Compositions and methods for targeting extracellular DNA

Abstract

Provided herein are methods of internalizing anti-DNA antibodies or DNA-binding antibody fragments into a cell. The methods can be used for disease diagnosis and therapeutic treatments.

Description

COMPOSITIONS AND METHODS FOR TARGETING EXTRACELLULAR DNACROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 521,214, filed June 15, 2023, and U.S. Provisional Application No. 63 / 613,475, filed December 21, 2023, the disclosures of which are herein incorporated by reference in their entirety for all purposes.BACKGROUND

[0002] Stressed, damaged and dying cells release intracellular factors, including DNA, into their cell surface and / or local microenvironment, that activate immune components. Such extracellular DNA can lead to inflammation that contributes to pathogenesis of various autoimmune diseases.

[0003] It was recently reported that extracellular DNA is present on the surface of pancreatic cancer cells, but not on normal pancreas cells (Wen F, et al. Cancer Res. 2013;73(14):4256- 4266). The cancer cell-related extracellular DNA appeared to be involved in cell metastatic potential in vitro and DNase I treatment decreased cancer metastasis in an orthotopic xenograft pancreatic cancer mouse model. However, extracellular DNA carrying specific sequences derived from the genome of a target cell has not been used for cell targeting and the presence of such biomarkers has not been applied in diagnostic, prognostic and / or theranostic fields.SUMMARY

[0004] Provided herein are compositions and methods of internalizing anti-DNA antibodies or DNA-binding antibody fragments into a cell. The methods comprise contacting the cell with anti-DNA antibodies or DNA-binding antibody fragments that bind to extracellular DNA on the surface of the cell whereby the anti-DNA antibodies or DNA-binding antibody fragments can be internalized into the cell. The methods can be further optimized by, such as contacting the cell with nucleases, to facilitate the internalization. Such methods and related compositions can be used in various applications, including drug delivery, disease diagnosis and therapeutic treatments.

[0005] In one aspect, the present disclosure provides a method of internalizing one or more anti-DNA antibodies into a cell, the method comprising, contacting the cell with the one or more anti-DNA antibodies, wherein the one or more anti-DNA antibodies bind to extracellular DNA on the surface of the cell and are internalized into the cell.

[0006] In some embodiments, the contacting comprises contacting the cell with at least one, two, three, four, five, six, seven, eight or more different anti-DNA antibodies. In some embodiments, the at least one, two, three, four, five, six, seven, eight or more different anti- DNA antibodies comprises one, two, three, four, five, six, seven, eight or more different anti- DNA antibodies. In some embodiments, the method further comprises contacting the cell with one or more nucleases. In some embodiments, the one or more nucleases comprise DNA nucleases. In some embodiments, the one or more DNA nucleases comprise a single stranded DNA nuclease, a double-stranded DNA nuclease, or a combination thereof.

[0007] In some embodiments, the one or more nucleases and the one or more anti-DNA antibodies are contacted to the cell at the same time, optionally wherein the one or more nucleases are attached to the one or more anti-DNA antibodies. In some instances, the one or more nucleases are contacted to the cell before the one or more anti-DNA antibodies. In other instances, the one or more nucleases are contacted to the cell after the one or more anti-DNA antibodies. In some embodiments, the one or more nucleases are DNase I, a restriction enzyme, and / or Benzonase.

[0008] In some embodiments, the one or more anti-DNA antibodies are covalently or non- covalently linked to at least one payload. In some instances, the at least one payload is covalently linked to the anti-DNA antibody. In other instances, the one or more anti-DNA antibodies 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 is linked to one or more secondary antibodies, wherein the one or more secondary antibodies bind to the anti-DNA antibody.

[0009] In some embodiments, the one or more anti-DNA antibodies and / or the one or more secondary antibodies are linked to the at least one payload via a linker. In some instances, the linker comprises a non-cleavable linker. In other instances, the linker comprises a cleavable linker. In some embodiments, the linker is cleaved after contacting the cell with the one or more anti-DNA antibodies, thereby releasing the payload on or in the cell. In some embodiments, the cleavable linker comprises a protease-sensitive linker, a pH-sensitive linker, a radiation-sensitive linker, a glutathione-sensitive linker, a disulfide linker, or a combinationthereof. In some embodiments, the cleavable linker comprises a protease-sensitive linker comprising a sequence of sortase recognition motif LPXTG (SEQ ID NO: 1). In some embodiments, the sequence of sortase recognition motif is LPETG (SEQ ID NO: 2).

[0010] 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.

[0011] 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.

[0012] 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 active agent; 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.

[0013] In some embodiments, the antitumor agent is selected from the group consisting of 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 inhibitor, and any combination 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, radioactive label, or a combination thereof. In some instances, the cell is in vitro. In other instances, the cell is in vivo.

[0015] In some embodiments, the one or more anti-DNA antibodies bind to a known DNA sequence. In other embodiments, the one or more anti-DNA antibodies bind to an unknown DNA sequence. Alternatively, the one or more anti-DNA antibodies bind to both known and unknown DNA sequences.

[0016] In some embodiments, the one or more anti-DNA antibodies are sequence specific. In other embodiments, the one or more anti-DNA antibodies are non-sequence specific. Alternatively, the one or more anti-DNA antibodies comprise a combination of sequence specific and non-sequence specific anti-DNA antibodies.

[0017] In another aspect, the present disclosure provides a method of internalizing one or more anti-DNA antibodies into a mammalian cell, the method comprising, contacting the cell with the one or more anti-DNA antibodies, wherein the one or more anti-DNA antibodies bind to extracellular DNA on the surface of the cell and are internalized into the cell. In some embodiments, the contacting comprises contacting the cell with at least one, two, three, four, five, six, seven, eight or more different anti-DNA antibodies. In some embodiments, the at least one, two, three, four, five, six, seven, eight or more different anti-DNA antibodies comprises one, two, three, four, five, six, seven, eight or more different anti-DNA antibodies.

[0018] In some embodiments, the method further comprises contacting the cell with one or more nucleases. In some embodiments, the one or more nucleases comprise DNA nucleases. In some embodiments, the one or more 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 transposase, 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.

[0019] In some embodiments, the one or more nucleases and the one or more anti-DNA antibodies are contacted to the cell at the same time, optionally wherein the one or more nucleases are attached to the one or more anti-DNA antibodies. In some embodiments, the one or more nucleases are contacted to the cell before the one or more anti-DNA antibodies. In some embodiments, the one or more nucleases are contacted to the cell after the one or more anti-DNA antibodies.

[0020] In some embodiments, the one or more anti-DNA antibodies are covalently or non- covalently linked to at least one payload. In some embodiments, the one or more anti-DNA antibodies are bound by a secondary antibody. In some embodiments, the secondary antibody is covalently or non-covalently linked to at least one payload. In some embodiments, both the secondary antibody and the anti-DNA antibody are covalently or non-covalently linked to at least one payload.

[0021] In some embodiments, the anti-DNA antibody or the secondary antibody is covalently linked to the payload. In some embodiments, the anti-DNA antibody or the secondary antibody is linked to the payload via a linker. In some embodiments, the linker comprises a non- cleavable linker. In some embodiments, the non-cleavable linker comprises a maleimide alkane linker, or a maleimide cyclohexane linker. In some embodiments, the linker comprises a cleavable linker. In some embodiments, the linker is cleaved after contacting the cell with the one or more anti-DNA antibodies, thereby releasing the payload on or in the cell. In some embodiments, the cleavable linker comprises a hydrazone linker, a cathepsin B-responsive linker, a disulfide linker, or a pyrophosphate diester linker, or a combination thereof. In some embodiments, the cleavable linker comprises a protease-sensitive linker, a pH-sensitive linker, a radiation-sensitive linker, a disulfide linker, or a glutathione-sensitive linker, or a combination thereof.

[0022] In some embodiments, the anti-DNA antibody or the secondary antibody is non- covalently linked to the payload. In some embodiments, the anti-DNA antibody or the secondary antibody is 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.

[0023] 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.

[0024] 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 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.

[0025] 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 comprises a tubulin inhibitor, a DNA inhibitor, and / or an RNA inhibitor.

[0026] In some embodiments, the tubulin 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 someembodiments, the DNA inhibitor is selected from the group consisting of an alkylator, duocarmycin, duocarmycin DM (DMDM), calicheamicin, pyrrolobenzodiazepine (PDB), enediyne, uncialamycin, a topoisomerase inhibitor, topotecan, camptothecin (CPT), exatecan, and any derivative thereof. In some embodiments, the RNA inhibitor is selected from the group consisting of an RNA splicing inhibitor, RNA polymerase II inhibitor, thailanstatin, amatoxin, and any derivative thereof.

[0027] 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, radioactive label, or a combination thereof.

[0028] In some embodiments, the one or more anti-DNA antibodies are sequence specific. In some embodiments, the one or more anti-DNA antibodies are non-sequence specific. In some embodiments, the one or more anti-DNA antibodies comprise a combination of sequence specific and non-sequence specific anti-DNA antibodies. In some embodiments, the one or more anti-DNA antibodies comprise anti-dsDNA antibodies, anti-ssDNA antibodies, or a combination thereof.

[0029] In some embodiments, the one or more anti-DNA antibodies are coated on a gold nanoparticle when entering the cell. In some embodiments, at least two anti-DNA antibodies are covalently or non-covalently linked when entering the cell.

[0030] In another aspect, provided herein is a method of delivering a therapeutic agent inside of a cell, the method comprising, contacting the cell with a therapeutic agent covalently or non- covalently linked to an anti-DNA antibody, wherein the anti-DNA antibody binds to extracellular DNA on the surface of the cell and delivers the therapeutic agent inside of the cell. In some embodiments, the therapeutic agent is covalently linked to the anti-DNA antibody. In some embodiments, the therapeutic agent is non-covalently linked to the anti- DNA antibody. In some embodiments, the one or more anti-DNA antibodies are bound by one or more secondary antibodies, and wherein the therapeutic drug is conjugated to the one or more secondary antibodies.

[0031] In some embodiments, the method further comprises contacting the cell with one or more nucleases, optionally 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.

[0032] In some embodiments, the cell has an aneuploidy and / or DNA repair deficiency. In some embodiments, the cell comprises a functionally-impaired transcription factor selected from the group consisting of p53, NF-Kb, AP-1, E2F1, and BRCA1. In some embodiments, the functional impairment of the transcription factor is caused by one or more gene mutations, loss of the gene or a chromosomal region, and / or deficient expression of the protein. In some embodiments, the cell comprises a functionally-impaired transcription factor p53, optionally wherein the impairment of p53 comprises a mutation.

[0033] In some embodiments, the cell is a cancer cell and wherein the therapeutic drug kills or inhibits growth or division of the cancer cell. In some embodiments, the cancer’s type 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 skincarcinoma; 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.

[0034] In some embodiments, the cancer 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. In some embodiments, the cancer cell is from a cancer in a subject.

[0035] In yet another aspect, the present disclosure provides a method of treating in a subject in need thereof, the method comprising, administering to the subject a composition comprising a therapeutic drug linked to an anti-DNA antibody, wherein the therapeutic drug is delivered inside of a cell of the subject using the method disclosed herein, wherein the delivery of the therapeutic drug is effective to treat the subject. In some embodiments, the composition further comprises a DNA nuclease. In some embodiments, the subject has a cancer and wherein the cell is a cancer cell.

[0036] In some embodiments, the method further comprises determining, prior to the administering, whether the cell has an aneuploidy, a DNA repair deficiency, and / or a functionally-impaired transcription factor selected from the group consisting of p53, NF-Kb, AP-1, E2F1, and BRCA1. In some embodiments, the functional impairment of the transcription factor is caused by one or more gene mutations, loss of the gene or a chromosomal region, and / or deficient expression of the protein. In some embodiments, the cell comprises a functionally-impaired transcription factor p53, optionally wherein the impairment of p53 comprises a mutation.

[0037] In yet another aspect, the present disclosure provides a composition comprising one or more anti-DNA antibodies and one or more nucleases, optionally wherein the one or more nucleases comprise one or more DNA nucleases. In some embodiments, the one or more anti- DNA antibodies are bound by a secondary antibody. In some embodiments, the one or more anti-DNA antibodies are coated on a gold nanoparticle. In some embodiments, at least two anti-DNA antibodies are linked together as a polymer. In some embodiments, the polymer comprises a dimer, a trimer, a tetramer, a pentamer, or has more than five anti-DNA antibodies. In some embodiments, the composition comprises two, three, four, five, six, seven, eight or more different anti-DNA antibodies. In some embodiments, at least two different anti-DNA antibodies are crosslinked together.

[0038] In some embodiments, the composition further comprising a therapeutic agent covalently or non-covalently linked to the anti-DNA antibody and / or the secondary antibody. 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, antiprotozoalagent, antiviral agent, bioactive peptide, steroid hormone, photosensitizer substance, radiopharmaceutical, anti-prion agent, and any combination thereof.

[0039] 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 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.

[0040] 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.

[0041] In some embodiments, the antitumor agent comprises a tubulin inhibitor, a DNA inhibitor, and / or an RNA inhibitor. In some embodiments, the tubulin 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. In some embodiments, the DNA inhibitor is selected from the group consisting of an alkylator, duocarmycin, duocarmycin DM (DMDM), calicheamicin, pyrrolobenzodiazepine (PDB), enediyne, uncialamycin, a topoisomerase inhibitor, topotecan, camptothecin (CPT), exatecan, and any derivative thereof.

[0042] In some embodiments, the RNA inhibitor is selected from the group consisting of an RNA splicing inhibitor, RNA polymerase II inhibitor, thailanstatin, amatoxin, and anyderivative thereof. In some embodiments, the one or more 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 transposase, 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.

[0043] The present also disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of the composition disclosed herein, and a pharmaceutically acceptable excipient, carrier, and / or diluent. The present disclosure further provides a method of treating or ameliorating a disease or disorder in a subject in need thereof, comprising administering the composition to the subject, optionally wherein the disease or disorder comprises a cancer.

[0044] In some embodiments, the method further comprising determining, prior to the administering, whether the cancer has an aneuploidy, a DNA repair deficiency, and / or a functionally-impaired transcription factor selected from the group consisting of p53, NF-Kb, AP-1, E2F1, and BRCA1. In some embodiments, the functional impairment of the transcription factor is caused by one or more gene mutations, loss of the gene or a chromosomal region, and / or deficient expression of the protein. In some embodiments, the cancer comprises a functionally-impaired transcription factor p53 resulting from a deficiency of the TP53 gene, optionally wherein the impairment of p53 comprises a mutation. 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.

[0045] The present disclosure further provides a first composition comprising one or more anti-DNA antibodies. In some embodiments, at least one of the one or more anti-DNA antibodies are covalently or non-covalently linked to at least one payload. In some embodiments, at least one of the one or more anti-DNA antibodies are bound to one or more secondary antibodies. In some embodiments, at least one of the one or more secondary antibodies are covalently or non-covalently linked to at least one payload. In some embodiments, the one or more anti-DNA antibodies and / or the one or more secondary antibodies are covalently linked to the at least one payload.

[0046] In some embodiments, the one or more anti-DNA antibodies and / or the one or more secondary antibodies are linked to the at least one payload via one or more linker. In some embodiments, the one or more linkers comprises a non-cleavable linker. In some embodiments, the non-cleavable linker comprises a maleimide alkane linker, or a maleimide cyclohexane linker. In some embodiments, the one or more linkers comprises a cleavable linker. In some embodiments, the cleavable linker comprises a hydrazone linker, a cathepsin B-responsive linker, a disulfide linker, or a pyrophosphate diester linker, or a combination thereof. In some embodiments, the cleavable linker comprises a protease-sensitive linker, a pH-sensitive linker, a radiation-sensitive linker, a disulfide linker, or a glutathione-sensitive linker, or a combination thereof. In some embodiments, the one or more anti-DNA antibodies and / or the one or more secondary antibodies are attached to a biotin moiety, and the at least one payload is attached to a streptavidin.

[0047] 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.

[0048] 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.

[0049] 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 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, acompound targeting / decreasing protein or lipid phosphatase activity, any further anti- angiogenic compound, and any combination thereof.

[0050] 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.

[0051] In some embodiments, the antitumor agent comprises a tubulin inhibitor, a DNA inhibitor, and / or an RNA inhibitor. In some embodiments, the tubulin 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. In some embodiments, the DNA inhibitor is selected from the group consisting of an alkylator, duocarmycin, duocarmycin DM, calicheamicin, pyrrolobenzodiazepine (PDB), enediyne, uncialamycin, a topoisomerase inhibitor, topotecan, camptothecin (CPT), exatecan, and any derivative thereof. In some embodiments, the RNA inhibitor is selected from the group consisting of an RNA splicing inhibitor, RNA polymerase II inhibitor, thailanstatin, amatoxin, and any derivative thereof.

[0052] 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, radioactive label, or a combination thereof.

[0053] In some embodiments, the one or more anti-DNA antibodies are sequence specific for the target DNA. In some embodiments, the one or more anti-DNA antibodies are nonsequence specific for the target DNA. In some embodiments, the one or more anti-DNA antibodies comprise a combination of sequence specific and non-sequence specific anti-DNA antibodies. In some embodiments, the one or more anti-DNA antibodies bind to double stranded DNA (dsDNA), single stranded DNA (ssDNA), or a combination thereof.

[0054] In some embodiments, the one or more anti-DNA antibodies are coated on a gold nanoparticle. In some embodiments, at least two anti-DNA antibodies are linked together as a polymer. In some embodiments, the polymer comprises a dimer, a trimer, a tetramer, a pentamer, or has more than five anti-DNA antibodies. In some embodiments, the composition comprises two, three, four, five, six, seven, eight or more different anti-DNA antibodies. In some embodiments, at least two different anti-DNA antibodies are crosslinked together.

[0055] The present disclosure further provides a second composition comprising one or more nucleases. In some embodiments, the one or more nucleases comprise DNA nucleases. In some embodiments, the one or more 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 transposase, 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.

[0056] The present disclosure further provides a method of internalizing one or more anti- DNA antibodies into a human cell, the method comprising, contacting the cell with the first composition disclosed herein, wherein the one or more anti-DNA antibodies bind to extracellular DNA on the surface of the cell and are internalized into the cell.

[0057] The present disclosure further provides a method of delivering a payload inside of a cell, the method comprising, contacting the cell with the first composition disclosed herein, wherein the one or more anti-DNA antibodies binds to extracellular DNA on the surface of the cell and delivers the payload inside of the cell. In some embodiments, the payload comprises a therapeutic agent disclosed herein. In some embodiments, the method further comprises contacting the cell with the second composition disclosed herein. In some embodiments, the first composition and second composition are contacted to the cell at the same time, optionally wherein the first composition and second composition are the same composition. In some embodiments, the one or more nucleases are attached to the one or more anti-DNA antibodies. In some embodiments, the first composition is contacted to the cell before the second composition. In some embodiments, the first composition is contacted to the cell after the second composition. In some embodiments, the linker is cleaved after contacting the cell with the one or more anti-DNA antibodies, thereby releasing the payload on or in the cell. In someembodiments, 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 selected from the group consisting of p53, NF-Kb, AP-1, E2F1, and BRCA1. In some embodiments, the functional impairment of the transcription factor is caused by one or more gene mutations, loss of the gene or a chromosomal region, and / or deficient expression of the protein. In some embodiments, the cell comprises a functionally-impaired transcription factor p53, optionally wherein the impairment of p53 comprises a mutation. In some embodiments, the cell is a cancer cell and wherein the payload kills or inhibits growth or division of the cancer cell. In some embodiments, the cancer cell is from a cancer in a subject.

[0058] The present disclosure further provides a method comprising administering to a subject the first composition as disclosed herein, and optionally further comprising administering to the subject the second composition as disclosed herein. In some embodiments, the first composition and the second composition are the same composition. In some embodiments, the first composition and the second composition are administered at the same time, the first composition is administered before the second composition, or the first composition is administered after the second composition. In some embodiments, the subject has a cancer, and the administering is performed at a dosage effective to treat the cancer. In some embodiments, the anti-DNA antibody within the first composition comprises a payload.

[0059] In some embodiments, the method further comprises determining, prior to the administering, whether the cancer has an aneuploidy, a DNA repair deficiency, and / or a functionally-impaired transcription factor selected from the group consisting of p53, NF-Kb, AP-1, E2F1, and BRCA1. In some embodiments, the functional impairment of the transcription factor is caused by one or more gene mutations, loss of the gene or a chromosomal region, and / or deficient expression of the protein.

[0060] In some embodiments, the cancer comprises a functionally-impaired transcription factor p53, optionally wherein the impairment of p53 comprises a mutation. In some embodiments, the cancer’s type 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 pelvisand ureter; trophoblastic tumor; ureter cancer; urethral cancer; uterine cancer; uterine sarcoma; vaginal cancer; vulvar cancer; Waldenstrom macroglobulinemia; or Wilm’s tumor.

[0061] In some embodiments, the cancer 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.

[0062] 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.

[0063] The present disclosure further provides a method of internalizing one or more anti- DNA antibodies into a cell. In some embodiments, the method comprises contacting the cell with the one or more anti-DNA antibodies, wherein the one or more anti-DNA antibodies comprise means for binding extracellular DNA, wherein the one or more anti-DNA antibodies bind to the extracellular DNA on the surface of the cell and the one or more anti- DNA antibodies are internalized into the cell.

[0064] The present disclosure further provides a method of delivering a therapeutic agent inside of a cell. In some embodiments, the method comprises contacting the cell with a therapeutic agent covalently or non-covalently linked to an anti-DNA antibody, wherein the one or more anti-DNA antibodies comprise means for binding extracellular DNA, wherein the anti-DNA antibody binds to the extracellular DNA on the surface of the cell and delivers the therapeutic agent inside of the cell.

[0065] The present disclosure further provides a composition comprising one or more anti- DNA antibodies and one or more nucleases, optionally wherein the one or more nucleases comprise one or more DNA nucleases, wherein the one or more anti-DNA antibodies comprise means for binding extracellular DNA.

[0066] The present disclosure further provides, in a method of contacting an antibody to a cell, an improvement that comprises internalizing one or more anti-DNA antibodies into a cell by contacting the cell with the one or more anti-DNA antibodies, wherein the one or more anti-DNA antibodies bind to extracellular DNA on the surface of the cell and the one or more anti-DNA antibodies are internalized into the cell.

[0067] The present disclosure further provides, in a method of treating in a subject having a cancer, an improvement that comprises administering to the subject a composition comprising a therapeutic agent linked to an anti-DNA antibody, wherein the therapeutic agent is delivered inside of a cell of the subject, wherein the delivery of the therapeutic agent is effective to treat the subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0068] FIGS. 1A-C depict schematic drawings of binding and internalization of anti-DNA antibody constructs to a cell. FIG. 1A shows an anti-DNA antibody binding to extracellular DNA on a cell surface, and the one or more anti-DNA antibodies are also recognized and linked to a 2ndantibody conjugated with a desired payload, such as a therapeutic drug or fluorescent label. Internalization of the complex of extracellular DNA with the anti-DNA 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 payload is directly attached to the anti- DNA antibody. FIG. 1C shows a similar schematic as FIG. IB wherein the extracellular DNA is packaged around extracellular nucleosomes. In addition, DNA nuclease is shown cleaving the extracellular DNA. See related discussion herein. FIG. 1C also shows that a secondary antibody could be used in some embodiments, such as in FIG. 1A. FIG. ID depicts a schematic drawing of possible formats of anti-DNA ADCs / ADC complexes.

[0069] FIGS. 2A-2E depict cytotoxicity of anti-DNA antibody with drug conjugates in cancer cells: human pancreatic cancer cells AsPC-1 (FIG. 2A), human HER2+ breast cancer cells AU565 (FIG. 2B), human pancreatic cancer cells SW1990-1 (FIG. 2C), and human triple-negative breast cancer cells HCC1395 (FIG. 2D), and in human non-tumorigenicepithelial cells MCF10A (FIG. 2E). The cytotoxicity assays are shown after six days of treatment. All the cancer cells (FIGS. 2A-2D) treated with anti-DNA antibody and a 2ndantibody-drug conjugate (2ndADC) were less viable compared to the control cells which were treated with a mouse IgG isotype antibody with 2ndantibody-drug conjugate. There is no significant difference between the test antibody and the control in the non-cancer cell, MCF 10 A (FIG. 2E), indicating that the cytotoxicity of anti-DNA antibody with drug conjugate is specifically to cancer cells.

[0070] FIGS. 3A-3B depict cytotoxicity of anti-DNA antibody without drug conjugates in AU565 cells (FIG. 3A) and HCC1395 cells (FIG. 3B), indicating that the cytotoxicity was not caused by the anti-DNA antibody alone.

[0071] FIGS. 4A-4D show that cytotoxicity of anti-DNA antibody with drug conjugates can be increased by nuclease treatment. HCC1395 cells (FIGS. 4A and 4C) and AU565 cells (FIGS. 4B and 4D) showed a significantly stronger cytotoxicity response in the treatment with anti-DNA antibody / ADC and nuclease, in comparison to the cells treated with anti-DNA antibody / ADC without nuclease. FIGS. 4A and 4B depict the cytotoxicity results of the cells treated with DNase I. FIGS. 4C and 4D depict the cytotoxicity results of the cells treated with Benzonase, a recombinant DNA / RNA endonuclease.

[0072] FIGS. 5A-5K illustrate the effect of nucleases on cytotoxicity of the anti-DNA ADCs provided herein. FIGS. 5A-5B depict cytotoxicity of DNase I with different concentrations in non-tumorigenic human cells MCF10A (FIG. 5A) and human cancer cells HCC1395 (FIG. 5B), indicating that cytotoxicity was not caused by DNase I. FIGS. 5C-5D show cytotoxicity of non-anti-DNA ADC constructs in the absence (FIG. 5C) or presence (FIG. 5D) of DNase I. These figures show that DNase I did not sensitize cells to antibody drug conjugates (ADCs) that are not targeting extracellular DNA on the surface of cancer cells. FIG. 5E shows similar results using an anti-CD71 with secondary ADC. FIG. 5F shows that the cytotoxicity of anti- DNA ADC with nuclease treatment is dose and / or duration dependent, wherein more cytotoxicity was observed without pre-incubation of the cells (middle bar) with DNase prior to addition of the anti-DNA ADC as compared to cells pre-incubated with DNase (bars 4 and 5). FIGS. 5G-5J show that various DNA nucleases sensitize cells to the anti-DNA ADCs, including bovine DNase I (FIG. 5G), recombinant human DNase I (FIG. 5H), Benzonase (FIG. 51), and micrococcal nuclease (MNase) (FIG. 5J). However, RNase did not sensitize the cells. See FIG. 5K.

[0073] FIGS. 6A-6B illustrate that cytotoxicity of anti-DNA ADC with nuclease treatment specifically targets cancer cells (human HER2+ breast cancer cells AU565 and human triplenegative breast cancer cells HCC139) as compared to normal cells (non-cancerous breast epithelial cell MCF10A).

[0074] FIG. 7 depicts specific cytotoxicity towards cancer cells with three different anti- DNA antibody / ADC with nuclease treatments. As indicated in the figure, the antibodies are Abeam 3519, Millipore BV16-13, and Millipore 16-19. AU565 is a cancer cell line (derived from a patient with HER2+ breast adenocarcinoma) and MCF 10A is a non-cancer control (non- cancerous breast epithelial cell). Treatment composition is indicated in the figure.

[0075] FIGS. 8A-8B indicate that cytotoxicity of anti-DNA ADC and DNase I associates with TP53 mutation in cancer cells. In FIG. 8A, each group of bars are ordered from left to right in the same ordering as the list of cell lines on the left from top to bottom. FIG. 8B is the same as the last grouping in FIG. 8A but also shows detail about the TP53 status of the cell lines. In FIG. 8B, the bars from left to right across the entire plot have the same ordering as the list of cell lines on the left from top to bottom (which is also the same ordering as each grouping in FIG. 8A). FIG. 8A shows that both DNase I (grouping labeled DNase I) and anti- DNA Ab+ 2ndADC (grouping labeled Anti-DNA ADC) had minimum cytotoxicity to the cells alone, while substantial levels of cytotoxicity were observed when combining DNase I (at the testing concentration 0.2ug / ml) together with anti-DNA Ab+ 2ndADC in TP53 mutated cells (grouping labeled Anti-DNA ADC + DNase I). The data in FIG. 8B shows that anti-DNA Ab + 2ndADC + DNase I had greater cytotoxicity to a variety of cell lines harboring TP53 loss of function mutations (grouping labeled TP53 mutated) when compared to non-cancer cell lines or TP53 WT cell lines (grouping labeled TP53 WT).

[0076] FIGS. 9A-9B depict cytotoxicity of three therapeutic payloads when delivered by anti-DNA antibodies together with DNase I treatment after three days (FIG. 9A) or six days (FIG. 9B). Although cytotoxicity was observed in all settings, the payload monomethylauristatin F (MMAF) exhibited higher potency with anti-DNA Ab as compared to Duocarmycin DM (DMDM) or Extecan.

[0077] FIG. 10 shows the cytotoxicity of different anti-DNA antibodies and related ADC constructs. The constructs are depicted above each bar. In these experiments, we observed high cytotoxicity with ADCs comprising a complex of an anti-DNA Ab bound with a 2ndAb, whether the drug was conjugated to the anti-DNA Ab and / or the 2ndAb. The highestcytotoxicity was observed in the group treated with drug conjugate on both the anti-DNA antibodies and the secondary antibodies.

[0078] FIGS. 11A-11G show a comparison of the efficacy of anti-DNA ADC compared to trastuzumab deruxtecan, an ADC comprised of the humanized monoclonal antibody trastuzumab covalently linked to the topoisomerase I inhibitor deruxtecan. The plots in FIGS. 11A-11E show cell viability versus antibody concentrations using the ADCs, with or without DNase I, as indicated in the legends for various cell lines: HER2+ breast cancer cell line AU565 (FIG. 11 A); HER2+ breast cancer cell line BT474 (FIG. 11B); HER2 low breast cancer cell line HCC1395 (FIG. 11C); HER2 low breast cancer cell line MDA-MB-468 (FIG. 11D). The upper plot in FIG. HE is the same as FIG. 11C for HCC1395, whereas the lower plot is related normal adjacent cell line HCC1395 BL. The vertical line shows the IC50 in the cancer (upper) and normal (lower) cells. FIGS. 11F-11G show that HER2+ cell lines AU565 (FIG. HF) and BT474 (FIG. 11G), unlike anti-DNA ADC, were not sensitized to trastuzumab deruxtecan by 2ndAb or DNase I.DETAILED DESCRIPTIONI. Introduction

[0079] The present disclosure provides compositions and methods of targeting anti-DNA antibodies to cells of interest. The antibodies may be internalized into the target cells. In addition, contacting the cell with one or more nucleases can facilitate this internalization. In some embodiments, complexes of two or more anti-DNA antibodies are internalized. When the one or more anti-DNA antibodies are conjugated with one or more payloads, including without limitation therapeutic agents and / or detectable labels, the one or more anti-DNA antibodies can be used to deliver the desired payloads to or into the cells. Therefore, the anti- DNA antibodies with drug conjugates or detectable labels are particularly useful for use in diagnostic and therapeutic applications.II. Definitions

[0080] 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.

[0081] 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.

[0082] 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). As disclosed herein, “One or more” may be used interchangeably herein with “at least one.” For example, 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).

[0083] 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.

[0084] 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 refers 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.

[0085] 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.

[0086] “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers 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.

[0087] 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 tablesproviding 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.

[0088] 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)).

[0089] 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.

[0090] 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, multispecific 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, unlike conventional 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 humanantibody, 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.

[0091] 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.

[0092] 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.

[0093] 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 covalently linked 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.

[0094] 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 fordifferent 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.

[0095] As disclosed herein, the term “secondary antibody” or “2ndantibody” refers to an antibody that binds to another antibody, typically referred to as the “primary antibody”. Whereas the primary antibody recognizes the target antigen, the secondary antibody recognizes the primary antibody. Secondary antibodies are commonly raised against the species and isotype of the primary antibody. As a non-limiting example, a primary antibody may be a murine IgG monoclonal antibody specific to a biomarker of interest, and the secondary antibody is an anti-murine IgG antibody. Secondary antibodies have many uses, including detection or isolation or primary antibodies alone or bound to their antigen, or delivery of payloads to the site of the primary antibody’s antigen. For example, a secondary antibody may carry a detectable label, and thereby facilitate indirect detection via the label of antigen that is bound by the primary antibody. As another example, a secondary antibody may carry a payload such as a therapeutic agent, and thereby facilitate indirect delivery of the therapeutic agent to antigen that is bound by the primary antibody. As used herein, the secondary antibody carrying a therapeutic agent may be referred to as a secondary antibody-drug conjugate, secondary ADC, 2ndantibody drug conjugate, 2ndADC, or the like. For example, as used herein “anti- DNA Ab + 2ndADC” refers to an anti-DNA primary antibody together with a secondary antibody that carries a cytotoxic payload. See, e.g., FIGS. 1A-1D and related discussion.

[0096] 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 DNA. In particular embodiments, the antigen is extracellular DNA. 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.

[0097] 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 and numbers + / - 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

[0098] The present disclosure provides method of targeting cells of interest using anti-DNA antibodies. Such antibodies may be internalized after contact with the target cells. In an aspect, the present disclosure provides a method of internalizing one or more anti-DNA antibodies (which is understood to include DNA-binding antibody fragments herein) into a cell, the method comprising contacting the cell with the one or more anti-DNA antibodies or DNA- binding antibody fragments that bind to extracellular DNA on the surface of the cell and are internalized into the cell after the binding. In some embodiments, the cell is a mammalian cell, including without limitation a human cell. In some embodiments, the method further comprises contacting the cell with one or more nucleases before, after or at the same time as the one or more anti-DNA antibodies or DNA-binding antibody fragments disclosed herein. Furthermore, the one or more anti-DNA antibodies or DNA-binding antibody fragments 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.ANTI-DNA ANTIBODY

[0099] As used herein, an “anti-DNA antibody” or “DNA-binding antibody” refers to an antibody or antibody fragment that can recognize and bind to sites on the phosphodiester backbone of single-stranded DNA (ssDNA) and / or double-stranded DNA (dsDNA). Such binding may be to nucleotide sequences, including such sequences in higher-order structures such as nucleosomes. See, e.g., FIG. 1C. In some instances, the anti-DNA antibodies or DNA- binding antibody fragments recognize and bind to single-stranded DNA (ssDNA). In other instances, the anti-DNA antibodies or DNA-binding antibody fragments recognize and bind to double-stranded DNA (dsDNA). In yet other instances, the anti-DNA antibodies or DNA- binding antibody fragments recognize and bind to both ssDNA and dsDNA. In some embodiments, the anti-DNA antibodies or DNA-binding antibody fragments recognize and bind to extracellular DNA on the surface of a cell. In some embodiments, the anti-DNA antibodies or DNA-binding antibody fragments recognize and bind to extracellular DNA in the vicinity around a cell. As a non-limiting example, the vicinity of the cell could be a tumor microenvironment.

[0100] The anti-DNA antibodies or fragments provided herein can bind to specific or unspecified sequences as desired. In some instances, the one or more anti-DNA antibodies orDNA-binding antibody fragments bind to one or more known DNA sequences. In other instances, the one or more anti-DNA antibodies or DNA-binding antibody fragments bind to one or more unknown DNA sequences. In yet other instances, the one or more anti-DNA antibodies or DNA-binding antibody fragments bind to both known and unknown DNA sequences. In addition, in some instances, the one or more anti-DNA antibodies or DNA- binding antibody fragments are sequence specific. In other instances, the one or more anti- DNA antibodies or DNA-binding antibody fragments are non-sequence specific. As used herein, a non-sequence specific antibody refers to an antibody that binds to more than one epitope of an antigen or binds to more than one antigen. As a non-limiting example, the non- sequence specific constructs could comprise a polyclonal antibody composition. In yet other instances, the one or more anti-DNA antibodies or DNA-binding antibody fragments comprise a combination of sequence specific and non-sequence specific anti-DNA antibodies.

[0101] In some cases, the target DNA sequences recognized by the anti-DNA antibodies or fragments thereof are less than 10 base pair long. In some cases, the target DNA sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more base pair long. In some cases, the target DNA sequence is about 20, 50, 100, 1000, 5000, 10,000, or more base pair long. As disclosed herein, the anti-DNA antibody can bind to a specific DNA fragment. Alternatively, the anti- DNA antibody can bind to two or more DNA fragments.

[0102] As disclosed herein, a plurality of anti-DNA antibodies that form an antibody polymer, complex, or cluster, can be internalized into a 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 has more than five anti-DNA antibodies. The polymer can have any desired number of antibodies. In some embodiments, such polymers are coated on a carrier such as a nanoparticle, including without limitation gold nanoparticles. In some embodiments, the polymers comprise multi-paratopic antibodies which bind to distinct epitopes of the same DNA. In some embodiments, the polymers form antibody cocktails. Without being bound by theory, a polymer having multiple target sequences may facilitate cellular recognition and subsequent internalization. See, e.g., FIG. 10 and related discussion.

[0103] As disclosed herein, the anti-DNA antibody can covalently or non-covalently link to at least one payload. The payload may comprise any useful molecule or entity, including without limitation one or more 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-DNA antibody or DNA-binding antibody fragment. In some embodiments, the anti-DNA antibody or DNA-binding antibody fragment is bound by a secondary antibody, and the payload is conjugated to the secondary antibody. In some embodiments, the one or more payloads are bound to both the anti-DNA antibodies and the secondary antibody. Non-limiting exemplary anti-DNA ADCs / ADC complexes are shown in FIG. ID

[0104] In some embodiments, multiple anti-DNA antibodies are attached to a carrier such as a nanoparticle to form an antibody complex / cluster (see, e.g., FIG. ID, anti-DNA ADC conjugated on nanoparticle), the nanoparticle can be any useful nanoparticle, including without limitation a polymer-based, non-polymeric, or lipid-based nanoparticle. Polymer-based nanoparticles include without limitation dendrimers, nanoparticles, micelles, nanogels, protein nanoparticles, and drug conjugates. Non-polymeric nanoparticles include without limitation carbon nanotubes, nanodiamonds, metallic nanoparticles, quantum dots, and silica-based nanoparticles. Lipid-based nanoparticles include without limitation liposomes, vesicles (e.g., microvesicles or exosomes) and solid lipid nanoparticles. See, e.g., 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, 2, 3, 4, 5, 6, 7, 8, or more anti-DNA antibodies are coated on a nanoparticle. In some embodiments, the nanoparticle is 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, identical anti-DNA antibodies can be coated onto one such carrier. In some embodiments, different anti-DNA antibodies are coated on the same carrier.

[0105] In some embodiments, the one or more anti-DNA antibodies are a multi-paratopic antibody. See, e.g., Ludwig SD, et al. Multiparatopic antibodies induce targeted downregulation of programmed death-ligand 1. Cell Chem Biol. 2024 May 16;31(5):904- 919. el 1. Multiparatropic anti-DNA antibodies may bind to two or more distinct sites within one DNA fragment. In some cases, the multi-paratopic antibodies may bind to the same or different DNA fragments and form an antibody cluster that may promote cellular internalization. In some cases, the anti-DNA multi -paratopic antibody is a bi-paratopic antibody, which recognizes two target sequences. In some cases, the anti-DNA multi-paratopicantibody is a tri-paratopic antibody, which recognizes three target sequences. The multiparatopic can be configured to recognize any desired number of targets. Some exemplary biparatopic anti-DNA ADCs or ADC complexes are demonstrated in FIG. ID.

[0106] The compositions and methods disclosed herein can also be used to internalize any desired number of anti-DNA antibodies. In some embodiments, 2, 3, 4, 5, 6, 7, 8, or more anti- DNA antibodies are used. In some instances, the multiple anti-DNA antibodies entering the cell are same kind of anti-DNA antibody which recognize and bind a same DNA epitope / site / fragment. In other instances, the multiple anti-DNA antibodies entering the cell are different anti-DNA antibodies. The term “different anti-DNA antibodies” refers to a group of anti-DNA antibodies that bind to different DNA epitopes / sites / fragments. In some instances, multiple different anti-DNA antibodies bind to different DNA epitopes / sites / fragments in one DNA sequence. In other instances, multiple different anti-DNA antibodies bind to different DNA sequences. In yet other instances, some different anti-DNA antibodies bind to different DNA epitopes / sites / fragments in one DNA sequence and some other different anti-DNA antibodies bind to different DNA sequences on the cell surface. In some cases, the multiple anti-DNA antibodies binding the cell are a combination of same and different anti-DNA antibodies.

[0107] As disclosed herein, multiple anti-DNA antibodies can form a cluster / complex that can bind to extracellular DNAs on the cell surface and enter the cell. In some instances, the cluster / complex can be formed before or after the multiple anti-DNA antibodies bind to extracellular DNAs. In some instances, the cluster / complex can be formed at the same time that the multiple anti-DNA antibodies bind to extracellular DNAs. In some instances, the multiple anti-DNA antibodies can form a polymer, such as a dimer, a trimer, a tetramer, a pentamer, or a polymer with any desired number of antibodies. In some instances, the multiple anti-DNA antibodies can bind to 2ndantibodies. In some instances, the multiple anti-DNA antibodies can be coated on a carrier. In some instances, the multiple anti-DNA antibodies can be crosslinked to each other. In some instances, the multiple anti-DNA antibodies can be mixed as an antibody cocktail.

[0108] A variety of anti-DNA antibodies are known and in some embodiments are used in the compositions and methods described herein. Examples of commercially-available anti- DNA antibodies which can be used in the compositions and methods provided herein include, but are not limited to, antibody 121-3 (Abeam, part of Danaher Corporation, Washington, DC),antibody 3519 DNA (Abeam), antibody SPM603 (Abeam), antibody DSD958 (Abeam), antibody BV16-13 (MilliporeSigma, part of Merck KGaA, Darmstadt, Germany), antibody AE-2 (MilliporeSigma), antibody 4565 (NeoBiotechnologies, Union City, CA), antibody TNT- 3 (MilliporeSigma), antibody 16-19 (MilliporeSigma), and antibody F7-26 (MilliporeSigma).

[0109] In some instances, the anti-DNA antibodies provided herein recognize at least one specific known extracellular DNA sequence. In some embodiments, the anti-DNA antibodies recognize and bind to extracellular DNA sequences encoding segments of cancer related genes. Such cancer related genes include without limitation, KRAS, TP53, BRAF, PIK3CA and IDH1. The anti-DNA antibodies may be specific to wildtype sequences of the cancer genes of interest, or mutant forms thereof such asG12D KRAS, R175H TP53, V600E BRAF, E545K PIK3CA, or R132H IDH1. Other exemplary cancer related genes that could be targeted by the anti-DNA antibody compositions and methods provided herein can be found using the COSMIC (Catalogue of Somatic Mutations in Cancer) database, available at cancer.sanger.ac.uk / cosmic.NUCLEASE

[0110] As disclosed herein, the method of anti-DNA antibody internalization can further include contacting the target cell with one or more agents that break extracellular DNA into pieces. Without being bound by theory, smaller DNA fragments may promote internalization of the anti-DNA antibody or fragment into the cell. In some embodiments, such agents comprise one or more nucleases. The terms “nuclease,” “nucleotide polymerase” 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 exo-nuclease functions. As disclosed herein, the DNA nuclease has DNA cleaving activity but may also be able to cleave RNA. Any useful combination of exonucleases, endonucleases, exo-endonucleases, and DNA / RNA nucleases may be used. See FIGS. 5A-5K and elsewhere herein.[OHl] In some embodiments, the one or more nuclease comprises an endonuclease selected from the group consisting of a deoxyribonuclease (DNase), Benzonase®, a serratia marcescensDNA / RNA nuclease, a micrococcal nuclease (MNase), a transposase, 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 functional derivative, fragment or fusion thereof.

[0112] In some embodiments, the one or more nucleases comprise a DNase. In some embodiments, the DNase is 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 I is a recombinant human DNase I. In some embodiments, the DNase I is a Dornase alfa (brand name Pulmozyme), an FDA approved drug for cystic fibrosis treatment.

[0113] 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.

[0114] 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.

[0115] 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 otherinstances, the recognition and cleavage sites of the restriction enzyme are separate from one another.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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 at least one, two, three, four, five, six, seven, eight or more different anti-DNA antibodies. In some embodiments, the at least one, two, three, four, five, six, seven, eight or more different anti-DNA antibodies comprises one, 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 one or more anti-DNA 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-DNA antibodies or DNA-binding antibody fragments. In yet other instances, the one or more nucleases (e.g., DNA nucleases) are contacted to the cell after the anti-DNA antibodies or DNA-binding antibody fragments.

[0120] 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 one or more nuclease is or comprises DNase I, which may be from human or non-human sources (e.g., bovine, murine). In other instances, the one or more nuclease is or comprises Benzonase. In other instances, the one or more nuclease is or comprises a bacterial nuclease (e.g., MNase). See, e.g., FIGS. SASK and related discussion herein. The nucleases may be recombinant and / or humanized.PAYLOAD

[0121] As disclosed herein, the method of internalizing anti-DNA antibodies can be used to deliver desired payload, linked to the antibodies, to the target cells, including without limitation therapeutic agents and / or detectable labels. In some embodiments, the one or more anti-DNA antibodies are attached to at least one payload. See, e.g., FIG. IB and related discussion herein. In some embodiments, the one or more anti-DNA antibodies or DNA-binding antibody fragments are bound by one or more secondary antibodies, wherein the one or more secondary antibodies are attached to at least one payload. See, e.g., FIGS. 1A and 1C and related discussion herein. Such attachments can be covalent, non-covalent, direct, indirect, or any useful combination thereof. In some instances, the one or more anti-DNA antibodies and / or the one or more secondary antibodies are covalently linked to the at least one payload. In some instances, the one or more anti-DNA antibodies and / or the one or more secondary antibodies are non-covalently linked to the at least one payload. Non-limiting examples of non-covalent attachment include use of binding agents, e.g., antibodies and / or aptamers, or via other strong and specific associations such as avidin-biotin. For example, in some embodiments, the anti- DNA antibody or the secondary antibody is attached to a biotin moiety, and the at least one payload is attached to a streptavidin or avidin, thereby the antibody and the payload is non- covalently linked. The anti-DNA 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-DNA antibody are produced as a translational fusion. In some embodiments, the anti-DNA antibody is prepared and then attached to the payload via a chemical linker. In some embodiments, the compositions and methods herein contemplate direct and / or indirect payload attachment. An example of direct attachment includes conjugation of a small molecule payload to the polypeptide itself. Alternately, the small molecule payload could be indirectly attached to the polypeptide such as via a linker or encapsulated within a particle (e.g., a nanoparticle, liposome, microvesicle, bead, etc.) that isattached to the polypeptide. Payloads can be attached through any useful combination of covalent, non-covalent, direct and indirect mechanisms.

[0122] As noted, the method provided herein can be used to deliver one or more payloads to the target cell. In some embodiments, one anti-DNA 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-DNA antibody delivers multiple copies of a payload molecule into the cell. In other instances, one anti-DNA 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, directly or indirectly, to the anti-DNA antibody. The binding of one or more payloads to the anti-DNA antibody will not impact the binding affinity of the antibody to the target DNA.

[0123] Any useful and desired payload can be delivered using the method provided herein. Such flexibility allows the anti-DNA 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.

[0124] 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-DNA antibody, such a tumor cell. Such an application may be intended to provide a therapeutic effect.

[0125] Examples of therapeutic agents that may be attached as payload to the anti-DNA 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, radiopharmaceuticals, antiprion agents, and combinations thereof.

[0126] For example, the therapeutic agent may be an antitumor agent comprising an aromatase inhibitor; an anti-estrogen; an anti-androgen; 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 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, or any desired combinations thereof.

[0127] 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, or any desired combinations thereof. Additional examples of antitumor and other therapeutic agents are known in the art.

[0128] In some embodiments, the antitumor agent comprises 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 tubulin inhibitors include, but are not limited to, 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. 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. Thesetubulin 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 antibody-drug 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 tubulin 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-DNA antibody as disclosed herein.

[0129] In some embodiments, the antitumor agent comprises 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, an alkylator, duocarmycin, duocarmycin DM (DMDM), calicheamicin, pyrrolobenzodiazepine (PDB), enediyne, uncialamycin, a topoisomerase inhibitor, topotecan, camptothecin (CPT), exatecan, and any derivative thereof.

[0130] In some embodiments, the antitumor agent comprises 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, an RNA splicing inhibitor, RNA polymerase II inhibitor, thailanstatin, amatoxin, and any derivative thereof.

[0131] 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, radioactive label, or a combination thereof.

[0132] 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.

[0133] In some cases, the payload may be attached to the anti-DNA antibody covalently, including without limitation direct conjugation to the DNA-binding antibody, via a linker entity, or both. In other cases, the payload may be attached to the anti-DNA antibody non- covalently. In a non-limiting example, the anti-DNA antibody may be conjugated to a biotin moiety, and the payload could be attached to a streptavidin or avidin. In this example, the biotin-streptavidin bond or the biotin-avidin bond would provide the non-covalent attachment between the anti-DNA antibody and the payload. In another example of non-covalent binding between the anti-DNA antibody and the payload, the payload is conjugated to a secondary antibody, and the secondary antibody binds to the anti-DNA antibody. In yet other cases, e.g., in the case of multiple payloads linked to the anti-DNA antibodies, the payloads may be attached both covalently and non-covalently.LINKER

[0134] As disclosed herein, the payloads may be linked to the anti-DNA antibodies or DNA- binding antibody fragments via a linker. At noted, multiple linkers can be used to connect the antibodies and the payloads. In some embodiments, one anti-DNA antibody is linked to one or more payloads via one, two, three, four, five, six, seven, eight or more identical or different linkers. In some instances, the anti-DNA antibody is linked to the one or more payloads using the same linker. In other instances, the anti-DNA antibody is linked to the one or more payloads using different linkers. In some embodiments, one secondary antibody is linked to one or more payloads via one, two, three, four, five, six, seven, eight or more identical or different linkers. In some instances, the secondary antibody is linked to the one or more payloads using the same linker. In other instances, the secondary antibody is linked to the one or more payloads using different linkers. As used herein, a linker can be any desired molecule, complex, or other entity that links an antibody (an anti-DNA antibody or a secondary antibody) and a payload.

[0135] In some instances, the linker comprises 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 comprises 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).

[0136] In other instances, the linker comprises a cleavable linker. In such instances, the linker is cleaved after contacting the cell with the one or more anti-DNA antibodies, thereby releasing the payload on or in the cell. In some embodiments, the cleavable linker comprises 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. Antibodydrug 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 glutathionesensitive linker, a disulfide linker, and a combination thereof. In some instances, the cleavable linker comprises 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-DNA antibody is stable in extracellular fluid but is cleaved by cathepsin B once 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-DNA 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- DNA antibodies.

[0137] As noted, the method provided herein can be used to deliver one or more payloads to the target cell. In some embodiments, one anti-DNA 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-DNA antibody delivers multiple copies of a payload molecule into the cell. In otherinstances, one anti-DNA 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, directly or indirectly, to the anti-DNA antibody. The binding of one or more payloads to the anti-DNA antibody will not impact the binding affinity of the antibody to the target DNA.EXTRACELLULAR DNA

[0138] The anti-DNA antibodies provided herein recognize extracellular DNA on the cell surface. In contrast to intracellular DNA, which is DNA located within cell membranes, extracellular DNA refers the DNA located outside cell membranes, for example DNA on the cell surface. Extracellular DNA is present abundantly on the surface and vicinity of abnormal cells (e.g., cancer cells, diseased cells), especially cells having impaired regulation including but not limit to cell cycle progression, senescence, DNA repair, cell death, cell metabolism, development, aging, cell differentiation, but is absent from normal cells. As described herein, the extracellular DNA can carry information about the cell of interest from which the nucleic acid is derived. In some embodiments, the extracellular DNA is derived from nuclear or mitochondrial DNA. In some embodiments, the extracellular DNA is derived from the microenvironment of a cell. In some embodiments, the extracellular DNA is packaged around one or more extracellular nucleosomes. In such embodiments, the anti-DNA antibodies can recognize and bind to the extracellular DNA of the one or more extracellular nucleosomes. See. e.g., FIG. 1C and related discussion herein.

[0139] In some instances, the anti-DNA antibodies provided herein recognize and bind to at least one specific known extracellular DNA (i.e., a known DNA sequence). In such instances, the target extracellular DNA can be chosen to allow the anti-DNA antibody to identify one or more cell of interest. In preferred embodiments, the target extracellular DNA originates within the target cell. For example, the target extracellular DNA can carry one or more mutation that identifies the target cell as a mutated or diseased cell, including without limitation a cancer cell. The present disclosure further contemplates that the target extracellular DNA is derived from the target cell’s microenvironment. In a non-limiting example, consider that the target cell of the anti-DNA antibody is a cell within a tissue, such as a tumor tissue. If the target cell is necrotic or apoptotic, it may release nucleic acids into its microenvironment, in this example the tumor microenvironment. As a result, other cells within the microenvironment may also become target cells if the released nucleic acids attach thereto. The disclosure also envisions that the target cell actively releases nucleic acid into its microenvironment, e.g., in an area ofinflammation. Such necrosis, apoptosis, inflammation, or other cell damage or response may be induced by the cell or its environment (e.g., due to immune response), or both.

[0140] Particular nucleic acids derived from a cell can be used to specifically target the desired cell in this method. In some embodiments, the target nucleic acid has a wild-type (WT) sequence. In some embodiments, the target nucleic acid has a sequence comprising one or more mutations. As used herein, unless stated otherwise a mutation can refer to any sequence other than a “normal” wild type sequence. For example, a mutation can be a single nucleotide variant sequence (whether pathogenic or not), more than one such variant, an insertion, a deletion, a substitution, inversion, translocation, fusion, break, loss, duplication, amplification, or repeat. The anti-DNA antibody used in the method provided herein can recognize cells whose genomic DNA differs by a single point mutation. The anti-DNA antibody can also be targeted to a sequence that occurs from a genomic alteration, such as a sequence created by a translocation, break or loss in a sequence. As described herein, cells may export nucleic acids such as gDNA due to inflammation, disease, or cellular damage. Thus, the levels of nucleic acids may be used to target the cell of interest. In a non-limiting example, an amplification event in cancer may produce abnormally high levels of a certain sequence. The anti-DNA antibody provided herein may target such amplified nucleic acids.

[0141] In other instances, the anti-DNA antibodies used in the method provided herein can recognize and bind to unknown extracellular DNA, i.e., where the target sequence or precise epitope is not known. In other words, the target epitope need not be known so long as the antibody binds DNA that can be used to target the cells of interest. Such anti-DNA antibodies can 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). In yet other instances, the anti-DNA antibodies provided herein can recognize and bind to both known and unknown extracellular DNA sequences. In some embodiments, the anti-DNA antibodies recognize and bind to extracellular DNA sequences encoding wildtype or mutant KRAS, wildtype or mutant TP53, wildtype or mutant BRAF, wildtype or mutant PIK3CA, or wildtype or mutant IDH1. In some embodiments, the mutant gene comprises G12D KRAS, Q61H KRAS, R175H TP53, R273H TP53, V600E BRAF, E545K PIK3CA, or R132H IDH1.TARGET CELLS

[0142] As described herein, the method provided herein can be used in various applications. In non-limiting examples, the anti-DNA 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.

[0143] 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.

[0144] 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; extragonadalgerm 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.

[0145] In some embodiments, the cancer 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 cellcancer (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.

[0146] 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.

[0147] In some embodiments, the cell has an aneuploidy and / or DNA repair deficiency. Without being bound by theory or intending to limit the scope of the invention, it is believed that such cells may have extracellular DNA on their surface due to the aneuploidy, mutations or lower expression of DNA repair enzymes. In some embodiments, the cell has an aneuploidy. In some embodiments, the cell has abnormal or low efficiency of DNA damage repair 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 Rad3 related (ATR) kinases, and poly (ADP- ribose) polymerases (PARPs). In some embodiments, the cell comprises a functionally impaired transcription factor selected from the group consisting of p53, NF-Kb, AP-1, E2F1, breast cancer-associated protein 1 (BRCA1), and breast cancer-associated protein 2 (BRCA2). In some embodiments, the functional impairment of the transcription factor is caused by one or more gene mutations, loss of one or more gene, loss of one or more chromosomal region, and / or deficient expression of one or more proteins. 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 cells have at least onemutation, deficient expression, loss, or other deficiency in one or more mismatch repair (MMR) gene, including without limitation the MLH1, MLH2, MLH3, MSH2, MSH6, PMS1 and / or PMS2 genes. In some embodiments, the cell comprises one or more mutations or other deficiencies in one or more 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.

[0148] In particular embodiments, the cell comprises a functionally impaired transcription factor p53, optionally wherein the impairment of p53 comprises a mutation. p53, also known as Tumor protein P53, TP53, 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 one or more deficiencies of the TP53 gene (such as one or more mutations of the TP53 gene), reduced p53 expression, and / or an inhibited p53 activity. In some embodiments, the functionally impaired p53 results in cellular aneuploidy. In some embodiments, the functionally impaired p53 is present in an abnormal cell such as a cancer cell or other diseased cell. In some embodiments, the functionally impaired p53 is present in a cell with extracellular DNA 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, R273, and / or R282. In particular embodiments, the p53 mutation is a R175H, Y220C, G245S, R248Q, R248W, R249S, R273H, R273C, or R282W mutation. See, e.g., FIGS. 8A-8B and related discussion.METHODSMethod of binding

[0149] Provided herein is a method of binding one or more anti-DNA antibodies to extracellular DNA on a cell surface comprising contacting the cell with the one or more anti- DNA antibodies provided herein. The method can employ various configurations of the anti-DNA antibodies, fragments, and related ADC constructs as desired for a given application. Such various configurations of the anti-DNA antibodies, fragments, and related ADC constructs can be as provided herein. In some embodiments, the anti-DNA antibody is attached to a cytotoxic payload and binding of the anti-DNA antibody kills the cell. In some embodiments, the anti-DNA antibody is attached to a detectable payload and the method further comprises detecting binding of the anti-DNA 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 cells in a biological specimen, wherein the one or more anti-DNA antibodies are bound to or internalized within the target cells. 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

[0150] 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-DNA antibodies as provided herein, and detecting the anti-DNA antibodies bound to and / or internalized into the at least one cell or tissue. In some embodiments, the one or more anti-DNA antibodies are 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-DNA 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 wildtype extracellular DNA on the surface, wherein the anti-DNA antibody disclosed herein binds to the mutant or wildtype extracellular DNA. In some embodiments, the anti-DNA antibody is specific for either the mutant or wildtype form of the DNA. As a non-limiting example, such anti-DNA antibody could be used to image cells harboring a particular mutation of interest, such as a cancer mutation.

[0151] 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, animmune 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 any cancer of interest, including without limitation other cancers provided herein.Method of delivering

[0152] Also provided herein is a method of delivering a payload inside of a cell comprising the extracellular DNA. Any useful and desired payload can be delivered, including without limitation therapeutic agents or other payloads as described herein. In an aspect, the method comprises contacting the cell with the anti-DNA antibody linked with a therepeutic agent, including but not limted to a cytotoxic drug, wherein the anti-DNA antibody binds to the extracellular DNA on the cell and internalizes the therepeutic agent inside of the cell. In some embodiments, the anti-DNA antibody is directly linked to the therapeutic agent, covalently or non-covalently. In some embodiments, the anti-DNA antibody is indirectly linked to the therepeutic agent. For example, the anti-DNA antibody can be bound by a secondary antibody, wherein the secondary antibody is covalently or non-covalently linked to the therepeutic agent. Such anti-DNA antibody constructs or complexes linked to one or more therapeutic agent may be referred to herein as anti-DNA antibody-drug conjugates, or anti-DNA ADCs.

[0153] In some embodiments, the method further comprises contacting the cell with one or more nucleases, thereby facilitating internalization of the anti-DNA antibody and corresponding payload. See, e.g., FIGS. 5A-5K herein and related discussion. The nuclease can be an endonuclease, an exonuclease, or a combination thereof. In some embodiments, the one or more nucleases comprise one or more single stranded DNA (ssDNA) nuclease, one or more double-stranded DNA (dsDNA) nuclease, or a combination thereof. In some embodiments, the one or more nucleases and the anti-DNA antibodies conjugated with payloads, including without limitation anti-DNA ADCs, are contacted to the cell at the same time. In some embodiments, the one or more nucleases are attached to the anti-DNA antibodies.In some embodiments, the one or more nucleases are contacted to the cell before the anti-DNA antibodies conjugated with payloads, including without limitation anti-DNA ADCs. In some embodiments, the one or more nucleases are contacted to the cell after the anti-DNA antibodies carrying payloads, including without limitation anti-DNA ADCs. As desired, the one or more nucleases can be contacted with the cell on a schedule comprising one or more contacts of the nucleases including before, during and / or after the anti-DNA antibodies carrying payloads.

[0154] As described herein, the payload conjugated with the anti-DNA 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 payload comprises a liposome or nanoparticle. In such cases, the liposome or nanoparticle may carry the desired therapeutic agent inside. The anti-DNA 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 further described herein, 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

[0155] Further provided herein is a method of treating or ameliorating a disease or disorder in a human subject in need thereof, comprising administering a pharmaceutically effective amount of a composition comprising an anti-DNA antibody to the subject. In preferred embodiments, the anti-DNA antibody comprises an anti-DNA ADC. As disclosed herein, the anti-DNA antibody binds extracellular DNA 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 DNA and therefore specifically kill the target cells. In some embodiments, the composition further comprises one or more nucleases. In some embodiments, the one or more nucleases comprise DNA nucleases. As disclosed herein, the nuclease can be an endonuclease, an exonuclease, or a combination thereof. In some embodiments, the one or more nucleases and the anti-DNA antibodies with therapeutic payloads are contacted to the cell at the same time. In some embodiments, the one or more nucleases are attached to the anti-DNA antibodies.In some embodiments, the one or more nucleases are contacted to the cell before the anti-DNA antibodies with therapeutic payloads. In some embodiments, the one or more nucleases are contacted to the cell after the anti-DNA antibodies with therapeutic payloads. In some embodiments, the one or more nucleases are contacted with the cell on a schedule comprising one or more contacts before, during, or after contact of the anti-DNA antibodies carrying therapeutic payloads.

[0156] 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. 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 or other caregiver 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 therapeutically effective 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 or other caregiver upon the appreciation of the disclosure set forth herein.

[0157] 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 subject 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. In some embodiments, the anti-DNA antibodies provided herein are used in the treatment of cancer.

[0158] 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.

[0159] As used herein, the term “diagnostic agent” refers to any chemical used in the imaging of diseased tissue, such as, e.g., a tumor. Non-limiting examples of imaging agents and detectable labels are provided herein.

[0160] In some embodiments, the disease or disorder comprises 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 pharmaceutically effective amount of the composition provided herein is administered to a subject, thereby allowing targeting of cancer cells.

[0161] 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, including but not limited to a transcription factor selected from the group consisting of p53, NF-Kb, AP-1, E2F1, BRCA1 and BRCA2. In some instances, the determining step comprises detecting one or more mutations or other deficiencies (e.g., loss or deficient expression) of the transcription factor gene. In other instances, the determining step comprises detecting one or more mutations or other deficiencies of other DNA repair related genes. In some embodiments, the cells have at least one mutation or other deficiencies in oneor more mismatch repair (MMR) genes, including without limitation the MLH1, MLH2, MLH3, MSH2, MSH6, PMS1 and / or PMS2 genes. In some embodiments, the determining step comprises detecting one or more mutations or other deficiencies in one or more 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; polfi; 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 deficiency of the TP53 gene (such as 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

[0162] In an aspect, provided herein is a composition comprising one or more anti-DNA antibodies disclosed herein. In some embodiments, the composition further comprises one or more nucleases. In some embodiments, the one or more nucleases comprise DNA nucleases. In some embodiments, the one or more anti-DNA antibodies are bound by a secondary antibody. In some embodiments, the one or more anti-DNA antibodies are coated on a carrier such as a gold nanoparticle or other carrier such as described herein. In some embodiments, at least two anti-DNA 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-DNA antibodies are different anti-DNA antibodies. In some cases, the different anti-DNA antibodies are crosslinked together. Examples of antibody crosslinking can include for example chemical cross-linking, for example but not limited to as in Ueda et al^IA J Mol Sci. 2020 Feb; 21(3): 711. In some cases, the different anti-DNA antibodies comprise multi-paratopic antibodies. Various embodiments comprising carriers and polymeric constructs are described further herein. See also FIG. ID and related discussion.

[0163] In some embodiments, the composition further comprises at least one payload covalently or non-covalently linked to the one or more anti-DNA antibodies. In some embodiments, the at least one payload is directly conjugated to the one or more anti-DNA antibodies. In some embodiments, the one or more anti-DNA antibodies are bound by one or more secondary antibodies, and the at least one payload is linked, e.g., directly conjugated, to the secondary antibodies. In some embodiments, multiple payloads are linked to the one or more anti-DNA antibodies and / or the one or more secondary antibodies. As a non-limitingexample, at least one payload is conjugated to the anti-DNA antibody and at least one payload is conjugated to the secondary antibody. In some instances, the anti-DNA antibody and / or the secondary antibody are non-covalently linked to the payload. For example, in some embodiments, the anti-DNA antibody or the secondary antibody is attached to a biotin moiety, and the at least one payload is attached to a streptavidin or avidin, thereby the antibody and the payload are non-covalently linked. Various embodiments comprising anti-DNA antibodies and payloads are disclosed further herein. See also FIG. ID and related discussion.

[0164] As provided herein, a pharmaceutical composition may comprise a therapeutically effective amount of the composition disclosed above, and a pharmaceutically acceptable excipient, carrier, and / or diluent.

[0165] Also provided herein is a pharmaceutical composition comprising a therapeutically effective amount of the anti-DNA antibody and at least one payload, such as a therapeutic agent. In some embodiments, the therapeutic agent comprises 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, or any desired combination thereof. In some embodiments, the antitumor agent comprises a tubulin inhibitor, a DNA inhibitor, and / or an RNA inhibitor. In some embodiments, the tubulin 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.

[0166] In some embodiments, the pharmaceutical composition further comprises one or more nucleases. In some embodiments, the one or more nucleases comprise DNA nucleases. In some embodiments, the 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 transposase, a restriction enzyme, nuclease SI, nuclease Pl, a sequence specific endonuclease, or a sequence nonspecific endonuclease. In some embodiments, the one or more DNA nucleases comprise a single stranded DNA (ssDNA) nuclease, a double-stranded DNA (dsDNA) nuclease, or acombination thereof. Further embodiments comprising nucleases are disclosed herein. See also FIGS. 5A-5J and related discussion.

[0167] 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-DNA antibodies. In some embodiments, the additional agent comprises non-targeted therapies. As a non-limiting example, an anti-DNA ADC 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, a topoisomerase inhibitors and / or corticosteroids.

[0168] 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-DNA antibody, a detection reagent, a secondary detection reagent, a wash buffer, an elution buffer, a solid support, and any combination thereof.Administration

[0169] 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.

[0170] 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-DNA antibody required to be administered may depend on the judgment of the treating physician or other caregiver.

[0171] 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.

[0172] 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 administration 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.

[0173] As used herein, “pharmaceutical formulations” include formulations for human and veterinary use with acceptable levels of adverse toxicological effects, if any. “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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] Sterile injectable solutions are prepared by incorporating the active ingredients (i.e. anti-DNA 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.

[0178] 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.

[0179] 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.

[0180] 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-DNA 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-DNA 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 an anti- DNA antibody engineered to target an alternate target nucleic acid sequence.

[0181] In some embodiments, the pharmaceutical composition provided herein is administered contemporaneously with at least one nuclease. As used herein, contemporaneous administration indicates that the pharmaceutical composition and at least one nuclease 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-DNA antibody at least one nuclease may be coadministered or administered sequentially. The timing of the administration of the anti-DNA antibody and at least one nuclease 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. In preferred embodiments, at least some doses of the at least one nuclease are administered before, e.g., at least 5 min, 10 min, 15 min, 20 min, 30 min, Ih, 2h, or 3h before, administration of the anti-DNA antibody. The regimen of timing can be determined by the treating physician.IV. Examples

[0182] 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

[0183] Human breast cancer cell lines (AU565, SK-BR-3, HCC1395, MDA-MB-468, HCC1806, Hs 578T, MDA-MB-453, MCF-7, DU4475, BT474), human pancreatic cancer cell lines (SW1990-1, AsPC-1), human lung cancer cell line (NCI-H460) and non-tumorigenic human cell lines (MCF10A, HCC1395 BL) were purchased from ATCC (Manassas, VA).AU565, HCC1395, HCC1806, DU4475, BT474, AsPC-1 and SW1990 cells were cultured at 37 °C with 5% CO2 in Roswell Park Memorial Institute 1640 Medium (RMPI1640) supplemented with 10% Fetal Bovine Serum (FBS). MDA-MB-468, Hs 578T, MDA-MB-453, MCF-7 were cultured at 37 °C with 5% CO2 in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS. SK-BR-3 was cultured at 37 °C with 5% CO2 in McCoy's 5A Medium supplemented with 10% FBS. HCC1395 BL was cultured at 37 °C with 5% CO2 in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 20% FBS. MCF10A cells were cultured at 37 °C with 5% CO2 in Human Mammary Epithelial Cell (HuMEC) supplemented with HuMEC supplement kit. RPMH640, DMEM, McCoy's 5 A, IMDM media and FBS were purchased from ATCC. HuMEC media and HuMEC supplement kit were purchased from Thermo Fisher Scientific Inc. (Waltham, MA).Fluorescent imaging

[0184] Cancer cells were plated in 18-well glass-bottom chambered coverslips (Cat. No. 81817, ibidi USA, Inc. (Fitchburg, WI)) at a density of 4,000 cells per well and incubated for 3 days. The anti-DNA antibody was pre-incubated with an anti-mouse IgG labeled with Alexa Fluor 647 conjugate. The molar ratio of the two components was 1 : 1, and the pre-incubation was carried out in the dark at room temperature for 1 hour. The antibody complex was introduced to the cells at a concentration of 2 pg / ml anti-DNA antibody. Half of the wells were also treated with 20 units / ml of DNase I (M0303S, New England Biolabs Inc (Ipswich, MA)). The cells were then cultured for an additional 3 days at 37°C. Afterward, the cells were washed with PBS, fixed with 4% formaldehyde in PBS, and permeabilized with 0.1% Triton-XIOO. The cell membrane was stained with WGA488 (W11261, Invitrogen, a part of Thermo Fisher), the nucleus / DNA was stained with NucBlue (R37605, Invitrogen), and an antibody against LAMP-1 (NB021157, Thermo Fisher Scientific) was used as a marker for lysosomes. Images were taken using a confocal fluorescent microscope (FV3000, Olympus).Antibody conjugation

[0185] Conjugation of anti-DNA antibody, isotype control antibody and secondary antibody

[0186] An anti-DNA antibody (ab27156, Abeam Limited (Boston, MA)), an isotype control antibody (BE0085, Bio X Cell (Lebanon, NH)) and an anti-mouse IgG secondary antibody (115-005-071, Jackson Immuno Research Labs (West Grove, PA)) were conjugated with MC- Val-Cit-PAB-MMAF (BP-27843, BroadPharm (San Diego, CA)) by DTT partial reduction and thiol-maleimide reaction. The antibodies were treated with 4 molar equivalents of DTT in PBSwith 5 mM EDTA for 2 h at 37 °C. The excess DTT was removed from the partially reduced antibodies through buffer exchange, performed 10 times with an Amicon Ultra-0.5 centrifugal filter unit featuring a 10k molecular weight cut-off (UFC5010, MilliporeSigma). The partially reduced antibody was then alkylated with 5 molar equivalents of MC-Val-Cit-PAB-MMAF at 10°C for 30 minutes. To quench any unreacted, excess MC-Val-Cit-PAB-MMAF, 10 molar equivalents of cysteine hydrochloride (44889, Thermo Fisher Scientific) were added. The MMAF-conjugated antibody was further purified by buffer exchange to PBS 10 times, again using an Amicon Ultra-0.5 centrifugal filter unit with a 10k molecular weight cut-off. The absorbance of the flow-through at 248 nm was monitored to ensure the complete removal of the unconjugated payload. The drug-to-antibody ratio (DAR) of the conjugated antibody was estimated by calculating the UV absorbance ratio (R) between 248 nm and 280 nm using the following equations.R = (A248) / ( A280)DAR = (21 x R - 9) / (1.615 - 0.1425 x R)The three conjugated antibodies had a DAR of approximately 4.Cytotoxicity assay

[0187] Determination of IC50 of anti-DNA antibody + 2ndADC

[0188] Cancer cells (AU565, HCC1395, AsPC-1, SW1990) and non-tumorigenic breast cell line MCF10A were plated in 96-well tissue-culture (TC) treated optical -bottom white plates (165306, Thermo Fisher) at a density of 1,500 cells per well and incubated for 3 days. An anti- DNA antibody (ab27156, Abeam) or an isotype control antibody (AM26775LE-N, Origene Technologies Inc (Rockville, MD)) was pre-incubated with an anti -mouse IgG secondary antibody (115-005-071, Jackson Immuno Research) conjugated with MMAF using a Valine- Citrulline (Val-Cit; or VC) linker (conjugated in house). Antibodies were mixed at a 1 : 1 molar ratio and pre-incubated at room temperature for 1 hour. Subsequently, the antibody complex was added to the cells at various concentrations ranging from 5pg / ml to 1.22ng / ml of anti- DNA antibody. Cells treated with PBS were used as a control. Each condition was tested in triplicate. The cells were then cultured for 6 days at 37°C before assessing viability using the CellTiter-Glo 2.0 assay (G9242, Promega Corporation (Madison, WI)). The amount of luminescence emitted from each well was measured using a multimode microplate reader (Synergy Hl, BioTek Instruments (Winooski, VT)). The percentage of viable cells wascalculated using the luminescence readings of treated wells divided to the luminescence reading from control wells. The IC50 values of each antibody in each cell line were calculated using Prism 10 (GraphPad Software Inc (San Diego, CA)).

[0189] Determination of IC50 of anti-DNA antibody + 2ndADC + DNase I / Benzonase

[0190] Cancer cells (AU565, HCC1395) and non-tumorigenic breast cell line MCF10A were plated in 96-well tissue-culture (TC) treated optical -bottom white plates (165306, Thermo Fisher) at a density of 1,500 cells per well and incubated for 3 days. An anti-DNA antibody (ab27156, Abeam) or an isotype control antibody (AM26775LE-N, Origene) was preincubated with an anti-mouse IgG secondary antibody (115-005-071, Jackson Immuno Research) conjugated with MMAF using a Valine-Citrulline (Val-Cit) linker (conjugated in house). Antibodies were mixed at a 1 : 1 molar ratio and pre-incubated at room temperature for 1 hour. Half of the wells were exposed to 40 units / ml of DNase I (M0303S, New England Biolabs) or Benzonase (E8263, MilliporeSigma) for 1 hour before antibody complex was added. Subsequently, the antibody complex was added to the cells at various concentrations ranging from 5pg / ml to 1.22ng / ml of anti-DNA antibody. Addition of antibody diluted the DNase I or Benzonase to a final concentration of 20 units / ml. Cells treated with PBS were used as control. Each condition was tested in triplicate. The cells were then cultured for 6 days at 37°C before assessing viability using the CellTiter-Glo 2.0 assay (G9242, Promega). The amount of luminescence emitted from each well was measured using a 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. The IC50 values of each anti-DNA antibody in each cell line were calculated using Prism 10 (GraphPad).

[0191] Determination of IC50 of MMAF

[0192] Cancer cell lines (AU565, SK-BR-3, HCC1395, MDA-MB-468, HCC1806, Hs-578t, MDA-MB-453, MCF-7, DU4475, NCI-H460) and non-tumorigenic cell lines (MCF10A, HCC1395 BL) were seeded in 96-well plates (165306, Thermo Fisher) with a density of 1,500 cells per well and allowed to incubate for 3 days. Cells were then treated with varying concentrations of MMAF at 37°C for 3 days, ranging from 25pM to 1.6nM before viability assessment using the CellTiter-Glo 2.0 assay (G9242, Promega). Each condition was tested in triplicate. The amount of luminescence emitted from each well was measured using a multimode microplate reader (Synergy Hl, BioTek). The percentage of viable cells wascalculated using the luminescence readings of treated wells divided to the luminescence reading from control wells. The IC50 values of each anti-DNA antibody in each cell line were calculated using Prism 10 (GraphPad).

[0193] Determination of cytotoxicity of different anti-DNA antibodies + 2ndADC + DNase I

[0194] Cancer cell lines (AU565, SK-BR-3, HCC1395, MDA-MB-468, HCC1806, Hs-578t, MDA-MB-453, MCF-7, DU4475, NCI-H460) and non-tumorigenic cell lines (MCF10A, HCC1395 BL) were plated in 96-well tissue-culture (TC) treated optical -bottom white plates (165306, Thermo Fisher) at a density of 1,500 cells per well and incubated for 3 days. Each of 3 different commercially available anti-DNA antibodies or an isotype control antibody (AM26775LE-N, Origene) were pre-incubated with an anti-mouse IgG secondary antibody (115-005-071, Jackson Immuno Research) conjugated with MMAF using a Valine-Citrulline (Val-Cit) linker (conjugated in house; Val-Cit linker with MMAF may be referred to herein as VC-MMAF). 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 0.2pg / ml of anti-DNA antibody, 0.2pg / ml 2ndADC, and 20 units / ml DNase I. Cells treated with PBS were used as a control. Each condition was tested in triplicate. The cells were then cultured for 3 days at 37°C before assessing viability using the CellTiter-Glo 2.0 assay (G9242, Promega). The amount of luminescence emitted from each well was measured using a multimode microplate reader (Synergy Hl, BioTek). The percentage of viable cells was calculated using the luminescence readings of treated wells divided by the luminescence reading from control wells.

[0195] Determination of IC50 of anti-DNA ADC + 2ndantibody

[0196] HER2 (human epidermal growth factor receptor 2) positive breast cancer cells (AU565, BT474), triple negative breast cell lines (MDA-MB-468, HCC1395) and a non- tumorigenic B lymphoblast cell line (HCC1395 BL) from the same patient of the breast cancer cell line HCC1395 were plated in 96-well tissue-culture (TC) treated optical-bottom white plates (165306, Thermo Fisher) at a density of 1,500 cells per well and incubated for 3 days. An anti-DNA antibody (ab27156, Abeam) or an isotype control antibody (BE0085, Bio X Cell) conjugated with MMAF using a Valine-Citrulline (Val-Cit) linker (conjugated in house) were pre-incubated with an anti-mouse IgG secondary antibody (115-005-071, Jackson Immuno Research). Antibodies were mixed at a 1 : 1 molar ratio and pre-incubated at room temperaturefor 1 hour. Subsequently, the anti-DNA antibody complex was added to the cells at various concentrations ranging from 5pg / ml to 1.22ng / ml of anti-DNA ADC. The isotype control antibody complex was added to the cells at various concentrations ranging from 20pg / ml to 1.28ng / ml of isotype ADC. Half of the wells were also treated with 20 units / ml of DNase I (M0303S, New England Biolabs). Cells treated with PBS were used as control. Each condition was tested in quadruplicate. The cells were then cultured for 6 days at 37°C before assessing viability using the CellTiter-Glo 2.0 assay (G9242, Promega). The 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. The ICso values of each antibody drug conjugate, both with and without DNase I, were calculated for each cell line using Prism 10 (GraphPad).

[0197] Determination of ICso of trastuzumab deruxtecan

[0198] HER2+ breast cancer cells (AU565, BT474), triple negative breast cell lines (MDA- MB-468, HCC1395) and non-tumorigenic B lymphoblast cell from the same patient of the breast cancer cell line HCC1395 were plated in 96-well tissue-culture (TC) treated optical- bottom white plates (165306, Thermo Fisher) at a density of 1,500 cells per well and incubated for 3 days. Cells were then treated with trastuzumab deruxtecan (HY-138298, MedchemExpress (Monmouth Junction, NJ)) at various concentrations ranging from 20pg / ml to 1.28ng / ml. Half of the wells were also treated with 20 units / ml of DNase I (M0303S, New England Biolabs). Cells treated with PBS were used as control. Each condition was tested in quadruplicate. The cells were then cultured for 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. The ICso values of Trastuzumab deruxtecan, both with and without DNase I, were calculated for each cell line using Prism 10. (GraphPad).

[0199] Determination of cytotoxicity of trastuzumab deruxtecan in combination with 2ndantibody and DNase I

[0200] To rule out the possibility that using 2ndantibody, DNase I, or both would confer an enhanced cytotoxicity to ADCs in general. HER2 positive breast cancer cells AU565 and BT474 were treated with 0.2 pg / ml of Trastuzumab deruxtecan alone, with 0.2 pg / ml of 2ndantibody (109-005-170, Jackson ImmunoResearch), with 20 units / ml of DNase I, or with both, using cells treated with PBS were used as control. Each condition was tested in quadruplicate. The cells were cultured for 3 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.

[0201] Determination of cytotoxicity of different anti-DNA antibodies 2ndADC in combination with different nucleases

[0202] To evaluate whether the sensitization depended on nuclease activity or specifically on the DNase I molecule, AU565 cells were treated with 0.2 pg / ml of anti-DNA antibody + 0.2pg / ml 2ndADC in combination with three DNA nucleases: recombinant bovine DNase I (M0303S, New England Biolabs), recombinant human DNase I (ENZ-319, ProSpec), and micrococcal nuclease: MNase (N3755, Sigma-Aldrich). Additionally, we test one non-specific recombinant endonuclease: Benzonase (E8263, Sigma-Aldrich), which cleaves both DNA and RNA, and one RNA nuclease: RNase A (556746, Sigma-Aldrich). Recombinant bovine DNase I that was substantially heat-inactivated by heating at 75°C for 5 minutes was also included in the tests. Cells treated with PBS were used as control. Each condition was tested in quadruplicate. The cells were cultured for 3 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.EXAMPLE 2: INTERNALIZATION OF ANTI-DNA ANTIBODIES

[0203] This example shows that anti-DNA antibodies can bind to extracellular DNA on the surface of the cells and internalize into the cells, and the internalization can be increased by DNase I treatment.

[0204] The binding and internalization of anti-DNA antibodies were observed by fluorescent imaging in human breast cancer cell lines HCC1395 and AU565 after incubation with the antibodies. After 3-day incubation, the DNA binding and internalization of the antibodies were observed in all the cell samples incubated with various anti-DNA antibodies: anti-dsDNA antibody [121-3] (Abeam), anti-dsDNA antibody [3519 DNA] (Abeam), anti-dsDNA antibodyBV16-13 (MilliporeSigma), anti-dsDNA antibody AE-2 (Millipore), or anti-dsDNA antibody rDSD / 4565 (Thermo Fisher), but not in the cells with isotype IgG2a and IgG3 controls. Data not shown.

[0205] Furthermore, fluorescent imaging showed a significant increase in the internalization of anti-DNA antibodies in the cells with DNase I treatment. The AU565 cells were treated with 40 units / ml DNase I for 1 hour before incubation with anti-DNA antibodies, and DNase I concentration was maintained at 20 units / ml during the 3 -day incubation. With all five anti- DNA antibodies, significantly more antibodies were observed inside of the nuclease-treated cells compared with the cells without DNase I treatment. This result indicates that nuclease treatment can increase the internalization of anti-DNA antibodies.EXAMPLE 3: CYTOTOXICITY OF ANTI-DNA ANTIBODY WITH DRUGCONJUGATES IN CANCER CELLS

[0206] This example illustrates that anti-DNA antibody with drug conjugates can kill cancer cells and the cytotoxicity increases with nuclease treatment.

[0207] The cytotoxicity of anti-DNA antibodies with drug conjugates were tested in two human breast cancer cell lines (HCC1395 and AU565), two human pancreatic cancer cell lines (SW 1990-1 and AsPC-1), and a human normal (non-cancer) cell line MCFlOA. The anti-DNA antibody [3519 DNA] (Abeam) and anti -mouse IgG-VC-MMAF complex were incubated with the cells for 6 days before testing cell viability. As shown in FIGS. 2A-2D, all the cancer cells treated with anti-DNA antibody 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 which had non-specific cytotoxicity at high concentration. There is no significant difference between the test antibody and the control in the non-cancer cell, MCF10A (FIG. 2E), indicating that cancer cells were more susceptible to the cytotoxicity of anti-DNA antibody with drug conjugate than normal cells. The data in FIGS. 3A and 3B indicates that the cytotoxicity of anti-DNA ADC was not caused by the bare anti-DNA antibody alone.

[0208] Consistent with the internalization results of anti-DNA antibody shown by fluorescent imaging (see Example 2), the cytotoxicity of anti-DNA antibody [3519 DNA] (Abeam) and anti-mouse IgG-VC-MMAF can be increased by various nuclease treatments. As shown in FIGS. 4A-4D, higher cytotoxicity was observed with both HCC1395 and AU565 cells when treated with anti-DNA antibody / 2ndADC and nuclease in comparison to the cells treated with anti-DNA antibody / 2ndADC only. FIGS. 4A and 4B show the cytotoxicity resultswhen the nuclease used was DNase I. FIGS. 4C and 4D show the cytotoxicity results when the nuclease used was Benzonase.

[0209] Next, we tested whether nuclease alone causes cytotoxicity. MCF10A and HCC1395 cells were treated by different concentrations of DNase I for 6 days. As shown in FIGS. 5A and 5B, DNase I alone did not result in cytotoxicity in either normal or cancer cells at concentrations ranging from 25 units / ml to 100 units / ml, indicating that the cytotoxicity of the anti-DNA antibody / 2ndADC with DNase I combination was not caused by DNase I alone.

[0210] Further, we examined whether the nuclease could sensitize cells to non-anti-DNA antibodies. Cell viability was assayed at 3-day and 6-day time points. As shown in FIGS. 5C and 5D, when AU565 breast cancer cells were treated with 3 isotype antibodies (IgG2a, IgG3 or IgGl as indicated in the plots) with 0.2pg / ml 2ndantibody drug conjugate in combination with 20 units / ml DNase I cytotoxicity was not observed from any of the samples regardless of the presence (FIG. 5C) or absence (FIG. 5D) of DNase I. We also tested in other cell lines (e.g., MCF10A), with or without DNase I treatment, and no cytotoxicity was observed in any samples (data not shown). When AU565 cells were treated with anti-CD71 antibody and 2ndADC at 0.2pg / ml, a high degree of cytotoxicity was observed but there was no significant difference between experiments with or without addition of 20 units / ml DNase I. See FIG. 5E. DNase I also failed to enhance cytotoxicity of trastuzumab or trastuzumab deruxtecan as further described below in Example 8. On the other hand, cells were significantly sensitized to anti- DNA antibody / 2ndADC in the presence of DNase I and other DNA nucleases. See, e.g., FIGS. 4A-4D; FIGS. 5F-5J. However, the effect may require DNA nuclease activity as we observed no significant effect on cytotoxicity with RNase A. See FIG. 5K. Taken together, these results indicate that DNA nuclease does not sensitize cells to ADC that does not target extracellular DNA.

[0211] We further tested whether the cytotoxicity of anti-DNA ADC with nuclease treatment is dose and / or duration dependent. AU565 cells were treated with 0.2pg / ml anti-DNA antibody and 0.2pg / ml 2ndADC for 3 days with different amounts of DNase I added at different time points. As shown in FIG. 5F, little toxicity was observed without adding DNase I. The highest cytotoxicity was observed when 20 unit / ml DNase I was added together with anti-DNA ADC complex. However, when cells were treated with 20 unit / ml DNase I for 24 hours before adding anti-DNA ADC complex, the cytotoxicity was reduced as compared to the experiments where ADC and DNase I were added at the same time. Cytotoxicity was further reduced in the cellstreated with a double amount of DNase I (40 unit / ml) DNase I for 24 hours before adding anti- DNA ADC complex. These data indicate that nuclease sensitized cells to anti-DNA ADC, but excessive nuclease activity or extended period of nuclease reaction time reduced the cytotoxicity of the anti-DNA antibody. Without being bound by theory, this may be due to increased degradation of extracellular DNA into fragments smaller than the antibody's binding capacity, thereby eliminating the target of the anti-DNA ADC complex. For anti-DNA antibody [3519 DNA] (Abeam), the minimal size of DNA binding is >16 bases pairs.EXAMPLE 4: TARGET SPECIFICITY OF ANTI-DNA ANTIBODY WITH DRUG CONJUGATES

[0212] This example illustrates cancer cells are more sensitive to anti-DNA antibody with drug conjugates than normal cells.

[0213] It was demonstrated in FIGS. 2A-2E that anti-DNA antibody with drug conjugates show more selectivity to cancer cells (AsPC-1, AU565, SW1990-1 and HCC1395; FIGS. 2A- 2D, respectively) than normal cells (MCF10A; FIG. 2E), FIG. 6A shows the ICso of the anti- dsDNA ADC with DNase I in the indicated cancer (AU565, HCC1395) and normal cells (MCF10A). FIG. 6B shows cytotoxicity in the same setting over a range of antibody concentrations. These data further confirm a significant cytotoxic difference between cancer cells and non-cancer cells with anti-DNA antibody / ADC with nuclease treatment. Cytotoxicity appears to be dosage dependent.

[0214] To examine whether the observations in FIGS. 6A-6B were antibody specific instead of target specific, we tested two additional commercially available anti-DNA antibodies in combination with 2ndADC and DNase I and found similar responses. See FIG. 7. As shown in the figure, significant cytotoxicity towards cancer cells was achieved with 3 different anti- DNA antibodies in combination with 2ndADC and DNase I.EXAMPLE 5: CYTOTOXICITY OF ANTI-DNA ADC IN TP53 MUTANT CELLS

[0215] This example illustrates the cytotoxicity of anti-DNA antibody with drug conjugates associates with the presence of TP53 mutations.

[0216] In this example, we analyzed the reactions to anti-DNA antibody paired with 2ndADC and DNase I across a wider range of cancer and normal cells. We tested 12 cell lines including nine human breast cancer cell lines (AU565, SK-BR-3, HCC1395, MDA-MB-468, HCC1806, Hs 578T, MDA-MB-453, MCF-7, DU4475), one human lung cancer cell line (NCI-H460) and two non-tumorigenic human cell lines (MCF10A, HCC1395 BL). The cells were treatedseparately with 20 units / ml DNase I, 0.2pg / ml of anti-DNA antibody + 0.2pg / ml 2ndADC, or in combination. As shown in FIG. 8A, little to no cytotoxicity was observed when cells were treated separately with DNase I (second grouping) or anti-DNA antibody + 2ndADC (third grouping). When DNase I and anti-DNA ADC complex were used in combination, about 50% of cell death (ranging from 43% to 68%) was observed in TP53 mutated cells, whereas less effect was observed in TP53 wild cells, whether cancer or normal (FIGs. 8A and 8B). Table 1 lists the 12 cell lines and their TP53 gene status. These experiments suggest that TP53 mutation status could be used as a marker to predict if a cancer cell will be sensitive to anti- DNA ADC + DNase I. Table 1 : Cell lines and TP53 gene.*HER2: human epidermal growth factor receptor 2, TNA: Triple negative type A; TNB: Triple negative type B.

[0217] Next, we examined the effects of anti-DNA +2ndADCs in the 12 cells lines comparing to MMAF alone.

[0218] Table 2 presents the ICso values of MMAF in each cell line and the viability of each cell line after treatment with antibody drug conjugate complex composed of 0.2pg / ml anti- DNA antibody + 0.2pg / ml secondary antibody conjugated with MMAF (equivalent to about 6nM MMAF) + 20 units / ml DNase I. Among the six cell lines harboring TP53 loss of functionmutations, approximately 6nM of MMAF conjugated to the antibody was sufficient to achieve approximately 50% cell death (ranging from 43% to 68%). Conversely, to achieve the same level of cell death using MMAF as a free drug (i.e., not conjugated), notably higher concentrations of MMAF were required (ranging from 15.66nM to 2783nM). Particularly in MDA-MB-468 cells, the combination of the antibody and DNase I boosted the sensitivity of MMAF by more than 450-fold. However, the anti-DNA ADC combination demonstrated minimal toxicity to non-cancer cells and TP53 wild-type cancer cells. The sensitivity of each cell line to MMAF free drug did not exhibit a correlation with TP53 status, suggesting the correlation we observed between TP53 status and the cytotoxicity of the anti-DNA 2ndADC is antibody dependent rather than payload dependent.Table 2: MMAF ICso values vs. ADC combination toxicity on 12 cell lines.EXAMPLE 6: CYTOTOXIC EFFECTS WITH DIFFERENT PAYLOADS

[0219] This example illustrates the anti-DNA ADC can use different anti-tumor molecules with different mechanisms of action as payloads.

[0220] To test if the combination of anti-DNA ADC complex and DNase I can work with other anti-tumor agents as payload other than MMAF, human breast cancer cell lines AU565 and SK-BR-3 were treated with 0.2pg / ml of anti-DNA antibody, 0.2pg / ml 2ndantibody conjugated with MMAF, DMDM or Extecan, along with 20 units / ml DNase I. Each drug has a different mode of action: MMAF inhibits tubulin polymerization, DMDM alkylates DNA,and Exetecan is a topoisomerase I inhibitor. As shown in FIGS. 9A-9B, with 3 -day treatment, significant cytotoxicity was achieved with MMAF conjugates in both cell lines, with detectable but lesser cytotoxicity with DMDM and Exetecan. With 6-day treatment, substantial cytotoxicity was achieved from combinations using any of the three cytotoxic payloads. These results indicate the anti-DNA ADCs can use different anti-tumor molecule with different mechanism of action as payloads.EXAMPLE 7: CYTOTOXICITY OF ADC CONFIGURATIONS

[0221] This example compares the cytotoxicity of anti-DNA antibody in various configurations.

[0222] As shown in FIG. 10, comparison was made between: anti-DNA antibodies alone without payload; secondary antibodies alone without payload; anti-DNA and secondary antibody without payload; anti-DNA antibody with secondary antibody conjugated to payload (MMAF); anti-DNA ADC with payload (MMAF) directly conjugated; anti-DNA ADC direct conjugate with unconjugated secondary antibody; and anti-DNA ADC with secondary antibody conjugated to MMAF. In these experiments, MMAF was directly conjugated to anti- DNA antibody via a Valine-Citrulline (Val-Cit) linker by use of a maleimide-thiol conjugation reaction. No cytotoxicity was observed without cytotoxic payload present, suggesting the cytotoxicity resulted from the payload conjugated to antibodies rather than the antibodies alone. See FIG. 10, bars 2-4. We found that the directly conjugated ADC with DNase I (6thbar) had reduced cytotoxicity compared to all configurations comprising a secondary antibody (bars 5, 7 and 8). However, cytotoxicity of the anti-DNA ADC was regained by adding a bare 2ndantibody or 2ndADC. Compare bar 6 to bars 7 and 8. Without being bound by theory, these results suggest that the internalization of anti-DNA antibody was enhanced with increased cluster size or molecular weight of the cell targeting construct. As disclosed herein, such anti- DNA antibody constructs can be configured using multiple antibodies and / or carrier molecule or other entities such as nanoparticles. See, e.g., FIG. ID and related discussion.EXAMPLE 8: CYTOTOXIC EFFECTS COMPARING TO TRASTUZUMAB DERUXTECAN

[0223] Trastuzumab deruxtecan (T-DXd) is one of the most successful antibody-drug conjugates (ADCs) for treating cancer. It was initially approved by FDA in December 2019 for patients with unresectable or metastatic HER2+ breast cancer. In August 2022, FDA further approved Trastuzumab deruxtecan for treatment of HER2-low breast cancer. And in March2024, the FDA granted a broader approval for trastuzumab deruxtecan for any HER2-positive solid tumors regardless of cancer type. This example compares the cytotoxicity of anti-DNA 2ndADC to T-DXd in HER2 positive (HER2+) and HER2 low triple negative cancer cells.

[0224] In the HER2+ breast cancer cell line AU565, both anti-DNA 2ndADC and T-DXd exhibited high potency of cell killing, with T-DXd having slightly lower ICso. See FIG. 11 A. In another HER2+ breast cancer cell line, BT474, anti-DNA 2ndADC showed significantly lower ICso comparing to T-DXd. See FIG. 11B. Unlike anti-DNA 2ndADC, 50% cytotoxicity of T-DXd was not observed even at the highest dose tested (20 pg / ml). Similarly, in two triple negative cancer cell lines, HCC1395 (FIG. 11C) and MDA-MB-468 (FIG. 11D), 50% cell killing by T-DXd was not achieved at the 20 pg / ml. In contrast, anti-DNA 2ndADC complex had an IC50 at approximately 0.3 pg / ml in HCC1395 cells (FIG. 11C). Anti-DNA 2ndADC and T-DXd had ICsos of approximately 1.3 pg / ml and 9.3 pg / ml in MDA-MB-468 cells, respectively. See FIG. 11D. To assess whether the potency from anti-DNA 2ndADC is nonspecific to cancer cells, HCC1395 BL, a non-tumorigenic B lymphoblast cell derived from the same patient of the breast cancer cell line HCC1395, was tested with both anti-DNA 2ndADC and T-DXd. Unlike HCC1395 cancer cells, the ADCs resulted in cytotoxicity with non-cancer HCC1395 BL cells only at highest concentrations. See FIG. HE. Comparing results from HCC1395 (FIG. HE upper plot) and HCC1395 BL (FIG. HE lower plot), the IC50 with HCC1395 was lOx lower than with HCC1395 BL. In all comparisons, we observed that DNase I significantly sensitized cells to anti-DNA 2ndADC, but not T-DXd. See FIGS. 11A-11G. T- DXd was also unaffected by 2ndantibody. See FIGS. 11F-11G. An isotype control ADC complex, which comprises an MMAF conjugated isotype antibody and a secondary antibody, didn’t exhibit significant cytotoxicity under any condition. See FIGS. 11A-11E.

[0225] Taken together, the results in this example suggest that the anti-DNA ADC constructs provided herein can be as effective as the FDA approved T-DXd ADC in some settings, and further may be more effective in some settings.

[0226] 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 example only. 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 intendedthat 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 anti-DNA antibodies into a cell, the method comprising, contacting the cell with the one or more anti-DNA antibodies, wherein the one or more anti-DNA antibodies bind to extracellular DNA on the surface of the cell and the one or more anti-DNA antibodies are internalized into the cell.

2. The method of claim 1, wherein the contacting comprises contacting the cell with at least one, two, three, four, five, six, seven, eight or more different anti-DNA antibodies, optionally wherein the at least one, two, three, four, five, six, seven, eight or more different anti-DNA antibodies comprises one, two, three, four, five, six, seven, eight or more different anti-DNA antibodies.

3. The method of any one of claims 1-2, further comprising contacting the cell with one or more nucleases, optionally wherein the one or more nucleases comprise DNA nucleases.

4. The method of claim 3, wherein the one or more nucleases comprise an endonuclease, an exonuclease, or a combination thereof.

5. The method of claim 4, wherein the endonuclease is a deoxyribonuclease (DNase), a serratia marcescens nuclease (Benzonase), a micrococcal nuclease (MNase), a transposase, a restriction enzyme, nuclease SI, nuclease Pl, a sequence specific endonuclease, or a sequence non-specific endonuclease.

6. The method of claim 3, wherein the one or more DNA nucleases comprise a single stranded DNA (ssDNA) nuclease, a double-stranded DNA (dsDNA) nuclease, or a combination thereof.

7. The method of any one of claims 3 to 6, wherein the one or more nucleases and the one or more anti-DNA antibodies are contacted to the cell at the same time, optionally wherein the one or more nucleases are attached to the one or more anti-DNA antibodies.

8. The method of any one of claims 3 to 6, wherein the one or more nucleases are contacted to the cell before the one or more anti-DNA antibodies.

9. The method of any one of claims 3 to 6, wherein the one or more nucleases are contacted to the cell after the one or more anti-DNA antibodies.

10. The method of any one of claims 1 -9, wherein the one or more anti-DNA antibodies are covalently or non-covalently linked to at least one payload.

11. The method of any one of claims 1 -9, wherein the one or more anti-DNA antibodies are bound by one or more secondary antibody.

12. The method of claim 11, wherein the one or more secondary antibodies are covalently or non-covalently linked to at least one payload.

13. The method of any one of claims 1-11, wherein the anti-DNA antibody is covalently or non-covalently linked to at least one payload.

14. The method of any one of claims 11 to 13, wherein the one or more anti- DNA antibodies and / or the one or more secondary antibodies are covalently linked to at least one payload.

15. The method of any one of claims 11-14, wherein the one or more anti- DNA antibodies and / or the one or more secondary antibodies are linked to at least one payload via a linker.

16. The method of claim 15, wherein the linker comprises a non-cleavable linker.

17. The method of claim 16, wherein the non-cleavable linker comprises a maleimide alkane linker, or a maleimide cyclohexane linker.

18. The method of claim 15, wherein the linker comprises a cleavable linker.

19. The method of claim 18, wherein the linker is cleaved after contacting the cell with the one or more anti-DNA antibodies, thereby releasing the payload on or in the cell.

20. The method of claim 18 or 19, wherein the cleavable linker comprises a hydrazone linker, a cathepsin B-responsive linker, a disulfide linker, a pyrophosphate diester linker, or a combination thereof.

21. The method of any one of claims 18 to 20, wherein the cleavable linker comprises a protease-sensitive linker, a pH-sensitive linker, a radiation-sensitive linker, a disulfide linker, a glutathione-sensitive linker, or a combination thereof.

22. The method of any one of claims 10 to 13, wherein the one or more anti- DNA antibodies and / or the one or more secondary antibodies are attached to a biotin moiety, and the at least one payload is attached to a streptavidin.

23. The method of any one of claims 10-22, 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.

24. The method of claim 23, 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.

25. The method of claim 24, 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 compoundtargeting / decreasing protein or lipid kinase activity, a compound targeting / decreasing protein or lipid phosphatase activity, an anti-angiogenic compound, and any combination thereof.

26. The method of claim 24 or 25, 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.

27. The method of any one of claims 24 to 26, wherein the antitumor agent comprises a tubulin inhibitor, a DNA inhibitor, and / or an RNA inhibitor.

28. The method of claim 27, wherein the tubulin 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.

29. The method of claim 27, wherein the DNA inhibitor is selected from the group consisting of an alkylator, duocarmycin, duocarmycin DM (DMDM), calicheamicin, pyrrolobenzodiazepine (PDB), enediyne, uncialamycin, a topoisomerase inhibitor, topotecan, camptothecin (CPT), exatecan, and any derivative thereof.

30. The method of claim 27, wherein the RNA inhibitor is selected from the group consisting of an RNA splicing inhibitor, RNA polymerase II inhibitor, thailanstatin, amatoxin, and any derivative thereof.

31. The method of claim 23, 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, radioactive label, or a combination thereof.

32. The method of any one of claims 1-31, wherein the one or more anti- DNA antibodies are sequence specific.

33. The method of any one of claims 1-31, wherein the one or more anti- DNA antibodies are non-sequence specific.

34. The method of any one of claims 1-31, wherein the one or more anti- DNA antibodies comprise a combination of sequence specific and non-sequence specific anti- DNA antibodies.

35. The method of any one of claims 1-34, wherein the one or more anti- DNA antibodies comprise anti-dsDNA antibodies, anti-ssDNA antibodies, or a combination thereof.

36. The method of any one of claims 1-35, wherein the one or more anti- DNA antibodies are coated on a gold nanoparticle when entering the cell.

37. The method of any one of claims 1-35, wherein the one or more anti- DNA antibodies are covalently or non-covalently linked when entering the cell.

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 1-37, wherein the cell comprises a functionally-impaired transcription factor 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 is caused by one or more gene mutations, loss, and / or deficient expression.

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

42. A method of delivering a therapeutic agent inside of a cell, the method comprising,contacting the cell with a therapeutic agent covalently or non-covalently linked to an anti-DNA antibody, wherein the anti-DNA antibody binds to extracellular DNA on the surface of the cell and delivers the therapeutic agent inside of the cell.

43. The method of claim 42, wherein the therapeutic agent is covalently linked to the anti-DNA antibody.

44. The method of claim 42, wherein the therapeutic agent is non-covalently linked to the anti-DNA antibody.

45. The method of claim 44, wherein the anti-DNA antibody is bound by a secondary antibody, and wherein the therapeutic agent is conjugated to the secondary antibody.

46. The method of any one of claims 42 to 45, wherein the method further comprises contacting the cell with one or more nucleases, optionally wherein the one or more nucleases comprise a single stranded DNA (ssDNA) nuclease, a double-stranded DNA (dsDNA) nuclease, or a combination thereof.

47. The method of any one of claims 42-46, wherein the cell is in vitro.

48. The method of any one of claims 42-46, wherein the cell is in vivo.

49. The method of any one of claims 42-48, wherein the cell has an aneuploidy and / or DNA repair deficiency.

50. The method of any one of claims 42-49, wherein the cell comprises a functionally-impaired transcription factor selected from the group consisting of p53, NF-Kb, AP-1, E2F1, and BRCA1.

51. The method of claim 50, wherein the functional impairment of the transcription factor is caused by one or more gene mutations, loss, and / or deficient expression.

52. The method of claim 50 or 51, wherein the cell comprises a functionally- impaired transcription factor p53, optionally wherein the impairment of p53 comprises a mutation.

53. The method of any one of claims 42-52, wherein the cell is a cancer cell and wherein the therapeutic agent kills or inhibits growth or division of the cancer cell.

54. The method of claim 53, wherein the cancer type 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; pleuropulmonaryblastoma; 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.

55. The method of claim 53, wherein the cancer 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.

56. The method of any one of claims 53 to 55, wherein the cancer cell is from a cancer in a subject.

57. A method of treating in a subject in need thereof, the method comprising, administering to the subject a composition comprising a therapeutic agent linked to an anti-DNA antibody, wherein the therapeutic agent is delivered inside of a cell of the subject using the method of any one of claims 42 to 46, wherein the delivery of the therapeutic agent is effective to treat the subject.

58. The method of claim 57, wherein the composition further comprises aDNA nuclease.

59. The method of claim 57 or 58, wherein the subject has a cancer and wherein the cell is a cancer cell.

60. The method of any one of claims 57 to 59, further comprising determining, prior to the administering, whether the cell has an aneuploidy, a DNA repair deficiency, and / or a functionally-impaired transcription factor 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 is caused by one or more gene mutations, loss, and / or deficient expression.

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

63. A composition comprising one or more anti -DNA antibodies and one or more nucleases, optionally wherein the one or more nucleases comprise one or more DNA nucleases.

64. The composition of claim 63, wherein the one or more anti-DNA antibodies are bound by one or more secondary antibodies.

65. The composition of claim 63 or 64, wherein the one or more anti-DNA antibodies are coated on a gold nanoparticle.

66. The composition of claim 63 or 64, wherein the one or more anti-DNA antibodies comprise at least two anti-DNA antibodies that are linked together as a polymer.

67. The composition of claim 66, wherein the polymer comprises a dimer, a trimer, a tetramer, a pentamer, or has more than five anti-DNA antibodies.

68. The composition of any one of claims 63 to 67, wherein the composition comprises two, three, four, five, six, seven, eight or more different anti-DNA antibodies.

69. The composition of claim 68, wherein at least two different anti-DNA antibodies are crosslinked together.

70. The composition of any one of claims 63 to 69, further comprising a therapeutic agent covalently or non-covalently linked to the one or more anti-DNA antibodies and / or the one or more secondary antibodies.

71. The composition of claim 70, 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.

72. The composition of claim 71, 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.

73. The composition of claim 71 or 72, 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.

74. The composition of any one of claims 71 to 73, wherein the antitumor agent comprises a tubulin inhibitor, a DNA inhibitor, and / or an RNA inhibitor.

75. The composition of claim 74, wherein the tubulin 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.

76. The composition of claim 74, wherein the DNA inhibitor is selected from the group consisting of an alkylator, duocarmycin, duocarmycin DM (DMDM), calicheamicin, pyrrolobenzodiazepine (PDB), enediyne, uncialamycin, a topoisomerase inhibitor, topotecan, camptothecin (CPT), exatecan, and any derivative thereof.

77. The composition of claim 74, wherein the RNA inhibitor is selected from the group consisting of an RNA splicing inhibitor, RNA polymerase II inhibitor, thailanstatin, amatoxin, and any derivative thereof.

78. The composition of any one of claims 63 to 77, wherein the one or more DNA nucleases comprise an endonuclease, an exonuclease, or a combination thereof.

79. The composition of claim 78, wherein the endonuclease is a deoxyribonuclease (DNase), a serratia marcescens nuclease (Benzonase), a micrococcal nuclease (MNase), a transposase, a restriction enzyme, nuclease SI, nuclease Pl, a sequence specific endonuclease, or a sequence non-specific endonuclease.

80. The composition of any one of claims 63 to 79, wherein the one or more DNA nucleases comprise a single stranded DNA (ssDNA) nuclease, a double-stranded DNA (dsDNA) nuclease, or a combination thereof.

81. A pharmaceutical composition comprising a therapeutically effective amount of the composition according to any one of claims 63 to 80, and a pharmaceutically acceptable excipient, carrier, and / or diluent.

82. A method of treating or ameliorating a disease or disorder in a subject in need thereof, comprising administering the composition of claim 81 to the subject, optionally wherein the disease or disorder comprises a cancer.

83. The method of claim 82, further comprising determining, prior to the administering, whether the cancer has an aneuploidy, a DNA repair deficiency, and / or a functionally-impaired transcription factor selected from the group consisting of p53, NF-Kb, AP-1, E2F1, and BRCA1.

84. The method of claim 83, wherein the functional impairment of the transcription factor is caused by one or more gene mutations, loss, and / or deficient expression.

85. The method of any one of claims 82 to 84, wherein the cancer comprises a functionally-impaired transcription factor p53 resulting from a deficiency of the TP53 gene, optionally wherein the deficiency of the TP53 gene comprises a mutation.

86. The method of any one of claims 82 to 85, 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.

87. A method of internalizing one or more anti-DNA antibodies into a cell, the method comprising, contacting the cell with the one or more anti-DNA antibodies, wherein the one or more anti-DNA antibodies comprise means for binding extracellular DNA, wherein the one or more anti-DNA antibodies bind to the extracellular DNA on the surface of the cell and the one or more anti-DNA antibodies are internalized into the cell.

88. A method of delivering a therapeutic agent inside of a cell, the method comprising, contacting the cell with a therapeutic agent covalently or non-covalently linked to an anti-DNA antibody,wherein the one or more anti-DNA antibodies comprise means for binding extracellular DNA, wherein the anti-DNA antibody binds to the extracellular DNA on the surface of the cell and delivers the therapeutic agent inside of the cell.

89. A composition comprising one or more anti-DNA antibodies and one or more nucleases, optionally wherein the one or more nucleases comprise one or more DNA nucleases, wherein the one or more anti-DNA antibodies comprise means for binding extracellular DNA.

90. In a method of contacting an antibody to a cell, the improvement comprising internalizing one or more anti-DNA antibodies into a cell by contacting the cell with the one or more anti-DNA antibodies, wherein the one or more anti-DNA antibodies bind to extracellular DNA on the surface of the cell and the one or more anti-DNA antibodies are internalized into the cell.

91. In a method of treating in a subject having a cancer, the improvement comprising administering to the subject a composition comprising a therapeutic agent linked to an anti-DNA antibody, wherein the therapeutic agent is delivered inside of a cell of the subject, wherein the delivery of the therapeutic agent is effective to treat the subject.

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