strand breakage, alkylation, chimerism, crosslinking, causing cytotoxic effects, and having therapeutic effect on solid tumors. Specific examples of DNA inhibitors include, but are not limited to, alkylator, duocarmycin, duocarmycin DM, calicheamicin, pyrrolobenzodiazepine (PDB), enediyne, uncialamycin, topoisomerase inhibitor, topotecan, camptothecin (CPT), exatecan, and any derivative thereof.

[0103] In some embodiments, the antitumor agent is an RNA inhibitor. RNA inhibitors are small molecule agents that specifically target RNA to kill both dividing and dormant tumor cells. RNA inhibitors can be used as ADC payloads effective in both fast and slow- proliferating cells and against tumor drug resistance and tumor recurrence. Specific examples of RNA inhibitors include, but are not limited to, RNA splicing inhibitor, RNA polymerase II inhibitor, thailanstatin, amatoxin, and any derivative thereof.

[0104] In other embodiments, the method may be used to detect the diseased cells, such as cancer cells. In such cases, detectable labels may be desired payload. In some embodiments, the detectable label comprises at least one magnetic label, fluorescent moiety, enzyme, light emitting particle, chemiluminescent probe, metal particle, non-metal colloidal particle, polymeric dye particle, pigment molecule, electrochemically active species, semiconductor nanocrystal, nanoparticle, quantum dot, gold particles, fluorophore, or radioactive label.

[0105] In still other embodiments, payload may be used for both detection for diagnostic purposes and simultaneously for therapeutic purposes. As a non-limiting example, a radioactive label could be used to detect and / or kill target cells.

[0106] In some cases, the payload may be attached to the anti-nucleosome antibody covalently, including without limitation direct conjugation to the antibody, via a linker entity, or both. In other cases, the payload may be attached to the anti-nucleosome antibody non- covalently. In a non-limiting example, the anti-nucleosome antibody may be conjugated to a biotin moiety, and the payload could be attached to a streptavidin. In this example, the biotin-streptavidin bond would provide the non-covalent attachment between the anti-nucleosome antibody and the payload. In another example of non-covalent binding between the antinucleosome antibody and the payload, the payload is conjugated to a second antibody, and the second antibody binds to the anti-nucleosome antibody. In yet other cases, e.g., in the case of multiple payloads linked to the anti-nucleosome antibodies, the payloads may be attached both covalently and non-covalently.LINKER

[0107] As disclosed herein, the payload may be linked to the anti-nucleosome antibody via a linker.

[0108] In some instances, the linker is a non-cleavable linker. The term “non-cleavable linker” refers to a linker that do not have a designated weak point in its structure that can lead to cleavage by proteases, hydrolases or chemically by pH changes. In some cases, the non- cleavable linker is a maleimide alkane linker, a maleimide cyclohexane (MCC) linker, or any derivative, fragment, or fusion thereof (see, McCombs JR, Owen SC. Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry. AAPS J. 2015; 17(2):339-351 , for a more detailed description of ADC linker selections). In some cases, the non-cleavable linker includes a flexible peptide linker such as a GS linker or a proline rich rigid linker. In some embodiments, the non-cleavable linker is a GS flexible linker with a sequence of GGGGS (SEQ ID NO: 1), GGGGSGGGGS (SEQ ID NO: 2) or GGGGSGGGGSGGGGS (SEQ ID NO: 3). In some embodiments, the non-cleavable linker is a proline rich rigid linker with a sequence of PAPAPPAPAP (SEQ ID NO: 4).

[0109] In other instances, the linker is a cleavable linker. In such instances, the linker is cleaved following contacting the cell with the antibody, thereby releasing the payload on or in the cell. In some embodiments, the cleavable linker is a hydrazone linker, a cathepsin B- responsive linker, a disulfide linker, a pyrophosphate diester linker, or any derivative, fragment, or fusion thereof. (See, Tsuchikama K, An Z. Antibody-drug conjugates: recent advances in conjugation and linker chemistries. Protein Cell. 2018;9(l):33-46.) In some embodiments, the cleavable linker includes, but not limited to, a protease-sensitive linker, a pH-sensitive linker, a radiation-sensitive linker, a glutathione-sensitive linker, a disulfide linker, and a combination thereof. In some instances, the cleavable linker is a Valine-Citrulline (Val-Cit) linker sensitive to cathepsin B. In such instances, the linker between the payload (such as MMAF or MMAE) and the anti-nucleosome antibody is stable in extracellular fluid but is cleaved by cathepsin Bonce the antibody-payload conjugate has entered a target cell (such as a cancer cell), thus releasing the payload inside of the cell. In some embodiments, the cleavable linker is a protease-sensitive linker comprising a sequence of a sortase recognition motif (e.g., LPXTG (SEQ ID NO: 5)). In a particular embodiment, the anti-nucleosome antibody is designed to comprise a sortase recognition sequence (LPETG, SEQ ID NO: 6) for site-specific payload conjugation. In this example, a small molecule (e.g., a tubulin polymerization inhibitor such as a monomethyl auristatin F (MMAF), a monomethylauristatin E (MMAE)) or a maytansine) is modified by addition of a pentaglycine peptide to make them suitable substrates for sortase A-mediated drug conjugation to the anti-nucleosome antibodies.TARGET CELLS

[0110] As described herein, the method provided herein can be used in various applications. In non-limiting examples, the anti-nucleosome antibody can be used to label the target cell or kill the target cell as desired. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is in vitro. In some embodiments, the cell is in vivo.[OHl] In some embodiments, the target cell comprises a diseased cell. The diseased cell can be within a tissue, such as a solid tumor, or it may be circulating within a body, including without limitation a human body. In various embodiments, the disease comprises a cancer, a premalignant condition, an inflammatory disease, an immune disease, an autoimmune disease or disorder, a cardiovascular disease or disorder, a neurological disease or disorder, an infectious disease or pain. Cancer cells display a mutator phenotype and may harbor thousands of mutations.

[0112] Any cancer (e.g., human cancer) cell of interest can be the target cell. In some embodiments, the cancer comprises bladder cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, ovarian cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, lymphoma, pancreatic cancer, prostate cancer or thyroid cancer. In some embodiments, the cancer comprises an acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical carcinoma; AIDS-related cancer; AIDS-related lymphoma; anal cancer; appendix cancer; astrocytomas; atypical teratoid / rhabdoid tumor; basal cell carcinoma; bladder cancer; brain stem glioma; brain tumor (e.g., brain stem glioma, central nervous system atypical teratoid / rhabdoid tumor, central nervous system embryonal tumors, astrocytomas, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma,medulloepithelioma, pineal parenchymal tumors of intermediate differentiation, supratentorial primitive neuroectodermal tumors and pineoblastoma); breast cancer; bronchial tumors; Burkitt lymphoma; cancer of unknown primary (CUP) site; carcinoid tumor; carcinoma of unknown primary site; central nervous system atypical teratoid / rhabdoid tumor; central nervous system embryonal tumors; cervical cancer; childhood cancers; chordoma; chronic lymphocytic leukemia; chronic myelogenous leukemia; chronic myeloproliferative disorders; colon cancer; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; endocrine pancreas islet cell tumors; endometrial cancer; ependymoblastoma; ependymoma; esophageal cancer; esthesioneuroblastoma; Ewing sarcoma; extracranial germ cell tumor; extragonadal germ cell tumor; extrahepatic bile duct cancer; gallbladder cancer; gastric (stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal cell tumor; gastrointestinal stromal tumor (GIST); gestational trophoblastic tumor; glioma; hairy cell leukemia; head and neck cancer; heart cancer; Hodgkin lymphoma; hypopharyngeal cancer; intraocular melanoma; islet cell tumors; Kaposi sarcoma; kidney cancer; Langerhans cell histiocytosis; laryngeal cancer; lip cancer; liver cancer; lung cancer; malignant fibrous histiocytoma bone cancer; medulloblastoma; medulloepithelioma; melanoma; Merkel cell carcinoma; Merkel cell skin carcinoma; mesothelioma; metastatic squamous neck cancer with occult primary; mouth cancer; multiple endocrine neoplasia syndromes; multiple myeloma; multiple myeloma / plasma cell neoplasm; mycosis fungoides; myelodysplastic syndromes; myeloproliferative neoplasms; nasal cavity cancer; nasopharyngeal cancer; neuroblastoma; Non-Hodgkin lymphoma; nonmelanoma skin cancer; non-small cell lung cancer; oral cancer; oral cavity cancer; oropharyngeal cancer; osteosarcoma; other brain and spinal cord tumors; ovarian cancer; ovarian epithelial cancer; ovarian germ cell tumor; ovarian low malignant potential tumor; pancreatic cancer; papillomatosis; paranasal sinus cancer; parathyroid cancer; pelvic cancer; penile cancer; pharyngeal cancer; pineal parenchymal tumors of intermediate differentiation; pineoblastoma; pituitary tumor; plasma cell neoplasm / multiple myeloma; pleuropulmonary blastoma; primary central nervous system (CNS) lymphoma; primary hepatocellular liver cancer; prostate cancer; rectal cancer; renal cancer; renal cell (kidney) cancer; renal cell cancer; respiratory tract cancer; retinoblastoma; rhabdomyosarcoma; salivary gland cancer; Sezary syndrome; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma; squamous neck cancer; stomach (gastric) cancer; supratentorial primitive neuroectodermal tumors; T-cell lymphoma; testicular cancer; throat cancer; thymic carcinoma; thymoma; thyroid cancer; transitional cell cancer; transitional cell cancer of the renal pelvis and ureter; trophoblastic tumor; ureter cancer; urethral cancer; uterine cancer; uterine sarcoma;vaginal cancer; vulvar cancer; Waldenstrom macroglobulinemia; or Wilm’s tumor. In some embodiments, the cancer is a breast cancer.

[0113] In some embodiments, the cancer’s type comprises an acute myeloid leukemia (AML), breast carcinoma, cholangiocarcinoma, colorectal adenocarcinoma, extrahepatic bile duct adenocarcinoma, female genital tract malignancy, gastric adenocarcinoma, gastroesophageal adenocarcinoma, gastrointestinal stromal tumor (GIST), glioblastoma, head and neck squamous carcinoma, leukemia, liver hepatocellular carcinoma, low grade glioma, lung bronchioloalveolar carcinoma (BAC), non-small cell lung cancer (NSCLC), lung small cell cancer (SCLC), lymphoma, male genital tract malignancy, malignant solitary fibrous tumor of the pleura (MSFT), melanoma, multiple myeloma, neuroendocrine tumor, nodal diffuse large B-cell lymphoma, non-epithelial ovarian cancer (non-EOC), ovarian surface epithelial carcinoma, pancreatic adenocarcinoma, pituitary carcinomas, oligodendroglioma, prostatic adenocarcinoma, retroperitoneal or peritoneal carcinoma, retroperitoneal or peritoneal sarcoma, small intestinal malignancy, soft tissue tumor, thymic carcinoma, thyroid carcinoma, or uveal melanoma.

[0114] The cancer may be in an individual diagnosed with, suffering from, at risk of developing, or suspected of having cancer. The cancer may be selected from the group comprising bladder urothelial carcinoma, breast invasive carcinoma, colon adenocarcinoma, colorectal adenocarcinoma, oseophageal carcinoma, head and neck squamous cell carcinoma, kidney rental clear cell carcinoma, kidney renal papillar cell carcinoma, liver hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, prostate adenocarcinoma, stomach and esophageal carcinoma, thyroid carcinoma, uterine corpus endometrial carcinoma, and chronic lymphocytic leukemia. In some embodiments, the cancer harbors wild type or a mutation in KRAS, TP53, BRAF, PIK3CA, and / or IDH1.

[0115] In some embodiments, the cell has an aneuploidy and / or DNA repair deficiency. Without intending to limit the scope of the invention, it is believed that due to aneuploidy, mutations or lower expression of DNA repair enzymes, cells have extracellular nucleosome on their surface. In some embodiments, the cell has an aneuploidy. In some embodiments, the cell has abnormal or low efficiency of DNA damage response. In some embodiments, the cell has abnormal or clogged DNA repair pathways. In some embodiments, the cells have mutated or inadequate DNA repair proteins / enzymes such as phosphatidylinositol-3 (PI3) kinases. PI3 kinases include but not limited to ataxia telangiectasia mutated (ATM) kinases, ATM and Rad3related (ATR) kinases, and poly (ADP -ribose) polymerases (PARPs). In some embodiments, the cell comprises a functionally impaired transcription factor or regulator selected from the group consisting of p53, NF-Kb, AP-1, E2F1, and breast cancer-associated protein 1 (BRCA1). In some embodiments, the functional impairment of the transcription factor or regulator is caused by a gene mutation. In some instances, the gene mutation can be a mutation of the transcription factor gene. In other instances, the gene mutation can be a mutation of other DNA repair related genes. In some embodiments, the cell comprises one or more mutations in a DNA repair gene selected from the group consisting of apexl; ddbl; ddb2; erccl;fenl; karpl; ligl; mgmt; mpg; mlhl; msh2; neill; oggl; pcna; pms2; poll; polfl; polH; polK; rev3; trexl; xrccl; xpc; xpf; and xpg. See, e.g., Christmann M, Kaina B. Transcriptional regulation of human DNA repair genes following genotoxic stress: trigger mechanisms, inducible responses and genotoxic adaptation. Nucleic Acids Res. 2013;41(18):8403-8420.

[0116] In particular embodiments, the cell comprises a functionally impaired transcription factor p53. p53, also known as Tumor protein P53, cellular tumor antigen p53, or transformation-related protein 53 (TRP53) is a transcription factor that plays a major role in the regulation of DNA repair, apoptosis and cell cycle progression. p53 is often mutated and / or functionally impaired in cancer cells. As disclosed herein, the functionally impaired p53 can be resulted from a mutation of the TP53 gene, a reduced p53 expression, and / or an inhibited p53 activity. In some embodiments, the functionally impaired p53 results cell aneuploidy. In some embodiments, the functionally impaired p53 is present in an abnormal cell such as a cancer cell or a diseased cell. In some embodiments, the functionally impaired p53 is a gain- of-function mutation that promotes tumor growth and progression. In some embodiments, the gain-of-function mutation of the functionally impaired p53 modulates tumor immune microenvironment, upregulates chemokines, enhances inflammation, creates an immunosuppressive environment within tumors, influences treatment outcomes, and / or links to poorer prognosis. In some embodiments, the functionally impaired p53 is present in a cell with extracellular nucleosome on the cell surface. In some embodiments, the functionally impaired p53 is caused by a missense mutation of the TP53 gene. In some embodiments, the functionally impaired p53 comprises one or more missense mutations in the DNA-binding domain of the protein. In some embodiments, the p53 mutations locate in residues R175, Y220, G245, R248, R249, R270, R273, and / or R282. In particular embodiments, the p53 mutations comprise R175H, Y220C, G245S, R245W, R248Q, R248W, R249S, R270H, R273H, R273C, and / or R282W mutations.METHODSMethod of binding

[0117] Provided herein is a method of binding the anti-nucleosome antibody to extracellular nucleosome on a cell surface comprising contacting the cell with the anti-nucleosome antibody provided herein. In some embodiments, the anti-nucleosome antibody is attached to a cytotoxic payload and binding of the anti-nucleosome antibody kills the cell. In some embodiments, the anti-nucleosome antibody is attached to a detectable payload and the method further comprises detecting binding of the anti-nucleosome antibody to the cell by detecting the detectable payload. In non-limiting examples, the method can be used to detect a presence or level of one or more target cell in a biological specimen, wherein the anti-nucleosome antibody is bound to or internalized within the target cell. The method can be applied in various settings as desired. For example, the contacting can be performed in vivo or in vitro depending on the desired application of the method.Method of imaging

[0118] Further provided herein is a method of imaging at least one cell or tissue, comprising contacting the at least one cell or tissue with the anti-nucleosome antibody as provided herein, and detecting the anti-nucleosome antibody internalized into the at least one cell or tissue. In some embodiments, the anti-nucleosome antibody is administered to a subject prior to the detecting. The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, rats, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed. In some embodiments, the detecting is performed in vitro. As desired, these methods can be combined. For example, the anti-nucleosome antibody can be administered to a subject, and then a sample can be taken from the subject for subsequent analysis in vitro. In some embodiments, the at least one cell or tissue comprises cells displaying mutant or wild type extracellular nucleosome on the surface, wherein anti-nucleosome antibody disclosed herein binds to the mutant or wild type extracellular DNA.

[0119] In some embodiments, the at least one cell or tissue is from a subject suspected of having or being predisposed to a disease or disorder. In non-limiting examples, the disease or disorder may comprise a cancer, a premalignant condition, an inflammatory disease, an immune disease, an autoimmune disease or disorder, a cardiovascular disease or disorder,neurological disease or disorder, infectious disease or pain. In some embodiments, the at least one cell or tissue comprises neoplastic, malignant, tumor, hyperplastic, dysplastic, and / or metastatic cells. In the case of tumor cells, the tumor can be a primary tumor or a metastatic tumor. The tumor can be related to any type of cancer as desired. In some embodiments, the target cells or tissue comprise a bladder cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, ovarian cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, lymphoma, pancreatic cancer, prostate cancer or thyroid cancer cell. The cancer can also comprise other cancers such as provided herein.Method of delivering

[0120] Also provided herein is a method of delivering one or more payloads inside of a cell comprising the extracellular DNA. The method comprises contacting the cell with the antinucleosome antibody directly or indirectly linked to with a payload, such as a therepeutic or cytotoxic drug, wherein the anti-nucleosome antibody binds to the extracellular nucleosome on the cell and internalizes the payload inside of the cell. In some embodiments, the antinucleosome antibody is directly linked to the therapeutic drug, covalently or non-covalently. In some embodiments, the anti-nucleosome antibody is indirectly linked to the payload. For example, the anti-nucleosome antibody is bound by a secondary antibody, wherein the secondary antibody is covalently or non-covalently linked to the payload.

[0121] In some embodiments, the method further comprises contacting the cell with one or more DNA nucleases facilitating drug internalization. The DNA nuclease can be an endonuclease, an exonuclease, or a combination thereof. In some embodiments, the one or more DNA nucleases comprise a single stranded DNA (ssDNA) nuclease, a double-stranded DNA (dsDNA) nuclease, or a combination thereof. In some embodiments, the one or more DNA nucleases and the anti-nucleosome antibodies conjugated with therapeutic drug are contacted to the cell at the same time, optionally wherein the one or more DNA nucleases are attached to the anti-nucleosome antibodies. In some embodiments, the one or more DNA nucleases are contacted to the cell before or after the anti-nucleosome antibodies conjugated with therapeutic drug.

[0122] As described herein, the payload conjugated with the anti-nucleosome antibody can be selected to achieve a desired activity, such as a therapeutic effect. In some embodiments, the payload comprises a small molecule, drug, protein, nucleic acid, toxin, chemotherapeutic agent, or other therapeutic agent, such as described herein. In some embodiments, the payloadcomprises a liposome or nanoparticle. In such cases, the liposome or nanoparticle may carry the desired therapeutic agent inside. The anti-nucleosome antibody and / or payload may be internalized into the target cell. In some embodiments, the target cell is a cancer cell. In some embodiments, the therapeutic drug kills or inhibits growth or division of the cancer cell. In some embodiments, the cancer cell is from a cancer in a subject. As described, the payload may be released in the cell to provide a therapeutic effect, e.g., via cleavage of a linker between the binding portion of the construct and the payload, via proteolytic cleavage of the binding portion, or other mechanism.Method of treatment

[0123] Further provided herein is a method of treating or ameliorating a disease or disorder in a human subject in need thereof, comprising administering a pharmaceutical effective amount of composition comprising an anti-nucleosome antibody to the subject. As disclosed herein, the anti-nucleosome antibody binds extracellular nucleosome on the surface of the diseased cell and is attached to at least one toxic payload such as but not limited to a small molecule drug. Administration of the pharmaceutical composition may result in delivery of the payload to cells comprising the extracellular nucleosome and therefore specifically kill the target cells. In particular embodiments, the composition further comprises one or more DNA nucleases. As disclosed herein, the DNA nuclease can be an endonuclease, an exonuclease, or a combination thereof. In some embodiments, the one or more DNA nucleases and the antinucleosome antibodies with therapeutic payloads are contacted to the cell at the same time, optionally wherein the one or more DNA nucleases are attached to the anti-nucleosome antibodies. In some embodiments, the one or more DNA nucleases are contacted to the cell before or after the anti-nucleosome antibodies with therapeutic payloads.

[0124] As used herein “therapeutically effective amount” refers to an amount of a composition that relieves (to some extent, as judged by a skilled medical practitioner) one or more symptoms of the disease or condition in a mammal. Additionally, by “therapeutically effective amount” of a composition is meant an amount that returns to normal, either partially or completely, physiological or biochemical parameters associated with or causative of a disease or condition. A clinician skilled in the art can determine the therapeutically effective amount of a composition in order to treat or prevent a particular disease condition, or disorder when it is administered, such as intravenously, subcutaneously, intraperitoneally, orally, or through inhalation. The precise amount of the composition required to be therapeuticallyeffective will depend upon numerous factors, e.g., such as the specific activity of the active agent, the delivery device employed, physical characteristics of the agent, purpose for the administration, in addition to many patient specific considerations. But a determination of a therapeutically effective amount is within the skill of an ordinarily skilled clinician upon the appreciation of the disclosure set forth herein.

[0125] The terms “treating,” “treatment,” “therapy,” and “therapeutic treatment” as used herein refer to curative therapy, prophylactic therapy, or preventative therapy. An example of “preventative therapy” is the prevention or lessening the chance of a targeted disease (e.g., cancer or other proliferative disease) or related condition thereto. Those in need of treatment include those already with the disease or condition as well as those prone to have the disease or condition to be prevented. The terms “treating,” “treatment,” “therapy,” and “therapeutic treatment” as used herein also describe the management and care of a mammal for the purpose of combating a disease, or related condition, and includes the administration of a composition to alleviate the symptoms, side effects, or other complications of the disease, condition. Therapeutic treatment for cancer includes, but is not limited to, surgery, chemotherapy, radiation therapy, gene therapy, and immunotherapy.

[0126] As used herein, the term “agent” or “drug” or “therapeutic agent” or “therapeutic drug” refers to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues that are suspected of having therapeutic properties. The agent or drug can be purified, substantially purified or partially purified. An “agent” according to the present invention, also includes a “radiation therapy agent” or a “chemotherapeutic agent.” As used herein, the term “chemotherapeutic agent” refers to an agent with activity against cancer, neoplastic, and / or proliferative diseases, or that has ability to kill cancerous cells directly.

[0127] As used herein, the term “diagnostic agent” refers to any chemical used in the imaging of diseased tissue, such as, e.g., a tumor.

[0128] In some embodiments, the disease or disorder may comprise a cancer, a premalignant condition, an inflammatory disease, an immune disease, an autoimmune disease or disorder, a cardiovascular disease or disorder, neurological disease or disorder, infectious disease or pain. In some embodiments, the target cell comprises a neoplastic, malignant, tumor, hyperplastic, dysplastic, and / or metastatic cell. In particular embodiments, the disease or disorder is a cancer.In some embodiments, the cancer is a bladder cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, ovarian cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, lymphoma, pancreatic cancer, prostate cancer or thyroid cancer. The cancer can also comprise other cancers such as provided herein. In some embodiments, the subject is a human. In some embodiments, the subject has a cancer. In some embodiments, the pharmaceutical effective amount of composition is administered into a cancer cell of the subject.

[0129] In some embodiments, the method further comprises, prior to the administering, determining whether the cell has an aneuploidy, a DNA repair deficiency, and / or a functionally impaired transcription factor or regulator selected from the group consisting of p53, NF-Kb, AP-1, E2F1, and BRCA1. In some instances, the determining step comprises detecting one or more mutations of the transcription factor gene. In other instances, the determining step comprises detecting one or more mutations of other DNA repair related genes. In some embodiments, the determining step comprises detecting one or more mutations in a DNA repair gene selected from the group consisting of apexl; ddbl; ddb2; erccl; fenl; karpl; ligl; mgmt; mpg; mlhl; msh2; neill; oggl; pcna; pms2; poll; polfl; polH; polK; rev3; trexl; xrccl; xpc; xpf; and xpg. In particular embodiments, the method comprises detecting a functionally impaired transcription factor p53 in the cell of the subject. The functionally impaired p53 can be resulted from a mutation of the TP53 gene, a reduced p53 expression, and / or an inhibited p53 activity. In some cases, the mutation of the TP53 gene is an R175H mutation.COMPOSITIONS

[0130] In one aspect, a composition comprises one or more anti-nucleosome antibodies disclosed herein and one or more DNA nucleases. In some embodiments, the one or more antinucleosome antibodies are bound by a secondary antibody. In some embodiments, the one or more anti-nucleosome antibodies are coated on a carrier such as a gold nanoparticle. In some embodiments, at least two anti-nucleosome antibodies are linked together as a polymer such as a dimer, a trimer, a tetramer, or a pentamer, or a polymer with any desired number of antibodies. In some embodiments, at least two anti-nucleosome antibodies are different anti-nucleosome antibodies. In some cases, the different anti-nucleosome antibodies are crosslinked together. Examples of antibody cross-linking can include for example chemical cross-linking, for example but not limited to as in Ueda et a Int J Mol Sci. 2020 Feb; 21(3): 711. In some cases, the different anti-nucleosome antibodies are multi-paratopic antibodies clustering together.

[0131] As provided herein, the composition further comprises a payload covalently or non- covalently linked to the anti-nucleosome antibody. As disclosed herein, the payload can be a small molecule, peptide, protein, nucleic acid, toxin, therapeutic agent, drug, chemotherapeutic agent, liposome, nanoparticle, dendrimer, detectable label, or any derivative, fragment, or combination thereof. In some embodiments, the payload is directly conjugated to the antinucleosome antibody. In some embodiments, the anti-nucleosome antibody is bound by a secondary antibody, and the payload is directly conjugated to the secondary antibody. In some embodiments, multiple payloads are linked to the anti-nucleosome antibody. For example, at least one payload is conjugated to the anti-nucleosome antibody and at least one payload is conjugated to the secondary antibody. In some instances, the anti-nucleosome antibody or the secondary antibody is non-covalently linked to the payload. For example, in some embodiments, the anti-nucleosome antibody or the secondary antibody is attached to a biotin moiety, and the at least one payload is attached to a streptavidin, thereby the antibody and the payload is non-covalently linked.

[0132] As provided herein, a pharmaceutical composition may a therapeutically effective amount of the composition disclosed above, and a pharmaceutically acceptable excipient, carrier, and / or diluent.

[0133] Also provided herein, a pharmaceutical composition comprises a therapeutically effective amount of the anti-nucleosome antibody and at least one therapeutic agent. In some embodiments, the therapeutic agent is selected from the group consisting of an antitumor agent, antineoplastic agent, prodrug, lysosome destabilizing agent (e.g., chloroquine), alkylating agent, alkaloid, allosteric inhibitor, anti-folic, anti-inflammatory agent, antibiotics antibacterial, antifungal, antifibrotic agent, anti-infective agent, anti-parasitic agent, antiviral agent, antimycobacterial agent, antineoplastic agent, antiprotozoal agent, antiviral agent, bioactive peptide, steroid hormone, photosensitizer substance, radio-pharmaceutical, anti-prion agent, and any combination thereof. In some embodiments, the antitumor agent is a tubule inhibitor, a DNA inhibitor, or an RNA inhibitor. In some embodiments, the tubule inhibitor is selected from the group consisting of monomethyl auristatin F (MMAF), monomethyl auri statin E (MMAE), maytansine, maytansinoid, mertansine (emtansine, DM1), ravtansine (soravtansine, DM4), tubulysin, halichondrin (eribulin), cryptophycin, EG5 inhibitor, and any derivative thereof.

[0134] In some embodiments, the pharmaceutical composition further comprises one or more DNA nucleases. In some embodiments, the DNA nuclease can be an endonuclease, an exonuclease, or a combination thereof. In some embodiments, the endonuclease is a deoxyribonuclease (DNase), a serratia marcescens nuclease (Benzonase), a micrococcal nuclease (MNase), a restriction enzyme, nuclease SI, nuclease Pl, a sequence specific endonuclease, or a sequence non-specific endonuclease. In some embodiments, the one or more DNA nucleases comprise a single stranded DNA (ssDNA) nuclease, a double-stranded DNA (dsDNA) nuclease, or a combination thereof.

[0135] The pharmaceutical composition may comprise at least one of a pharmaceutically acceptable excipient, carrier, and / or diluent. It is contemplated that other agents may be used in combination with the pharmaceutical composition to improve the therapeutic efficacy of treatment. These additional agents include chemotherapeutic agents such as small molecule drugs or other biological agents. As desired, such additional agents may target the same biomarker as the anti-nucleosome antibodies. In some embodiments, the additional agent comprises non-targeted therapies. As a non-limiting example, an anti-nucleosome antibody with drug conjugates directed to target cells may be administered concurrently or sequentially with other related therapies (e.g., immunotherapy, CAR-T therapy, other antibody therapy, cellular therapy), and / or traditional chemotherapy, including without limitation alkylating agents, plant alkaloids, antimetabolites, anthracyclines, topoisomerase inhibitors and / or corticosteroids.

[0136] Relatedly, provided herein is a kit comprising at least one reagent for carrying out the methods provided herein, such as those described above. Also provided herein is use of at least one reagent for carrying out the methods. Any useful reagent can be a component of the kit or use. In some embodiments, the at least one reagent comprises the anti-nucleosome antibody, a detection reagent, a secondary detection reagent, a wash buffer, an elution buffer, a solid support, and any combination thereof.Administration

[0137] Administration of the pharmaceutical compositions provided herein can be via any desired and useful route. This includes, but is not limited to parenteral, orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal, or intravenous injection. In some embodiments, the route of administration comprises at least one of intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intracerebral,intravaginal, transdermal, rectal, by inhalation, topical administration, or any useful combination thereof.

[0138] Typically, the pharmaceutical compositions provided herein are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective. The quantity to be administered depends on the subject to be treated. Precise amounts of the anti-nucleosome antibody required to be administered may depend on the judgment of the treating physician or other caregiver.

[0139] The manner of application may be varied widely. Various methods for administration of pharmaceutical compositions comprising protein components are applicable. The dosage of the pharmaceutical composition will depend on the route of administration and can vary according to the size and health of the subject.

[0140] In many instances, it will be desirable to have multiple administrations of at most about or at least about 3, 4, 5, 6, 7, 8, 9, 10 or more administrations. The timing of the administrations may vary over a time course. In some embodiments, the timing of the administrations ranges from 2-day to 12-week intervals, e.g., one to two week intervals. The course of the administrations can be followed by assays to monitor the presence and / or level of the target cells in the patients. The monitoring may be performed as described herein.

[0141] As used herein, “pharmaceutical formulations” include formulations for human and veterinary use with no significant adverse toxicological effect. “Pharmaceutically acceptable formulation” as used herein refers to a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity.

[0142] The phrase “pharmaceutically acceptable” as used herein refer to molecular entities and compositions that do not produce unacceptably adverse, allergic, or other untoward reaction when administered to a subject, e.g., a human in need of treatment for a disease or disorder. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for formulating pharmaceutical active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. The pharmaceutical compositions of the current disclosure are pharmaceutically acceptable compositions.

[0143] The compositions of the disclosure can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, or intraperitoneal routes. Such compositions can be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared. The preparations can also be emulsified.

[0144] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol. The pharmaceutical forms should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0145] Sterile injectable solutions are prepared by incorporating the active ingredients (i.e. anti-nucleosome antibodies provided herein) in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.

[0146] An effective amount of a composition is determined based on the intended goal. The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the pharmaceutical composition calculated to produce the desired responses discussed herein in association with its administration, i.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the result and / or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual.

[0147] Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations can be administered in a variety of dosage forms, such as the type of injectable solutions described above.

[0148] In some embodiments, the pharmaceutical composition provided herein is administered contemporaneously with at least one other therapeutic agent. As used herein, contemporaneous administration indicates that the pharmaceutical composition and alternate treatments may be part of the same treatment regimen for a patient, but the precise timing of such administrations can be optimized. For example, the anti-nucleosome antibody and alternate treatment such as a drug or biologic may be co-administered or administered sequentially. The timing of the administration of the anti-nucleosome antibody and alternate treatment can be offset, e.g., by at least 5 min, 10 min, 15 min, 20 min, 30 min, Ih, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, lOh, l lh, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h, 30h, 36h, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 18 days, 3 weeks, 4 weeks or more. The timing can be determined by the treating physician. In some embodiments, the at least one other therapeutic agent comprises a anti-nucleosome antibody engineered to target an alternate target nucleic acid sequence.IV. Examples

[0149] The following examples, along with the methods described herein are presently representative of preferred embodiments, are provided only as examples, and are not intended as limitations on the scope of the compositions and methods provided herein. Changes therein and other uses which are encompassed within the spirit of the disclosure as defined by the scope of the claims will occur to those skilled in the art.EXAMPLE 1: MATERIALS AND METHODSCancer cell lines

[0150] Human breast cancer cells (AU565 and BT474), and human non-small cell lung carcinoma cells (A549) were purchased from ATCC (Manassas, VA). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. AU565 cells were cultured in Roswell Park Memorial Institute 1640 Medium (RMPI1640) with 10% fetal bovine serum (FBS). BT474 cells were grown in Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM / F-12) with 10% FBS, while A549 cells were maintained in in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% FBS. Media and FBS were purchased from ATCC.Fluorescent imaging

[0151] Cancer cells were seeded onto 18-well glass-bottom chambered coverslips (81817, ibidi) at a density of 4,000 cells per well and incubated for 72 hours. Anti-histone H3 antibody was pre-mixed with an Alexa Fluor 647-labeled anti-mouse IgG 2ndantibody at a 1 :1 molar ratio and incubated at room temperature in the dark for one hour. The antibody complex was then applied to the cells at a final concentration of 1 pg / ml anti-histone H3 antibody, with half of the wells additionally treated with 20 units / ml of DNase I. The cells were cultured for 72 hours at 37°C before imaging. Afterward, the cells were washed with PBS, fixed with 4% formaldehyde in PBS, and permeabilized with 0.1% Triton-XIOO. To stain cellular components, WGA488 (W11261, Invitrogen) was used for the cell membrane, and NucBlue (R37605, Invitrogen) for nuclei / DNA. Images were captured using a confocal fluorescent microscope (FV3000, Olympus).Cytotoxicity assay

[0152] Determination of cytotoxicity of 0.2 pg / ml different anti-nucleosome antibodies + 0.2 pg / ml 2ndADC + 20 units / ml DNase I. Cancer cell lines (AU565, BT474, A549) were plated in 96-well tissue-culture treated optical-bottom white plates (165306, Thermofisher) at a density of 1,500 cells per well and incubated for 72 hours. Each of the anti-nucleosome antibodies or an isotype control antibody (BE0085, Bioxcell) were pre-incubated with an antimouse IgG 2ndantibody (NC9254315, Jackson ImmunoResearch) conjugated in house with MMAF through a Valine-Citrulline (Val-Cit) linker (HY-112786, MedChemExpress). Antibodies were mixed at a 1 : 1 molar ratio and pre-incubated at room temperature for 1 hour. Subsequently, the antibody complex and DNase I were added to the cells at 0.2 pg / ml of antinucleosome antibody + 0.2 pg / ml 2ndADC with or without 20 units / ml DNase I. Cells treated with PBS or 20 units / ml DNase I were used as control. Each condition was tested in triplicates. The cells were then cultured for 3 or 6 days at 37°C before assessing viability using the CellTiter-Glo 2.0 assay (G9242, Promega). Amount of luminescence emitted from each well was measured using multimode microplate reader, (Synergy Hl, BioTek). The percentage of viable cells was calculated using the luminescence readings of treated wells divided to the luminescence reading from control wells.Western blot assay

[0153] Pellets of cancer cells were collected and lysed using RIPA buffer supplemented with protease inhibitors. The lysates were centrifuged to remove insoluble material, and protein concentrations in the supernatants were measured using a BCA protein assay kit. A total of 40pg of protein per sample was loaded onto SDS-PAGE gels for electrophoretic separation. Proteins were then transferred onto nitrocellulose membranes (IB23002, ThermoFisher), blocked with StartingBlock T20 blocking buffer (37543, Thermo Fisher), and incubated overnight at 4°C with 1 pg / ml of either anti-DNA antibody or anti-histone H3 antibody, both prepared in blocking buffer. Following washes, the membranes were treated with an HRP- conjugated secondary antibody (115-035-071, Jackson ImmunoResearch) for 1 hour at room temperature. Membranes were then washed three times with TBS-T (Tris-buffered saline containing 0.1% Tween-20), before protein bands were visualized using enhanced chemiluminescent (ECL) horseradish peroxidase (HRP) substrate (34580, ThermoFisher) and visualized with a chemiluminescence imaging system.Dot blot DNA binding assay

[0154] Gnomic DNA samples were isolated from cancer cells using QIAamp DNA Kits (51304, QIAGEN) and quantified using a NanoDrop spectrophotometer. For dot blot analysis, 10 pL aliquots containing various amounts of gDNA (ranging from 50 ng to 400 ng) were carefully spotted onto a positively charged nylon membrane (77016, ThermoFisher) using a narrow-mouth pipette tip. Membranes were air-dried overnight at room temperature. The dried membranes were blocked by immersion in StartingBlock T20 blocking buffer with gentle agitation for 1 hour at room temperature. Blocked membranes were then incubated with primary antibodies (anti-DNA antibody or anti-histone H3 antibody) at a concentration of 1 pg / ml in blocking buffer for 1 hour at room temperature. After incubation, membranes were washed three times with TBS-T. Membranes were then incubated with HRP-conjugated secondary antibody in blocking buffer for 1 hour at room temperature. Membranes were washed three times with TBS-T, before signals were developed using ECL substrate and visualized using a chemiluminescence imaging system.EXAMPLE 2: INTERNALIZATION OF ANTI-NUCLEOSOME ANTIBODIES

[0155] This example illustrates that anti-nucleosome antibodies can bind to extracellular nucleosome on the surface of the cells and internalize into the cells, and the internalization can be increased by DNase I treatment.

[0156] The binding and internalization of anti-nucleosome antibodies were observed by fluorescent imaging in human breast cancer cell line AU565 after incubation with the antibodies: anti-histone H3 antibody + 2ndantibody conjugated with Alexa Fluor 647. As shown in FIGs. 2A-2D, after 72-hour incubation, the cell surface binding and internalizationof the antibodies were observed in all the cell samples incubated with anti-histone H3 antibodies (FIGs. 2B and 2D, arrows indicate detection of anti-histone H3 Ab via the Alexa Fluor 647-labeled 2ndAb), but not in the cells with isotype antibodies (FIGs. 2A and 2C).

[0157] Furthermore, fluorescent imaging also showed a significant increase in the internalization of anti-histone H3 antibodies in the cells with DNase I treatment. The AU565 cells were treated with 20 units / ml DNase I along with anti-nucleosome antibodies for 3 days. Significantly more antibodies were observed inside of the nuclease-treated cells (FIG. 2D) compared with the cells without DNase I treatment (FIG. 2B). This result indicates presentence of nuclease activity can increase the internalization of anti-histone H3 antibodies.

[0158] This unique effect was not observed or reported with other antibodies or ADCs. To eliminate the possibility of shared off-target interactions producing similar effects, we further confirmed the specificity of these two antibodies for their respective targets using Western blot and dot blot DNA binding assays.

[0159] The Western blot analysis, performed using whole protein lysates from AU565 and MCF7 cells, revealed that the Histone H3 protein was detected by anti-histone antibody at the expected molecular weight in both lysates, with no additional bands observed. Conversely, the anti-DNA antibody did not detect any protein bands, even under conditions of high protein loading (FIG. 3A). Furthermore, the DNA dot blot assay showed that the anti-DNA antibody exhibited dose-dependent binding to gDNA, whereas the anti-histone antibody showed no DNA binding activity (FIG. 3B). These findings demonstrate that extracellular DNA (exDNA) is internalized into cancer cells upon binding by either anti-DNA or anti-histone antibodies, and that this process can be facilitated by DNase I, likely by digesting exDNA into smaller nucleosome units that are more suitable for endocytosis.EXAMPLE 3: CYTOTOXICITY OF ANTI-NUCLEOSOME ANTIBODY WITH DRUG CONJUGATES IN CANCER CELLS

[0160] This example illustrates that anti-nucleosome antibody with drug conjugates can kill cancer cells, and the cytotoxicity is increase with nuclease treatment.

[0161] The cytotoxicity of anti-nucleosome antibody with drug conjugates were tested in human breast cancer cell lines AU565 and BT474. The anti-DNA antibody [3519 DNA] (Abeam) or anti-histone H3 antibody [1B1B2] (Abeam) were incubated, together with the 2ndantibody-drug conjugate: anti-mouse IgG-vc-MMAF complex in cell samples for 3 or 6 days before testing cell viability. As shown in FIGS. 2A-2B, all the cancer cells treated with anti-nucleosome antibody (either anti-DNA antibody

[3519] or anti -histone H3 antibody [1B1B2]) and the antibody-drug conjugate were less viable compared to the control cells which were treated with a mouse IgG isotype antibody with the antibody-drug conjugate in both cell lines.

[0162] Consistent with the internalization results of anti-histone H3 antibody shown by fluorescent imaging, the cytotoxicity of histone H3 antibody and anti-mouse IgG-vc-MMAF can be increased by nuclease treatments. As also shown in FIGs. 4A-4D, AU565 and BT474 cells were more sensitive to the treatment with anti-histone H3 antibody / ADC and DNase I, in comparison to the cells treated with histone H3 antibody / ADC only.

[0163] We further tested whether the cytotoxicity of anti-nucleosome ADC with nuclease treatment is dose and / or duration dependent of DNase I. As shown in FIG. 5A and 5B, AU565 cells were treated with 0.2 pg / ml anti -nucleosome antibody (either anti-DNA antibody

[3519] or anti-histone H3 antibody [1B1B2]) and 0.2 pg / ml 2ndADC for 3 days (FIG. 5A) or 6 days (FIG. 5B) with different amount of DNase I added at different time. Low toxicity was observed without adding DNase I. Highest cytotoxicity was observed when 20 unit / ml DNase I was added together with anti -nucleosome ADC complex (either anti-DNA antibody

[3519] as shown in FIG. 5A or anti-histone H3 antibody [1B1B2] as shown in FIG. 5B). When cells were treated with 20 unit / ml DNase I for 24 hours before adding anti-nucleosome ADC complex, cytotoxicity was reduced comparing to adding ADC and DNase I at the same time. Cytotoxicity was further reduced in the cells treated with double amount of DNase I (40 unit / ml) DNase I for 24 hours before adding anti-nucleosome ADC complex. This data indicated that nuclease sensitized anti-nucleosome ADC, but excessive nuclease activity or extended period of reaction time reduced the anti-tumor activity of anti-nucleosome antibody. This was due to increased degradation of extracellular nucleosomes reduced targets for antinucleosome ADC on the surface of cancer cells.EXAMPLE 4: CYTOTOXICITY OF ANTI-NUCLEOSOME ADC VS. TP53 MUTATION

[0164] This example illustrates the cytotoxicity of anti-nucleosome antibody with drug conjugates correlates with the presence of TP53 mutations in cells.

[0165] We further analyzed the reactions to anti-nucleosome antibody (either anti-DNA antibody or anti-histone antibody) paired with 2ndADC and DNase I in human breast cancer cells (AU565 carries TP53 R175H mutation; BT474 carries TP53 E285K mutation) and human non-small cell lung cancer cells (A549, which carries wild type TP53). The tested anti-nucleosome antibodies included anti-dsDNA antibodies (Abeam 3519), anti -histone H2A antibodies, anti-histone H2AX antibodies, anti-histone H2B antibodies, anti-histone H3 antibodies, and anti-histone H4 antibodies. Cells were treated with 0.2 pg / ml anti-nucleosome antibody + 0.2 pg / ml 2ndADC, with or without 20 units / ml DNase I. When the cells were treated with anti-DNA antibody 3519 or anti-histone H3 antibody, together with 2ndADC and DNase I, significant cell death was observed in TP53 mutated cells (AU565, as shown in FIG. 6)) but not in TP53 wild type cells (A549, as shown in FIG. 7). This finding indicates TP53 mutation status could be used as one of the markers to predict if a cancer cell will be sensitive to anti-nucleosome ADC + DNase I combination.EXAMPLE 5: SIZE OF ANTI-NUCLEOSOME ANTIBODY IMPACTS ITS ADC ENDOCYTOSIS

[0166] This example illustrates the internalization of anti-nucleosome antibody can be enhanced with increased cluster size or molecular weight by adding a secondary antibody.

[0167] Through maleimide-thiol conjugation reaction, MMAF can be directly conjugated to an anti-nucleosome antibody (e.g., anti-histone H3 antibody) via a Valine-Citrulline (Val-Cit) linker. Based on our previous study on anti-DNA antibody for drug delivery, we expect the cells (e.g., AU565) treated with 0.2 pg / ml anti-histone H3 ADC + 20 units / ml DNase shows a lower anti-tumor activity in comparison to the cells treated with 0.2 pg / ml anti-histone H3 antibody + 0.2 pg / ml 2ndADC + 20 units / ml DNase I. We also expect the cytotoxicity of the anti-nucleosome ADC can be regained by adding a bare 2ndantibody or 2ndADC. Bare anti- nucleosome antibody, 2ndantibody or together may not show any cytotoxicity in combination with 20 units / ml DNase, indicating the cytotoxicity is from payload conjugated to antibodies instead of antibodies alone. These results also indicate that the internalization of antinucleosome antibody can be enhanced with increased cluster size or molecular weight, which can be achieved by binding to, or link to, or conjugate to other antibodies or molecules as illustrated in FIG. IF. The enhanced ADC endocytosis can be resulted from 1) that the increased cluster size or molecular weight enhances the internalization of anti-nucleosome antibody, or 2) that larger ADCs have less steric hindrance or competitive effect to nucleases, allowing nucleases to more efficiently reduce the size of extracellular DNA, which enhances internalization.

[0168] While embodiments of the present disclosure have been described herein, it is to be understood by those skilled in the art that such embodiments are provided by way of exampleonly. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the compositions and methods provided herein. It should be understood that various alternatives to the embodiments provided herein may be employed. It is intended that the following claims define the scope thereof and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

WHAT IS CLAIMED IS:

1. A method of internalizing one or more antibodies into a mammalian cell, comprising, contacting the cell with the one or more antibodies, wherein the one or more antibodies bind to a nucleosome protein on the surface of the cell and the one or more antibodies are internalized into the cell.

2. The method of claim 1, wherein the nucleosome protein comprises a nuclear protein, a protein bound to the nuclear protein, a DNA binding protein, or a protein bound to the DNA binding protein.

3. The method of claim 1 or 2, wherein the nucleosome protein comprises histone Hl, histone H2A, histone H2B, histone H3, histone H4, a modified histone protein, a histone modifying enzyme, a histone acetyltranferase, a histone deacetylase, a histone lysine methyl-transferase, a histone lysine demethylase, a transcription factor, a DNA ligase, nucleophosmin, nucleolin, Ku70, Ku80, centromere protein A (CENPA), high mobility group protein 14 (HMG14), high mobility group protein 17 (HMG17 or HMGN2), retinoblastomabinding protein 5 (RBBP5), Holliday junction recognition protein (HJURP), a DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a transcription factor co-activator, or a transcription factor co-repressor.

4. The method of any one of claims 1-3, wherein the antibodies are bound by a secondary antibody.

5. The method of any one of claims 1-4, wherein the antibodies are coated on a gold nanoparticle.

6. The method of any one of claims 1-5, wherein at least two antibodies are linked together as a polymer.

7. The method of claim 6, wherein the polymer is a dimer, a trimer, a tetramer, or a pentamer.

8. The method of any one of claims 1-7, wherein at least two antibodies are crosslinked together.

9. The method of any one of claims 1-8, further comprising contacting the cell with one or more DNA nucleases.

10. The method of claim 9, wherein the one or more DNA nucleases comprises an endonuclease, an exonuclease, or a combination thereof.

11. The method of claim 10, wherein the endonuclease is a deoxyribonuclease (DNase), a serratia marcescens nuclease (Benzonase), a micrococcal nuclease (MNase), a restriction enzyme, nuclease SI, nuclease Pl, a sequence specific endonuclease, or a sequence non-specific endonuclease.

12. The method of any one of claims 9-11, wherein the one or more DNA nucleases comprise a single stranded DNA (ssDNA) nuclease, a double-stranded DNA (dsDNA) nuclease, or a combination thereof.

13. The method of any one of claims 9-12, wherein the DNA nucleases and the antibodies are contacted to the cell at the same time, optionally wherein the DNA nucleases are attached to the antibodies.

14. The method of any one of claims 9-12, wherein the DNA nucleases are contacted to the cell before the antibodies.

15. The method of any one of claims 1-14, wherein the antibodies are covalently or non-covalently linked to at least one payload.

16. The method of claim 15, wherein the antibodies are bound by a secondary antibody, and wherein the secondary antibody is covalently or non-covalently linked to at least one payload.

17. The method of claim 15 or 16, wherein the antibodies and / or the secondary antibody are linked to the payload via a linker.

18. The method of claim 17, wherein the linker is a non-cleavable linker.

19. The method of claim 18, wherein the non-cleavable linker is a maleimide alkane linker, or a maleimide cyclohexane linker.

20. The method of claim 17, wherein the linker is a cleavable linker.

21. The method of claim 20, wherein the linker is cleaved following contacting the cell with the one or more antibodies, thereby releasing the payload on or in the cell.

22. The method of claim 20 or 21, wherein the cleavable linker is a hydrazone linker, a cathepsin B-responsive linker, a disulfide linker, or a pyrophosphate diester linker.

23. The method of any one of claims 20 to 22, wherein the cleavable linker is a protease-sensitive linker, a pH-sensitive linker, a radiation-sensitive linker, a disulfide linker, or a glutathione-sensitive linker.

24. The method of any one of claims 20 to 23, wherein the cleavable linker is a Valine-Citrulline (Val-Cit) linker.

25. The method of claim 15 or 16, wherein the antibodies and / or the secondary antibody are attached to a biotin moiety, and the at least one payload is attached to a streptavidin.

26. The method of any one of claims 15-25, wherein the at least one payload comprises a small molecule, peptide, protein, nucleic acid, toxin, therapeutic agent, drug, chemotherapeutic agent, liposome, nanoparticle, dendrimer, detectable label, or any derivative, fragment, or combination thereof.

27. The method of claim 26, wherein the therapeutic agent is selected from the group consisting of an antitumor agent, antineoplastic agent, prodrug, lysosome destabilizing agent (e.g., chloroquine), alkylating agent, alkaloid, allosteric inhibitor, anti-folic, anti-inflammatory agent, antibiotics anti-bacterial, antifungal, antifibrotic agent, anti-infective agent, anti-parasitic agent, antiviral agent, antimycobacterial agent, antineoplastic agent, antiprotozoal agent, antiviral agent, bioactive peptide, steroid hormone, photosensitizer substance, radio-pharmaceutical, anti-prion agent, and any combination thereof.

28. The method of claim 27, wherein the antitumor agent is selected from the group consisting of an aromatase inhibitor; an anti-estrogen; an anti-androgen; a gonadorelin agonist; a topoisomerase I inhibitor; a topoisomerase II inhibitor; a microtubule inhibitor; an alkylating agent; a retinoid; a carotenoid; a tocopherol; a cyclooxygenaseinhibitor; an MMP inhibitor; a mTOR inhibitor; an antimetabolite; a platin compound; a methionine aminopeptidase inhibitor; a bisphosphonate; an antiproliferative antibody; a heparinase inhibitor; an inhibitor of Ras oncogenic isoforms; a telomerase inhibitor; a proteasome inhibitor; a Fit- 3 inhibitor; an Hsp90 inhibitor; a kinesin spindle protein inhibitor; a MEK inhibitor; a PARP inhibitor; a Tyrosine kinase inhibitor; a PI3K inhibitor; an AKT inhibitor; an EGFR inhibitor; an antitumor antibiotic; a nitrosourea; a compound targeting / decreasing protein or lipid kinase activity; a compound targeting / decreasing protein or lipid phosphatase activity; any further anti -angiogenic compound; and any combination thereof.

29. The method of claim 27 or 28, wherein the antitumor agent is selected from the group consisting of azacitidine, axathioprine, bleomycin, capecitabine, carboplatin, chlorabucil, cisplatin, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, etoposide, fenretinide, fluorouracil, gemcitabine, herceptin, idarubicin, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, tafluposide, teniposide, tioguanine, retinoic acid, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, receptor tyrosine kinase inhibitor, and any combination thereof.

30. The method of any one of claims 27 to 29, wherein the antitumor agent is a tubule inhibitor, a DNA inhibitor, or an RNA inhibitor.

31. The method of claim 30, wherein the tubule inhibitor is selected from the group consisting of monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), maytansine, maytansinoid, mertansine (emtansine, DM1), ravtansine (soravtansine, DM4), tubulysin, halichondrin (eribulin), cryptophycin, EG5 inhibitor, and any derivative thereof.

32. The method of claim 30, wherein the DNA inhibitor is selected from the group consisting of alkylator, duocarmycin, duocarmycin DM, calicheamicin, pyrrolobenzodiazepine (PDB), enediyne, uncialamycin, topoisomerase inhibitor, topotecan, camptothecin (CPT), exatecan, and any derivative thereof.

33. The method of claim 30, wherein the RNA inhibitor is selected from the group consisting of RNA splicing inhibitor, RNA polymerase II inhibitor, thailanstatin, amatoxin, and any derivative thereof.

34. The method of claim 26, wherein the detectable label is selected from the group consisting of magnetic label, fluorescent moiety, enzyme, light emitting particle, chemiluminescent probe, metal particle, non-metal colloidal particle, polymeric dye particle, pigment molecule, electrochemically active species, semiconductor nanocrystal, nanoparticle, quantum dot, gold particles, fluorophore, or radioactive label.

35. The method of any one of claims 1-34, wherein the cell is a human cell.

36. The method of any one of claims 1-35, wherein the cell is in vitro.

37. The method of any one of claims 1-35, wherein the cell is in vivo.

38. The method of any one of claims 1-37, wherein the cell has an aneuploidy and / or DNA repair deficiency.

39. The method of any one of claims 1-38, wherein the cell comprises a functionally impaired transcription factor or regulator selected from the group consisting of p53, NF -Kb, AP-1, E2F1, and BRCA1.

40. The method of claim 39, wherein the functional impairment of the transcription factor or regulator is caused by a gene mutation.

41. The method of claim 39 or 40, wherein the cell comprises a functionally impaired transcription factor p53.

42. A method of delivering one or more payloads inside of a cell, the method comprising, contacting the cell with the one or more payloads covalently or non-covalently linked to an antibody, wherein the antibody binds to a nucleosome protein on the surface of the cell and delivers the one or more payloads inside of the cell.

43. The method of claim 42, wherein the nucleosome protein comprises histone Hl, histone H2A, histone H2B, histone H3, histone H4, centromere protein A (CENPA), high mobility group protein 14 (HMG14), high mobility group protein 17 (HMG17 or HMGN2), retinoblastoma-binding protein 5 (RBBP5), or Holliday junction recognition protein (HJURP).

44. The method of claim 42 or 43, wherein the one or more payloads comprise a small molecule, peptide, protein, nucleic acid, toxin, therapeutic agent, drug, chemotherapeutic agent, liposome, nanoparticle, dendrimer, detectable label, or any derivative, fragment, or combination thereof.

45. The method of claim 44, wherein the therapeutic agent is selected from the group consisting of an antitumor agent, antineoplastic agent, prodrug, lysosome destabilizing agent (e.g., chloroquine), alkylating agent, alkaloid, allosteric inhibitor, anti-folic, anti-inflammatory agent, antibiotics anti-bacterial, antifungal, antifibrotic agent, anti-infective agent, anti-parasitic agent, antiviral agent, antimycobacterial agent, antineoplastic agent, antiprotozoal agent, antiviral agent, bioactive peptide, steroid hormone, photosensitizer substance, radio-pharmaceutical, anti-prion agent, and any combination thereof.

46. The method of claim 45, wherein the antitumor agent is selected from the group consisting of an aromatase inhibitor; an anti-estrogen; an anti-androgen; a gonadorelin agonist; a topoisomerase I inhibitor; a topoisomerase II inhibitor; a microtubule inhibitor; an alkylating agent; a retinoid; a carotenoid; a tocopherol; a cyclooxygenase inhibitor; an MMP inhibitor; a mTOR inhibitor; an antimetabolite; a platin compound; a methionine aminopeptidase inhibitor; a bisphosphonate; an antiproliferative antibody; a heparinase inhibitor; an inhibitor of Ras oncogenic isoforms; a telomerase inhibitor; a proteasome inhibitor; a Fit- 3 inhibitor; an Hsp90 inhibitor; a kinesin spindle protein inhibitor; a MEK inhibitor; a PARP inhibitor; a Tyrosine kinase inhibitor; a PI3K inhibitor; an AKT inhibitor; an EGFR inhibitor; an antitumor antibiotic; a nitrosourea; a compound targeting / decreasing protein or lipid kinase activity; a compound targeting / decreasing protein or lipid phosphatase activity; any further anti -angiogenic compound; and any combination thereof.

47. The method of claim 45 or 46, wherein the antitumor agent is selected from the group consisting of azacitidine, axathioprine, bleomycin, capecitabine, carboplatin, chlorabucil, cisplatin, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, etoposide, fenretinide, fluorouracil, gemcitabine, herceptin, idarubicin, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, tafluposide, teniposide, tioguanine, retinoic acid, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, receptor tyrosine kinase inhibitor, and any combination thereof.

48. The method of any one of claims 45 to 47, wherein the antitumor agent is a tubule inhibitor, a DNA inhibitor, or an RNA inhibitor.

49. The method of claim 48, wherein the tubule inhibitor is selected from the group consisting of monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), maytansine, maytansinoid, mertansine (emtansine, DM1), ravtansine (soravtansine, DM4), tubulysin, halichondrin (eribulin), cryptophycin, EG5 inhibitor, and any derivative thereof.

50. The method of claim 49, wherein the DNA inhibitor is selected from the group consisting of alkylator, duocarmycin, duocarmycin DM, calicheamicin, pyrrolobenzodiazepine (PDB), enediyne, uncialamycin, topoisomerase inhibitor, topotecan, camptothecin (CPT), exatecan, and any derivative thereof.

51. The method of claim 49, wherein the RNA inhibitor is selected from the group consisting of RNA splicing inhibitor, RNA polymerase II inhibitor, thailanstatin, amatoxin, and any derivative thereof.

52. The method of claim 44, wherein the detectable label is selected from the group consisting of magnetic label, fluorescent moiety, enzyme, light emitting particle, chemiluminescent probe, metal particle, non-metal colloidal particle, polymeric dye particle, pigment molecule, electrochemically active species, semiconductor nanocrystal, nanoparticle, quantum dot, gold particles, fluorophore, or radioactive label.

53. The method of any one of claims 42 to 52, wherein the one or more payloads are covalently linked to the antibody.

54. The method of any one of claims 42 to 52, wherein the one or more payloads are non-covalently linked to the antibody.

55. The method of claim 54, wherein the antibody is bound by a secondary antibody, and wherein the one or more payloads are conjugated to the second antibody.

56. The method of any one of claims 42 to 55, wherein the method further comprises contacting the cell with one or more nucleases, wherein the one or more nucleases comprise a single stranded DNA (ssDNA) nuclease, a double-stranded DNA (dsDNA) nuclease, or a combination thereof.

57. The method of any one of claims 42-56, wherein the cell is in vitro.

58. The method of any one of claims 42-56, wherein the cell is in vivo.

59. The method of any one of claims 42-58, wherein the cell has an aneuploidy and / or DNA repair deficiency.

60. The method of any one of claims 42-59, wherein the cell comprises a functionally impaired transcription factor or regulator selected from the group consisting of p53, NF -Kb, AP-1, E2F1, and BRCA1.

61. The method of claim 60, wherein the functional impairment of the transcription factor or regulator is caused by a gene mutation.

62. The method of claim 60 or 61, wherein the cell comprises a functionally impaired transcription factor p53.

63. The method of any one of claims 42-62, wherein the cell is a cancer cell and wherein the one or more payloads kill or inhibit growth or division of the cancer cell.

64. The method of claim 63, wherein the cancer comprises an acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical carcinoma; AIDS-related cancer; AIDS-related lymphoma; anal cancer; appendix cancer; astrocytomas; atypical teratoid / rhabdoid tumor; basal cell carcinoma; bladder cancer; brain stem glioma; brain tumor; brain stem glioma; central nervous system atypical teratoid / rhabdoid tumor; central nervous system embryonal tumors; astrocytomas; craniopharyngioma; ependymoblastoma; ependymoma; medulloblastoma; medulloepithelioma; pineal parenchymal tumors of intermediate differentiation; supratentorial primitive neuroectodermal tumors and pineoblastoma; breast cancer; bronchial tumors; Burkitt lymphoma; cancer of unknown primary site (CUP); carcinoid tumor; carcinoma of unknown primary site; central nervous system atypical teratoid / rhabdoid tumor; central nervous system embryonal tumors; cervical cancer; childhood cancers; chordoma; chronic lymphocytic leukemia; chronic myelogenous leukemia; chronic myeloproliferative disorders; colon cancer; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; endocrine pancreas islet cell tumors; endometrial cancer; ependymoblastoma; ependymoma; esophageal cancer; esthesioneuroblastoma; Ewing sarcoma; extracranial germ cell tumor; extragonadal germ celltumor; extrahepatic bile duct cancer; gallbladder cancer; gastric (stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal cell tumor; gastrointestinal stromal tumor (GIST); gestational trophoblastic tumor; glioma; hairy cell leukemia; head and neck cancer; heart cancer; Hodgkin lymphoma; hypopharyngeal cancer; intraocular melanoma; islet cell tumors; Kaposi sarcoma; kidney cancer; Langerhans cell histiocytosis; laryngeal cancer; lip cancer; liver cancer; lung cancer; malignant fibrous histiocytoma bone cancer; medulloblastoma; medulloepithelioma; melanoma; Merkel cell carcinoma; Merkel cell skin carcinoma; mesothelioma; metastatic squamous neck cancer with occult primary; mouth cancer; multiple endocrine neoplasia syndromes; multiple myeloma; multiple myeloma / plasma cell neoplasm; mycosis fungoides; myelodysplastic syndromes; myeloproliferative neoplasms; nasal cavity cancer; nasopharyngeal cancer; neuroblastoma; Non-Hodgkin lymphoma; nonmelanoma skin cancer; non-small cell lung cancer; oral cancer; oral cavity cancer; oropharyngeal cancer; osteosarcoma; other brain and spinal cord tumors; ovarian cancer; ovarian epithelial cancer; ovarian germ cell tumor; ovarian low malignant potential tumor; pancreatic cancer; papillomatosis; paranasal sinus cancer; parathyroid cancer; pelvic cancer; penile cancer; pharyngeal cancer; pineal parenchymal tumors of intermediate differentiation; pineoblastoma; pituitary tumor; plasma cell neoplasm / multiple myeloma; pleuropulmonary blastoma; primary central nervous system (CNS) lymphoma; primary hepatocellular liver cancer; prostate cancer; rectal cancer; renal cancer; renal cell (kidney) cancer; renal cell cancer; respiratory tract cancer; retinoblastoma; rhabdomyosarcoma; salivary gland cancer; Sezary syndrome; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma; squamous neck cancer; stomach (gastric) cancer; supratentorial primitive neuroectodermal tumors; T-cell lymphoma; testicular cancer; throat cancer; thymic carcinoma; thymoma; thyroid cancer; transitional cell cancer; transitional cell cancer of the renal pelvis and ureter; trophoblastic tumor; ureter cancer; urethral cancer; uterine cancer; uterine sarcoma; vaginal cancer; vulvar cancer; Waldenstrom macroglobulinemia; or Wilm’s tumor.

65. The method of claim 63, wherein the cancer comprises an acute myeloid leukemia (AML), breast carcinoma, cholangiocarcinoma, colorectal adenocarcinoma, extrahepatic bile duct adenocarcinoma, female genital tract malignancy, gastric adenocarcinoma, gastroesophageal adenocarcinoma, gastrointestinal stromal tumor (GIST), glioblastoma, head and neck squamous carcinoma, leukemia, liver hepatocellular carcinoma, low grade glioma, lung bronchioloalveolar carcinoma (BAC), non-small cell lung cancer (NSCLC), lung small cell cancer (SCLC), lymphoma, male genital tract malignancy, malignantsolitary fibrous tumor of the pleura (MSFT), melanoma, multiple myeloma, neuroendocrine tumor, nodal diffuse large B-cell lymphoma, non-epithelial ovarian cancer (non-EOC), ovarian surface epithelial carcinoma, pancreatic adenocarcinoma, pituitary carcinomas, oligodendroglioma, prostatic adenocarcinoma, retroperitoneal or peritoneal carcinoma, retroperitoneal or peritoneal sarcoma, small intestinal malignancy, soft tissue tumor, thymic carcinoma, thyroid carcinoma, or uveal melanoma.

66. The method of any of claims 42-65, wherein the cell is in a human subject having a disease, and wherein the delivery of the one or more therapeutic drugs are effective to treat the disease.

67. The method of claim 66, further comprising administering one or more DNA nucleases to the human subject.

68. The method of claim 66 or 67, wherein the human subject has a cancer and wherein the cell is a cancer cell.

69. The method of any one of claims 42-68, further comprising determining, prior to the contacting, whether the cell has aneuploidy, a DNA repair deficiency, and / or a functionally impaired transcription factor or regulator selected from the group consisting of p53, NF -Kb, AP-1, E2F1, and BRCA1.

70. The method of claim 69, wherein the functional impairment of the transcription factor or regulator is caused by a gene mutation.

71. The method of claim 69 or 70, wherein the cell comprises a functionally impaired transcription factor p53.

72. A composition comprising an antibody that binds to a nucleosome protein.

73. The composition of claim 72, wherein the nucleosome protein comprises a nuclear protein, a protein bound to the nuclear protein, a DNA binding protein, or a protein bound to the DNA binding protein.

74. The composition of claim 72 or 73, wherein the nucleosome protein comprises histone Hl, histone H2A, histone H2B, histone H3, histone H4, a modified histoneprotein, a histone modifying enzyme, a histone acetyltranferase, a histone deacetylase, a histone lysine methyl-transferase, a histone lysine demethylase, a transcription factor, a DNA ligase, nucleophosmin, nucleolin, Ku70, Ku80, centromere protein A (CENPA), high mobility group protein 14 (HMG14), high mobility group protein 17 (HMG17 or HMGN2), retinoblastoma-binding protein 5 (RBBP5), Holliday junction recognition protein (HJURP), a DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a transcription factor coactivator or a transcription factor co-repressor.

75. The composition of any one of claims 72-74, wherein the composition comprises a secondary antibody that binds the antibody.

76. The composition of any one of claims 72-75, wherein the antibody is coated on a gold nanoparticle.

77. The composition of any one of claims 72-76, wherein at least two antibodies that bind to the nucleosome protein are linked together as a polymer.

78. The composition of claim 77, wherein the polymer is a dimer, a trimer, a tetramer, or a pentamer.

79. The composition of any one of claims 72-78, wherein at least two antibodies that bind to the nucleosome protein are crosslinked together.

80. The composition of any one of claims 72 to 79, further comprising one or more payloads covalently or non-covalently linked to the antibody and / or the secondary antibody.

81. The composition of claim 80, wherein the one or more payloads comprise a small molecule, peptide, protein, nucleic acid, toxin, therapeutic agent, drug, chemotherapeutic agent, liposome, nanoparticle, dendrimer, detectable label, or any derivative, fragment, or combination thereof.

82. The composition of claim 81, wherein the therapeutic agent is selected from the group consisting of an antitumor agent, antineoplastic agent, prodrug, lysosome destabilizing agent (e.g., chloroquine), alkylating agent, alkaloid, allosteric inhibitor, anti-folic, anti-inflammatory agent, antibiotics anti-bacterial, antifungal, antifibrotic agent, anti-infective agent, anti-parasitic agent, antiviral agent, antimycobacterial agent, antineoplastic agent,antiprotozoal agent, antiviral agent, bioactive peptide, steroid hormone, photosensitizer substance, radio-pharmaceutical, anti-prion agent, and any combination thereof.

83. The composition of claim 82, wherein the antitumor agent is selected from the group consisting of an aromatase inhibitor; an anti-estrogen; an anti-androgen; a gonadorelin agonist; a topoisomerase I inhibitor; a topoisomerase II inhibitor; a microtubule inhibitor; an alkylating agent; a retinoid, a carotenoid, or a tocopherol; a cyclooxygenase inhibitor; an MMP inhibitor; a mTOR inhibitor; an antimetabolite; a platin compound; a methionine aminopeptidase inhibitor; a bisphosphonate; an antiproliferative antibody; a heparinase inhibitor; an inhibitor of Ras oncogenic isoforms; a telomerase inhibitor; a proteasome inhibitor; a Fit- 3 inhibitor; an Hsp90 inhibitor; a kinesin spindle protein inhibitor; a MEK inhibitor; a PARP inhibitor; a Tyrosine kinase inhibitor; a PI3K inhibitor; an AKT inhibitor; an EGFR inhibitor; an antitumor antibiotic; a nitrosourea, a compound targeting / decreasing protein or lipid kinase activity, a compound targeting / decreasing protein or lipid phosphatase activity, any further anti-angiogenic compound, and any combination thereof.

84. The composition of claim 82 or 83, wherein the antitumor agent is selected from the group consisting of azacitidine, axathioprine, bleomycin, capecitabine, carboplatin, chlorabucil, cisplatin, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, etoposide, fenretinide, fluorouracil, gemcitabine, herceptin, idarubicin, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, tafluposide, teniposide, tioguanine, retinoic acid, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, receptor tyrosine kinase inhibitor, and any combination thereof.

85. The composition of any one of claims 82 to 84, wherein the antitumor agent is a tubule inhibitor, a DNA inhibitor, or an RNA inhibitor.

86. The composition of claim 85, wherein the tubule inhibitor is selected from the group consisting of monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), maytansine, maytansinoid, mertansine (emtansine, DM1), ravtansine (soravtansine, DM4), tubulysin, halichondrin (eribulin), cryptophycin, EG5 inhibitor, and any derivative thereof.

87. The composition of claim 85, wherein the DNA inhibitor is selected from the group consisting of alkylator, duocarmycin, duocarmycin DM, calicheamicin, pyrrolobenzodiazepine (PDB), enediyne, uncialamycin, topoisomerase inhibitor, topotecan, camptothecin (CPT), exatecan, and any derivative thereof.

88. The composition of claim 85, wherein the RNA inhibitor is selected from the group consisting of RNA splicing inhibitor, RNA polymerase II inhibitor, thailanstatin, amatoxin, and any derivative thereof.

89. The composition of claim 81, wherein the detectable label is selected from the group consisting of magnetic label, fluorescent moiety, enzyme, light emitting particle, chemiluminescent probe, metal particle, non-metal colloidal particle, polymeric dye particle, pigment molecule, electrochemically active species, semiconductor nanocrystal, nanoparticle, quantum dot, gold particles, fluorophore, or radioactive label.

90. The composition of any one of claims 72-89, further comprising one or more DNA nucleases.

91. The composition of claim 90, wherein the one or more DNA nucleases comprise an endonuclease, an exonuclease, or a combination thereof.

92. The composition of claim 91, wherein the endonuclease is a deoxyribonuclease (DNase), a serratia marcescens nuclease (Benzonase), a micrococcal nuclease (MNase), a restriction enzyme, nuclease SI, nuclease Pl, a sequence specific endonuclease, or a sequence non-specific endonuclease.

93. The composition of any one of claims 90 to 92, wherein the one or more DNA nucleases comprise a single stranded DNA (ssDNA) nuclease, a double-stranded DNA (dsDNA) nuclease, or a combination thereof.

94. A pharmaceutical composition comprising a therapeutically effective amount of the composition according to any one of claims 80 to 93, and a pharmaceutically acceptable excipient, carrier, and / or diluent.

95. A method of treating or ameliorating a disease or disorder in a human subject in need thereof, comprising administering the pharmaceutical composition of claim 94 to the subject, optionally wherein the disease or disorder comprises a cancer.

96. The method of claim 95, further comprising determining, prior to the administering, whether the cancer has an aneuploidy, a DNA repair deficiency, and / or a functionally impaired transcription factor or regulator selected from the group consisting of p53, NF -Kb, AP-1, E2F1, and BRCA1.

97. The method of claim 96, wherein the functional impairment of the transcription factor or regulator is caused by a gene mutation.

98. The method of any one of claims 95 to 97, wherein the cancer comprises a functionally impaired transcription factor p53 resulting from a mutation of the TP53 gene.

99. The method of any one of claims 95 to 98, wherein the administering comprises at least one of intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intracerebral, intravaginal, transdermal, rectal, by inhalation, topical administration, or any combination thereof.

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