Compositions and methods for targeting extracellular DNA

Abstract

This specification provides a method for internally transferring an anti-DNA antibody or a DNA-binding antibody fragment into a cell. This method can be used for the diagnosis and therapeutic treatment of diseases. TIFF2026522339000004.tif104170

Description

Technical Field

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

Background Art

[0002] Background Stressed, damaged, and dying cells release intracellular factors, including DNA, to their cell surface and / or the local microenvironment, activating immune components. Such extracellular DNA can cause inflammation, which contributes to the pathogenesis of various autoimmune diseases.

[0003] Recently, it has been reported that extracellular DNA is present on the surface of pancreatic cancer cells but not on normal pancreatic cells (Wen F, et al. Cancer Res. 2013;73(14):4256 - 4266 (Non - Patent Document 1)). Cancer - associated extracellular DNA is thought to be involved in cell migration ability in vitro, and DNase I treatment reduced cancer metastasis in an orthotopic xenograft pancreatic cancer mouse model. However, extracellular DNA carrying specific sequences derived from the genome of target cells has not been used in cell targeting, and the existence of such biomarkers has not been applied in the fields of diagnosis, prognosis, and / or theranostics.

Prior Art Documents

Non - Patent Documents

[0004]

Non - Patent Document 1

Summary of the Invention

[0005] overview This specification provides compositions and methods for the internal delivery of anti-DNA antibodies or DNA-binding antibody fragments into cells. The method comprises contacting cells with an anti-DNA antibody or DNA-binding antibody fragment that binds to extracellular DNA on the cell surface, thereby enabling the anti-DNA antibody or DNA-binding antibody fragment to be internally delivered into the cells. The method can be further optimized to facilitate internal delivery, for example, by contacting the cells with a nuclease. Such methods and related compositions can be used in a variety of applications, including drug delivery, disease diagnosis, and therapeutic treatment.

[0006] In one aspect, the present disclosure provides a method for internally transferring one or more anti-DNA antibodies into a cell, comprising the step of contacting a cell with 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 internally transfer into the cell.

[0007] In some embodiments, the contacting step includes contacting cells with at least one, two, three, four, five, six, seven, eight or more different anti-DNA antibodies. In some embodiments, at least one, two, three, four, five, six, seven, eight or more different anti-DNA antibodies comprise one, two, three, four, five, six, seven, eight or more different anti-DNA antibodies. In some embodiments, the method further includes contacting cells with one or more nucleases. In some embodiments, one or more nucleases comprise DNA nucleases. In some embodiments, one or more DNA nucleases comprise single-stranded DNA nucleases, double-stranded DNA nucleases, or a combination thereof.

[0008] In some embodiments, one or more nucleases and one or more anti-DNA antibodies are simultaneously contacted with the cell, and optionally, one or more nucleases are bound to one or more anti-DNA antibodies. In some examples, one or more nucleases are contacted with the cell before one or more anti-DNA antibodies. In other examples, one or more nucleases are contacted with the cell after one or more anti-DNA antibodies. In some embodiments, one or more nucleases are DNase I, restriction enzymes, and / or benzoases.

[0009] In some embodiments, one or more anti-DNA antibodies are covalently or non-covalently linked to at least one payload. In some examples, at least one payload is covalently linked to an anti-DNA antibody. In other examples, one or more anti-DNA antibodies are bound to a biotin moiety, and at least one payload is bound to streptavidin. In some embodiments, at least one payload is linked to one or more secondary antibodies, and one or more secondary antibodies are bound to an anti-DNA antibody.

[0010] In some embodiments, one or more anti-DNA antibodies and / or one or more secondary antibodies are linked to at least one payload via a linker. In some examples, the linker includes a non-cleaving linker. In other examples, the linker includes a cleaving linker. In some embodiments, the linker is cleaved after a step of contacting a cell with one or more anti-DNA antibodies, thereby releasing the payload onto or into the cell. In some embodiments, the cleaving linker includes a protease-sensitive linker, a pH-sensitive linker, a radiosensitive linker, a glutathione-sensitive linker, a disulfide linker, or a combination thereof. In some embodiments, the cleaving linker includes a protease-sensitive linker containing the sequence LPXTG (SEQ ID NO: 1) of a saltase-recognition motif. In some embodiments, the sequence of the saltase-recognition motif is LPETG (SEQ ID NO: 2).

[0011] In some embodiments, at least one payload comprises small molecules, peptides, proteins, nucleic acids, toxins, therapeutic substances, drugs, chemotherapeutic agents, liposomes, nanoparticles, dendrimers, detectable labels, or any derivatives, fragments, or combinations thereof.

[0012] In some embodiments, therapeutic substances are selected from the group consisting of antitumor agents, anticancer agents, prodrugs, lysosomal destabilizers (e.g., chloroquine), alkylating agents, alkaloids, allosteric inhibitors, antifolic acid agents, anti-inflammatory agents, antibiotics, antibacterial agents, antifungal agents, antifibrotic agents, antiinfective agents, antiparasitic agents, antiviral agents, antimycobacterial agents, anticancer agents, antiprotozoal agents, antiviral agents, physiologically active peptides, steroid hormones, photosensitive substances, radiopharmaceuticals, antiprion agents, and any combination thereof.

[0013] In some embodiments, antitumor agents include aromatase inhibitors; anti-estrogens; anti-androgens; gonadrelin agonists; topoisomerase I inhibitors; topoisomerase II inhibitors; microtubule activators; alkylating agents; retinoids, carotenoids, or tocopherols; cyclooxygenase inhibitors; MMP inhibitors; mTOR inhibitors; antimetabolites; platinum compounds; methionine aminopeptidase inhibitors; bisphosphonates; antiproliferative antibodies; heparinase inhibitors; inhibitors of Ras oncogenic isoforms; and terrorists. Melase inhibitors; proteasome inhibitors; Flt-3 inhibitors; Hsp90 inhibitors; kinesin spindle protein inhibitors; MEK inhibitors; PARP inhibitors, tyrosine kinase inhibitors, PI3K inhibitors, AKT inhibitors, EGFR inhibitors, antitumor antibiotics; nitrosoureas, compounds that target / reduce protein or lipid kinase activity, compounds that target / reduce protein or lipid phosphatase activity, any further anti-angiogenic compounds, and any combination thereof are selected from the group.

[0014] In some embodiments, the antitumor agent is selected from the group consisting of azacitidine, azathioprine, bevacizumab, bleomycin, capecitabine, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, etoposide, fenretinide, fluorouracil, gemcitabine, herceptin, idarubicin, mechloretamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, tafluposide, teniposide, thioguanine, retinoic acid, barrubicin, vinblastine, vincristine, vindesine, vinorelbine, receptor tyrosine kinase inhibitors, and any combination thereof.

[0015] In some embodiments, the detectable label is selected from the group consisting of magnetic labels, fluorescent moieties, enzymes, luminescent particles, chemiluminescent probes, metallic particles, nonmetallic colloidal particles, polymer dye particles, dye molecules, electrochemically active species, semiconductor nanocrystals, nanoparticles, quantum dots, gold particles, fluorophores, radioactive labels, or combinations thereof. In some examples, the cells are in vitro. In other examples, the cells are in vivo.

[0016] In some embodiments, one or more anti-DNA antibodies bind to a known DNA sequence. In other embodiments, one or more anti-DNA antibodies bind to an unknown DNA sequence. Alternatively, one or more anti-DNA antibodies bind to both a known DNA sequence and an unknown DNA sequence.

[0017] In some embodiments, one or more anti-DNA antibodies are sequence-specific. In other embodiments, one or more anti-DNA antibodies are non-sequence-specific. Alternatively, one or more anti-DNA antibodies comprise a combination of sequence-specific anti-DNA antibodies and non-sequence-specific anti-DNA antibodies.

[0018] In another aspect, the Disclosure provides a method for internally transferring one or more anti-DNA antibodies into a mammalian cell, comprising the step of contacting a cell with one or more anti-DNA antibodies, wherein one or more anti-DNA antibodies bind to extracellular DNA on the surface of the cell and internally transfer into the cell. In some embodiments, the contacting step comprises the step of contacting a cell with at least one, two, three, four, five, six, seven, eight or more different anti-DNA antibodies. In some embodiments, 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.

[0019] In some embodiments, the method further comprises the step of contacting cells with one or more nucleases. In some embodiments, one or more nucleases include DNA nucleases. In some embodiments, one or more nucleases include endonucleases, exonucleases, or combinations 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 S1, nuclease P1, a sequence-specific endonuclease, or a sequence-nonspecific endonuclease. In some embodiments, one or more DNA nucleases include single-stranded DNA (ssDNA) nucleases, double-stranded DNA (dsDNA) nucleases, or combinations thereof.

[0020] In some embodiments, one or more nucleases and one or more anti-DNA antibodies are simultaneously brought into contact with the cell, and optionally, one or more nucleases are bound to one or more anti-DNA antibodies. In some embodiments, one or more nucleases are brought into contact with the cell before one or more anti-DNA antibodies. In some embodiments, one or more nucleases are brought into contact with the cell after one or more anti-DNA antibodies.

[0021] In some embodiments, one or more anti-DNA antibodies are covalently or non-covalently linked to at least one payload. In some embodiments, one or more anti-DNA antibodies are linked 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.

[0022] In some embodiments, an anti-DNA antibody or secondary antibody is covalently linked to the payload. In some embodiments, the anti-DNA antibody or secondary antibody is linked to the payload via a linker. In some embodiments, the linker includes a non-cleaving linker. In some embodiments, the non-cleaving linker includes a maleimide alkane linker or a maleimide cyclohexane linker. In some embodiments, the linker includes a cleaving linker. In some embodiments, the linker is cleaved after a step of contacting a cell with one or more anti-DNA antibodies, thereby releasing the payload onto or into the cell. In some embodiments, the cleaving linker includes a hydrazone linker, a cathepsin B-responsive linker, a disulfide linker, or a pyrophosphate diester linker, or a combination thereof. In some embodiments, the cleaving linker includes a protease-sensitive linker, a pH-sensitive linker, a radiosensitive linker, a disulfide linker, or a glutathione-sensitive linker, or a combination thereof.

[0023] In some embodiments, an anti-DNA antibody or secondary antibody is non-covalently linked to the payload. In some embodiments, the anti-DNA antibody or secondary antibody is bound to a biotin moiety, and at least one payload is bound to streptavidin. In some embodiments, at least one payload comprises small molecules, peptides, proteins, nucleic acids, toxins, therapeutic substances, drugs, chemotherapeutic agents, liposomes, nanoparticles, dendrimers, detectable labels, or any derivatives, fragments, or combinations thereof.

[0024] In some embodiments, therapeutic substances are selected from the group consisting of antitumor agents, anticancer agents, prodrugs, lysosomal destabilizers (e.g., chloroquine), alkylating agents, alkaloids, allosteric inhibitors, antifolic acid agents, anti-inflammatory agents, antibiotics, antibacterial agents, antifungal agents, antifibrotic agents, antiinfective agents, antiparasitic agents, antiviral agents, antimycobacterial agents, anticancer agents, antiprotozoal agents, antiviral agents, physiologically active peptides, steroid hormones, photosensitive substances, radiopharmaceuticals, antiprion agents, and any combination thereof.

[0025] In some embodiments, antitumor agents include aromatase inhibitors; anti-estrogens; anti-androgens; gonadrelin agonists; topoisomerase I inhibitors; topoisomerase II inhibitors; microtubule inhibitors; alkylating agents; retinoids, carotenoids, or tocopherols; cyclooxygenase inhibitors; MMP inhibitors; mTOR inhibitors; antimetabolites; platinum compounds; methionine aminopeptidase inhibitors; bisphosphonates; antiproliferative antibodies; heparinase inhibitors; inhibitors of Ras oncogenic isoforms; and terrorists. Melase inhibitors; proteasome inhibitors; Flt-3 inhibitors; Hsp90 inhibitors; kinesin spindle protein inhibitors; MEK inhibitors; PARP inhibitors, tyrosine kinase inhibitors, PI3K inhibitors, AKT inhibitors, EGFR inhibitors, antitumor antibiotics; nitrosoureas, compounds that target / reduce protein or lipid kinase activity, compounds that target / reduce protein or lipid phosphatase activity, any further anti-angiogenic compounds, and any combination thereof are selected from the group.

[0026] In some embodiments, the antitumor agent is selected from the group consisting of azacitidine, azathioprine, bleomycin, capecitabine, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, etoposide, fenretinide, fluorouracil, gemcitabine, herceptin, idarubicin, mechloretamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, tafluposide, teniposide, thioguanine, retinoic acid, barrubicin, vinblastine, vincristine, vindesine, vinorelbine, receptor tyrosine kinase inhibitors, and any combination thereof. In some embodiments, the antitumor agent includes tubulin inhibitors, DNA inhibitors, and / or RNA inhibitors.

[0027] In some embodiments, tubulin inhibitors are selected from the group consisting of monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), maytansine, maytansinoids, meltansine (emtansine, DM1), labtansine (sorabtansine, DM4), tubricin, halichondrin (eribulin), cryptophycin, EG5 inhibitors, and any derivatives thereof. In some embodiments, DNA inhibitors are selected from the group consisting of alkylating agents, duocalmycin, duocalmycin DM (DMDM), calicheamicin, pyrrolobenzodiazepine (PDB), enediyne, unciaramycin, topoisomerase inhibitors, topotecan, camptothecin (CPT), exatecan, and any derivatives thereof. In some embodiments, RNA inhibitors are selected from the group consisting of RNA splicing inhibitors, RNA polymerase II inhibitors, tylanstatin, amatoxin, and any derivatives thereof.

[0028] In some embodiments, the detectable labels are selected from the group consisting of magnetic labels, fluorescent moieties, enzymes, luminescent particles, chemiluminescent probes, metallic particles, nonmetallic colloidal particles, polymer dye particles, dye molecules, electrochemically active species, semiconductor nanocrystals, nanoparticles, quantum dots, gold particles, fluorophores, radioactive labels, or combinations thereof.

[0029] In some embodiments, one or more anti-DNA antibodies are sequence-specific. In some embodiments, one or more anti-DNA antibodies are non-sequence-specific. In some embodiments, one or more anti-DNA antibodies comprise a combination of a sequence-specific anti-DNA antibody and a non-sequence-specific anti-DNA antibody. In some embodiments, one or more anti-DNA antibodies comprise an anti-dsDNA antibody, an anti-ssDNA antibody, or a combination thereof.

[0030] In some embodiments, one or more anti-DNA antibodies are coated onto gold nanoparticles upon entering a cell. In some embodiments, at least two anti-DNA antibodies are covalently or noncovalently linked upon entering a cell.

[0031] In another aspect, this specification provides a method for delivering a therapeutic substance into a cell, comprising the step of contacting the cell with the therapeutic substance, which is covalently or non-covalently linked to an anti-DNA antibody, such that the anti-DNA antibody binds to extracellular DNA on the cell surface and delivers the therapeutic substance into the cell. In some embodiments, the therapeutic substance is covalently linked to the anti-DNA antibody. In some embodiments, the therapeutic substance is non-covalently linked to the anti-DNA antibody. In some embodiments, one or more anti-DNA antibodies are conjugated by one or more secondary antibodies, and the therapeutic agent is conjugated to one or more secondary antibodies.

[0032] In some embodiments, the method further comprises the step of contacting cells with one or more nucleases, optionally, one or more nucleases including single-stranded DNA (ssDNA) nucleases, double-stranded DNA (dsDNA) nucleases, or a combination thereof. In some embodiments, the cells are in vitro. In some embodiments, the cells are in vivo.

[0033] In some embodiments, the cells have aneuploidy and / or DNA repair defects. In some embodiments, the cells contain a functionally impaired transcription factor selected from the group consisting of p53, NF-κb, AP-1, E2F1, and BRCA1. In some embodiments, the transcription factor dysfunction is caused by one or more gene mutations, loss of a gene or chromosomal region, and / or protein expression defects. In some embodiments, the cells contain the functionally impaired transcription factor p53, and optionally, the p53 dysfunction includes mutations.

[0034] In some aspects, the cells are cancer cells, and the therapeutic agent kills the cancer cells or inhibits their proliferation or division. In some aspects, the types of cancer are acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical carcinoma; AIDS-related cancer; AIDS-related lymphoma; anal cancer; appendiceal cancer; astrocytoma; atypical teratoid / rhabdoid tumor; basal cell carcinoma; bladder cancer; brainstem glioma; brain tumor, brainstem glioma, atypical teratoid / rhabdoid tumor of the central nervous system, embryonic tumor of the central nervous system, astrocytoma, craniopharyngioma, ependymoblastoma, ependymodium, medulloblastoma, medullary epithelioma, intermediate pineal parenchymal tumor, tentorium Primitive neuroectodermal tumors and pineal blastomas; breast cancer; bronchial tumors; Burkitt lymphoma; cancer of unknown primary origin (CUP); carcinoid tumors; carcinomas of unknown primary origin; atypical teratoid / rhabdoid tumors of the central nervous system; embryonal tumors of the central nervous system; cervical cancer; childhood cancer; chordoma; chronic lymphocytic leukemia; chronic myeloid leukemia; chronic myeloproliferative disorders; colon cancer; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; endocrine islet cell tumors; endometrial cancer; ependymoblastoma; ependymoma; esophageal cancer; nose Cavity neuroblastoma; Ewing's sarcoma; extracranial germ cell tumor; extragonadal germ cell tumor; extrahepatic cholangiocarcinoma; gallbladder cancer; gastric (stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal cell tumor; gastrointestinal stromal tumor (GIST); gestational choriocarcinoma; glioma; hairy cell leukemia; head and neck cancer; heart cancer; Hodgkin's lymphoma; hypopharyngeal cancer; intraocular melanoma; pancreatic islet cell tumor; Kaposi's sarcoma; kidney cancer; Langerhans cell histiocytosis; laryngeal cancer; lip cancer; Liver cancer; lung cancer; malignant fibrous histiocytoma bone cancer; medulloblastoma; medullary epithelioma; melanoma; Merkel cell carcinoma; Merkel cell carcinoma; mesothelioma; metastatic squamous cell carcinoma of unknown primary origin for the neck; mouth cancer; multiple endocrine neoplasia syndrome; multiple myeloma; multiple myeloma / plasmacytic neoplasm; mycosis fungoides; myelodysplastic syndrome; myeloproliferative neoplasm; nasal cavity cancer; nasopharyngeal cancer; neuroblastoma; non-Hodgkin lymphoma; non-melanoma 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 carcinoma; ovarian germ cell tumor; low-grade ovarian tumor; pancreatic cancer; papilloma; paranasal sinus cancer; parathyroid cancer; pelvic cancer; penile cancer; pharyngeal cancer; intermediate pineal parenchymal tumor; pineoblastoma;Pituitary tumors; plasma cell tumors / multiple myeloma; pleuroblastoma; primary central nervous system (CNS) lymphoma; primary hepatocellular carcinoma; prostate cancer; rectal cancer; kidney cancer; renal cell carcinoma; respiratory cancer; retinoblastoma; rhabdomyosarcoma; salivary gland cancer; Sézary syndrome; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma; squamous cervical cancer; stomach cancer; supratentorial primitive neuroectodermal tumor; T-cell lymphoma; testicular cancer; throat cancer; thymic cancer; thymoma; thyroid cancer; transitional cell carcinoma; transitional cell carcinoma of the renal pelvis and ureter; choriocarcinoma; ureteral cancer; urethral cancer; uterine cancer; uterine sarcoma; vaginal cancer; vulvar cancer; Waldenström macroglobulinemia; or Wilms' tumor.

[0035] In some aspects, cancer types include acute myeloid leukemia (AML), breast cancer, cholangiocarcinoma, colorectal adenocarcinoma, extrahepatic cholangiocarcinoma, female reproductive tract malignancies, gastric adenocarcinoma, gastroesophageal adenocarcinoma, gastrointestinal stromal tumors (GIST), glioblastoma, head and neck squamous cell carcinoma, leukemia, hepatocellular carcinoma, low-grade glioma, bronchioloalveolar carcinoma (BAC), non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), lymphoma, and male This includes malignancies of the genitourinary tract, malignant solitary fibrous neoplasm of the pleura (MSFT), melanoma, multiple myeloma, neuroendocrine tumors, diffuse large B-cell lymphoma, non-epithelial ovarian cancer (non-EOC), ovarian surface epithelial carcinoma, pancreatic adenocarcinoma, pituitary cancer, oligodendroglioma, prostate adenocarcinoma, retroperitoneal or peritoneal cancer, retroperitoneal or peritoneal sarcoma, small intestinal malignancies, soft tissue tumors, thymic carcinoma, thyroid cancer, or uveal melanoma. In some embodiments, cancer cells originate from cancer in the subject.

[0036] In yet another aspect, the Disclosure provides a method for treating a subject in need of treatment, comprising the step of administering to the subject a composition comprising a therapeutic agent linked to an anti-DNA antibody, wherein the therapeutic agent is delivered into the cells of the subject using the method disclosed herein, and the delivery of the therapeutic agent is effective in treating the subject. In some embodiments, the composition further comprises a DNA nuclease. In some embodiments, the subject has cancer, and the cells are cancer cells.

[0037] In some embodiments, the method further includes a step of determining, prior to the administration step, whether the cells have aneuploidy, DNA repair defects, and / or functionally impaired transcription factors selected from the group consisting of p53, NF-κb, AP-1, E2F1, and BRCA1. In some embodiments, the dysfunction of a transcription factor is caused by one or more gene mutations, loss of a gene or chromosomal region, and / or impaired protein expression. In some embodiments, the cells contain the functionally impaired transcription factor p53, and optionally, the dysfunction of p53 includes mutations.

[0038] In yet another aspect, the disclosure provides compositions comprising one or more anti-DNA antibodies and one or more nucleases, wherein the one or more nucleases optionally comprise one or more DNA nucleases. In some embodiments, one or more anti-DNA antibodies are conjugated by a secondary antibody. In some embodiments, one or more anti-DNA antibodies are coated onto gold nanoparticles. In some embodiments, at least two anti-DNA antibodies are linked together as a polymer. In some embodiments, the polymer comprises a dimer, trimer, tetramer, pentamer, or has six or more 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.

[0039] In some embodiments, the composition further comprises a therapeutic substance covalently or noncovalently linked to an anti-DNA antibody and / or a secondary antibody. In some embodiments, the therapeutic substance is selected from the group consisting of antitumor agents, anti-cancer agents, prodrugs, lysosomal destabilizers (e.g., chloroquine), alkylating agents, alkaloids, allosteric inhibitors, antifolic acid agents, anti-inflammatory agents, antibiotics, antibacterial agents, antifungal agents, antifibrotic agents, anti-infective agents, antiparasitic agents, antiviral agents, antimycobacterial agents, anti-cancer agents, antiprotozoal agents, antiviral agents, physiologically active peptides, steroid hormones, photosensitive substances, radiopharmaceuticals, antiprion agents, and any combination thereof.

[0040] In some embodiments, antitumor agents include aromatase inhibitors; anti-estrogens; anti-androgens; gonadrelin agonists; topoisomerase I inhibitors; topoisomerase II inhibitors; microtubule inhibitors; alkylating agents; retinoids, carotenoids, or tocopherols; cyclooxygenase inhibitors; MMP inhibitors; mTOR inhibitors; antimetabolites; platinum compounds; methionine aminopeptidase inhibitors; bisphosphonates; antiproliferative antibodies; heparinase inhibitors; inhibitors of Ras oncogenic isoforms; and terrorists. Melase inhibitors; proteasome inhibitors; Flt-3 inhibitors; Hsp90 inhibitors; kinesin spindle protein inhibitors; MEK inhibitors; PARP inhibitors, tyrosine kinase inhibitors, PI3K inhibitors, AKT inhibitors, EGFR inhibitors, antitumor antibiotics; nitrosoureas, compounds that target / reduce protein or lipid kinase activity, compounds that target / reduce protein or lipid phosphatase activity, any further anti-angiogenic compounds, and any combination thereof are selected from the group.

[0041] In some embodiments, the antitumor agent is selected from the group consisting of azacitidine, azathioprine, bleomycin, capecitabine, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, etoposide, fenretinide, fluorouracil, gemcitabine, herceptin, idarubicin, mechloretamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, tafluposide, teniposide, thioguanine, retinoic acid, barrubicin, vinblastine, vincristine, vindesine, vinorelbine, receptor tyrosine kinase inhibitors, and any combination thereof.

[0042] In some embodiments, the antitumor agent includes 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, meltansine (emtansine, DM1), labtansine (solabtansine, DM4), tubulinin, halichondrin (eribulin), cryptophycin, EG5 inhibitors, and any derivatives thereof. In some embodiments, the DNA inhibitor is selected from the group consisting of alkylating agents, duocalmycin, duocalmycin DM (DMDM), calicheamycin, pyrrolobenzodiazepine (PDB), enediyne, unciaramycin, topoisomerase inhibitors, topotecan, camptothecin (CPT), exatecan, and any derivatives thereof.

[0043] In some embodiments, the RNA inhibitor is selected from the group consisting of RNA splicing inhibitors, RNA polymerase II inhibitors, tylanstatins, amatoxins, and any derivatives thereof. In some embodiments, one or more nucleases include endonucleases, exonucleases, or combinations 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 S1, nuclease P1, a sequence-specific endonuclease, or a sequence-nonspecific endonuclease. In some embodiments, one or more DNA nucleases include single-stranded DNA (ssDNA) nucleases, double-stranded DNA (dsDNA) nucleases, or combinations thereof.

[0044] This disclosure also provides a pharmaceutically effective amount of the composition disclosed herein and a pharmaceutically acceptable excipient, carrier, and / or diluent, comprising a pharmaceutically acceptable pharmaceutically acceptable excipient, carrier, and / or diluent. This disclosure further provides a method for treating or relieving a disease or disorder in a subject where there is a need, comprising the step of administering the composition to the subject, optionally, the disease or disorder including cancer.

[0045] In some embodiments, the method further includes a step of determining, prior to the administration step, whether the cancer has aneuploidy, DNA repair defects, and / or a functionally impaired transcription factor selected from the group consisting of p53, NF-κb, AP-1, E2F1, and BRCA1. In some embodiments, the transcription factor dysfunction is caused by one or more gene mutations, loss of a gene or chromosomal region, and / or impaired protein expression. In some embodiments, the cancer includes a functionally impaired transcription factor p53 resulting from a deficiency of the TP53 gene, and optionally, the p53 dysfunction includes mutations. In some embodiments, the administration step includes at least one of intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intracerebral, vaginal, transdermal, rectal, inhalation, topical, or any combination thereof.

[0046] This 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 is covalently or non-covalently linked to at least one payload. In some embodiments, at least one of the one or more anti-DNA antibodies is bound to one or more secondary antibodies. In some embodiments, at least one of the one or more secondary antibodies is covalently or non-covalently linked to at least one payload. In some embodiments, one or more anti-DNA antibodies and / or one or more secondary antibodies are covalently linked to at least one payload.

[0047] In some embodiments, one or more anti-DNA antibodies and / or one or more secondary antibodies are linked to at least one payload via one or more linkers. In some embodiments, one or more linkers include non-cleaving linkers. In some embodiments, the non-cleaving linkers include maleimide alkane linkers or maleimide cyclohexane linkers. In some embodiments, one or more linkers include cleaving linkers. In some embodiments, the cleaving linkers include hydrazone linkers, cathepsin B-responsive linkers, disulfide linkers, or pyrophosphate diester linkers, or a combination thereof. In some embodiments, the cleaving linkers include protease-sensitive linkers, pH-sensitive linkers, radiosensitive linkers, disulfide linkers, or glutathione-sensitive linkers, or a combination thereof. In some embodiments, one or more anti-DNA antibodies and / or one or more secondary antibodies are bound to a biotin moiety, and at least one payload is bound to streptavidin.

[0048] In some embodiments, at least one payload comprises small molecules, peptides, proteins, nucleic acids, toxins, therapeutic substances, drugs, chemotherapeutic agents, liposomes, nanoparticles, dendrimers, detectable labels, or any derivatives, fragments, or combinations thereof.

[0049] In some embodiments, therapeutic substances are selected from the group consisting of antitumor agents, anticancer agents, prodrugs, lysosomal destabilizers (e.g., chloroquine), alkylating agents, alkaloids, allosteric inhibitors, antifolic acid agents, anti-inflammatory agents, antibiotics, antibacterial agents, antifungal agents, antifibrotic agents, antiinfective agents, antiparasitic agents, antiviral agents, antimycobacterial agents, anticancer agents, antiprotozoal agents, antiviral agents, physiologically active peptides, steroid hormones, photosensitive substances, radiopharmaceuticals, antiprion agents, and any combination thereof.

[0050] In some embodiments, antitumor agents include aromatase inhibitors; anti-estrogens; anti-androgens; gonadrelin agonists; topoisomerase I inhibitors; topoisomerase II inhibitors; microtubule inhibitors; alkylating agents; retinoids, carotenoids, or tocopherols; cyclooxygenase inhibitors; MMP inhibitors; mTOR inhibitors; antimetabolites; platinum compounds; methionine aminopeptidase inhibitors; bisphosphonates; antiproliferative antibodies; heparinase inhibitors; inhibitors of Ras oncogenic isoforms; and terrorists. Melase inhibitors; proteasome inhibitors; Flt-3 inhibitors; Hsp90 inhibitors; kinesin spindle protein inhibitors; MEK inhibitors; PARP inhibitors, tyrosine kinase inhibitors, PI3K inhibitors, AKT inhibitors, EGFR inhibitors, antitumor antibiotics; nitrosoureas, compounds that target / reduce protein or lipid kinase activity, compounds that target / reduce protein or lipid phosphatase activity, any further anti-angiogenic compounds, and any combination thereof are selected from the group.

[0051] In some embodiments, the antitumor agent is selected from the group consisting of azacitidine, azathioprine, bleomycin, capecitabine, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, etoposide, fenretinide, fluorouracil, gemcitabine, herceptin, idarubicin, mechloretamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, tafluposide, teniposide, thioguanine, retinoic acid, barrubicin, vinblastine, vincristine, vindesine, vinorelbine, receptor tyrosine kinase inhibitors, and any combination thereof.

[0052] In some embodiments, the antitumor agent includes 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), meitansine, meitansinoids, meltansine (emtansine, DM1), labtansine (sorabtansine, DM4), tubulinin, halichondrin (eribulin), cryptophycin, EG5 inhibitors, and any derivatives thereof. In some embodiments, the DNA inhibitor is selected from the group consisting of alkylating agents, duocalmycin, duocalmycin DM, calicheamicin, pyrrolobenzodiazepine (PDB), enediyne, unciaramycin, topoisomerase inhibitors, topotecan, camptothecin (CPT), exatecan, and any derivatives thereof. In some embodiments, the RNA inhibitor is selected from the group consisting of RNA splicing inhibitors, RNA polymerase II inhibitors, tylanstatin, amatoxin, and any derivatives thereof.

[0053] In some embodiments, the detectable labels are selected from the group consisting of magnetic labels, fluorescent moieties, enzymes, luminescent particles, chemiluminescent probes, metallic particles, nonmetallic colloidal particles, polymer dye particles, dye molecules, electrochemically active species, semiconductor nanocrystals, nanoparticles, quantum dots, gold particles, fluorophores, radioactive labels, or combinations thereof.

[0054] In some embodiments, one or more anti-DNA antibodies are sequence-specific to target DNA. In some embodiments, one or more anti-DNA antibodies are non-sequence-specific to target DNA. In some embodiments, one or more anti-DNA antibodies comprise a combination of sequence-specific anti-DNA antibodies and non-sequence-specific anti-DNA antibodies. In some embodiments, one or more anti-DNA antibodies bind to double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), or a combination thereof.

[0055] In some embodiments, one or more anti-DNA antibodies are coated onto gold nanoparticles. In some embodiments, at least two anti-DNA antibodies are linked together as a multimer. In some embodiments, the multimer comprises a dimer, trimer, tetramer, pentamer, or has six or more 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.

[0056] This 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 endonucleases, exonucleases, or a combination thereof. In some embodiments, the endonucleases are deoxyribonucleases (DNases), Serratia marcescens nucleases (benzoases), micrococcal nucleases (MNases), transposases, restriction enzymes, nuclease S1, nuclease P1, sequence-specific endonucleases, or sequence-nonspecific endonucleases. In some embodiments, the one or more DNA nucleases comprise single-stranded DNA (ssDNA) nucleases, double-stranded DNA (dsDNA) nucleases, or a combination thereof.

[0057] The disclosure further provides a method for internally transferring one or more anti-DNA antibodies into human cells, comprising the step of contacting the cells with a first composition disclosed herein, wherein the one or more anti-DNA antibodies bind to extracellular DNA on the surface of the cells and are internally transferred into the cells.

[0058] This disclosure further provides a method for delivering a payload into a cell, comprising the step of contacting the cell with a first composition disclosed herein, wherein one or more anti-DNA antibodies bind to extracellular DNA on the surface of the cell and deliver the payload into the cell. In some embodiments, the payload comprises a therapeutic substance disclosed herein. In some embodiments, the method further comprises the step of contacting the cell with a second composition disclosed herein. In some embodiments, the first and second compositions are contacted to the cell simultaneously, and optionally, the first and second compositions are the same composition. In some embodiments, one or more nucleases are bound to 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, a linker is cleaved after the step of contacting the cell with one or more anti-DNA antibodies, thereby releasing the payload onto or into the cell. In some embodiments, the cell is in vitro. In some embodiments, the cell is in vivo. In some embodiments, the cells have aneuploidy and / or DNA repair defects. In some embodiments, the cells contain a functionally impaired transcription factor selected from the group consisting of p53, NF-κb, AP-1, E2F1, and BRCA1. In some embodiments, the transcription factor dysfunction is caused by one or more gene mutations, loss of a gene or chromosomal region, and / or protein expression defects. In some embodiments, the cells contain the functionally impaired transcription factor p53, and optionally, the p53 dysfunction includes mutations. In some embodiments, the cells are cancer cells, and the payload kills the cancer cells or inhibits their proliferation or division. In some embodiments, the cancer cells originate from cancer in the subject.

[0059] This disclosure further provides a method comprising the step of administering a first composition disclosed herein to a subject, and optionally further comprising the step of administering a second composition disclosed herein to a subject. 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 simultaneously, or 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 cancer, and the administration step is carried out in a dose effective to treat the cancer. In some embodiments, the anti-DNA antibody in the first composition constitutes a payload.

[0060] In some embodiments, the method further includes a step of determining, prior to the administration step, whether the cancer has aneuploidy, DNA repair defects, and / or functionally impaired transcription factors selected from the group consisting of p53, NF-κb, AP-1, E2F1, and BRCA1. In some embodiments, the transcription factor dysfunction is caused by one or more gene mutations, loss of a gene or chromosomal region, and / or impaired protein expression.

[0061] In some aspects, cancer includes a functionally impaired transcription factor p53, and optionally, the impairment of p53 includes mutations. In some aspects, cancer types include acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical carcinoma; AIDS-related cancer; AIDS-related lymphoma; anal cancer; appendiceal cancer; astrocytoma; atypical teratomatous / rhabdoid tumor; basal cell carcinoma; bladder cancer; brainstem glioma; brain tumor, brainstem glioma, atypical teratomatous / rhabdoid tumor of the central nervous system, embryonic tumor of the central nervous system, astrocytoma, craniopharyngioma, ependymoblastoma, ependymodium, medulloblastoma, medullary epithelioma, intermediate pineal parenchymal tumor, ten Primitive neuroectodermal tumors and pineal blastomas; breast cancer; bronchial tumors; Burkitt lymphoma; cancer of unknown primary origin (CUP); carcinoid tumors; carcinomas of unknown primary origin; atypical teratoid / rhabdoid tumors of the central nervous system; embryonal tumors of the central nervous system; cervical cancer; childhood cancer; chordoma; chronic lymphocytic leukemia; chronic myeloid leukemia; chronic myeloproliferative disorders; colon cancer; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; endocrine islet cell tumors; endometrial cancer; ependymoblastoma; ependymoma; esophageal cancer ; Nasal cavity neuroblastoma; Ewing's sarcoma; Extracranial germ cell tumor; Extragonadal germ cell tumor; Extrahepatic cholangiocarcinoma; Gallbladder cancer; Gastric (stomach) cancer; Gastrointestinal carcinoid tumor; Gastrointestinal stromal cell tumor; Gastrointestinal stromal tumor (GIST); Gestational choriocarcinoma; Glioma; Hairy cell leukemia; Head and neck cancer; Heart cancer; Hodgkin's lymphoma; Hypopharyngeal cancer; Intraocular melanoma; Pancreatic islet cell tumor; Kaposi's sarcoma; Kidney cancer; Langerhans cell histiocytosis; Laryngeal cancer; Mouth Lip cancer; liver cancer; lung cancer; malignant fibrous histiocytoma bone cancer; medulloblastoma; medullary epithelioma; melanoma; Merkel cell carcinoma; Merkel cell cutaneous carcinoma; mesothelioma; metastatic squamous cell carcinoma of unknown primary origin in the neck; oral cancer; multiple endocrine neoplasia syndrome; multiple myeloma; multiple myeloma / plasmacytic neoplasm; mycosis fungoides; myelodysplastic syndrome; myeloproliferative neoplasm; nasal cavity cancer; nasopharyngeal cancer; neuroblastoma; non-Hodgkin lymphoma; non-melanoma skin cancer; non-small cell lung cancer; oral cancer; oral cancer; oropharyngeal cancer; osteosarcoma; other brain and spinal cord tumors; ovarian cancer; ovarian epithelial carcinoma; ovarian germ cell tumor; low-grade ovarian tumor; pancreatic cancer; papilloma; paranasal sinus cancer; parathyroid cancer; pelvic cancer; penile cancer; pharyngeal cancer; intermediate pineal parenchymal tumor; pineoblastoma; pituitary tumor; plasma cell neoplasm / multiple myeloma; pleuroblastoma;Primary central nervous system (CNS) lymphoma; primary hepatocellular carcinoma; prostate cancer; rectal cancer; kidney cancer; renal cell carcinoma; respiratory cancer; retinoblastoma; rhabdomyosarcoma; salivary gland cancer; Sézary syndrome; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma; squamous cervical cancer; stomach cancer; supratentorial primitive neuroectodermal tumor; T-cell lymphoma; testicular cancer; throat cancer; thymic cancer; thymoma; thyroid cancer; transitional cell carcinoma; transitional cell carcinoma of the renal pelvis and ureter; choriocarcinoma; ureteral cancer; urethral cancer; uterine cancer; uterine sarcoma; vaginal cancer; vulvar cancer; Waldenström macroglobulinemia; or Wilms' tumor.

[0062] In some aspects, cancer types include acute myeloid leukemia (AML), breast cancer, cholangiocarcinoma, colorectal adenocarcinoma, extrahepatic cholangiocarcinoma, female reproductive tract malignancies, gastric adenocarcinoma, gastroesophageal adenocarcinoma, gastrointestinal stromal tumors (GIST), glioblastoma, head and neck squamous cell carcinoma, leukemia, hepatocellular carcinoma, low-grade glioma, bronchioloalveolar carcinoma (BAC), non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), lymphoma, and male This includes malignancies of the genitourinary tract, malignant solitary fibrous neoplasms of the pleura (MSFT), melanoma, multiple myeloma, neuroendocrine tumors, diffuse large B-cell lymphoma nodosa, non-epithelial ovarian cancer (non-EOC), ovarian surface epithelial carcinoma, pancreatic adenocarcinoma, pituitary cancer, oligodendroglioma, prostate adenocarcinoma, retroperitoneal or peritoneal cancer, retroperitoneal or peritoneal sarcoma, small intestinal malignancies, soft tissue tumors, thymic carcinoma, thyroid cancer, or uveal melanoma.

[0063] In some embodiments, the administration step includes at least one of the following: intradermal administration, intramuscular administration, intraperitoneal administration, intravenous administration, subcutaneous administration, intranasal administration, epidural administration, oral administration, sublingual administration, intracerebral administration, vaginal administration, transdermal administration, rectal administration, administration by inhalation, topical administration, or any combination thereof.

[0064] This disclosure further provides a method for internally transferring one or more anti-DNA antibodies into a cell. In some embodiments, the method comprises the step of contacting a cell with one or more anti-DNA antibodies, the one or more anti-DNA antibodies comprising means for binding to extracellular DNA, the one or more anti-DNA antibodies binding to extracellular DNA on the surface of the cell, and the one or more anti-DNA antibodies internally transferring into the cell.

[0065] The disclosure further provides a method for delivering a therapeutic substance into the interior of a cell. In some embodiments, the method comprises the step of contacting a cell with a therapeutic substance covalently or noncovalently linked to an anti-DNA antibody, one or more anti-DNA antibodies comprising means for binding to extracellular DNA, the anti-DNA antibody binding to extracellular DNA on the cell surface to deliver the therapeutic substance into the interior of the cell.

[0066] The disclosure further provides compositions comprising one or more anti-DNA antibodies and one or more nucleases, wherein the one or more nucleases optionally comprises one or more DNA nucleases, and the one or more anti-DNA antibodies comprises means for binding to extracellular DNA.

[0067] The disclosure further provides an improvement in a method for contacting an antibody with a cell, comprising contacting the cell with one or more anti-DNA antibodies to cause one or more anti-DNA antibodies to be internally delivered into the cell, wherein one or more anti-DNA antibodies bind to extracellular DNA on the surface of the cell, and one or more anti-DNA antibodies are internally delivered into the cell.

[0068] The disclosure further provides an improvement in a method of treating a subject having cancer, comprising administering to the subject a composition comprising a therapeutic substance conjugated to an anti-DNA antibody, wherein the therapeutic substance is delivered into the cells of the subject, and the delivery of the therapeutic substance is effective in treating the subject. [Brief explanation of the drawing]

[0069] [Figure 1A] Figures 1A–C illustrate schematic diagrams of the binding and internal migration of anti-DNA antibody constructs to cells. Figure 1A shows the binding of an anti-DNA antibody to extracellular DNA on the cell surface, where one or more anti-DNA antibodies are also recognized and ligated to a second antibody conjugated with a desired payload, such as a therapeutic agent or fluorescent label. Internal migration of the complex of the extracellular DNA, anti-DNA antibody, and second ADC results in intracellular payload delivery, e.g., cell death (if the conjugate is therapeutic) or cell imaging (if the conjugate is fluorescent label). Figure 1B shows a schematic diagram similar to Figure 1A, except that the payload is directly bound to the anti-DNA antibody. Figure 1C shows a schematic diagram similar to Figure 1B, where the extracellular DNA is packaged around an extracellular nucleosome. In addition, it is shown that DNA nucleases cleave the extracellular DNA. See relevant discussions in this specification. Figure 1C also shows that a secondary antibody may be used in some embodiments, for example, in Figure 1A. Figure 1D illustrates schematic diagrams of possible formats for anti-DNA ADC / ADC complexes. [Figure 1B] See the explanation in Figure 1A. [Figure 1C] See the explanation in Figure 1A. [Figure 1D] See the explanation in Figure 1A. [Figure 2-1]Figures 2A-2E illustrate the cytotoxicity of anti-DNA antibodies with drug conjugates in cancer cells: human pancreatic cancer cells AsPC-1 (Figure 2A), human HER2+ breast cancer cells AU565 (Figure 2B), human pancreatic cancer cells SW1990-1 (Figure 2C), and human triple-negative breast cancer cells HCC1395 (Figure 2D), as well as human non-tumor-forming epithelial cells MCF10A (Figure 2E). Cytotoxicity assays are shown 6 days after treatment. All cancer cells treated with anti-DNA antibodies and 2nd antibody-drug conjugates (2nd ADCs) (Figures 2A-2D) had lower survival rates compared to control cells treated with mouse IgG isotype antibodies along with the 2nd antibody-drug conjugate. In non-cancer cells, MCF10A, there was no significant difference between the test antibody and the control (Figure 2E), suggesting that the cytotoxicity of anti-DNA antibodies with drug conjugates is specific to cancer cells. [Figure 2-2] See the explanation in Figure 2-1. [Figure 2-3] See the explanation in Figure 2-1. [Figure 3] Figures 3A-3B illustrate the cytotoxicity of anti-DNA antibodies without drug conjugates in AU565 cells (Figure 3A) and HCC1395 cells (Figure 3B), suggesting that the cytotoxicity was not caused by the anti-DNA antibody alone. [Figure 4] Figures 4A–4D show that the cytotoxicity of anti-DNA antibodies with drug conjugates can be increased by nuclease treatment. HCC1395 cells (Figures 4A and 4C) and AU565 cells (Figures 4B and 4D) showed significantly stronger cytotoxic responses with anti-DNA antibody / ADC and nuclease compared to cells treated with anti-DNA antibody / ADC without nuclease. Figures 4A and 4B illustrate the cytotoxic results of cells treated with DNase I. Figures 4C and 4D illustrate the cytotoxic results of cells treated with benzonase, a recombinant DNA / RNA endonuclease. [Figure 5A]Figures 5A–5K illustrate the effect of nucleases on the cytotoxicity of anti-DNA ADCs provided herein. Figures 5A–5B illustrate the cytotoxicity of DNase I at various concentrations in non-tumoric human cells MCF10A (Figure 5A) and human cancer cells HCC1395 (Figure 5B), suggesting that the cytotoxicity was not caused by DNase I. Figures 5C–5D show the cytotoxicity of non-anti-DNA ADC constructs in the absence (Figure 5C) or presence (Figure 5D) of DNase I. These figures show that DNase I did not enhance the sensitivity of cells to antibody-drug conjugates (ADCs) that do not target extracellular DNA on the surface of cancer cells. Figure 5E shows similar results using anti-CD71 with a secondary ADC. Figure 5F shows that the cytotoxicity of anti-DNA ADCs with nuclease treatment is dose and / or duration-dependent, with greater cytotoxicity observed when cells were not pre-incubated with DNase before the addition of anti-DNA ADCs compared to cells pre-incubated with DNase (bars 4 and 5) (center bar). Figures 5G–5J show that various DNA nucleases, including bovine DNase I (Figure 5G), recombinant human DNase I (Figure 5H), benzonase (Figure 5I), and micrococcal nuclease (MNase) (Figure 5J), enhance the sensitivity of cells to anti-DNA ADCs. However, RNase did not enhance the sensitivity of cells. See Figure 5K. [Figure 5B] See the explanation in Figure 5A. [Figure 5C] See the explanation in Figure 5A. [Figure 5D] See the explanation in Figure 5A. [Figure 5E] See the explanation in Figure 5A. [Figure 5F] See the explanation in Figure 5A. [Figure 5G] See the explanation in Figure 5A. [Figure 5H] See the explanation in Figure 5A. [Figure 5I] See the explanation in Figure 5A. [Figure 5J]See the explanation in Figure 5A. [Figure 5K] See the explanation in Figure 5A. [Figure 6] Figures 6A-6B illustrate that the cytotoxicity of anti-DNA ADCs treated with nucleases specifically targets cancer cells (human HER2+ breast cancer cells AU565 and human triple-negative breast cancer cells HCC139) compared to normal cells (non-cancerous breast epithelial cells MCF10A). [Figure 7] The specific cytotoxicity of cancer cells by three different anti-DNA antibody / ADCs treated with nucleases is illustrated. As shown in the figure, the antibodies are Abcam 35I9, Millipore BV16-13, and Millipore 16-19. AU565 is a cancer cell line (derived from a patient with HER2+ breast cancer), and MCF10A is a non-cancer control (non-cancerous breast epithelial cells). The treatment compositions are shown in the figure. [Figure 8A]Figures 8A and 8B show that the cytotoxicity of anti-DNA ADC and DNase I is associated with TP53 mutations in cancer cells. In Figure 8A, each group of bars is arranged from left to right in the same order as the list of cell lines on the left, from top to bottom. Figure 8B is the same as the last grouping in Figure 8A, but also provides more detail on the TP53 status of the cell lines. In Figure 8B, the bars from left to right across the entire plot have the same order as the list of cell lines on the left, from top to bottom (which is also the same order as each grouping in Figure 8A). Figure 8A shows that both DNase I (the group labeled DNase I) and anti-DNA Ab+2nd ADC (the group labeled anti-DNA ADC) exhibited minimal cytotoxicity to cells when used alone, whereas when DNase I (test concentration 0.2 ug / ml) was combined with anti-DNA Ab+2nd ADC (the group labeled anti-DNA ADC+DNase I) in TP53 mutant cells, a considerable level of cytotoxicity was observed. The data in Figure 8B show that anti-DNA Ab+2nd ADC+DNase I exhibited greater cytotoxicity against various cell lines carrying TP53 loss-of-function mutations (the group labeled as TP53 mutations) compared to non-cancer cell lines or TP53 WT cell lines (the group labeled as TP53 WT). [Figure 8B] See the explanation in Figure 8A. [Figure 9] Figures 9A–9B illustrate the cytotoxicity of three therapeutic payloads when delivered by anti-DNA antibody in conjunction with DNase I treatment at 3 days (Figure 9A) or 6 days (Figure 9B). Cytotoxicity was observed in all conditions, but the monomethyl auristatin F (MMAF) payload showed higher efficacy with anti-DNA antibody compared to duocalmycin DM (DMDM) or exatecan (Extecan). [Figure 10]The cytotoxicity of various anti-DNA antibodies and associated ADC constructs is shown. The constructs are illustrated at the top of each bar. In these experiments, the inventors observed high cytotoxicity with ADCs containing anti-DNA Ab complexes conjugated to 2nd Ab, regardless of whether the drug was conjugated to anti-DNA Ab and / or 2nd Ab. The highest cytotoxicity was observed in the group treated with drug conjugates in both anti-DNA antibody and secondary antibody. [Figure 11-1] Figures 11A–11G show a comparison of the efficacy of anti-DNA ADCs compared to trastuzumab deruxtecan, an ADC composed of trastuzumab, a humanized monoclonal antibody covalently linked to the topoisomerase I inhibitor deruxtecan. The plots in Figures 11A–11E show cell viability against antibody concentration using ADCs, with or without DNase I, for various cell lines: HER2+ breast cancer cell line AU565 (Figure 11A); HER2+ breast cancer cell line BT474 (Figure 11B); HER2 low breast cancer cell line HCC1395 (Figure 11C); HER2 low breast cancer cell line MDA-MB-468 (Figure 11D), as indicated in the legend. The upper plot in Figure 11E is the same as in Figure 11C for HCC1395, while the lower plot relates to the adjacent normal cell line HCC1395 BL. The vertical lines show IC50 in cancer cells (top) and normal cells (bottom). Figures 11F-11G show that HER2+ cell lines AU565 (Figure 11F) and BT474 (Figure 11G) did not show increased sensitivity to trastuzumab deruxtecan by 2nd Ab or DNase I, unlike anti-DNA ADCs. [Figure 11-2] See the explanation in Figure 11-1. [Figure 11-3] See the explanation in Figure 11-1. [Figure 11-4] See the explanation in Figure 11-1. [Modes for carrying out the invention]

[0070] Detailed explanation I. Introduction This disclosure provides compositions and methods for targeting anti-DNA antibodies to cells of interest. The antibodies can be internally transported into the target cells. In addition, this internal transport can be facilitated by contacting the cells with one or more nucleases. In some embodiments, a complex of two or more anti-DNA antibodies is internally transported. When one or more anti-DNA antibodies are conjugated with one or more payloads, including but not limited to therapeutic substances and / or detectable labels, the one or more anti-DNA antibodies can be used to deliver the desired payload to or into cells. Therefore, anti-DNA antibodies with drug conjugates or detectable labels are particularly useful for use in diagnostic and therapeutic applications.

[0071] II. Definition Throughout this disclosure, various quantities, such as amount, size, dimensions, and ratios, are presented in the form of ranges. The presentation of quantities in range form is for convenience and brevity only and should not be interpreted as a strict limitation on the range of any particular aspect. Therefore, unless clearly indicated otherwise in the context, a range statement should be considered to specifically disclose all possible subranges within that range, as well as all individual numerical values. For example, a range statement such as 1–6 should be considered to specifically disclose subranges such as 1–3, 1–4, 1–5, 2–4, 2–6, 3–6, as well as individual values ​​within that range, such as 1.1, 2, 2.3, 4.62, 5, and 5.9. This applies regardless of the breadth of the range. These intervening upper and lower limits may be independently contained within smaller ranges and are included within this disclosure, subject to any particularly excluded limits within the stated range. If the described scope includes either or both of the upper and lower limits, the scope excluding either or both of these included upper and lower limits is also included in this disclosure unless the context clearly indicates otherwise.

[0072] The terms used herein are intended solely to describe specific aspects and are not intended to limit any particular aspect. Where used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Where used herein, the terms “include,” “comprise,” “including,” and / or “comprising” identify the presence of a described feature, integer, process, operation, element, and / or component, but are further understood not to exclude the presence or addition of one or more other features, integers, processes, operations, elements, components, and / or groups thereof.

[0073] As used herein, the term “and / or” includes any one or more of the related enumerated items and all combinations thereof. In addition, it should be understood that an enumeration in the form of “at least one of A, B, and C” may 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 with “at least one” herein. For example, an enumeration in the form of “at least one of A, B, or C” may mean (A);(B);(C);(A and B);(B and C);(A and C); or (A, B, and C).

[0074] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to polymers of amino acid residues. These terms include amino acid polymers, in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, as well as naturally occurring and unnatural amino acid polymers.

[0075] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimes that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as subsequently modified amino acids, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, namely, an α-carbon bonded to a hydrogen, carboxyl group, amino group, and R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have a modified R group (e.g., norleucine) or a modified peptide skeleton, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimes refer to chemical compounds that have a different structure from the general chemical structure of amino acids, but function similarly to naturally occurring amino acids.

[0076] In this specification, amino acids may be referred to by either the commonly known three-letter or one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Nucleotides may similarly be referred to by their commonly accepted one-letter codes.

[0077] The term "conservatively modified variant" applies to both amino acid sequences and nucleic acid sequences. With respect to a particular nucleic acid sequence, a conservatively modified variant means a nucleic acid that codes for the same or essentially the same amino acid sequence, or, if the nucleic acid does not code for an amino acid sequence, for an essentially identical sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids code for any given protein. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Therefore, at all positions where alanine is identified by a codon, the codon can be changed to any of the corresponding codons listed without altering the encoded polypeptide. Such nucleic acid mutations are "silent mutations" and are a type of conservatively modified mutation. All nucleic acids in this specification that code for polypeptides also describe all possible silent mutations of the nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (with the exception of AUG, which is usually the sole codon for methionine, and TGG, which is usually the sole codon for tryptophan) can be modified to obtain a functionally identical molecule. Therefore, each silent mutation in the nucleic acid encoding the polypeptide is implied in each described sequence for the expression product, but not in the actual probe sequence.

[0078] With respect to amino acid sequences, those skilled in the art recognize that individual substitutions, deletions, or additions to nucleic acids, peptides, polypeptides, or protein sequences, which alter, add, or delete a single amino acid or a small proportion of amino acids in the encoded sequence, are “conservatively modified variants” in which the alteration results in the substitution of an amino acid with a chemically similar amino acid. Tables of conservative substitutions resulting in functionally similar amino acids are well known in the art. Such conservatively modified variants are additional to, and do not preclude, the polymorphic variants, interspecific homologs, and alleles of the present invention.

[0079] The following eight groups each contain amino acids that are conserved substitutions with each other: 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, for example, Creighton, Proteins (1984)).

[0080] Antibodies can consist of one or more polypeptides substantially encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene. Recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and muon constant region genes, as well as numerous immunoglobulin variable region genes. The light chain is classified as either kappa or lambda. The heavy chain is classified as gamma, muon, alpha, delta, or epsilon, which define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. An “antibody” is structurally defined as a binding protein that contains the amino acid sequence of the framework region of an immunoglobulin-coding gene of an antibody produced by an animal, or an amino acid sequence derived therefrom.

[0081] In this application, the term “antibody” is used in its broadest sense (unless otherwise specified) and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies containing two light chains and two heavy chains), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single-domain antibodies. The term “monoclonal antibody” generally refers to a substantially homogeneous group of antibodies, i.e., antibodies obtained from a cluster in which multiple antibodies are identical except for a few possible natural variants. Monoclonal antibodies generally have high specificity for a single antigen site. Furthermore, unlike conventional polyclonal antibody preparations (which generally contain different antibodies directed to different determinants), each monoclonal antibody is directed to a single determinant on an antigen. The term “chimeric antibody” generally refers to an antibody in which the variable region originates from one species and the constant region originates from another species. Generally, the variable region is derived from antibodies in experimental animals such as rodents ("parental antibodies"), and the constant region is derived from human antibodies, reducing the likelihood of the resulting chimeric antibody causing a harmful immune response in individual humans compared to the parental (e.g., mouse-derived) antibody. The term "humanized antibody" generally refers to an antibody in which some or all of the amino acids other than the CDR of a non-human antibody (such as a mouse antibody) are replaced by corresponding amino acids derived from human immunoglobulin. In the CDR, slight additions, deletions, insertions, substitutions, or modifications to amino acids may also be acceptable, as long as the antibody still retains its ability to bind to a specific antigen. Humanized antibodies may optionally contain at least a portion of the constant region of human immunoglobulin. "Humanized antibodies" retain similar antigen specificity to that of the original antibody. The "humanized" form of a non-human antibody (e.g., a mouse antibody) may contain a minimum amount of chimeric antibodies derived from the non-human immunoglobulin sequence. In some cases, CDR residues in human immunoglobulins (receptor antibodies) may be substituted with CDR residues from non-human species (donor antibodies) (such as mice, rats, rabbits, or non-human primates) that possess the desired properties, affinity, and / or capabilities.In some cases, FR residues of human immunoglobulins may be substituted with corresponding non-human residues. In addition, humanized antibodies may contain amino acid modifications not present in receptor or donor antibodies. These modifications may be made to further improve antibody properties such as binding affinity. The term "fully human antibody" generally refers to an antibody obtained by transplanting a human antibody-coding gene into a genetically modified antibody gene-deficient animal, enabling the animal to express it. All parts of the antibody (including the variable and constant regions) are encoded by genes of human origin. Fully human antibodies can significantly reduce the immunological side effects caused by heterologous antibodies against the human body. Methods for obtaining fully human antibodies in the art include phage display technology, transgenic mouse technology, ribosome display technology, and RNA polypeptide technology.

[0082] The structural unit of an exemplary immunoglobulin (antibody) is a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kD) and one "heavy" chain (approximately 50-70 kD). The N-terminus of each chain defines a variable region of approximately 100-110 or more amino acids, primarily responsible for antigen recognition. L ) and variable heavy chain (V H The terms ) refer to these heavy and light chains, respectively.

[0083] As used herein, the term "antibody" includes antibody fragments that retain binding specificity. For example, there are a plurality of well - characterized antibody fragments. Thus, for example, pepsin digests the antibody C - terminus to the disulfide bond in the hinge region, generating F(ab)'2, which is a dimer of Fab, and the light chain itself is linked to VH - CH1 by a disulfide bond. F(ab)'2 can be reduced under mild conditions to break the disulfide bond in the hinge region, thereby converting the (Fab')2 dimer to Fab' monomers. Fab' monomers are essentially Fab with a 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). Although various antibody fragments are defined from the perspective of digestion of intact antibodies, those skilled in the art understand that the fragments can be synthesized de novo by chemical or recombinant DNA methods. Thus, the term antibody as used herein also includes antibody fragments generated by modification of the whole antibody or synthesized using recombinant DNA methods.

[0084] Antibodies can contain V H -V L dimers, including single - chain antibodies (antibodies that exist as a single polypeptide chain), such as single - chain Fv antibodies (sFv or scFv), in which the variable heavy - chain region and the variable light - chain region are linked together (either directly or through a peptide linker) to form a continuous polypeptide. Single - chain Fv antibodies are covalently linked V H -V L and can be expressed from nucleic acids containing the V H coding sequence and the V L coding sequence (e.g., Huston, et al. Proc. Nat. Acad. Sci. USA, 85:5879 - 5883, 1988). V H and V L are each linked as a single polypeptide chain, VH Domain and V L The domains associate non-covalently. Alternatively, the antibody may be another fragment. Other fragments can be generated, for example, using recombinant technology as soluble proteins or as fragments obtained from display methods. Antibodies may also include diantibodies and miniantibodies. Antibodies may also include heavy chain dimers, such as camel-derived antibodies, or antibodies such as nanobodies.

[0085] Where disclosed herein, the term “antibody” also includes multiparatopic antibodies (e.g., biparatopic antibodies, triparatopic antibodies) that bind to two or more different epitopes of the same antigen. Binding of one antibody leaves further epitopes on the target exposed, allowing for the binding of further antibodies and resulting in cross-linking of antigen-antibody clustering. In such cases, multiple multiparatopic antibodies of the same type (e.g., biparatopic antibodies, triparatopic antibodies, etc.) can cluster together through binding to the same antigen. Alternatively, two, three or more different antibodies (i.e., specific to different binding sites / epitopes) against the same antigen, mixed as an antibody cocktail, can form a larger antibody complex through binding to the same antigen.

[0086] Where disclosed herein, the term “secondary antibody” or “2 ndThe term "antibody" typically refers to an antibody that binds to another antibody, usually called a "primary antibody." Primary antibodies recognize target antigens, while secondary antibodies recognize primary antibodies. Secondary antibodies are usually produced for the species and isotype of the primary antibody. As a non-limiting example, the primary antibody may be a mouse IgG monoclonal antibody specific to a biomarker of interest, and the secondary antibody may be an anti-mouse IgG antibody. Secondary antibodies have many applications, including detection or isolation, delivery of a payload to the antigen site of a primary antibody, either alone or bound to its antigen, or to the antigen site of the primary antibody. For example, a secondary antibody may harbor a detectable label, thereby facilitating indirect detection of the antigen to which the primary antibody is bound via labeling. Alternatively, a secondary antibody may harbor a payload, such as a therapeutic agent, thereby facilitating the indirect delivery of the therapeutic agent to the antigen to which the primary antibody is bound. As used herein, a secondary antibody harboring a therapeutic agent is referred to as a secondary antibody-drug conjugate, secondary ADC, or 2. nd Antibody-drug conjugate, or 2 nd It may be referred to as ADC, etc. For example, as used herein, "anti-DNA Ab+2 nd "ADC" refers to an anti-DNA primary antibody accompanied by a secondary antibody with a cytotoxic payload. See, for example, Figures 1A-1D and related discussions.

[0087] The term "antigen" refers to a molecule, part, foreign particulate matter, or allergen that can bind to a specific antibody or T cell receptor. Antigens can be proteins, peptides (amino acid chains), polysaccharides (monosaccharide chains), lipids, or nucleic acids (e.g., DNA). Antigens are present on normal cells, cancer cells, parasites, viruses, fungi, and bacteria. In some embodiments, the antigen is DNA. In certain embodiments, the antigen is extracellular DNA. In some cases, antibodies are antigen-specific, meaning that an antibody can only react to and bind to one specific antigen. In other examples, antibodies can cross-react to bind to two or more antigens. The reaction between an antigen and an antibody is called an antigen-antibody reaction.

[0088] Unless otherwise specifically stated or evident from the context, the term “about” as used herein with respect to a number or range of numbers is understood to mean a number and a number plus or minus 10% thereof, or a value listed as a range that is 10% below the listed lower limit and 10% above the listed upper limit.

[0089] III. Detailed Description of the Embodiments This disclosure provides a method for targeting cells of interest using anti-DNA antibodies. Such antibodies can be internally transferred after contact with target cells. In one aspect, this disclosure provides a method for internally transferring one or more anti-DNA antibodies (understood herein to include DNA-binding antibody fragments) into cells, comprising the step of contacting cells with one or more anti-DNA antibodies or DNA-binding antibody fragments that bind to extracellular DNA on the surface of the cells and are internally transferred into the cells after binding. In some embodiments, the cells are mammalian cells, including but not limited to human cells. In some embodiments, the method further comprises the step of contacting the cells with one or more nucleases before, after, or concurrently with one or more anti-DNA antibodies or DNA-binding antibody fragments disclosed herein. Furthermore, one or more anti-DNA antibodies or DNA-binding antibody fragments can be covalently or acovalently bound to one or more payloads, including but not limited to therapeutic substances and detectable labels. Thus, the methods disclosed herein have a wide range of applications, including diagnostic and therapeutic applications.

[0090] anti-DNA antibody As used herein, “anti-DNA antibody” or “DNA-binding antibody” means an antibody or antibody fragment capable of recognizing and binding to a site on the phosphodiester skeleton of single-stranded DNA (ssDNA) and / or double-stranded DNA (dsDNA). Such binding may occur to nucleotide sequences, including sequences in higher-order structures such as nucleosomes. See, for example, Figure 1C. In some examples, an anti-DNA antibody or DNA-binding antibody fragment recognizes and binds to single-stranded DNA (ssDNA). In other examples, an anti-DNA antibody or DNA-binding antibody fragment recognizes and binds to double-stranded DNA (dsDNA). In yet another example, an anti-DNA antibody or DNA-binding antibody fragment recognizes and binds to both ssDNA and dsDNA. In some embodiments, an anti-DNA antibody or DNA-binding antibody fragment recognizes and binds to extracellular DNA on the surface of a cell. In some embodiments, an anti-DNA antibody or DNA-binding antibody fragment recognizes and binds to extracellular DNA in the vicinity of a cell. As a non-limiting example, the vicinity of a cell may be the tumor microenvironment.

[0091] The anti-DNA antibodies or fragments provided herein can, as desired, bind to specific or unspecified sequences. In some examples, one or more anti-DNA antibodies or DNA-binding antibody fragments bind to one or more known DNA sequences. In other examples, one or more anti-DNA antibodies or DNA-binding antibody fragments bind to one or more unknown DNA sequences. In yet another example, one or more anti-DNA antibodies or DNA-binding antibody fragments bind to both known and unknown DNA sequences. In addition, in some examples, one or more anti-DNA antibodies or DNA-binding antibody fragments are sequence-specific. In other examples, one or more anti-DNA antibodies or DNA-binding antibody fragments are non-sequence-specific. As used herein, a non-sequence-specific antibody means an antibody that binds to two or more epitopes of an antigen or to two or more antigens. As a non-limiting example, a non-sequence-specific construct may include a polyclonal antibody composition. In yet another example, one or more anti-DNA antibodies or DNA-binding antibody fragments include a combination of a sequence-specific anti-DNA antibody and a non-sequence-specific anti-DNA antibody.

[0092] In some cases, the target DNA sequence recognized by the anti-DNA antibody or its fragment is less than 10 base pairs long. In some cases, the target DNA sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more base pairs long. In some cases, the target DNA sequence is about 20, 50, 100, 1000, 5000, 10,000, or more base pairs 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.

[0093] As disclosed herein, multiple anti-DNA antibodies forming antibody multimers, complexes, or clusters can be internally transported into cells. Such antibody multimers, complexes, or clusters can be produced in various ways, such as by manipulating antibodies to form multimers such as dimers, trimers, tetramers, or pentamers, or by having six or more anti-DNA antibodies. A multimer can have any desired number of antibodies. In some embodiments, such multimers are coated on a carrier such as gold nanoparticles, but not limited to gold nanoparticles. In some embodiments, the multimer contains multiparatopic antibodies that bind to different epitopes of the same DNA. In some embodiments, the multimer forms an antibody cocktail. While not bound by theory, multimers with multiple target sequences may facilitate cell recognition and subsequent internal transport. See, for example, Figure 10 and related discussions.

[0094] As disclosed herein, an anti-DNA antibody can be covalently or non-covalently conjugated to at least one payload. The payload may include, but is not limited to, one or more small molecules, peptides, proteins, nucleic acids, toxins, therapeutic substances, diagnostic agents, drugs, chemotherapeutic agents, liposomes, nanoparticles, dendrimers, detectable labels, or any derivatives, fragments, or combinations thereof, of any useful molecule or entity. In some embodiments, the payload is directly conjugated to an anti-DNA antibody or DNA-binding antibody fragment. In some embodiments, the anti-DNA antibody or DNA-binding antibody fragment is conjugated by a secondary antibody, and the payload is conjugated to the secondary antibody. In some embodiments, one or more payloads are conjugated to both an anti-DNA antibody and a secondary antibody. A non-limiting exemplary anti-DNA ADC / ADC complex is shown in Figure 1D.

[0095] In some embodiments, multiple anti-DNA antibodies are conjugated to a carrier such as a nanoparticle to form an antibody complex / cluster (see, e.g., Figure 1D, anti-DNA ADCs conjugated on a nanoparticle). The nanoparticles can be any useful nanoparticle, including, but are not limited to, polymer-based nanoparticles, non-polymer nanoparticles, or lipid-based nanoparticles. Polymer-based nanoparticles include, but are not limited to, dendrimers, nanoparticles, micelles, nanogels, protein nanoparticles, and drug conjugates. Non-polymer nanoparticles include, but are not limited to, carbon nanotubes, nanodiamonds, metal nanoparticles, quantum dots, and silica-based nanoparticles. Lipid-based nanoparticles include, but are not limited to, 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, two, three, four, five, six, seven, eight, or more anti-DNA antibodies are coated onto the nanoparticles. In some embodiments, the nanoparticles are gold nanoparticles. The synthesis of spherical gold nanoparticles with functional surfaces via antibody-drug conjugates (ADCs) 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 onto the same carrier.

[0096] In some embodiments, one or more anti-DNA antibodies are multiparatopic antibodies. See, for example, Ludwig SD, et al. Multiparatopic antibodies induce targeted downregulation of programmed death-ligand 1. Cell Chem Biol. 2024 May 16;31(5):904-919.e11. Multiparatopic anti-DNA antibodies can bind to two or more different sites within a single DNA fragment. In some cases, multiparatopic antibodies can bind to the same or different DNA fragments, forming antibody clusters that can promote internal translocation within cells. In some cases, anti-DNA multiparatopic antibodies are biparatopic antibodies that recognize two target sequences. In some cases, anti-DNA multiparatopic antibodies are triparatopic antibodies that recognize three target sequences. Multiparatopic antibodies can be configured to recognize any desired number of targets. Some exemplary biparatopic anti-DNA ADCs or ADC complexes are shown in Figure 1D.

[0097] The compositions and methods disclosed herein can also be used to deliver any desired number of anti-DNA antibodies internally. In some embodiments, two, three, four, five, six, seven, eight, or more anti-DNA antibodies are used. In some examples, the multiple anti-DNA antibodies entering the cell are of the same type, recognizing and binding to the same DNA epitope / site / fragment. In other examples, the multiple anti-DNA antibodies entering the cell are different anti-DNA antibodies. The term “different anti-DNA antibodies” means a group of anti-DNA antibodies that bind to different DNA epitopes / sites / fragments. In some examples, multiple different anti-DNA antibodies bind to different DNA epitopes / sites / fragments within a single DNA sequence. In other examples, multiple different anti-DNA antibodies bind to different DNA sequences. In yet another example, some different anti-DNA antibodies bind to different DNA epitopes / sites / fragments within a single DNA sequence, and some other different anti-DNA antibodies bind to different DNA sequences on the cell surface. In some cases, multiple anti-DNA antibodies that bind to cells are a combination of the same anti-DNA antibody and different anti-DNA antibodies.

[0098] As disclosed herein, multiple anti-DNA antibodies can form clusters / complexes that can bind to extracellular DNA on the cell surface and enter the cell. In some examples, the clusters / complexes can be formed before or after the multiple anti-DNA antibodies bind to the extracellular DNA. In some examples, the clusters / complexes can be formed simultaneously with the binding of the multiple anti-DNA antibodies to the extracellular DNA. In some examples, multiple anti-DNA antibodies can form multimers, such as dimers, trimers, tetramers, pentamers, or multimers having any desired number of antibodies. In some examples, multiple anti-DNA antibodies can form 2 nd Antibodies can bind to other antibodies. In some cases, multiple anti-DNA antibodies can be coated onto a carrier. In some cases, multiple anti-DNA antibodies can be crosslinked with each other. In some cases, multiple anti-DNA antibodies can be mixed together as an antibody cocktail.

[0099] Various 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 that can be used in the compositions and methods provided herein include, but are not limited to, antibody 121-3 (Abcam, a subsidiary of Danaher Corporation, Washington, DC), antibody 35I9 DNA (Abcam), antibody SPM603 (Abcam), antibody DSD958 (Abcam), antibody BV16-13 (MilliporeSigma, a subsidiary 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).

[0100] In some examples, the anti-DNA antibodies provided herein recognize at least one specific known extracellular DNA sequence. In some embodiments, the anti-DNA antibody recognizes and binds to an extracellular DNA sequence encoding a segment of a cancer-related gene. Such cancer-related genes include, but are not limited to, KRAS, TP53, BRAF, PIK3CA, and IDH1. The anti-DNA antibody may be specific to the wild-type sequence or a variant of the oncogene of interest, such as G12D KRAS, R175H TP53, V600E BRAF, E545K PIK3CA, or R132H IDH1. Other exemplary cancer-related genes that can 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.

[0101] Nuclease As disclosed herein, a method for the internal delivery of an anti-DNA antibody may further include the step of contacting a target cell with one or more active agents that cleave extracellular DNA into small pieces. While not bound by theory, smaller DNA fragments may facilitate the internal delivery of the anti-DNA antibody or fragment into the cell. In some embodiments, such active agents comprise one or more nucleases. The terms “nuclease,” “nucleotide polymerase,” and “polynucleotidase” are used interchangeably herein and mean enzymes capable of cleaving 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 examples, the DNA nuclease is an exonuclease. In other examples, the DNA nuclease is an endonuclease. In further examples, DNA nucleases are exo-endonucleases that exhibit both endonuclease and exonuclease functions. As disclosed herein, DNA nucleases have DNA cleavage activity, but may also be capable of cleaving RNA. Any useful combination of exonucleases, endonucleases, exo-endonucleases, and DNA / RNA nucleases may be used. See Figures 5A–5K and other sections herein.

[0102] In some embodiments, one or more nucleases include endonucleases selected from the group consisting of deoxyribonuclease (DNase), benzonase (registered trademark), Serratia marcesens DNA / RNA nuclease, micrococcal nuclease (MNase), transposase, type I restriction enzyme, type II restriction enzyme, type III restriction enzyme, type IV restriction enzyme, type V restriction enzyme, nuclease S1, nuclease P1, sequence-specific endonucleases, sequence-nonspecific endonucleases, and any functional derivatives, fragments, or fusions thereof.

[0103] In some embodiments, one or more nucleases include DNase. In some embodiments, DNase is DNase I. DNase I is an endonuclease of the DNase family encoded by the gene DNASE1. DNase I preferentially cleaves DNA at phosphodiester bonds adjacent to pyrimidine nucleotides, yielding 5'-phosphate-terminated polynucleotides with a free hydroxyl group at the 3' position, generally producing tetranucleotides. It acts on ssDNA, dsDNA, and chromatin. In some embodiments, DNase I is recombinant human DNase I. In some embodiments, DNase I is dorunase alfa (trade name Pulmozyme), a drug approved by the FDA for the treatment of cystic fibrosis.

[0104] In some embodiments, the nuclease is a Serratia marcescens nuclease (benzonase). A Serratia marcescens nuclease (or Serratia nuclease) is a DNA / RNA nonspecific endonuclease that hydrolyzes both double-stranded and single-stranded substrate DNA or RNA to 5'-phosphomononucleotide and 5'-phosphooligonucleotide end products. Commercially available Serratia marcescens nucleases include, but are not limited to, benzonase, Basemuncher, Benzo Nuclease, Benz-Neburase, Decontaminase, Denarase, Dr. Nuclease, GENIUS Nuclease, Pierce Universal Nuclease, and TurboNuclease.

[0105] In some embodiments, the nuclease is a micrococcal nuclease (MNase). Micrococcal nucleases are endo-exonucleases that preferentially digest single-stranded nucleic acids. The cleavage rate is more than 30 times faster at the 5' side of A or T than at G or C, resulting in the production of mononucleotides and oligonucleotides with terminal 3'-phosphates. The enzyme is also active against double-stranded DNA and RNA, ultimately cleaving the entire sequence.

[0106] In some embodiments, a nuclease is a restriction enzyme (restriction endonuclease, REase). A restriction enzyme is an endonuclease that cleaves DNA into fragments at or near a specific recognition site within a molecule known as a restriction site. As disclosed herein, a restriction enzyme may 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 examples, a restriction enzyme cleaves its DNA substrate at its recognition site. In other examples, the recognition site and cleavage site of a restriction enzyme are separate from each other.

[0107] In some embodiments, the nuclease is nuclease S1 or nuclease P1. Nuclease S1 from Aspergillus oryzae and nuclease P1 from Penicillium citrinum are sequence-specific endonuclease enzymes that split single-stranded DNA (ssDNA) and RNA into oligonucleotides or mononucleotides. In some embodiments, nuclease S1 or nuclease P1 can also introduce single-strand breaks into double-stranded DNA or RNA, or DNA-RNA hybrids. In some embodiments, nuclease S1 or nuclease P1 hydrolyzes single-stranded regions in double-stranded DNA, such as loops or gaps. In some embodiments, nuclease S1 or nuclease P1 cleaves the strand opposite to a nick on the complementary strand.

[0108] In some embodiments, the nuclease is a sequence-specific endonuclease. In some embodiments, the nuclease is a sequence-nonspecific endonuclease. In some embodiments, the nuclease is any derivative, fragment, or fusion of the enzymes disclosed herein.

[0109] In some cases, a nuclease is a ribonuclease that targets RNA. In other cases, a nuclease is a deoxyribonuclease that targets DNA. In yet another case, like benzonase, a nuclease targets both RNA and DNA. In some cases, a nuclease is a single-stranded DNA nuclease, such as MNase and nuclease S1 / P1, which preferentially targets ssDNA. In other cases, a nuclease is a double-stranded DNA nuclease that preferentially targets dsDNA, such as most restriction enzymes. In yet another case, a nuclease targets both ssDNA and dsDNA.

[0110] In some embodiments, the methods disclosed herein include the step of contacting cells with one nuclease (e.g., DNA nuclease). In some embodiments, the methods disclosed herein include the step of contacting cells with at least one, two, three, four, five, six, seven, eight or more different anti-DNA antibodies. In some embodiments, at least one, two, three, four, five, six, seven, eight or more different anti-DNA antibodies include one, two, three, four, five, six, seven, eight or more different nucleases. In some examples, one or more nucleases (e.g., DNA nuclease) and one or more anti-DNA antibodies are contacted to the cells simultaneously. In other examples, one or more nucleases (e.g., DNA nuclease) are contacted to the cells before the anti-DNA antibody or DNA-binding antibody fragment. In yet another example, one or more nucleases (e.g., DNA nuclease) are contacted to the cells after the anti-DNA antibody or DNA-binding antibody fragment.

[0111] In some embodiments, one or more DNA nucleases may include, but are not limited to, DNase I, or benzoases, or restriction enzymes. In some examples, one or more nucleases may be or contain DNase I, which may be derived from human or non-human sources (e.g., cattle, mouse). In other examples, one or more nucleases may be or contain benzoases. In other examples, one or more nucleases may be bacterial nucleases (e.g., MNases) or contain bacterial nucleases. See, for example, Figures 5A-5K and relevant discussions herein. The nucleases may be recombinant and / or humanized.

[0112] payload As disclosed herein, methods for internalizing anti-DNA antibodies can be used to deliver a desired payload to cells, including but not limited to therapeutic substances and / or detectable labels linked to the antibody. In some embodiments, one or more anti-DNA antibodies are bound to at least one payload. See, for example, Figure 1B and the relevant discussion herein. In some embodiments, one or more anti-DNA antibodies or DNA-binding antibody fragments are bound by one or more secondary antibodies, and one or more secondary antibodies are bound to at least one payload. See, for example, Figures 1A and 1C and the relevant discussion herein. Such binding can be covalent, non-covalent, direct, indirect, or any useful combination thereof. In some examples, one or more anti-DNA antibodies and / or one or more secondary antibodies are covalently linked to at least one payload. In some examples, one or more anti-DNA antibodies and / or one or more secondary antibodies are non-covalently linked to at least one payload. Non-limiting examples of non-covalent bonding include the use of binding agents, such as antibodies and / or aptamers, or through other potent and specific associations, such as avidin-biotin. For example, in some embodiments, an anti-DNA antibody or secondary antibody is bound to a biotin moiety, and at least one payload is bound to streptavidin or avidin, thereby linking the antibody and payload non-covalently. An anti-DNA antibody covalently or non-covalently linked to at least one payload can specifically target cells of interest and deliver one or more payloads to the cells. In some embodiments, the payload and anti-DNA antibody are generated as a translational fusion. In some embodiments, the anti-DNA antibody is prepared and then bound to the payload via a chemical linker. In some embodiments, the compositions and methods herein intend direct and / or indirect payload conjugation. An example of direct conjugation is the conjugation of a small molecule payload to the polypeptide itself.Alternatively, the small molecule payload may be indirectly bound to the polypeptide, for example via a linker, or encapsulated within particles (e.g., nanoparticles, liposomes, microvesicles, beads, etc.) that are bound to the polypeptide. The payload can be bound through any useful combination of covalent, non-covalent, direct, and indirect mechanisms.

[0113] As described above, the methods provided herein can be used to deliver one or more payloads to target cells. 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 examples, one anti-DNA antibody delivers multiple copies of a payload molecule into the cell. In other examples, 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 directly or indirectly bound to the anti-DNA antibody. The binding of one or more payloads to the anti-DNA antibody does not affect the antibody's binding affinity to the target DNA.

[0114] The methods provided herein can be used to deliver any useful and desired payload. Such flexibility allows the use of anti-DNA antibodies in multiple applications, such as diagnosis, prognosis prediction, or theranostic. The term “theranostic” means treatment-related diagnosis and includes, but is not limited to, predicting or monitoring drug responses using diagnostic information.

[0115] In some embodiments, at least one payload comprises small molecules, peptides, proteins, nucleic acids, toxins, chemotherapeutic agents, liposomes, nanoparticles, dendrimers, detectable labels, or any useful combination thereof. In some embodiments, at least one payload can be carried by drug-delivering particles such as (A) lipid-based nanocarriers; (B) inorganic nanoparticles; or (C) polymer nanoparticles, for example, but not limited to, Lobo, GCNB et al. Pharmaceutics 2021, 13, 1167. As a non-limiting example, small molecules could be therapeutic substances, such as drugs delivered specifically to cells harboring a particular mutation, e.g., tumor cells, using anti-DNA antibodies. Such applications may be intended to provide a therapeutic effect.

[0116] Examples of therapeutic substances that may be conjugated as payloads to anti-DNA antibodies provided herein include, but are not limited to, antitumor agents, anticancer agents, prodrugs, lysosomal destabilizers (e.g., chloroquine), alkylating agents, alkaloids, allosteric inhibitors, antifolic acid agents, anti-inflammatory agents, antibiotics, antibacterial agents, antifungal agents, antifibrotic agents, antiinfective agents, antiparasitic agents, antiviral agents, antimycobacterial agents, anticancer agents, antiprotozoal agents, antiviral agents, drugs, bioactive peptides, steroid hormones, nucleic acids, photosensitive substances, radiopharmaceuticals, antiprion agents, and combinations thereof.

[0117] For example, therapeutic substances include aromatase inhibitors; anti-estrogens; anti-androgens; gonadrelin agonists; topoisomerase I inhibitors; topoisomerase II inhibitors; microtubule activators (e.g., microtubule inhibitors); alkylating agents; retinoids, carotenoids, or tocopherols; cyclooxygenase inhibitors; MMP inhibitors; mTOR inhibitors; antimetabolites; platinum compounds; methionine aminopeptidase inhibitors; bisphosphonates; antiproliferative antibodies; heparanase inhibitors; inhibitors of Ras carcinogenic isoforms; and terror. The antitumor agents may include melanase inhibitors; proteasome inhibitors; Flt-3 inhibitors; Hsp90 inhibitors; kinesin spindle protein inhibitors; MEK inhibitors; PARP inhibitors, tyrosine kinase inhibitors, PI3K inhibitors, AKT inhibitors, EGFR inhibitors, antitumor antibiotics; nitrosoureas, compounds that target / reduce protein or lipid kinase activity, compounds that target / reduce protein or lipid phosphatase activity, any further anti-angiogenic compounds, or any desirable combination thereof.

[0118] Specific examples of antitumor agents, but not limited to these, include azacitidine, azathioprine, bevacizumab, bleomycin, capecitabine, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, etoposide, fenretinide, fluorouracil, gemcitabine, herceptin, idarubicin, mechloretamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, tafluposide, teniposide, thioguanine, retinoic acid, barrubicin, vinblastine, vincristine, vindesine, vinorelbine, receptor tyrosine kinase inhibitors, or any desirable combination thereof. Additional examples of antitumor agents and other therapeutic substances are known in the art.

[0119] In some embodiments, antitumor agents include tubulin inhibitors. The terms “tubulin inhibitor,” “microtubule inhibitor,” and “mitotic inhibitor” are used interchangeably herein to mean drugs that inhibit mitosis or cell division and are used to treat cancer and other diseases. Specific examples of tubulin inhibitors, but not limited to, include monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), maytansine, maytansinoids, meltansine (emtansine, DM1), labtansine (solabtansine, DM4), tubulinsine, halichondrin (eribulin), cryptophycin, EG5 inhibitors, and any derivatives thereof. Both MMAE and MMAF are derived from drastatin 10 and are antimitotic agents that inhibit cell division by blocking the polymerization of tubulin. MMAE is more hydrophobic than MMAF. MMAF, which has a charged C-terminal phenylalanine, exhibits reduced cytotoxic activity compared to its uncharged counterpart, MMAE. Maytansines such as DM1 and DM4 inhibit the polymerization of tubulin dimers by inhibiting the formation of mature microtubules. These tubulin inhibitors are common payloads used in clinical ADC drugs. For example, MMAF is found in the drug verantamab mahodotin, which is approved for multiple myeloma, and in some experimental anti-cancer antibody-drug conjugates (ADCs), such as borsetuzumab mahodotin and SGN-CD19A. MMAE is another anti-mitotic auristatin that is often conjugated to monoclonal antibodies (MAb) such as brentuximab (cAC10), glenbatumumab (CR011, CDX-011), AGS67E, sofituzumab, polatuzumab, enfortumab, pinatuzumab, rifastuzumab, brentuximab, glenbatumumab, tisotumab, and indusatumab.Any tubulin inhibitor and other ADC payloads 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 conjugated to the anti-DNA antibodies disclosed herein.

[0120] In some embodiments, antitumor agents include DNA inhibitors. DNA inhibitors act on the entire cell cycle by disrupting DNA through double-strand breaks, alkylation, chimeration, and crosslinking, causing cytotoxic effects and having therapeutic effects against solid tumors. Specific examples of DNA inhibitors, but not limited to these, include alkylating agents, duocalmycin, duocalmycin DM (DMDM), calicheamicin, pyrrolobenzodiazepine (PDB), enediyne, unciaramycin, topoisomerase inhibitors, topotecan, camptothecin (CPT), exatecan, and any derivatives thereof.

[0121] In some embodiments, antitumor agents include RNA inhibitors. RNA inhibitors are small molecule activators that specifically target RNA and kill both dividing and resting tumor cells. RNA inhibitors can be used as ADC payloads against tumor drug resistance and tumor recurrence, effective against both rapidly growing and slowly growing cells. Specific examples of RNA inhibitors, but not limited to these, include RNA splicing inhibitors, RNA polymerase II inhibitors, tylanstatins, amatoxins, and any derivatives thereof.

[0122] In other embodiments, the method may be used to detect cells in a disease state, such as cancer cells. In such cases, a detectable label may be a desirable payload. In some embodiments, the detectable label includes at least one magnetic label, a fluorescent moiety, an enzyme, a luminescent particle, a chemiluminescent probe, a metallic particle, a nonmetallic colloidal particle, a polymer dye particle, a dye molecule, an electrochemically active species, a semiconductor nanocrystal, a nanoparticle, a quantum dot, a gold particle, a fluorophore, a radioactive label, or a combination thereof.

[0123] In yet another embodiment, the payload may be used for both diagnostic and therapeutic detection. In a non-limiting example, radioactive labeling may be used to detect and / or kill target cells.

[0124] In some cases, the payload may be covalently bound to the anti-DNA antibody, including, but not limited to, direct conjugation to the DNA-binding antibody, binding via a linker entity, or both. In other cases, the payload may be non-covalently bound to the anti-DNA antibody. In a non-limiting example, the anti-DNA antibody may be conjugated to a biotin moiety, and the payload may be bound to streptavidin or avidin. In this example, the biotin-streptavidin or biotin-avidin binding provides a non-covalent bond between the anti-DNA antibody and the payload. Another example of a non-covalent bond between the anti-DNA antibody and the payload is when the payload is conjugated to a secondary antibody, and the secondary antibody binds to the anti-DNA antibody. In yet another case, for example, in the case of multiple payloads linked to an anti-DNA antibody, the payload may be bound both covalently and non-covalently.

[0125] Linker Where disclosed herein, a payload may be linked to an anti-DNA antibody or DNA-binding antibody fragment via a linker. As described above, multiple linkers can be used to link the antibody and the payload. 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 examples, the anti-DNA antibody is linked to one or more payloads using the same linker. In other examples, the anti-DNA antibody is linked to 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 examples, the secondary antibody is linked to one or more payloads using the same linker. In other examples, the secondary antibody is linked to one or more payloads using different linkers. Where used herein, a linker can be any desired molecule, complex, or entity linking the antibody (anti-DNA antibody or secondary antibody) and the payload.

[0126] In some cases, linkers include non-cleaving linkers. The term “non-cleaving linker” means a linker that does not have a specified weakness in its structure that could cause cleavage by proteases, hydrolases, or chemical cleavage due to changes in pH. In some cases, non-cleaving linkers include maleimide alkane linkers, maleimide cyclohexane (MCC) linkers, or any derivatives, fragments, or fusions 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 explanation of ADC linker selection). In some cases, non-cleaving linkers include mobile peptide linkers such as GS linkers or proline-rich rigid linkers. In some embodiments, the non-cutting linker is a GS movable linker having the sequence GGGGS (SEQ ID NO: 1), GGGGSGGGGS (SEQ ID NO: 2), or GGGGSGGGGSGGGGS (SEQ ID NO: 3). In some embodiments, the non-cutting linker is a proline-rich rigid linker having the sequence PAPAPPAPAP (SEQ ID NO: 4).

[0127] In other examples, the linker includes a cleavage linker. In such examples, the linker is cleaved after contacting the cell with one or more anti-DNA antibodies, thereby releasing the payload onto or into the cell. In some embodiments, the cleavage linker includes a hydrazone linker, a cathepsin B-responsive linker, a disulfide linker, a pyrophosphate diester linker, or any derivative, fragment, or fusion thereof (see Tsuchikama K, An Z. Antibody-drug conjugates: recent advances in conjugation and linker chemistries. Protein Cell. 2018;9(1):33-46). In some embodiments, the cleavage linker includes, but is not limited to, a protease-sensitive linker, a pH-sensitive linker, a radiosensitive linker, a glutathione-sensitive linker, a disulfide linker, and combinations thereof. In some examples, the cleavage linker includes a cathepsin B-sensitive valine-citrulline (Val-Cit) linker. In such examples, the linker between the payload (e.g., MMAF or MMAE) and the anti-DNA antibody is stable in the extracellular fluid, but is cleaved by cathepsin B when the antibody-payload conjugate enters a target cell (e.g., a cancer cell), thus releasing the payload into the cell. In some embodiments, the cleaving linker is a protease-sensitive linker containing a sequence of a saltase-recognition motif (e.g., LPXTG (SEQ ID NO: 5)). In certain embodiments, the anti-DNA antibody is designed to contain a saltase-recognition sequence (LPETG, SEQ ID NO: 6) for site-specific payload conjugation. In this example, a small molecule (e.g., monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE)) or a tubulin polymerization inhibitor such as maytansine) is modified by the addition of a pentaglysin peptide to become a substrate suitable for saltase A-mediated drug conjugation to the anti-DNA antibody.

[0128] As described above, the methods provided herein can be used to deliver one or more payloads to target cells. 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 examples, one anti-DNA antibody delivers multiple copies of the payload molecule into the cell. In other examples, 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 directly or indirectly bound to the anti-DNA antibody. The binding of one or more payloads to the anti-DNA antibody does not affect the antibody's binding affinity to the target DNA.

[0129] extracellular DNA The anti-DNA antibodies provided herein recognize extracellular DNA on the cell surface. In contrast to intracellular DNA, which is DNA located within the cell membrane, extracellular DNA refers to DNA located outside the cell membrane, such as DNA on the cell surface. Extracellular DNA is abundant on and near the surface of abnormal cells (e.g., cancer cells, diseased cells), particularly cells with impaired control of processes including but not limited to cell cycle progression, aging, DNA repair, cell death, cellular metabolism, development, and cell differentiation, but is absent in normal cells. As described herein, extracellular DNA can hold information about the cell of interest from which its nucleic acid originates. In some embodiments, extracellular DNA originates from nuclear DNA or mitochondrial DNA. In some embodiments, extracellular DNA originates from the cellular microenvironment. In some embodiments, extracellular DNA is packaged around one or more extracellular nucleosomes. In such embodiments, the anti-DNA antibody can recognize and bind to the extracellular DNA of one or more extracellular nucleosomes. See, for example, Figure 1C and the relevant discussion herein.

[0130] In some examples, 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 examples, the target extracellular DNA can be selected so that the anti-DNA antibody can identify one or more cells of interest. In a preferred embodiment, the target extracellular DNA originates within the target cell. For example, the target extracellular DNA may harbor one or more mutations that identify the target cell as a mutant cell or a cell in a disease state, including but not limited to cancer cells. The disclosure further assumes that the target extracellular DNA originates from the microenvironment of the target cell. In non-limiting examples, it is considered that the target cell of the anti-DNA antibody is a cell in a tissue such as tumor tissue. If the target cell is necrotic or apoptotic, the target cell may release nucleic acids into its microenvironment (in this example, the tumor microenvironment). As a result, if the released nucleic acids adhere to other cells in the microenvironment, those other cells in the microenvironment may also become target cells. The disclosure also assumes that the target cell actively releases nucleic acids into its microenvironment, for example, in an inflammatory region. Such necrosis, apoptosis, inflammation, or other cellular damage or reactions can be induced by the cell, its environment (e.g., by an immune response), or both.

[0131] In this method, specific nucleic acids derived from cells can be used to specifically target desired cells. In some embodiments, the target nucleic acid has a wild-type (WT) sequence. In some embodiments, the target nucleic acid has a sequence containing one or more mutations. As used herein, unless otherwise stated, a mutation can mean any sequence other than a “normal” wild-type sequence. For example, a mutation can be a single nucleotide variant sequence (with or without pathogenicity), two or more such variants, an insertion, deletion, substitution, inversion, translocation, fusion, cleavage, deletion, duplication, amplification, or repeat. Anti-DNA antibodies used in the methods provided herein can recognize cells whose genomic DNA is different due to a single point mutation. Anti-DNA antibodies can also target sequences resulting from genomic changes, such as sequences produced by translocations, cleavage, or deletions in the sequence. As described herein, cells may excrete nucleic acids such as gDNA due to inflammation, disease, or cell damage. Therefore, levels of nucleic acids may be used to target cells of interest. In non-limiting examples, amplification events in cancer may result in abnormally high levels of certain sequences. Anti-DNA antibodies provided herein may target such amplified nucleic acids.

[0132] In other examples, anti-DNA antibodies used in the methods provided herein can recognize and bind to unknown extracellular DNA, i.e., extracellular DNA whose target sequence or exact epitope is unknown. In other words, the target epitope does not need to be known, as long as the antibody can bind to DNA that can be used to target cells of interest. Examples of such anti-DNA antibodies include, but are not limited to, antibody 121-3 (Abcam), antibody 35I9 DNA (Abcam), antibody SPM603 (Abcam), antibody DSD958 (Abcam), 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 another example, anti-DNA antibodies provided herein can recognize and bind to both known and unknown extracellular DNA sequences. In some embodiments, the anti-DNA antibody recognizes and binds to extracellular DNA sequences encoding wild-type or mutant KRAS, wild-type or mutant TP53, wild-type or mutant BRAF, wild-type or mutant PIK3CA, or wild-type or mutant IDH1. In some embodiments, the mutant genes include G12D KRAS, Q61H KRAS, R175H TP53, R273H TP53, V600E BRAF, E545K PIK3CA, or R132H IDH1.

[0133] target cell As described herein, the methods provided herein can be used in a variety of applications. In non-limiting examples, anti-DNA antibodies can be used to label target cells or, if necessary, to kill target cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells. In some embodiments, the cells are in vitro. In some embodiments, the cells are in vivo.

[0134] In some embodiments, target cells include cells in a disease state. Disease-state cells may reside within tissues such as solid tumors, or they may circulate within the body, including but not limited to the human body. In various embodiments, diseases include cancer, precancerous conditions, inflammatory diseases, immune diseases, autoimmune diseases or disorders, cardiovascular diseases or disorders, neurological diseases or disorders, infectious diseases, or pain. Cancer cells may exhibit a mutagenic phenotype and may harbor thousands of mutations.

[0135] Any cancer cells of interest (e.g., human cancer) can become target cells. In some aspects, cancer includes 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 aspects, cancer includes acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical carcinoma; AIDS-related cancer; AIDS-related lymphoma; anal cancer; appendiceal cancer; astrocytoma; atypical teratomatous / rhabdoid tumor; basal cell carcinoma; bladder cancer; brainstem glioma; brain tumors (e.g., brainstem glioma, atypical teratomatous / rhabdoid tumor of the central nervous system, embryonic tumor of the central nervous system, astrocytoma, craniopharyngioma, ependymoblastoma, ependymodium, medulloblastoma, medullary epithelioma, intermediate pineal parenchymal tumor, ten Primitive neuroectodermal tumors and pineal blastomas; breast cancer; bronchial tumors; Burkitt lymphoma; primary site tumors (CUP); carcinoid tumors; carcinomas of unknown primary origin; atypical teratoid / rhabdoid tumors of the central nervous system; embryonal tumors of the central nervous system; cervical cancer; childhood cancer; chordoma; chronic lymphocytic leukemia; chronic myeloid leukemia; chronic myeloproliferative disorders; colon cancer; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; endocrine islet cell tumors; endometrial cancer; ependymoblastoma; ependymoma; Cholera cancer; nasal neuroblastoma; Ewing's sarcoma; extracranial germ cell tumor; extragonadal germ cell tumor; extrahepatic cholangiocarcinoma; gallbladder cancer; gastric (stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal cell tumor; gastrointestinal stromal tumor (GIST); gestational choriocarcinoma; glioma; hairy cell leukemia; head and neck cancer; heart cancer; Hodgkin's lymphoma; hypopharyngeal cancer; intraocular melanoma; pancreatic islet cell tumor; Kaposi's sarcoma; kidney cancer; Langerhans cell histiocytosis; laryngeal cancer; Lip cancer; liver cancer; lung cancer; malignant fibrous histiocytoma bone cancer; medulloblastoma; medullary epithelioma; melanoma; Merkel cell carcinoma; Merkel cell cutaneous cancer; mesothelioma; metastatic squamous cell carcinoma of unknown primary origin in the neck; oral cancer; multiple endocrine neoplasia syndrome; multiple myeloma; multiple myeloma / plasmacytic neoplasm; mycosis fungoides; myelodysplastic syndrome; myeloproliferative neoplasm; nasal cavity cancer; nasopharyngeal cancer; neuroblastoma; non-Hodgkin lymphoma; non-melanoma skin cancer; non-small cell lung cancer; oral cancer; oral cancer; oropharyngeal cancer; osteosarcoma; other brain and spinal cord tumors; ovarian cancer; ovarian epithelial carcinoma; ovarian germ cell tumor; low-grade ovarian tumor;Pancreatic cancer; papilloma; sinus cancer; parathyroid cancer; pelvic cancer; penile cancer; pharyngeal cancer; intermediate pineal parenchymal tumor; pineoblastoma; pituitary tumor; Plasma cell tumors / multiple myeloma; pleuropulmonary blastoma; primary central nervous system (CNS) lymphoma; primary hepatocellular carcinoma; prostate cancer; rectal cancer; kidney cancer; renal cell carcinoma; renal cell carcinoma; respiratory cancer; retinoblastoma; rhabdomyosarcoma; salivary gland cancer; Sézary syndrome; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma; squamous cervical cancer; stomach cancer; supratentorial primitive neuroectodermal tumor; T-cell lymphoma; testicular cancer; pharyngeal cancer; thymic cancer; thymoma; thyroid cancer; transitional cell carcinoma; transitional cell carcinoma of the renal pelvis and ureter; choriocarcinoma; ureteral cancer; urethral cancer; uterine cancer; uterine sarcoma; vaginal cancer; vulvar cancer; Waldenström macroglobulinemia; or Wilms' tumor. In some aspects, cancer is breast cancer.

[0136] In some aspects, cancer types include acute myeloid leukemia (AML), breast cancer, cholangiocarcinoma, colorectal adenocarcinoma, extrahepatic cholangiocarcinoma, female reproductive tract malignancies, gastric adenocarcinoma, gastroesophageal adenocarcinoma, gastrointestinal stromal tumors (GIST), glioblastoma, head and neck squamous cell carcinoma, leukemia, hepatocellular carcinoma, low-grade glioma, bronchioloalveolar carcinoma (BAC), non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), lymphoma, and male This includes malignancies of the genitourinary tract, malignant solitary fibrous neoplasms of the pleura (MSFT), melanoma, multiple myeloma, neuroendocrine tumors, diffuse large B-cell lymphoma nodosa, non-epithelial ovarian cancer (non-EOC), ovarian surface epithelial carcinoma, pancreatic adenocarcinoma, pituitary cancer, oligodendroglioma, prostate adenocarcinoma, retroperitoneal or peritoneal cancer, retroperitoneal or peritoneal sarcoma, small intestinal malignancies, soft tissue tumors, thymic carcinoma, thyroid cancer, or uveal melanoma.

[0137] Cancer may be present in individuals who have been diagnosed with cancer, have cancer, are at risk of developing cancer, or are suspected of having cancer. Cancer may be selected from a group that includes urothelial carcinoma of the bladder, invasive carcinoma of the mammary gland, adenocarcinoma of the colon, adenocarcinoma of the colorectal gland, esophageal cancer, squamous cell carcinoma of the head and neck, clear cell carcinoma of the kidney, papillary cell carcinoma of the kidney, hepatocellular carcinoma of the liver, adenocarcinoma of the lung, squamous cell carcinoma of the lung, adenocarcinoma of the prostate, gastric and esophageal cancer, thyroid cancer, endometrial cancer of the uterine body, and chronic lymphocytic leukemia. In some embodiments, cancer may harbor wild-type or mutated forms of KRAS, TP53, BRAF, PIK3CA, and / or IDH1.

[0138] In some embodiments, cells have aneuploidy and / or DNA repair defects. Without being bound by theory, and without intending to limit the scope of the invention, such cells may have extracellular DNA on their surface due to aneuploidy, mutation, or reduced expression of DNA repair enzymes. In some embodiments, cells have aneuploidy. In some embodiments, cells have abnormal or low efficiency in DNA damage repair responses. In some embodiments, cells have abnormal or stalled DNA repair pathways. In some embodiments, cells have mutated or insufficient DNA repair proteins / enzymes such as phosphatidylinositol-3 (PI3) kinases. PI3 kinases include, but are not limited to, ataxia vasodilator mutation (ATM) kinases, ATM and Rad3-related (ATR) kinases, and poly(ADP-ribose) polymerase (PARP). In some embodiments, cells contain functionally impaired transcription factors selected from the group consisting of p53, NF-κb, AP-1, E2F1, breast cancer-related protein 1 (BRCA1), and breast cancer-related protein 2 (BRCA2). In some embodiments, the transcription factor dysfunction is caused by one or more gene mutations, loss of one or more genes, loss of one or more chromosomal regions, and / or deficiency of the expression of one or more proteins. In some examples, the gene mutation may be a mutation in a transcription factor gene. In other examples, the gene mutation may be a mutation in another DNA repair-related gene. In some embodiments, cells have at least one mutation, deficiency, loss, or other deficiency in one or more mismatch repair (MMR) genes, including, but not limited to, the MLH1, MLH2, MLH3, MSH2, MSH6, PMS1, and / or PMS2 genes.In some embodiments, cells contain one or more mutations or other defects in one or more DNA repair genes selected from the group consisting of apex1;ddb1;ddb2;ercc1;fen1;karp1;lig1;mgmt;mpg;mlh1;msh2;neil1;ogg1;pcna;pms2;polI;polβ;polH;polK;rev3;trex1;xrcc1;xpc;xpf; and xpg. See, for example, 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.

[0139] In certain embodiments, cells contain the functionally impaired transcription factor p53, and optionally, the impairment of p53 includes mutations. p53, also known as the oncoprotein p53, TP53, cellular tumor antigen p53, or transformation-associated protein 53 (TRP53), is a transcription factor that plays a major role in regulating DNA repair, apoptosis, and cell cycle progression. p53 is often mutated and / or functionally impaired in cancer cells. As disclosed herein, functionally impaired p53 may result from one or more failures of the TP53 gene (e.g., one or more mutations in the TP53 gene), decreased p53 expression, and / or inhibition of p53 activity. In some embodiments, functionally impaired p53 results in aneuploidy of cells. In some embodiments, functionally impaired p53 is present in abnormal cells such as cancer cells or cells of other disease states. In some embodiments, functionally impaired p53 is present in cells that have extracellular DNA on the cell surface. In some embodiments, functionally impaired p53 is caused by missense mutations in the TP53 gene. In some embodiments, a functionally impaired p53 contains one or more missense mutations in the protein's DNA-binding domain. In some embodiments, the p53 mutation is located at residues R175, Y220, G245, R248, R249, R273, and / or R282. In specific embodiments, the p53 mutation is R175H, Y220C, G245S, R248Q, R248W, R249S, R273H, R273C, or R282W. See, for example, Figures 8A–8B and related discussions.

[0140] method Method of joining This specification provides a method for conjugating one or more anti-DNA antibodies to extracellular DNA on a cell surface, comprising the step of contacting cells with one or more anti-DNA antibodies provided herein. The method can utilize various configurations of anti-DNA antibodies, fragments, and associated ADC constructs desired in a given application. Such various configurations of anti-DNA antibodies, fragments, and associated ADC constructs may be as provided herein. In some embodiments, the anti-DNA antibody is bound to a cytotoxic payload, and the binding of the anti-DNA antibody kills the cell. In some embodiments, the anti-DNA antibody is bound to a detectable payload, and the method further includes detecting the 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 the presence or level of one or more target cells in a biological non-sample, and one or more anti-DNA antibodies bind to or migrate internally into the target cells. The method can be applied to a variety of desired situations. For example, the contacting step can be carried out in vivo or in vitro, depending on the desired application of the method.

[0141] Imaging methods Further provided herein are methods for imaging at least one cell or tissue, comprising the steps of contacting at least one cell or tissue with an anti-DNA antibody provided herein, and detecting the anti-DNA antibody bound to and / or internally migrated into at least one cell or tissue. In some embodiments, one or more anti-DNA antibodies are administered to a subject prior to the detection step. The terms “subject,” “individual,” and “patient” are used interchangeably herein and mean vertebrates, preferably mammals, more preferably humans. Mammals include, but are not limited to, mice, rats, monkeys, humans, livestock, sports animals, and companion animals. Tissues, cells, and their offspring of biological entities obtained in vivo or cultured in vitro are also included. In some embodiments, the detection step is carried out in vitro. Where desired, these methods can be combined. For example, anti-DNA antibodies can be administered to a subject, and then a sample can be taken from the subject for subsequent in vitro analysis. In some embodiments, at least one cell or tissue comprises a cell exhibiting mutant or wild-type extracellular DNA on its surface, and the anti-DNA antibody disclosed herein binds to the mutant or wild-type extracellular DNA. In some embodiments, the anti-DNA antibody is specific to either the mutant or wild-type DNA. As a non-limiting example, such an anti-DNA antibody could be used to image cells harboring a mutation of particular interest, such as a cancer mutation.

[0142] In some embodiments, at least one cell or tissue originates from a subject who has or is suspected of having a disease or disorder. In non-limiting examples, the disease or disorder may include cancer, precancerous conditions, inflammatory diseases, immune diseases, autoimmune diseases or disorders, cardiovascular diseases or disorders, neurological diseases or disorders, infectious diseases, or pain. In some embodiments, at least one cell or tissue includes neoplastic cells, malignant cells, tumor cells, hyperplastic cells, dysplastic cells, and / or metastatic cells. In the case of tumor cells, the tumor may be a primary tumor or a metastatic tumor. The tumor may, if desired, be associated with any type of cancer. In some embodiments, the target cells or tissue include 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 cells. Cancer may also include any cancer of interest, including, but not limited to, other cancers provided herein.

[0143] Delivery method This specification also provides a method for delivering a payload into the interior of a cell, including extracellular DNA. Any useful and desired payload, including but not limited to the therapeutic substances or other payloads described herein, can be delivered. In one aspect, the method comprises the step of contacting a cell with an anti-DNA antibody conjugated to a therapeutic substance, including but not limited to a cytotoxic drug, the anti-DNA antibody binding to extracellular DNA on the cell and causing the therapeutic substance to be delivered into the interior of the cell. In some embodiments, the anti-DNA antibody is directly conjugated to the therapeutic substance covalently or non-covalently. In some embodiments, the anti-DNA antibody is indirectly conjugated to the therapeutic substance. For example, the DNA antibody can be conjugated by a secondary antibody, which is covalently or non-covalently conjugated to the therapeutic substance. Such anti-DNA antibody constructs or complexes conjugated to one or more therapeutic substances may be referred to herein as anti-DNA antibody-drug conjugates or anti-DNA ADCs.

[0144] In some embodiments, the method further comprises the step of contacting cells with one or more nucleases, thereby facilitating the internal migration of anti-DNA antibodies and corresponding payloads. See, for example, Figures 5A–5K and related discussions herein. The nucleases may be endonucleases, exonucleases, or combinations thereof. In some embodiments, one or more nucleases include one or more single-stranded DNA (ssDNA) nucleases, one or more double-stranded DNA (dsDNA) nucleases, or combinations thereof. In some embodiments, one or more nucleases and anti-DNA antibodies (including but not limited to anti-DNA ADCs) conjugated with the payload are contacted with the cells simultaneously. In some embodiments, one or more nucleases are conjugated with the anti-DNA antibodies. In some embodiments, one or more nucleases are contacted with the cells before the anti-DNA antibodies (including but not limited to anti-DNA ADCs) conjugated with the payload. In some embodiments, one or more nucleases are contacted with the cells after the anti-DNA antibodies (including but not limited to anti-DNA ADCs) carrying the payload. If desired, one or more nucleases may be brought into contact with cells in a schedule involving one or more contacts of the nucleases, including before, during, and / or after, the application of an anti-DNA antibody containing the payload.

[0145] As described herein, the payload conjugated with an anti-DNA antibody can be selected to achieve a desired activity, such as a therapeutic effect. In some embodiments, the payload includes small molecules, drugs, proteins, nucleic acids, toxins, chemotherapeutic agents, or other therapeutic substances, as described herein. In some embodiments, the payload includes liposomes or nanoparticles. In such cases, the liposomes or nanoparticles may contain the desired therapeutic substance. The anti-DNA antibody and / or payload may be internally delivered into target cells. In some embodiments, the target cells are cancer cells. In some embodiments, the therapeutic agent kills cancer cells or inhibits their proliferation or division. In some embodiments, the cancer cells originate from cancer in the subject. As further described herein, the payload may be released in cells to provide a therapeutic effect, for example, via cleavage of a linker between the binding portion of the construct and the payload, via protein cleavage of the binding portion, or via other mechanisms.

[0146] Treatment method This specification further provides a method for treating or relieving a disease or disorder in a human subject where such treatment is necessary, the method comprising administering a pharmaceutically effective amount of a composition comprising an anti-DNA antibody to the subject. In a preferred embodiment, the anti-DNA antibody comprises an anti-DNA ADC. As disclosed herein, the anti-DNA antibody binds to extracellular DNA on the surface of diseased cells and to at least one toxic payload, such as a small molecule drug, but is not limited to this method. Administration of the pharmaceutical composition results in the delivery of the payload to cells containing extracellular DNA, thereby specifically killing the target cells. In some embodiments, the composition further comprises one or more nucleases. In some embodiments, one or more nucleases comprises DNA nucleases. As disclosed herein, the nucleases may be endonucleases, exonucleases, or a combination thereof. In some embodiments, one or more nucleases and the anti-DNA antibody having a therapeutic payload are simultaneously brought into contact with cells. In some embodiments, one or more nucleases are bound to the anti-DNA antibody. In some embodiments, one or more nucleases are contacted with cells before an anti-DNA antibody having a therapeutic payload. In some embodiments, one or more nucleases are contacted with cells after an anti-DNA antibody having a therapeutic payload. In some embodiments, one or more nucleases are contacted with cells in a schedule that includes one or more contacts before, during, or after contact with an anti-DNA antibody possessing a therapeutic payload.

[0147] As used herein, “therapeutic effective dose” means the amount of a composition that alleviates one or more symptoms of a disease or condition (to the extent determined by a skilled physician). In addition, “therapeutic effective dose” of a composition means the amount that partially or completely restores to normal the physiological or biochemical parameters associated with or causing the disease or condition. A clinician or other caregiver skilled in the art can determine the therapeutic effective dose of a composition to treat or prevent a particular disease condition or disorder when administered intravenously, subcutaneously, intraperitoneally, orally, or by inhalation. The exact amount of a composition required to be therapeutically effective depends on many factors, such as the specific activity of the activator, the delivery device used, the physical properties of the active ingredient, and the purpose of administration, in addition to numerous patient-specific considerations. However, determining the therapeutic effective dose is within the scope of the skills of a clinician or other caregiver with ordinary skills based on an understanding of the disclosures described herein.

[0148] As used herein, the terms “to treat,” “treatment,” “therapy,” and “therapeutic treatment” mean curative treatment, prophylactic therapy, or preventative therapy. An example of “preventative therapy” is the prevention or reduction of the likelihood of a target disease (e.g., cancer or other proliferative disorders) or a related condition. Persons requiring treatment include not only those who already have the disease or condition, but also those who are prone to having the disease or condition that should be prevented. As used herein, the terms “to treat,” “treatment,” “therapy,” and “therapeutic treatment” also describe the management and care of subjects aimed at combating a disease or related condition, which include the administration of compositions that alleviate the symptoms, side effects, or other complications of the disease or condition. Therapeutic treatments for cancer include, but are not limited to, surgery, chemotherapy, radiotherapy, gene therapy, and immunotherapy. In some embodiments, anti-DNA antibodies provided herein are used in the treatment of cancer.

[0149] As used herein, the terms “active substance,” “drug,” “therapeutic substance,” or “therapeutic agent” mean chemical compounds, mixtures of chemical compounds, biological polymers, or extracts made from biological materials such as cells or tissues of bacteria, plants, fungi, or animals (especially mammals) that are thought to have therapeutic properties. Active substances or drugs may be purified, substantially purified, or partially purified. The “active substances” according to the present invention also include “radiotherapy agents” or “chemotherapeutic agents.” As used herein, the term “chemotherapeutic agent” means an active substance that is active against cancer, neoplasms, and / or proliferative disorders, or has the ability to directly kill cancer cells.

[0150] As used herein, the term “diagnostic agent” means any chemical substance used in imaging tissue of a diseased condition, such as a tumor. Non-limiting examples of imaging agents and detectable labels are provided herein.

[0151] In some embodiments, the disease or disorder includes cancer, precancerous conditions, inflammatory diseases, immune diseases, autoimmune diseases or disorders, cardiovascular diseases or disorders, neurological diseases or disorders, infectious diseases, or pain. In some embodiments, the target cells include neoplastic cells, malignant cells, tumor cells, hyperplastic cells, dysplastic cells, and / or metastatic cells. In certain embodiments, the disease or disorder is cancer. In some embodiments, cancer is 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. Cancer may also include other cancers such as those provided herein. In some embodiments, the subject is human. In some embodiments, the subject has cancer. In some embodiments, a pharmaceutically effective amount of the composition provided herein is administered to the subject, thereby enabling targeting of cancer cells.

[0152] In some embodiments, the method further includes a step of determining, prior to the administration step, whether the cells have aneuploidy, DNA repair failure, and / or functionally impaired transcription factors, including but not limited to transcription factors selected from the group consisting of p53, NF-κb, AP-1, E2F1, BRCA1, and BRCA2. In some examples, the determination step includes detecting one or more mutations or other failures (e.g., loss or failure of expression) in transcription factor genes. In other examples, the determination step includes detecting one or more mutations or other failures in other DNA repair-related genes. In some embodiments, the cells have at least one mutation or other failure in one or more mismatch repair (MMR) genes, including but not limited to the MLH1, MLH2, MLH3, MSH2, MSH6, PMS1, and / or PMS2 genes. In some embodiments, the determination step includes detecting one or more mutations or other defects in one or more DNA repair genes selected from the group consisting of apex1;ddb1;ddb2;ercc1;fen1;karp1;lig1;mgmt;mpg;mlh1;msh2;neil1;ogg1;pcna;pms2;polI;polβ;polH;polK;rev3;trex1;xrcc1;xpc;xpf; and xpg. In certain embodiments, the method includes detecting functionally impaired transcription factor p53 in the cells of interest. Functionally impaired p53 may result from TP53 gene deficiency (such as a mutation in the TP53 gene), decreased p53 expression, and / or inhibition of p53 activity. In some cases, the TP53 gene mutation is an R175H mutation.

[0153] composition In one aspect, this specification provides compositions comprising one or more anti-DNA antibodies disclosed herein. In some embodiments, the composition further comprises one or more nucleases. In some embodiments, one or more nucleases comprises DNA nucleases. In some embodiments, one or more anti-DNA antibodies are conjugated by secondary antibodies. In some embodiments, one or more anti-DNA antibodies are coated onto a carrier such as gold nanoparticles or other carriers as described herein. In some embodiments, at least two anti-DNA antibodies are linked together as a multimer such as a dimer, trimer, tetramer, or pentamer, or a multimer of 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 include, but are not limited to, chemical crosslinking, such as in Ueda et al, Int J Mol Sci. 2020 Feb; 21(3): 711. In some cases, the different anti-DNA antibodies comprise multiparatopic antibodies. Various embodiments, including carriers and polymer constructs, are further described herein. See also Figure 1D and related discussions.

[0154] In some embodiments, the composition further comprises at least one payload covalently or non-covalently linked to one or more anti-DNA antibodies. In some embodiments, at least one payload is directly conjugated to one or more anti-DNA antibodies. In some embodiments, one or more anti-DNA antibodies are conjugated by one or more secondary antibodies, and at least one payload is linked to the secondary antibody, e.g., directly conjugated. In some embodiments, multiple payloads are linked to one or more anti-DNA antibodies and / or one or more secondary antibodies. As a non-limiting example, at least one payload is conjugated to an anti-DNA antibody, and at least one payload is conjugated to a secondary antibody. In some examples, the anti-DNA antibody and / or secondary antibody are non-covalently linked to the payload. For example, in some embodiments, the anti-DNA antibody or secondary antibody is bound to a biotin moiety, and at least one payload is bound to streptavidin or avidin, thereby linking the antibody and payload non-covalently. Various embodiments comprising anti-DNA antibodies and payloads are further disclosed herein. See also Figure 1D and related discussion.

[0155] Where provided herein, a pharmaceutical composition may comprise a therapeutically effective amount of the composition disclosed above, and pharmaceutically acceptable excipients, carriers, and / or diluents.

[0156] This specification also provides pharmaceutical compositions comprising a therapeutically effective amount of anti-DNA antibody and at least one payload, such as a therapeutic substance. In some embodiments, the therapeutic substance includes antitumor agents, anti-cancer agents, prodrugs, lysosomal destabilizers (e.g., chloroquine), alkylating agents, alkaloids, allosteric inhibitors, antifolic acid agents, anti-inflammatory agents, antibiotics, antibacterial agents, antifungal agents, antifibrotic agents, anti-infective agents, antiparasitic agents, antiviral agents, antimycobacterial agents, anti-cancer agents, antiprotozoal agents, antiviral agents, physiologically active peptides, steroid hormones, photosensitive substances, radiopharmaceuticals, antiprion agents, or any desired combination thereof. In some embodiments, the antitumor agent includes tubulin inhibitors, DNA inhibitors, and / or RNA inhibitors. In some embodiments, tubulin inhibitors are selected from the group consisting of monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), maytansine, maytansinoid, meltansine (emtansine, DM1), labtansine (solabtansine, DM4), tubulinin, halichondrin (eribulin), cryptophycin, EG5 inhibitors, and any derivatives thereof.

[0157] In some embodiments, the pharmaceutical composition further comprises one or more nucleases. In some embodiments, one or more nucleases comprises DNA nucleases. In some embodiments, the nucleases may be endonucleases, exonucleases, or combinations thereof. In some embodiments, the endonucleases may be deoxyribonucleases (DNases), Serratia marcescens nucleases (benzoases), micrococcal nucleases (MNases), transposases, restriction enzymes, nuclease S1, nuclease P1, sequence-specific endonucleases, or sequence-nonspecific endonucleases. In some embodiments, one or more DNA nucleases comprises single-stranded DNA (ssDNA) nucleases, double-stranded DNA (dsDNA) nucleases, or combinations thereof. Further embodiments comprising nucleases are disclosed herein. See also Figures 5A–5J and related discussions.

[0158] The aforementioned pharmaceutical composition may contain at least one of pharmaceutically acceptable excipients, carriers, and / or diluents. To enhance the therapeutic effect of the treatment, other active agents may be used in combination with the pharmaceutical composition. These additional active agents include chemotherapeutic agents such as small molecule drugs, or other biological agents. Where desired, such additional active agents may target the same biomarker as the anti-DNA antibody. In some embodiments, the additional active agents include non-targeted therapies. In non-limiting examples, anti-DNA ADCs directed at target cells may be administered concurrently or sequentially with other related therapies (e.g., immunotherapy, CAR-T therapy, other antibody therapies, cell therapies), as well as / or conventional chemotherapy including, but not limited to, alkylating agents, plant alkaloids, antimetabolites, anthracyclines, topoisomerase inhibitors, and / or corticosteroids.

[0159] In connection therewith, this specification provides a kit comprising at least one reagent for carrying out the method provided herein, as described above. Also provided herein is the use of at least one reagent for carrying out the method. Any useful reagent can be a component of the kit or use. In some embodiments, the at least one reagent comprises an anti-DNA antibody, a detection reagent, a secondary detection reagent, a washing buffer, an elution buffer, a solid support, and any combination thereof.

[0160] Administration The pharmaceutical compositions provided herein may be administered via any desired and useful route, including, but not limited to, parenteral, orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal, or intravenous injection. In some embodiments, the route of administration includes at least one of intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intracerebral, vaginal, transdermal, rectal, inhalation, topical, or any useful combination thereof.

[0161] Typically, the pharmaceutical compositions provided herein are administered in a manner and in a therapeutically effective amount appropriate to the administered formulation. The amount to be administered depends on the subject being treated. The exact amount of anti-DNA antibody required for administration may be determined by the judgment of the physician or other caregiver performing the treatment.

[0162] The methods of application can vary widely. Various methods of administering pharmaceutical compositions containing protein components are applicable. The dosage of the pharmaceutical composition depends on the route of administration and may vary depending on the size and health status of the subject.

[0163] In many cases, it is desirable to have multiple doses, at most about 3, 4, 5, 6, 7, 8, 9, or 10 times, or at least about 3, 4, 5, 6, 7, 8, 9, or 10 doses. The timing of administration may vary over time. In some embodiments, the timing of administration is in the range of intervals from 2 days to 12 weeks, for example, between 1 and 2 weeks. The course of administration can be tracked by assays to monitor the presence and / or levels of target cells in the patient. Monitoring may be carried out as described herein.

[0164] As used herein, “pharmaceutical formulation” includes formulations for human and veterinary use that have, if any, adverse toxicological effects at an acceptable level. “Pharmaceutically acceptable formulation” as used herein means a composition or formulation that enables the effective distribution of the nucleic acid molecules of the present invention at the physical location most suitable for its desired activity.

[0165] As used herein, the term "pharmaceutically acceptable" means molecular entities and compositions that, when administered to a subject, for example, a person in need of treatment for a disease or disorder, do not produce an unacceptable adverse reaction, allergic reaction, or other undesirable reaction. As used herein, "pharmaceutically acceptable carrier" includes any solvent, dispersion medium, coating agent, antimicrobial and antifungal agent, as well as isotonic and absorption retardant agents, etc. The use of such media and active ingredients for formulating pharmaceutically active substances is known in the art. Unless any conventional media or active ingredient is incompatible with the active ingredient, its use in immunogenic and therapeutic compositions is intended. The pharmaceutically acceptable compositions of this disclosure are pharmaceutically acceptable compositions.

[0166] The compositions of this disclosure can be formulated for parenteral administration, for example, for injection via intravenous, intramuscular, subcutaneous, or intraperitoneal routes. Such compositions can be prepared as liquid solutions or suspensions for injection. Solid forms suitable for use can also be prepared, which are prepared by adding liquids before injection to create a solution or suspension. The preparations can also be emulsified.

[0167] Suitable pharmaceutical forms for use by injection include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol. Pharmaceutical forms should be stable under manufacturing and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi.

[0168] Sterile injectable solutions are prepared by incorporating the required amount of the active ingredient (i.e., the anti-DNA antibody provided herein) into a suitable solvent along with the various other components listed above as needed, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle containing a basic dispersion medium and the other components required from those listed above.

[0169] The effective amount of the composition is determined based on the intended objective. The term “unit dose” or “dosage” refers to a physically distinct unit suitable for use in the subject, each unit containing a predetermined amount of the pharmaceutical composition calculated to produce the desired response as discussed herein in relation to its administration, i.e., the appropriate route and regimen. The amount to be administered is determined by the desired outcome and / or protection, depending on both the number of treatments and the unit dose. The exact amount of the composition is also determined by the practitioner's judgment and is specific to each individual.

[0170] Factors influencing dosage include the subject's physical and clinical condition, the route of administration, the treatment's objective (symptom relief versus cure), and the potency, stability, and toxicity of the specific composition. After formulation, the solution is administered in a manner appropriate to the dosage form and in a therapeutically or prophylactically effective amount. The formulation can be administered in various forms of administration, such as the types of injection solutions described above.

[0171] In some embodiments, the pharmaceutical compositions provided herein are administered concurrently with at least one other therapeutic substance. Where used herein, concurrent administration means that the pharmaceutical compositions and alternative treatments may be part of the same treatment regimen for a patient, but the precise timing of such administrations can be optimized. For example, an anti-DNA antibody and an alternative treatment, such as a drug or biological agent, may be administered concurrently or sequentially. The timing of administration of anti-DNA antibodies and alternative treatments can be staggered by, for example, at least 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 30 hours, 36 hours, 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 longer. The timing can be determined by the physician administering the treatment. In some embodiments, at least one other therapeutic substance comprises an anti-DNA antibody engineered to target an alternative target nucleic acid sequence.

[0172] In some embodiments, the pharmaceutical compositions provided herein are administered concurrently with at least one nuclease. Where used herein, concurrent administration means 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 administration can be optimized. For example, an anti-DNA antibody and at least one nuclease may be administered concurrently or sequentially. The timing of administration of anti-DNA antibodies and at least one nuclease can be staggered by, for example, at least 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 30 hours, 36 hours, 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 longer. In a preferred embodiment, at least a portion of the dose of at least one nuclease is administered prior to the administration of the anti-DNA antibody, for example, at least 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, or 3 hours prior. The timing regimen may be determined by the physician performing the procedure. [Examples]

[0173] IV. Examples The following examples, along with the methods described herein, are representative of preferred embodiments at present and are provided for illustrative purposes only, and are not intended to limit the scope of compositions and methods provided herein. Those skilled in the art will be able to conceive of modifications and other uses therein that fall within the spirit of the disclosure as defined by the claims.

[0174] Example 1: Materials and Method cancer cell lines 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-tumor-forming 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 and 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, and MCF-7 were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS at 37°C and 5% CO2. SK-BR-3 was cultured in McCoy's 5A medium supplemented with 10% FBS at 37°C and 5% CO2. HCC1395 BL was cultured in Iscove's modified Dulbecco medium (IMDM) supplemented with 20% FBS at 37°C and 5% CO2. MCF10A cells were cultured in human mammary epithelial cells (HuMEC) supplemented with a HuMEC supplementation kit at 37°C and 5% CO2. RPMI1640 medium, DMEM medium, McCoy's 5A medium, IMDM medium, and FBS were purchased from ATCC. We purchased HuMEC medium and HuMEC supplementation kits from Thermo Fisher Scientific Inc. (Waltham, MA).

[0175] Fluorescence imaging Cancer cells were plated at a density of 4,000 cells per well in 18-well glass-bottom chamber coverslips (Cat. No. 81817, ibidi USA, Inc. (Fitchburg, WI)) and incubated for 3 days. Anti-DNA antibody was pre-incubated with anti-mouse IgG labeled with Alexa Fluor 647 conjugate. The molar ratio of the two components was 1:1, and pre-incubation was performed in the dark at room temperature for 1 hour. The antibody conjugate was introduced into the cells at a concentration of 2 μg / 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 at 37°C for a further 3 days. Subsequently, the cells were washed with PBS, fixed with 4% formaldehyde in PBS, and permeabilized with 0.1% Triton-X100. Cell membranes were stained with WGA488 (W11261, Invitrogen, Thermo Fisher), nuclei / DNA were stained with NucBlue (R37605, Invitrogen), and an antibody against LAMP-1 (NB021157, Thermo Fisher Scientific) was used as a lysosome marker. Images were acquired using a confocal fluorescence microscope (FV3000, Olympus).

[0176] Antibody conjugation Conjugation of anti-DNA antibodies, isotype control antibodies, and secondary antibodies Anti-DNA antibody (ab27156, Abcam Limited (Boston, MA)), isotype control antibody (BE0085, Bio X Cell (Lebanon, NH)), and 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 partial reduction of DTT and thiol-maleimide reaction. The antibodies were treated with 4 molar equivalents of DTT in PBS containing 5 mM EDTA at 37°C for 2 hours. Excess DTT was removed from the partially reduced antibodies by 10 buffer exchanges using an Amicon Ultra-0.5 centrifugal filter unit (UFC5010, MilliporeSigma) featuring a 10k molecular weight cutoff. Next, the partially reduced antibody was alkylated with 5 molar equivalents of MC-Val-Cit-PAB-MMAF at 10°C for 30 minutes. To quench the 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 10 buffer changes to PBS using an Amicon Ultra-0.5 centrifugal filter unit with a 10k molecular weight cutoff. The flow-through absorbance at 248 nm was monitored to ensure 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 formula. R=(A 248 ) / (A 280 ) DAR=(21×R-9) / (1.615-0.1425×R) The three types of conjugated antibodies had approximately 4 DARs.

[0177] Cytotoxic assay Anti-DNA antibody +2nd ADC IC 50 decision Cancer cells (AU565, HCC1395, AsPC-1, SW1990) and the non-tumor-forming mammary cell line MCF10A were plated at a density of 1,500 cells per well into 96-well tissue culture (TC) treated optical-bottom white plates (165306, Thermo Fisher) and incubated for 3 days. Anti-DNA antibodies (ab27156, Abcam) or isotype control antibodies (AM26775LE-N, Origene Technologies Inc (Rockville, MD)) were pre-incubated with anti-mouse IgG secondary antibodies (115-005-071, Jackson Immuno Research) conjugated with MMAF using a valine-citrulline (Val-Cit; or VC) linker (conjugated in-house). The antibodies were mixed in a 1:1 molar ratio and pre-incubated at room temperature for 1 hour. Subsequently, antibody conjugates were added to cells at various concentrations ranging from 5 μg / ml to 1.22 ng / ml of anti-DNA antibody. Cells treated with PBS were used as a control. Each condition was tested in triplicate. Then, after culturing the cells at 37°C for 6 days, viability was evaluated 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 H1, BioTek Instruments (Winooski, VT)). The percentage of viable cells was calculated using the luminescence value of the treated wells, divided by the luminescence value of the control wells. IC50 of each antibody in each cell line. 50 The values ​​were calculated using Prism 10 (GraphPad Software Inc (San Diego, CA)).

[0178] Anti-DNA antibody +2 nd ADC+DNase I / benzonase IC 50 decision Cancer cells (AU565, HCC1395) and the non-tumor-forming mammary cell line MCF10A were plated at a density of 1,500 cells per well into 96-well tissue culture (TC) treated optical-bottom white plates (165306, Thermo Fisher) and incubated for 3 days. Anti-DNA antibody (ab27156, Abcam) or isotype control antibody (AM26775LE-N, Origene) was pre-incubated with anti-mouse IgG secondary antibody (115-005-071, Jackson Immuno Research) conjugated with MMAF using a valine-citrulline (Val-Cit) linker (conjugated in-house). The antibodies were mixed in 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, after which the antibody conjugate was added. Subsequently, the antibody conjugate was added to the cells at various concentrations ranging from 5 μg / ml to 1.22 ng / ml of anti-DNA antibody. The addition of the antibody diluted the DNase I or benzonase to a final concentration of 20 units / ml. Cells treated with PBS were used as a control. Each condition was tested in triplicates. Cells were then cultured at 37°C for 6 days, and viability was assessed 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 H1, BioTek). The percentage of viable cells was calculated using the luminescence value of the treated wells, divided by the luminescence value of the control wells. IC50 of each anti-DNA antibody in each cell line. 50 The values ​​were calculated using Prism 10 (GraphPad).

[0179] MMAF IC 50 decision Cancer cell lines (AU565, SK-BR-3, HCC1395, MDA-MB-468, HCC1806, Hs-578t, MDA-MB-453, MCF-7, DU4475, NCI-H460) and non-tumor-forming cell lines (MCF10A, HCC1395 BL) were seeded at a density of 1,500 cells per well in 96-well plates (165306, Thermo Fisher) and incubated for 3 days. The cells were then treated with MMAF at various concentrations ranging from 25 μM to 1.6 nM at 37°C for 3 days, and their viability was evaluated using the CellTiter-Glo 2.0 assay (G9242, Promega). Each condition was tested in triple cycles. The amount of luminescence emitted from each well was measured using a multimode microplate reader (Synergy H1, BioTek). The percentage of viable cells was calculated using the luminescence measurement of the treated wells, obtained by dividing the luminescence measurement of the control wells by the luminescence measurement of the control wells. IC of each anti-DNA antibody in each cell line. 50 The values ​​were calculated using Prism 10 (GraphPad).

[0180] Different anti-DNA antibodies + 2 nd Determination of the cytotoxicity of ADC + DNase I Cancer cell lines (AU565, SK-BR-3, HCC1395, MDA-MB-468, HCC1806, Hs-578t, MDA-MB-453, MCF-7, DU4475, NCI-H460) and non-tumor-forming cell lines (MCF10A, HCC1395 BL) were plated at a density of 1,500 cells per well into 96-well tissue culture (TC) treated optical-bottom white plates (165306, Thermo Fisher) and incubated for 3 days. Three different commercially available anti-DNA antibodies or isotype control antibodies (AM26775LE-N, Origene) were pre-incubated with anti-mouse IgG secondary antibodies (115-005-071, Jackson Immuno Research) conjugated with MMAF using a valine-citrulline (Val-Cit) linker (conjugated in-house; Val-Cit linkers with MMAF may be referred to herein as VC-MMAF). The antibodies were mixed in a 1:1 molar ratio and pre-incubated at room temperature for 1 hour. Subsequently, the antibody conjugates and DNase I were mixed with 0.2 μg / ml anti-DNA antibody and 0.2 μg / ml 2 nd Cells were treated with ADC and 20 units / ml DNase I. Cells treated with PBS were used as a control. Each condition was tested in three sets. Subsequently, the cells were cultured at 37°C for 3 days, and their viability was evaluated 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 H1, BioTek). The percentage of viable cells was calculated using the luminescence value of the treated wells, divided by the luminescence value of the control wells.

[0181] anti-DNA ADC+2 nd antibody IC 50 decision HER2 (human epidermal growth factor receptor 2)-positive breast cancer cells (AU565, BT474), triple-negative mammary cell lines (MDA-MB-468, HCC1395), and non-tumor-forming B lymphoblast cell lines (HCC1395 BL) from the same patients as the HCC1395 breast cancer cell line were plated at a density of 1,500 cells per well into 96-well tissue culture (TC) treated optical-bottom white plates (165306, Thermo Fisher) and incubated for 3 days. Anti-DNA antibodies (ab27156, Abcam) or isotype control antibodies (BE0085, Bio X Cell) conjugated with MMAF using a valine-citrulline (Val-Cit) linker (conjugated in-house) were pre-incubated with anti-mouse IgG secondary antibodies (115-005-071, Jackson Immuno Research). Antibodies were mixed in a 1:1 molar ratio and pre-incubated at room temperature for 1 hour. Then, anti-DNA antibody conjugates were added to cells at various concentrations ranging from 5 μg / ml to 1.22 ng / ml of anti-DNA ADC. Isotype control antibody conjugates were added to cells at various concentrations ranging from 20 μg / ml to 1.28 ng / 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 a control. Each condition was tested in four-sequence sets. Cells were then cultured at 37°C for 6 days, and viability was evaluated 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 H1, BioTek). The percentage of viable cells was calculated using the luminescence value of the treated wells, divided by the luminescence value of the control wells. IC of each antibody-drug conjugate with and without DNase I 50 The values ​​were calculated for each cell line using Prism 10 (GraphPad).

[0182] IC of Trastuzumab deruxtecan 50 decision HER2+ breast cancer cells (AU565, BT474), triple-negative breast cell lines (MDA-MB-468, HCC1395), and non-tumor-forming B lymphoblasts from the same patient as the HCC1395 breast cancer cell line were plated at a density of 1,500 cells per well into 96-well tissue culture (TC)-treated optical-bottom white plates (165306, Thermo Fisher) and incubated for 3 days. Cells were then treated with trastuzumab deruxtecan (HY-138298, MedchemExpress (Monmouth Junction, NJ)) at various concentrations ranging from 20 μg / ml to 1.28 ng / ml. Half of the wells were also treated with DNase I (M0303S, New England Biolabs) at 20 units / ml. Cells treated with PBS were used as a control. Each condition was tested in four consecutive sets. Next, cells were cultured at 37°C for 6 days, and their viability was evaluated 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 H1, BioTek). The percentage of viable cells was calculated using the luminescence value of the treated wells, obtained by dividing the luminescence value of the control wells by the luminescence value of the control wells. IC50 of trastuzumab deruxtecan was evaluated both with and without DNase I. 50 The values ​​were calculated for each cell line using Prism 10 (GraphPad).

[0183] 2 nd Determination of the cytotoxicity of trastuzumab deruxtecan in combination with antibodies and DNase I. 2 nd To rule out the possibility that the use of antibodies, DNase I, or both may confer an overall enhancement of ADC cytotoxicity, HER2-positive breast cancer cells AU565 and BT474 were treated with 0.2 μg / ml trastuzumab deruxtecan alone, and 0.2 μg / ml 2 ndCells treated with antibody (109-005-170, Jackson ImmunoResearch), 20 units / ml of DNase I, or both, and then treated with PBS were used as controls. Each condition was tested in four-cell sets. After culturing the cells at 37°C for 3 days, viability was evaluated 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 H1, BioTek). The percentage of viable cells was calculated using the luminescence value of the treated wells, divided by the luminescence value of the control wells.

[0184] Various anti-DNA antibodies in combination with various nucleases 2 nd Determination of the cytotoxicity of ADCs To evaluate whether increased sensitivity is dependent on nuclease activity or specifically on the DNase I molecule, 0.2 μg / ml anti-DNA antibody + 0.2 μg / ml 2 was administered 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). ndAU565 cells were treated with ADC. In addition, the inventors tested one nonspecific recombinant endonuclease that cleaves both DNA and RNA: benzonase (E8263, Sigma-Aldrich), and one RNA nuclease: RNase A (556746, Sigma-Aldrich). Recombinant bovine DNase I, substantially heat-inactivated by heating at 75°C for 5 minutes, was also included in the tests. Cells treated with PBS were used as a control. Each condition was tested in four-row sets. After culturing cells at 37°C for 3 days, viability was evaluated 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 H1, BioTek). The percentage of viable cells was calculated using the luminescence measurement of the treated wells divided by the luminescence measurement of the control wells.

[0185] Example 2: Internal transfer of anti-DNA antibody This embodiment demonstrates that anti-DNA antibodies can bind to extracellular DNA on the cell surface and move into the cell, and that this internal movement can be increased by DNase I treatment.

[0186] Fluorescence imaging of human breast cancer cell lines HCC1395 and AU565 after incubation with anti-DNA antibodies was used to observe the binding and internal migration of anti-DNA antibodies. After 3 days of incubation, DNA binding and internal migration of antibodies were observed in all cell samples incubated with various anti-DNA antibodies: anti-dsDNA antibody [121-3] (Abcam), anti-dsDNA antibody [35I9 DNA] (Abcam), anti-dsDNA antibody BV16-13 (MilliporeSigma), anti-dsDNA antibody AE-2 (Millipore), or anti-dsDNA antibody rDSD / 4565 (Thermo Fisher), but not in cells using isotype IgG2a and IgG3 controls. Data are not shown.

[0187] Furthermore, fluorescence imaging demonstrated a significant increase in the internal translocation of anti-DNA antibodies within cells following DNase I treatment. AU565 cells were treated with 40 units / ml of DNase I for 1 hour, then incubated with anti-DNA antibodies, maintaining the DNase I concentration at 20 units / ml for 3 days. Significantly higher levels of all five anti-DNA antibodies were observed within nuclease-treated cells compared to untreated cells. This suggests that nuclease treatment can increase the internal translocation of anti-DNA antibodies.

[0188] Example 3: Cytotoxicity of anti-DNA antibodies with drug conjugates in cancer cells This example demonstrates that anti-DNA antibodies conjugated with drugs can kill cancer cells, and that their cytotoxicity is increased by nuclease treatment.

[0189] The cytotoxicity of anti-DNA antibodies with drug conjugates was tested in two human breast cancer cell lines (HCC1395 and AU565), two human pancreatic cancer cell lines (SW1990-1 and AsPC-1), and the human normal (non-cancer) cell line MCF10A. Before testing cell viability, the anti-DNA antibody [35I9 DNA] (Abcam) and the anti-mouse IgG-VC-MMAF conjugate were incubated with cells for 6 days. As shown in Figures 2A-2D, cancer cells treated with anti-DNA antibodies and antibody-drug conjugates all had lower viability compared to control cells treated with mouse IgG isotype antibodies along with antibody-drug conjugates that exhibited high concentrations of nonspecific cytotoxicity. In the non-cancer cell line MCF10A, there was no significant difference between the test antibody and the control (Figure 2E), suggesting that cancer cells were more sensitive to the cytotoxicity of anti-DNA antibodies with drug conjugates than normal cells. The data in Figures 3A and 3B demonstrate that the cytotoxicity of anti-DNA ADCs was not caused by bare anti-DNA antibodies alone.

[0190] Consistent with the results of internal migration of anti-DNA antibodies as shown by fluorescence imaging (see Example 2), the cytotoxicity of the anti-DNA antibody [35I9 DNA] (Abcam) and anti-mouse IgG-VC-MMAF can be increased by various nuclease treatments. As shown in Figures 4A-4D, anti-DNA antibody / 2 nd When treated with ADC and nuclease, anti-DNA antibody / 2 nd Higher cytotoxicity was observed in both HCC1395 and AU565 cells compared to cells treated with ADC alone. Figures 4A and 4B show the cytotoxicity results when DNase I was used as the nuclease. Figures 4C and 4D show the cytotoxicity results when benzonase was used as the nuclease.

[0191] Next, the inventors tested whether nuclease alone could induce cytotoxicity. MCF10A cells and HCC1395 cells were treated with DNase I at various concentrations for 6 days. As shown in Figures 5A and 5B, DNase I alone did not induce cytotoxicity in either normal or cancer cells at concentrations ranging from 25 units / ml to 100 units / ml, and anti-DNA antibody / 2 nd The cytotoxicity of the ADC and DNase I combination was not caused by DNase I alone.

[0192] Furthermore, the inventors investigated whether the nuclease could increase the sensitivity of cells to antibodies other than anti-DNA antibodies. Cell viability was assayed at 3 and 6 days. As shown in Figures 5C and 5D, 0.2 μg / ml 2 2 units / ml DNase I was used in combination with 20 units / ml DNase I. ndWhen AU565 breast cancer cells were treated with three isotype antibodies (IgG2a, IgG3, or IgG1, as shown in the plot) along with an antibody-drug conjugate, no cytotoxicity was observed in any of the samples, regardless of the presence (Figure 5C) or absence (Figure 5D) of DNase I. The inventors also tested other cell lines (e.g., MCF10A) with and without DNase I treatment, and no cytotoxicity was observed in any of the samples (data not shown). AU565 cells were treated with 0.2 μg / ml of anti-CD71 antibody and 2 nd When treated with ADC, a high degree of cytotoxicity was observed, but there was no significant difference between experiments with and without the addition of 20 units / ml DNase I. See Figure 5E. DNase I also failed to enhance the cytotoxicity of trastuzumab or trastuzumab deruxtecan, as will be further described in Example 8. On the other hand, cells were enhanced in the presence of anti-DNA antibody / 2 in the presence of DNase I and other DNA nucleases. nd Sensitivity to ADCs was significantly increased. See, for example, Figures 4A-4D; Figures 5F-5J. However, since the inventors did not observe any significant effect on RNase A-mediated cytotoxicity, this effect may require DNA nuclease activity. See Figure 5K. In summary, these results indicate that DNA nucleases do not increase the sensitivity of cells to ADCs that do not target extracellular DNA.

[0193] The inventors further investigated whether the cytotoxicity of anti-DNA ADCs with nuclease treatment was dose- and / or duration-dependent. AU565 cells were treated with 0.2 μg / ml anti-DNA antibody and 0.2 μg / ml 2 ndCells were treated with ADC for 3 days, and varying amounts of DNase I were added at different time points. As shown in Figure 5F, little toxicity was observed when DNase I was not added. The highest cytotoxicity was observed when 20 units / ml DNase I was added together with the anti-DNA ADC complex. However, when cells were treated with 20 units / ml DNase I for 24 hours before adding the anti-DNA ADC complex, cytotoxicity was reduced compared to experiments where ADC and DNase I were added simultaneously. Cytotoxicity was further reduced in cells treated with twice the amount of DNase I (40 units / ml) for 24 hours before adding the anti-DNA ADC complex. These data suggest that the nuclease increased the sensitivity of cells to anti-DNA ADC, but excessive nuclease activity or prolonged nuclease reaction time reduced the cytotoxicity of the anti-DNA antibody. While not bound by theory, this may be because the target of the anti-DNA ADC complex is removed by increased degradation of extracellular DNA into fragments smaller than the antibody's binding capacity. In the anti-DNA antibody [35I9 DNA] (Abcam), the minimum size of DNA binding is >16 base pairs.

[0194] Example 4: Target specificity of anti-DNA antibodies with drug conjugates This example demonstrates that cancer cells are more sensitive to anti-DNA antibodies conjugated with drugs than normal cells.

[0195] Figures 2A-2E demonstrate that anti-DNA antibodies conjugated with drug conjugates show higher sensitivity to cancer cells (AsPC-1, AU565, SW1990-1, and HCC1395; Figures 2A-2D, respectively) than to normal cells (MCF10A; Figure 2E). Figure 6A shows the IC50 of anti-dsDNA ADCs conjugated with DNase I in the indicated cancer cells (AU565, HCC1395) and normal cells (MCF10A). 50Figure 6B shows cytotoxicity under the same settings across a range of antibody concentrations. These data further demonstrate a significant difference in cytotoxicity between cancer cells and non-cancer cells with anti-DNA antibody / ADCs accompanied by nuclease treatment. Cytotoxicity appears to be dose-dependent.

[0196] To investigate whether the observations in Figures 6A-6B were antibody-specific rather than target-specific, the inventors conducted a study. nd Two additional commercially available anti-DNA antibodies were tested in combination with ADC and DNase I, and similar responses were observed. See Figure 7. As shown in the figure, significant cytotoxicity against cancer cells was observed in 2 nd This was achieved with three different anti-DNA antibodies in combination with ADC and DNase I.

[0197] Example 5: Cytotoxicity of anti-DNA ADCs in TP53 mutant cells This example demonstrates that the cytotoxicity of anti-DNA antibodies with drug conjugates is associated with the presence of the TP53 mutation.

[0198] In this embodiment, the inventors have demonstrated a broader range of cancer cells and normal cells. nd The response to anti-DNA antibodies in combination with ADC and DNase I was analyzed. The inventors tested 12 cell lines, including 9 human breast cancer cell lines (AU565, SK-BR-3, HCC1395, MDA-MB-468, HCC1806, Hs 578T, MDA-MB-453, MCF-7, DU4475), 1 human lung cancer cell line (NCI-H460), and 2 non-tumor-forming human cell lines (MCF10A, HCC1395 BL). The treatment involved 20 units / ml DNase I, 0.2 μg / ml anti-DNA antibody + 0.2 μg / ml 2 nd Cells were treated with ADCs separately or in combination. As shown in Figure 8A, cells were treated with DNase I (second group) or anti-DNA antibody + 2 ndWhen treated separately with ADC (third group), little cytotoxicity was observed. When DNase I and the anti-DNA ADC complex were used in combination, approximately 50% cell death (ranging from 43% to 68%) was observed in TP53 mutant cells, compared to less activity in TP53 wild-type cells, regardless of whether they were cancerous or normal (Figures 8A and 8B). Table 1 lists 12 cell lines and their TP53 gene status. These experiments suggest that TP53 mutation status can be used as a predictive marker for whether cancer cells are sensitive to anti-DNA ADC + DNase I.

[0199] (Table 1) Cell lines and TP53 gene TIFF2026522339000002.tif103143 * HER2: Human epidermal growth factor receptor 2; TNA: Triple-negative type A; TNB: Triple-negative type B.

[0200] Next, the inventors compared anti-DNA+2 in 12 different cell lines compared to MMAF alone. nd The effect of ADC was investigated.

[0201] Table 2 shows the IC50 of MMAF in each cell after treatment with an antibody-drug conjugate conjugate consisting of 0.2 μg / ml anti-DNA antibody + MMAF conjugated with 0.2 μg / ml secondary antibody (equivalent to approximately 6 nM MMAF) + 20 units / ml DNase I. 50The values ​​and viability of each cell type are presented. In six cell lines carrying the TP53 loss-of-function mutation, approximately 6 nM of MMAF conjugated with an antibody was sufficient to achieve approximately 50% cell death (ranging from 43% to 68%). Conversely, significantly higher concentrations of MMAF were required (ranging from 15.66 nM to 2783 nM) to achieve the same level of cell death using MMAF as a free drug (i.e., unconjugated). In particular, in MDA-MB-468 cells, the antibody and DNase I combination boosted MMAF sensitivity by more than 450-fold. However, the anti-DNA ADC combination showed minimal toxicity to non-cancer cells and TP53 wild-type cancer cells. The sensitivity of each cell line to the free MMAF drug did not correlate with TP53 status, and there was no correlation between TP53 status and anti-DNA ADC. nd The correlation observed by the inventors between ADCs and cytotoxicity suggests that it is antibody-dependent rather than payload-dependent.

[0202] (Table 2) MMAF IC for 12 different cell lines 50 Value and ADC combination toxicity TIFF2026522339000003.tif94153

[0203] Example 6: Cytotoxic effects of different payloads This example demonstrates that anti-DNA ADCs can use different antitumor molecules with different mechanisms of action as payloads.

[0204] To test whether the anti-DNA ADC complex and DNase I combination can function as a payload with other antitumor agents besides MMAF, human breast cancer cell lines AU565 and SK-BR-3 were conjugated with 0.2 μg / ml anti-DNA antibody, MMAF, DMDM, or exatecan along with 20 units / ml DNase I. ndThe cells were treated with antibodies. Each drug has a different mechanism of action: MMAF inhibits tubulin polymerization, DMDM ​​alkylates DNA, and exetecan is a topoisomerase I inhibitor. As shown in Figures 9A-9B, after 3 days of treatment, significant cytotoxicity was achieved with the MMAF conjugate in both cell lines, while detectable but lower cytotoxicity was achieved with DMDM ​​and exetecan. After 6 days of treatment, considerable cytotoxicity was achieved from any combination of the three cytotoxic payloads. These results indicate that anti-DNA ADCs can utilize various antitumor molecules with different mechanisms of action as payloads.

[0205] Example 7: Cytotoxicity of ADC configuration This example compares the cytotoxicity of anti-DNA antibodies in various configurations.

[0206] As shown in Figure 10, comparisons were made between anti-DNA antibody alone without a payload; secondary antibody alone without a payload; anti-DNA and secondary antibody without a payload; anti-DNA antibody with secondary antibody conjugation conjugated to a payload (MMAF); anti-DNA ADC with direct conjugation of payload (MMAF); direct conjugation of anti-DNA ADC with unconjugated secondary antibody; and anti-DNA ADC with secondary antibody conjugated to MMAF. In these experiments, MMAF was directly conjugated to the anti-DNA antibody by using a maleimide-thiol conjugation reaction via a valine-citrulline (Val-Cit) linker. In the absence of a cytotoxic payload, no cytotoxicity was observed, suggesting that cytotoxicity was due to the payload conjugated to the antibody rather than the antibody alone. See bars 2-4 in Figure 10. We found that the ADC directly conjugated with DNase I (bar 6) exhibited reduced cytotoxicity compared to all configurations including a secondary antibody (bars 5, 7, and 8). However, the cytotoxicity of anti-DNA ADCs is exposed. nd Antibody or 2nd Recovery was achieved by adding ADC. Compare bar 6 with bars 7 and 8. While not bound by theory, these results suggest that the internal migration of anti-DNA antibodies was enhanced by an increase in the cluster size or molecular weight of the cell-targeting construct. As disclosed herein, such anti-DNA antibody constructs can be constructed using multiple antibodies and / or other entities such as carrier molecules or nanoparticles. See, for example, Figure 1D and related discussion.

[0207] Example 8: Cytotoxic effect compared to trastuzumab deruxtecan Trastuzumab deruxtecan (T-DXd) is one of the most successful antibody-drug conjugates (ADCs) for treating cancer. It was initially approved by the FDA in December 2019 for patients with unresectable or metastatic HER2-positive breast cancer. In August 2022, the FDA further approved trastuzumab deruxtecan for the treatment of HER2-low breast cancer. And in March 2024, the FDA granted broader approval for trastuzumab deruxtecan for any HER2-positive solid tumor, regardless of cancer type. This example demonstrates anti-DNA 2 in HER2-positive (HER2+) cells and HER2-low triple-negative cancer cells. nd We will compare the cytotoxicity of ADC with that of T-DXd.

[0208] In the HER2-positive breast cancer cell line AU565, anti-DNA 2 nd Both ADC and T-DXd showed high cell death efficacy, while T-DXd had slightly lower IC50. 50 It had anti-DNA 2. See Figure 11A. Another HER2+ breast cancer cell line, BT474, had anti-DNA 2. nd ADC has significantly lower IC compared to T-DXd. 50 This was shown. See Figure 11B. Anti-DNA 2 ndUnlike ADCs, 50% cytotoxicity of T-DXd was not observed even at the highest dose tested (20 μg / ml). Similarly, 50% cell death by T-DXd was not achieved at 20 μg / ml in two triple-negative cancer cell lines, HCC1395 (Figure 11C) and MDA-MB-468 (Figure 11D). In contrast, anti-DNA 2 nd The ADC complex was detected in HCC1395 cells at approximately 0.3 μg / ml IC50. 50 It had (Figure 11C). Anti-DNA 2 nd ADC and T-DXd resulted in IC50 concentrations of approximately 1.3 μg / ml and 9.3 μg / ml, respectively, in MDA-MB-468 cells. 50 It possessed. See Figure 11D. Anti-DNA 2 nd To evaluate whether the efficacy of ADC is nonspecific to cancer cells, HCC1395 BL, a non-tumor-forming B lymphoblast cell line derived from the same patient as the breast cancer cell line HCC1395, was subjected to anti-DNA 2 nd Both ADC and T-DXd were tested. Unlike HCC1395 cancer cells, ADC induced cytotoxicity in non-cancerous HCC1395 BL cells only at the highest concentration. See Figure 11E. Comparing the results for HCC1395 (upper panel of Figure 11E) and HCC1395 BL (lower panel of Figure 11E), IC25 in HCC1395 was observed. 50 It was 10 times lower than that in HCC1395 BL. In all comparisons, the inventors found that DNase I was anti-DNA 2 in cells. nd We observed that T-DXd significantly increased sensitivity to ADC, but not to the other group. See Figures 11A–11G. T-DXd also showed 2 nd The antibody did not affect the cells. See Figures 11F-11G. Isotype control ADC conjugates containing MMAF-conjugated isotype antibodies and secondary antibodies did not show significant cytotoxicity under any conditions. See Figures 11A-11E.

[0209] In summary, the results in this embodiment suggest that the anti-DNA ADC constructs provided herein may be as effective as, and potentially more effective than, FDA-approved T-DXd ADCs in some situations.

[0210] While aspects of this disclosure are described herein, it should be understood by those skilled in the art that such aspects are provided only as examples. Numerous variations, modifications, and substitutions will be conceivable by those skilled in the art without departing from the compositions and methods provided herein. It should be understood that various alternatives to the aspects provided herein may be available. The appended claims define the scope thereof, and methods and structures within the scope of these claims, as well as their equivalents, are intended to be included therein.

Claims

1. A method for introducing one or more anti-DNA antibodies into a cell, The step includes contacting the cells with one or more anti-DNA antibodies, 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 move into the cell. The method.

2. The method according to claim 1, wherein the contacting step comprises contacting the cells with at least 1, 2, 3, 4, 5, 6, 7, 8 or more different anti-DNA antibodies, and optionally, the at least 1, 2, 3, 4, 5, 6, 7, 8 or more different anti-DNA antibodies comprise 1, 2, 3, 4, 5, 6, 7, 8 or more different anti-DNA antibodies.

3. The method according to any one of claims 1 to 2, further comprising the step of contacting the cells with one or more nucleases, wherein the one or more nucleases optionally include DNA nucleases.

4. The method according to claim 3, wherein the one or more nucleases include an endonuclease, an exonuclease, or a combination thereof.

5. The method according to claim 4, wherein the endonuclease is a deoxyribonuclease (DNase), a nuclease (benzonase) of Serratia marcescens, a micrococcal nuclease (MNase), a transposase, a restriction enzyme, nuclease S1, nuclease P1, a sequence-specific endonuclease, or a sequence-nonspecific endonuclease.

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

7. The method according to any one of claims 3 to 6, wherein the one or more nucleases and the one or more anti-DNA antibodies are simultaneously in contact with the cells, and optionally, the one or more nucleases are bound to the one or more anti-DNA antibodies.

8. The method according to any one of claims 3 to 6, wherein the one or more nucleases are brought into contact with the cells before the one or more anti-DNA antibodies.

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

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

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

12. The method according to claim 11, wherein one or more secondary antibodies are covalently or noncovalently linked to at least one payload.

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

14. The method according to 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 according to any one of claims 11 to 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 according to claim 15, wherein the linker includes a non-cutting linker.

17. The method according to claim 16, wherein the non-cleaving linker comprises a maleimide alkane linker or a maleimide cyclohexane linker.

18. The method according to claim 15, wherein the linker includes a cutting linker.

19. The method according to claim 18, wherein the linker is cleaved after the step of contacting the cells with the one or more anti-DNA antibodies, thereby releasing the payload onto or into the cells.

20. The method according to claim 18 or 19, wherein the cleavage linker includes a hydrazone linker, a cathepsin B-responsive linker, a disulfide linker, a pyrophosphate diester linker, or a combination thereof.

21. The method according to any one of claims 18 to 20, wherein the cleavage linker includes a protease-sensitive linker, a pH-sensitive linker, a radiosensitive linker, a disulfide linker, a glutathione-sensitive linker, or a combination thereof.

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

23. The method according to any one of claims 10 to 22, wherein the at least one payload comprises a small molecule, peptide, protein, nucleic acid, toxin, therapeutic substance, drug, chemotherapeutic agent, liposome, nanoparticle, dendrimer, detectable label, or any derivative, fragment, or combination thereof.

24. The method according to claim 23, wherein the therapeutic substance is selected from the group consisting of antitumor agents, anticancer agents, prodrugs, lysosomal destabilizers (e.g., chloroquine), alkylating agents, alkaloids, allosteric inhibitors, antifolic acid agents, anti-inflammatory agents, antibiotics, antibacterial agents, antifungal agents, antifibrotic agents, antiinfective agents, antiparasitic agents, antiviral agents, antimycobacterial agents, anticancer agents, antiprotozoal agents, antiviral agents, physiologically active peptides, steroid hormones, photosensitive substances, radiopharmaceuticals, antiprion agents, and any combination thereof.

25. The aforementioned antitumor agents include aromatase inhibitors; anti-estrogens; anti-androgens; gonadrelin agonists; topoisomerase I inhibitors; topoisomerase II inhibitors; microtubule inhibitors; alkylating agents; retinoids, carotenoids, or tocopherols; cyclooxygenase inhibitors; MMP inhibitors; mTOR inhibitors; antimetabolites; platinum compounds; methionine aminopeptidase inhibitors; bisphosphonates; antiproliferative antibodies; heparinase inhibitors; inhibitors of Ras oncogenic isoforms; and telomerase inhibitors. The method according to claim 24, selected from the group consisting of: proteasome inhibitors; Flt-3 inhibitors; Hsp90 inhibitors; kinesin spindle protein inhibitors; MEK inhibitors; PARP inhibitors; tyrosine kinase inhibitors; PI3K inhibitors; AKT inhibitors; EGFR inhibitors; antitumor antibiotics; nitrosoureas; compounds that target / reduce protein or lipid kinase activity; compounds that target / reduce protein or lipid phosphatase activity; anti-angiogenic compounds; and any combination thereof.

26. The method according to claim 24 or 25, wherein the antitumor agent is selected from the group consisting of azacitidine, azathioprine, bleomycin, capecitabine, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, etoposide, fenretinide, fluorouracil, gemcitabine, herceptin, idarubicin, mechloretamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, tafluposide, teniposide, thioguanine, retinoic acid, barrubicin, vinblastine, vincristine, vindesine, vinorelbine, receptor tyrosine kinase inhibitors, and any combination thereof.

27. The method according to 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 according to claim 27, wherein the tubulin inhibitor is selected from the group consisting of monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), maytansine, maytansinoid, meltansine (emtansine, DM1), labtansine (solabtansine, DM4), tubulinin, halichondrin (eribulin), cryptophycin, EG5 inhibitors, and any derivatives thereof.

29. The method according to claim 27, wherein the DNA inhibitor is selected from the group consisting of alkylating agents, duocalmycin, duocalmycin DM (DMDM), calicheamicin, pyrrolobenzodiazepine (PDB), enediyne, unciaramycin, topoisomerase inhibitors, topotecan, camptothecin (CPT), exatecan, and any derivatives thereof.

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

31. The method according to claim 23, wherein the detectable label is selected from the group consisting of magnetic labels, fluorescent moieties, enzymes, luminescent particles, chemiluminescent probes, metal particles, nonmetallic colloidal particles, polymer dye particles, dye molecules, electrochemically active species, semiconductor nanocrystals, nanoparticles, quantum dots, gold particles, fluorophores, radioactive labels, or combinations thereof.

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

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

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

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

36. The method according to any one of claims 1 to 35, wherein the one or more anti-DNA antibodies are coated on gold nanoparticles when they enter the cells.

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

38. The method according to any one of claims 1 to 37, wherein the cells have aneuploidy and / or DNA repair defects.

39. The method according to any one of claims 1 to 37, wherein the cells contain a functionally impaired transcription factor selected from the group consisting of p53, NF-κb, AP-1, E2F1, and BRCA1.

40. The method according to claim 39, wherein the dysfunction of the transcription factor is caused by mutation, loss, and / or failure of expression of one or more genes.

41. The method according to claim 39 or 40, wherein the cells contain a functionally impaired transcription factor p53, and optionally the impairment of p53 includes a mutation.

42. A method for delivering therapeutic substances into the interior of cells, The process includes contacting the cells with a therapeutic substance in which an anti-DNA antibody is covalently or noncovalently linked, The anti-DNA antibody binds to extracellular DNA on the surface of the cell and delivers the therapeutic substance into the cell. The method.

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

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

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

46. The method according to any one of claims 42 to 45, further comprising the step of contacting the cells with one or more nucleases, wherein the one or more nucleases optionally include single-stranded DNA (ssDNA) nucleases, double-stranded DNA (dsDNA) nucleases, or a combination thereof.

47. The method according to any one of claims 42 to 46, wherein the cells are in vitro.

48. The method according to any one of claims 42 to 46, wherein the cells are in vivo.

49. The method according to any one of claims 42 to 48, wherein the cells have aneuploidy and / or DNA repair defects.

50. The method according to any one of claims 42 to 49, wherein the cells contain a functionally impaired transcription factor selected from the group consisting of p53, NF-κb, AP-1, E2F1, and BRCA1.

51. The method according to claim 50, wherein the dysfunction of the transcription factor is caused by mutation, loss, and / or failure of expression of one or more genes.

52. The method according to claim 50 or 51, wherein the cells contain a functionally impaired transcription factor p53, and optionally the impairment of p53 includes a mutation.

53. The method according to any one of claims 42 to 52, wherein the cells are cancer cells, and the therapeutic substance kills the cancer cells or inhibits their proliferation or division.

54. The aforementioned types of cancer include acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical carcinoma; AIDS-related cancer; AIDS-related lymphoma; anal cancer; appendiceal cancer; astrocytoma; atypical teratoid / rhabdoid tumor; basal cell carcinoma; bladder cancer; brainstem glioma; brain tumor, brainstem glioma, atypical teratoid / rhabdoid tumor of the central nervous system, embryonic tumor of the central nervous system, astrocytoma, craniopharyngioma, ependymoblastoma, ependymphoblastoma, medullary epithelioma, and Intertype pineal parenchymal tumors, supratentorial primitive neuroectodermal tumors and pineoblastomas; breast cancer; bronchial tumors; Burkitt lymphoma; cancer of unknown primary origin (CUP); carcinoid tumors; carcinomas of unknown primary origin; atypical teratoid / rhabdoid tumors of the central nervous system; embryonal tumors of the central nervous system; cervical cancer; childhood cancer; chordoma; chronic lymphocytic leukemia; chronic myeloid leukemia; chronic myeloproliferative disorders; colon cancer; colorectal cancer; craniopharyngioma; cutaneous T cells Lymphoma; endocrine islet cell tumor; endometrial cancer; ependymoblastoma; ependymoma; esophageal cancer; nasal neuroblastoma; Ewing's 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's disease Lymphoma; Hypopharyngeal cancer; Intraocular melanoma; Islet cell tumor; Kaposi's sarcoma; Kidney cancer; Langerhans cell histiocytosis; Laryngeal cancer; Lip cancer; Liver cancer; Lung cancer; Malignant fibrous histiocytoma; Bone cancer; Medulloblastoma; Medullary epithelioma; Melanoma; Merkel cell carcinoma; Merkel cell cutaneous cancer; Mesothelioma; Metastatic squamous cell carcinoma of unknown primary origin; Mouth cancer; Multiple endocrine neoplasia syndrome; Multiple myeloma; Multiple myeloma / plasmacytic neoplasia; mycosis fungoides; Myelodysplastic syndrome; myeloproliferative neoplasm; nasal cavity cancer; nasopharyngeal cancer; neuroblastoma; non-Hodgkin lymphoma; non-melanoma 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; low-grade ovarian tumor; pancreatic cancer; papilloma; paranasal sinus cancer; parathyroid cancer; pelvic cancer; penile cancer; pharyngeal cancer; intermediate pineal parenchymal tumor; pineoblastoma; pituitary tumor; plasma cell tumor / multiple myeloma; pleuropneumonoma; primary central nervous system (CNS) lymphoma; primary hepatocellular liver cancer; prostate cancer; rectal cancer; kidney cancer; renal cell carcinoma; renal cell carcinoma; The method according to claim 53, comprising: respiratory cancer; retinoblastoma; rhabdomyosarcoma; salivary gland cancer; Sézary syndrome; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma; squamous cervical cancer; stomach cancer; supratentorial primitive neuroectodermal tumor; T-cell lymphoma; testicular cancer; throat cancer; thymic cancer; thymoma; thyroid cancer; transitional cell carcinoma; transitional cell carcinoma of the renal pelvis and ureter; choriocarcinoma; ureteral cancer; urethral cancer; uterine cancer; uterine sarcoma; vaginal cancer; vulvar cancer; Waldenström macroglobulinemia; or Wilms' tumor.

55. The aforementioned cancer types include acute myeloid leukemia (AML), breast cancer, cholangiocarcinoma, colorectal adenocarcinoma, extrahepatic cholangiocarcinoma, female reproductive organ malignancies, gastric adenocarcinoma, gastroesophageal adenocarcinoma, gastrointestinal stromal tumors (GIST), glioblastoma, head and neck squamous cell carcinoma, leukemia, hepatocellular carcinoma, low-grade glioma, bronchioloalveolar carcinoma (BAC), non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), lymphoma, male reproductive organ malignancies, and pleura. The method according to claim 53, comprising malignant solitary fibrous tumor (MSFT), melanoma, multiple myeloma, neuroendocrine tumor, nodular diffuse large B-cell lymphoma, non-epithelial ovarian cancer (non-EOC), ovarian surface epithelial carcinoma, pancreatic adenocarcinoma, pituitary cancer, oligodendroglioma, prostate adenocarcinoma, retroperitoneal or peritoneal cancer, retroperitoneal or peritoneal sarcoma, small intestinal malignancy, soft tissue tumor, thymic carcinoma, thyroid cancer, or uveal melanoma.

56. The method according to any one of claims 53 to 55, wherein the cancer cells are derived from cancer in the subject.

57. A method of taking action in the area where it is necessary, The process includes administering a composition containing a therapeutic substance linked to an anti-DNA antibody to the subject, The therapeutic substance is delivered into the target cells using the method described in any one of claims 42 to 46. The delivery of the therapeutic substance is effective in treating the subject. The method.

58. The method according to claim 57, wherein the composition further comprises a DNA nuclease.

59. The method according to claim 57 or 58, wherein the subject has cancer and the cells are cancer cells.

60. The method according to any one of claims 57 to 59, further comprising the step of determining whether the cells have aneuploidy, DNA repair defects, and / or functionally impaired transcription factors selected from the group consisting of p53, NF-κb, AP-1, E2F1, and BRCA1, prior to the administration step.

61. The method according to claim 60, wherein the dysfunction of the transcription factor is caused by mutation, loss, and / or expression failure of one or more genes.

62. The method according to claim 60 or 61, wherein the cells contain a functionally impaired transcription factor p53, and optionally the impairment of p53 includes a mutation.

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

64. The composition according to claim 63, wherein one or more anti-DNA antibodies are conjugated by one or more secondary antibodies.

65. The composition according to claim 63 or 64, wherein one or more anti-DNA antibodies are coated on gold nanoparticles.

66. The composition according to claim 63 or 64, wherein the one or more anti-DNA antibodies comprises at least two anti-DNA antibodies linked together as a polymer.

67. The composition according to claim 66, wherein the polymer comprises a dimer, trimer, tetramer, pentamer, or six or more anti-DNA antibodies.

68. The composition according to any one of claims 63 to 67, comprising 2, 3, 4, 5, 6, 7, 8 or more different anti-DNA antibodies.

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

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

71. The composition according to claim 70, wherein the therapeutic substance is selected from the group consisting of antitumor agents, anticancer agents, prodrugs, lysosome destabilizers (e.g., chloroquine), alkylating agents, alkaloids, allosteric inhibitors, antifolic acid agents, anti-inflammatory agents, antibiotics, antibacterial agents, antifungal agents, antifibrotic agents, antiinfective agents, antiparasitic agents, antiviral agents, antimycobacterial agents, anticancer agents, antiprotozoal agents, antiviral agents, physiologically active peptides, steroid hormones, photosensitive substances, radiopharmaceuticals, antiprion agents, and any combination thereof.

72. The aforementioned antitumor agents include aromatase inhibitors; anti-estrogens; anti-androgens; gonadrelin agonists; topoisomerase I inhibitors; topoisomerase II inhibitors; microtubule inhibitors; alkylating agents; retinoids, carotenoids, or tocopherols; cyclooxygenase inhibitors; MMP inhibitors; mTOR inhibitors; antimetabolites; platinum compounds; methionine aminopeptidase inhibitors; bisphosphonates; antiproliferative antibodies; heparinase inhibitors; inhibitors of Ras oncogenic isoforms; telomerase inhibitors; proteo The composition according to claim 71, selected from the group consisting of asome inhibitors; Flt-3 inhibitors; Hsp90 inhibitors; kinesin spindle protein inhibitors; MEK inhibitors; PARP inhibitors; tyrosine kinase inhibitors; PI3K inhibitors; AKT inhibitors; EGFR inhibitors; antitumor antibiotics; nitrosoureas; compounds that target / reduce protein or lipid kinase activity; compounds that target / reduce protein or lipid phosphatase activity; any further anti-angiogenic compounds; and any combination thereof.

73. The composition according to claim 71 or 72, wherein the antitumor agent is selected from the group consisting of azacitidine, azathioprine, bleomycin, capecitabine, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, etoposide, fenretinide, fluorouracil, gemcitabine, herceptin, idarubicin, mechloretamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, tafluposide, teniposide, thioguanine, retinoic acid, barrubicin, vinblastine, vincristine, vindesine, vinorelbine, receptor tyrosine kinase inhibitors, and any combination thereof.

74. The composition according to 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 according to claim 74, wherein the tubulin inhibitor is selected from the group consisting of monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), maytansine, maytansinoid, meltansine (emtansine, DM1), labtansine (solabtansine, DM4), tubulinin, halichondrin (eribulin), cryptophysine, EG5 inhibitors, and any derivatives thereof.

76. The composition according to claim 74, wherein the DNA inhibitor is selected from the group consisting of alkylating agents, duocalmycin, duocalmycin DM (DMDM), calicheamycin, pyrrolobenzodiazepine (PDB), enediyne, unciaramycin, topoisomerase inhibitors, topotecan, camptothecin (CPT), exatecan, and any derivatives thereof.

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

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

79. The composition according to claim 78, wherein the endonuclease is a deoxyribonuclease (DNase), a nuclease (benzonase) of Serratia marcescens, a micrococcal nuclease (MNase), a transposase, a restriction enzyme, nuclease S1, nuclease P1, a sequence-specific endonuclease, or a sequence-nonspecific endonuclease.

80. The composition according to any one of claims 63 to 79, wherein the one or more DNA nucleases comprises 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 for treating or relieving a disease or disorder in a person who needs it, The step includes administering the composition according to claim 81 to the subject, Optionally, if the disease or disorder includes cancer, The method.

83. The method according to claim 82, further comprising the step of determining, before the administration step, whether the cancer has aneuploidy, DNA repair defects, and / or functionally impaired transcription factors selected from the group consisting of p53, NF-κb, AP-1, E2F1, and BRCA1.

84. The method according to claim 83, wherein the dysfunction of the transcription factor is caused by mutation, loss, and / or failure of expression of one or more genes.

85. The method according to 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, and optionally, the deficiency of the TP53 gene includes a mutation.

86. The method according to any one of claims 82 to 85, wherein the administration step includes at least one of intradermal administration, intramuscular administration, intraperitoneal administration, intravenous administration, subcutaneous administration, intranasal administration, epidural administration, oral administration, sublingual administration, intracerebral administration, vaginal administration, transdermal administration, rectal administration, administration by inhalation, local administration, or any combination thereof.

87. A method for introducing one or more anti-DNA antibodies into a cell, The step includes contacting the cells with one or more anti-DNA antibodies, The one or more anti-DNA antibodies include means for binding to extracellular DNA, 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 move into the cell. The method.

88. A method for delivering therapeutic substances into the interior of cells, The process includes contacting the cells with a therapeutic substance in which an anti-DNA antibody is covalently or noncovalently linked, One or more anti-DNA antibodies include means for binding to extracellular DNA, The anti-DNA antibody binds to the extracellular DNA on the surface of the cell and delivers the therapeutic substance into the cell. The method.

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

90. An improvement in a method for bringing antibodies into contact with cells, This includes bringing one or more anti-DNA antibodies into contact with cells, thereby causing the one or more anti-DNA antibodies to move internally into the cells. 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 move into the cell. said improvement.

91. Improvements in methods of treating subjects with cancer, The method includes administering to a subject a composition containing a therapeutic substance linked to an anti-DNA antibody. The therapeutic substance is delivered into the target cells, The delivery of the therapeutic substance is effective in treating the subject. said improvement.

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