Conjugate and methods of treatment

Abstract

The present disclosure relates to nuclear penetrating conjugates comprising anti-DNA binding proteins and methods of use thereof in applications such as cancer therapy.

Description

[0001] CONJUGATE AND METHODS OF TREATMENT

[0002] FIELD OF THE INVENTION

[0003] The present disclosure relates to nuclear penetrating conjugates comprising anti- DNA binding proteins and methods of use thereof in applications such as cancer therapy.

[0004] INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0005] PROVIDED AS A SEQUENCE LISTING XML FILE

[0006] A Sequence Listing is provided herewith as a Sequence Listing XML, “RICE-233WO_540629PCT_Sequence_listing.xml”, created on April 12, 2024 and having a size of 132,005 bytes. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety.

[0007] BACKGROUND OF THE INVENTION

[0008] Antinuclear antibodies (ANAs) are autoimmune markers of unknown function that assist clinicians in making a diagnosis in diseases such as systemic lupus erythematosus (SLE). A variety of ANAs penetrate live cell nuclei to bind nuclear antigens, and their impact on intranuclear processes such as DNA repair is hypothesized to contribute to initiation and / or propagation of immune dysregulation in SLE.

[0009] In contrast to ANAs, antibodies that bind to cell surface antigens are commonly taken up by endocytosis and subsequently degraded in lysosomes. This process has been exploited in the design of antibody-drug conjugates (ADCs) that bind tumor cell surface antigens such as HER2 and release cargo drugs upon breakage of chemical linkers during their degradation in lysosomes. Such ADCs, heralded as “magic bullets,” have improved outcomes for malignancies expressing the relevant surface antigens.

[0010] However, a limitation of this approach is the inapplicability of conventional ADCs to tumors (and other diseases) that lack specific surface targets. Accordingly, there remains a need in the art for conjugates that are not reliant on cell-surface targets.

[0011] SUMMARY OF THE INVENTION

[0012] It has been discovered that nuclear-penetrating anti-DNA binding proteins, such as DX3, can translocate cathepsins into the nucleus of the cell. Accordingly, the present disclosure underpins the concept of utilising nuclear penetrating drug conjugates comprising a cathepsin-cleavable linker. Such conjugates can localise to the nucleus where translocated nuclear cathepsins cleave the linker, resulting in release of the pay load within the nucleus. This is a significant advance in the art as it provides an i elegant approach for selective payload delivery to cell nuclei. Furthermore, the DNA binding aspect of the present disclosure avoids the limitations of current binding protein drug conjugates which are reliant on a cell-surface target.

[0013] Accordingly, the present disclosure provides a nuclear penetrating conjugate comprising a nuclear penetrating, anti-DNA binding protein attached to a payload via a cleavable linker, wherein the linker is capable of being cleaved by a cathepsin.

[0014] In an example, the cathepsin is a nuclear cathepsin. In an example, the cathepsin is cathepsin B (CatB) or cathepsin S (CatS). In an example, the cathepsin is CatB. In an example, the cathepsin is CatS.

[0015] In an example, the linker comprises a peptide unit. In an example, the linker comprises a dipeptide unit. In an example, the linker comprises a dipeptide unit selected from the group consisting of valine-citrulline; valine-alanine; and phenylalaninearginine. In an example, the linker comprises a valine-citrulline unit. In an example, the linker comprises a valine-alanine unit. In an example, the linker comprises a phenylalanine-arginine unit.

[0016] In an example, the linker comprises: a) a valine-citrulline unit and a pentafluorophenyl group; b) a valine-citrulline unit and a succinimide group; c) a valine-citrulline unit and a para aminobenzoic acid group; d) a valine-citrulline unit and a para aminobenzoic acid group and a pentafluorophenyl group; or e) a valine-citrulline unit, a para aminobenzoic acid group and a succinimide group.

[0017] In an example, the linker comprises a valine-citrulline unit and a pentafluorophenyl group. In an example, the linker comprises a valine-citrulline unit and a succinimide group. In an example, the linker comprises a valine-citrulline unit and a para aminobenzoic acid group. In an example, the linker comprises a valine-citrulline unit and a para aminobenzoic acid group and a pentafluorophenyl group. In an example, the linker comprises a valine-citrulline unit, a para aminobenzoic acid group and a succinimide group.

[0018] In an example, the binding protein is an autoantibody derived from a subject or an animal with an autoimmune disease, preferably wherein the autoantibody is derived from a subject with systemic lupus erythematous, or an animal model thereof.

[0019] In an example, the autoantibody is 3E10 or a humanised form thereof. In an example, the autoantibody is 3E10. In an example, autoantibody is a humanized form of In an example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 2, a CDR3 as shown in SEQ ID NO: 3 and a VL having a CDR 1 as shown in SEQ ID NO: 4, a CDR2 as shown in SEQ ID NO: 5 and a CDR3 as shown in SEQ ID NO: 6 or a humanized form thereof.

[0020] In an example, the binding protein comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 7 and a VL as shown in SEQ ID NO: 8 or a humanized form thereof.

[0021] In an example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 9 or SEQ ID NO: 10, a CDR2 as shown in SEQ ID NO: 11 or SEQ ID NO: 12, a CDR3 as shown in SEQ ID NO: 13 and a VL having a CDR1 as shown in SEQ ID NO: 14 or SEQ ID NO: 15, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH having a CDR1 as shown in SEQ ID NO: 9 or SEQ ID NO: 10, a CDR2 as shown in SEQ ID NO: 11 or SEQ ID NO: 12, a CDR3 as shown in SEQ ID NO: 13 and a VL having a CDR1 as shown in SEQ ID NO: 14 or SEQ ID NO: 15, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17.

[0022] In an example, the binding protein comprises a VH which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 18-21 and a VL as shown in any one of SEQ ID NOs: 22-25 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 18-21 and a VL as shown in any one of SEQ ID NOs: 22-25.

[0023] In an example, the binding protein is a di-scFv.

[0024] In an example, the payload is a gene editing agent. In an example, the payload is a gene editing agent selected from the group consisting of triplex-forming molecules, pseudocomplementary oligonucleotides, a CRISPR system, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and intron encoded meganucleases.

[0025] In an example, the payload is an oligonucleotide. In an example, the oligonucleotide is a siRNA, a mRNA, an aptamer, or an antisense oligonucleotide.

[0026] In an example, the payload is a drug. In an example, the drug is an anti-cancer drug, preferably a chemotherapeutic agent, a cytotoxic agent, and / or a radiosensitizer. In an example, the drug is a chemotherapeutic agent. In an example, the drug is a cytotoxic agent. In an example, the drug is a radiosensitizer.

[0027] In an example, the payload is a recombinant viral expression vector. In an example, the recombinant viral expression vector is a recombinant adeno-associated virus (AAV) vector, a recombinant adenovirus (AdV) vector, or a recombinant lentivirus (LV) vector.

[0028] In an example, the payload is a modulator of DNA methylation, a modulator of chromatin, or a modulator of DNA replication and repair, preferably wherein the payload is Lysine-specific histone demethylase 1 A (LSD 1 ), Enhancer of zeste homolog 2 (EZH2) histone-lysine-N-methyltransferase, DNA methyltransferase 1 (DNMT1), or a histone deacetylase (HD AC).

[0029] In an example, the payload is a cathepsin inhibitor, preferably a cathepsin B and / or cathepsin S inhibitor. In an example, the payload is a cathepsin B inhibitor. In an example, the payload is a cathepsin S inhibitor. In an example, the payload is an agent that inhibits cathepsin B and cathepsin S. In an example, the payload is an agent that inhibits cathepsin B or cathepsin S.

[0030] In an example, the payload is an inhibitor of a nucleoside transporter, preferably dipyridamole. In an example, the payload is an inhibitor of a nucleoside transporter. In an example, the payload is dipyridamole.

[0031] In an example, the disclosure provides a composition comprising a conjugate according to the disclosure and DNA. In an example, the composition comprises a conjugate according to the disclosure and DNA, wherein the payload of the conjugate is an oligonucleotide. In an example, the oligonucleotide a siRNA, a mRNA, an aptamer, or an antisense oligonucleotide.

[0032] In an example, the disclosure provides a pharmaceutical composition comprising the conjugate according to the disclosure and a pharmaceutically acceptable carrier. In an example, the disclosure provides a pharmaceutical composition comprising the conjugate according to the disclosure and DNA, and a pharmaceutically acceptable carrier.

[0033] The present disclosure also provides a method of delivering a payload to the nucleus of a cell, comprising contacting the cell with an effective amount of a nuclear penetrating conjugate comprising a nuclear penetrating, anti-DNA binding protein attached to a payload via a linker, wherein the linker is capable of being cleaved via cathepsin. In an example, the contacting step comprises administering the conjugate to a subject.

[0034] The present disclosure also provides a method of gene editing comprising, administering to a subject in need thereof an effective amount of a nuclear penetrating conjugate comprising a nuclear penetrating, anti-DNA binding protein attached to a payload via a linker, wherein the linker is capable of being cleaved via cathepsin, and wherein the payload is a gene editing agent selected from the group consisting of triplex - forming molecules, pseudocomplementary oligonucleotides, a CRISPR system, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and intron encoded meganucleases. In an example, the method comprises administering to the subject the composition comprising a conjugate according to the disclosure and DNA.

[0035] The present disclosure also provides a method of treating a disease associated with cellular accumulation of cathepsin, comprising administering to a subject in need thereof an effective amount of a nuclear penetrating conjugate comprising a nuclear penetrating, anti-DNA binding protein attached to a payload via a linker, wherein the linker is capable of being cleaved via cathepsin, and wherein the payload is a cathepsin inhibitor.

[0036] In an example, the cathepsin is cathepsin B.

[0037] In an example, the cathepsin inhibitor is a cathepsin B inhibitor. In an example, the cathepsin B inhibitor is a cystatin, a peptidyl aldehyde, an aziridinyl peptide, an epoxysuccinyl peptide, an epoxysuccinyl inhibitor, an aziridine, a nitrile, or a monoclonal antibody.

[0038] In an example, the disease is a neurological disease. In an example, the disease is Alzheimer’s disease, Parkinson’s disease, traumatic brain injury, or cerebral ischemia.

[0039] In an example, the disease is a cardiovascular disease. In an example, the cardiovascular disease is myocardial infarction, diabetes, diabetic cardiomyopathy, or stroke.

[0040] In an example, the disease is pancreatitis, liver disease, lysosomal storage disorders (LSDs), retinitis pigmentosa, Kawaski disease, or Sjogren syndrome.

[0041] The disclosure also provides a method of treating an autoimmune disease, comprising, administering to a subject in need thereof an effective amount of a nuclear penetrating conjugate comprising a nuclear penetrating, anti-DNA binding attached linked to a payload via a linker, wherein the linker is capable of being cleaved via cathepsin, and wherein the payload is an inhibitor of a nucleoside transporter.

[0042] In an example, the autoimmune disease is one or more of systemic lupus erythematosus (lupus or SLE), CNS, lupus, systemic sclerosis (scleroderma), Graves’ disease, myasthenia gravis, autoimmune hemolytic anemia, and pemphigus vulgaris, and additionally may contribute to the severity of disease in other autoimmune diseases such as rheumatoid arthritis, autoimmune thrombocytopenia, autoimmune atrophic gastritis of pernicious anemia, myasthenia gravis, Goodpasture’s syndrome, diabetes mellitus, multiple sclerosis, Hashimoto’s thyroiditis, Crohn’s disease, and Sjogren’s syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, POEMS syndrome, dermatomyositis, inclusion body myositis, inflammatory myopathies, vasculitis syndromes including but not limited to Churg-Strauss Syndrome, Wegener granulomatosis, Behcet’s disease, Buerger’s disease, Kawasaki disease, Takayasu’s arteritis, Henoch-Schonlein purpura, Giant cell arteritis, or polyarteritis nodosa.

[0043] The disclosure also provides a method of treating cancer, comprising, administering to a subject in need thereof an effective amount of a nuclear penetrating conjugate comprising a nuclear penetrating, anti-DNA binding protein attached to a payload via a linker, wherein the linker is capable of being cleaved via cathepsin, and wherein the payload is a chemotherapeutic agent, a cytotoxic agent, or a radiosensitiser.

[0044] In an example, the payload is olaparib, niraparib, or rucaparib. In an example, the payload is olaparib. In an example, the payload is niraparib. In an example, the payload is rucaparib.

[0045] In an example, methods of treatment according to the present disclosure further comprises the step of administering to the subject an effective amount of an inhibitor of a nucleoside transporter to reduce transcellular transport of the conjugate thus reducing nuclear delivery of the payload.

[0046] In an example, the inhibitor of the nucleoside transporter inhibits activity or expression of one or more of ENT1 , ENT2, ENT3, or ENT4, optionally at least or only ENT2. In an example, the inhibitor of the nucleoside transporter is dipyridamole or pharmaceutically acceptable salt thereof.

[0047] In an example, the conjugate and inhibitor of the nucleoside transporter are administered to a subject in need thereof by a parenteral, enteral, transdermal, or transmucosal route of administration. In an example, the subject is administered the conjugate separately or together with the inhibitor of the nucleoside transporter.

[0048] In an example, methods of treatment of the disclosure further comprise the step of administering to the subject an effective amount of a cathepsin inhibitor to reduce cleavage of the linker, thus reducing release of the payload.

[0049] In an example, the conjugate and cathepsin inhibitor are administered to a subject in need thereof by a parenteral, enteral, transdermal, or transmucosal route of administration. In an example, the subject is administered the conjugate separately or together with the cathepsin inhibitor.

[0050] In an example, the cathepsin inhibitor is a cathepsin B inhibitor. In an example, the cathepsin B inhibitor is a cystatin, peptidyl aldehyde, an aziridinyl peptide, an epoxysuccinyl peptide, an epoxysuccinyl inhibitor, an aziridine, a nitrile, or a monoclonal antibody. In an example, the cathepsin B inhibitor is an epoxysuccinyl inhibitor. In an example, the epoxysuccinyl inhibitor is E-64d, CA074 or CA074Me. In another example, the cathepsin B inhibitor is an epoxysuccinyl peptide. In an example, the epoxysuccinyl peptide is E-64.

[0051] The present disclosure also provides the use of a nuclear penetrating conjugate comprising a nuclear penetrating, anti-DNA binding protein attached to a payload via a linker in the manufacture of a medicament for gene editing, wherein the linker is capable of being cleaved via cathepsin, and wherein the pay load is a gene editing agent selected from the group consisting of triplex-forming molecules, pseudocomplementary oligonucleotides, a CRISPR system, zinc finger nucleases (ZFN), transcription activatorlike effector nucleases (TALEN), and intron encoded meganucleases.

[0052] The present disclosure also provides the use of a nuclear penetrating conjugate comprising a nuclear penetrating, anti-DNA binding protein attached to a payload via a linker in the manufacture of a medicament for treating a disease characterized by cellular accumulation of cathepsin, wherein the linker is capable of being cleaved via cathepsin, and wherein the payload is a cathepsin inhibitor.

[0053] The present disclosure also provides the use of a nuclear penetrating conjugate comprising a nuclear penetrating, anti-DNA binding protein attached to a payload via a linker in the manufacture of a medicament for treating an autoimmune disease, wherein the linker is capable of being cleaved via cathepsin, and wherein the payload is an inhibitor of a nucleoside transporter.

[0054] The present disclosure also provides the use of a nuclear penetrating conjugate comprising a nuclear penetrating, anti-DNA binding protein attached to a payload via a linker in the manufacture of a medicament for treating cancer, wherein the linker is capable of being cleaved via cathepsin, and wherein the payload is a chemotherapeutic agent, a cytotoxic agent, or a radiosensitiser.

[0055] In an example, methods and uses according to the present disclosure comprise a conjugate as disclosed herein, wherein the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 9 or SEQ ID NO: 10, a CDR2 as shown in SEQ ID NO: 11 or SEQ ID NO: 12, a CDR3 as shown in SEQ ID NO: 13 and a VL having a CDR1 as shown in SEQ ID NO: 14 or SEQ ID NO: 15, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH having a CDR1 as shown in SEQ ID NO: 9 or SEQ ID NO: 10, a CDR2 as shown in SEQ ID NO: 11 or SEQ ID NO: 12, a CDR3 as shown in SEQ ID NO: 13 and a VL having a CDR1 as shown in SEQ ID NO: 14 or SEQ ID NO: 15, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17.

[0056] In an example, the binding protein comprises a VH which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 18-21 and a VL as shown in any one of SEQ ID NOs: 22-25 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 18-21 and a VL as shown in any one of SEQ ID NOs: 22-25.

[0057] The present disclosure also provides a kit comprising the conjugate according to the disclosure, a composition according the disclosure comprising a conjugate and DNA, or a pharmaceutical composition according to the disclosure, and an inhibitor of a nucleoside transporter. In an example, the inhibitor of the nucleoside transporter is dipyridamole or pharmaceutically acceptable salt thereof.

[0058] The present disclosure also provides a method of treating neurodegeneration or neurological disease, comprising administering to subject a nuclear penetrating anti- DNA binding protein in an amount effective to attenuate nuclear cathepsin activity. In an example, the cathepsin is cathepsin B.

[0059] In an example, the neurodegeneration is Alzheimer’s disease, Parkinson’s disease, traumatic brain injury, cerebral ischemia, or aging.

[0060] In an example, the nuclear penetrating anti-DNA binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 2, a CDR3 as shown in SEQ ID NO: 3 and a VL having a CDR 1 as shown in SEQ ID NO: 4, a CDR2 as shown in SEQ ID NO: 5 and a CDR3 as shown in SEQ ID NO: 6 or a humanized form thereof.

[0061] In an example, the binding protein comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 7 and a VL as shown in SEQ ID NO: 8 or a humanized form thereof.

[0062] In an example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 9 or SEQ ID NO: 10, a CDR2 as shown in SEQ ID NO: 11 or SEQ ID NO: 12, a CDR3 as shown in SEQ ID NO: 13 and a VL having a CDR1 as shown in SEQ ID NO: 14 or SEQ ID NO: 15, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH having a CDR1 as shown in SEQ ID NO: 9 or SEQ ID NO: 10, a CDR2 as shown in SEQ ID NO: 11 or SEQ ID NO: 12, a CDR3 as shown in SEQ ID NO: 13 and a VL having a CDR1 as shown in SEQ ID NO: 14 or SEQ ID NO: 15, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17. In an example, the binding protein comprises a VH which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 18-21 and a VL as shown in any one of SEQ ID NOs: 22-25 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 18-21 and a VL as shown in any one of SEQ ID NOs: 22-25.

[0063] In an example, the binding protein is a di-scFv.

[0064] In an example, the binding protein is an intact IgG. In an example, the binding protein is a human IgGl.

[0065] In an example, the binding protein is an intact IgG or a di-scFv. In an example, the present disclosure provides the use of a nuclear penetrating anti-DNA binding protein in the manufacture of a medicament for treating neurodegeneration or a neurological disease.

[0066] BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0067] Figures 1A-1C. DX3 is a cell-penetrating ANA. (Fig. 1A) DX3 binds single- stranded DNA. SPR binding profiles of DX3 (titrated from 500 nM) to 30-mer DNA oligonucleotide show KD 113 nM. (Fig. IB) DX3 penetrates live cell nuclei. Single channel and merged images of DLD1 colon cancer cells treated with IgG control or DX3 immunostained to detect antibody uptake (anti-IgG) with DAPI nuclear counterstain are shown. DX3 penetrated live cell nuclei while IgG control showed minimal to no uptake into cells. Bars: 20 pm. (Fig. 1C) Representative images of DX3 penetration into U87 glioma cells and NHAs visualized by anti-IgG immunofluorescence are shown. Bars: 20 m.

[0068] Figures 2A-2C. Nuclear penetration by DX3 is linked with nucleoside salvage and lysosomal avoidance. (Fig. 2A, Fig. 2B) Nucleoside salvage facilitates DX3 nuclear penetration. Representative images of DX3 uptake into U87 glioma cell nuclei in the presence of control buffer, 100 pM nucleoside (adenosine) or 25 pM nucleoside transport inhibitor (dipyridamole) visualized by anti-IgG immunofluorescence are shown in (Fig. 2A). DX3 uptake was enhanced by addition of nucleoside and suppressed by nucleoside transport inhibition, as shown by quantification of fluorescence signals by ImageJ expressed relative to control in (Fig. 2B). **P<0101, ****P<0.0001, Tukey’s multiple comparisons test. (Fig. 2C) Representative immunofluorescence confocal microscopy images of DX3-treated U87 glioma cells show DX3 localizes exclusively into nuclei and avoids lysosomes. DX3 was detected by anti-IgG, lysosomes by anti-LAMPl, and nuclei by DAPI counterstain. Bars: 20 pm.

[0069] Figures 3A-3B. DX3 promotes CatB and CatS nuclear translocation in U87 glioma cells. (Fig. 3A) Representative immunofluorescence confocal microscopy images of CatB, CatK, CatL, and CatS in U87 cells treated with control buffer, IgG control, or DX3 are shown. (Fig. 3B) Anti-cathepsin immunofluorescence images of IgG control and DX3- treated U87 cells from (Fig. 3A) are merged with corresponding channels showing anti- IgG fluorescence signal and DAPI nuclear counterstain. IgG control did not penetrate cells and had no impact on cathepsin localization, while DX3 was taken into nuclei and induced nuclear translocation of CatB and Cats, but not CatK or CatL. Bars: 10 |im. Images are representative of three independent experiments.

[0070] Figures 4A-4B. DX3 promotes CatB and CatS nuclear translocation in NHAs. (Fig. 4A) Representative immunofluorescence confocal microscopy images of CatB, CatK, CatL, and CatS in NHAs treated with control buffer or DX3 are shown. (Fig 4B) Anti-cathepsin immunofluorescence images of DX3-treated NHAs from (Fig. 4A) are merged with corresponding channels showing anti-IgG fluorescence signal and DAPI nuclear counterstain. Consistent with the results in U87 cells in Figs. 3A-3B, DX3 was taken into nuclei and induced nuclear translocation of CatB and CatS, but not CatK or CatL. Bars: 10 pm. Images are representative of two independent experiments.

[0071] Figures 5A-5F. DX3 induces nuclear accumulation of pro-CatB and pro-CatS, activates CatS, and promotes BRCA1 loss. (Fig. 5 A, Fig. 5B) Nuclear extracts from U87 cells treated with 4 pM IgG control or DX3 analyzed by anti-cathepsin western blot show progressive accumulation of pro-CatB and pro-CatS (pro-forms identified by MW ~50 kDa). Blots are shown in (Fig. 5 A) and bands quantified by ImageJ and expressed relative to content at 15 min in (Fig. 5B). (Fig. 5C - Fig. 5F) DX3 activates CatS and promotes BRC A 1 loss. Total cell lysates of U87 cells treated with IgG control or DX3 were probed for active CatS (as distinguished from pro- form by molecular weight 24 kDa) and BRC Al, with GAPDH for loading control and over a time course. Representative blots are shown in (Fig. 5C, Fig. 5D), and active CatS and BRCA1 quantified by ImageJ in (Fig. 5E) and (Fig. 5F), respectively. DX3 was associated with an increase in active CatS and a reduction in BRCA1 by factors of 1.8+0.3 and 0.58+0.03 relative to IgG control, respectively (*P<0.05, ****P<0.0001, one-tailed student’s t-test).

[0072] Figures 6A-6D. DX3 is synthetically lethal to BRCA2-deficient tumors. (Fig. 6A - Fig. 6D) Tumor volumes and Kaplan-Meier plots of tumor tripling times in athymic nude mice with bilateral DLD1 flank tumors (left flank BRCA2-WT, right flank BRCA2- deficient) treated with buffer control or DX3 are shown. DX3 did not significantly impact BRCA2-WT tumor growth (P = ns, one-tailed student’s t-test) (Fig. 6A), with median tripling times of 9 days in mice treated with buffer or DX3 (P = ns, log-rank test) (Fig. 6B). In contrast, DX3 significantly suppressed the contralateral BRCA2-deficient tumors (*P<0.05, one-tailed student’s t-test) (Fig. 6C), with median tripling times of 9 days and not reached in mice treated with buffer or DX3, respectively (P<0.05, log-rank test) (Fig. 6D).

[0073] Figures 7A-7D. ANADC penetrates U87 glioma cells and induces CatB and CatS nuclear translocation. (Fig. 7A) Schematic illustrating a cathepsin-labile dipeptide VC linker joining MMAE to DX3 to form ANADC. (Fig. 7B) U87 cells treated with 4 pM IgG control, ADC control, unconjugated DX3, or ANADC were visualized by anti-lgG immunofluorescence. Representative images at 60 minutes after treatment show minimal to no uptake of IgG control and ADC control, while DX3 and ANADC showed significant penetration into cells. Compared to DX3, cells treated with ANADC showed morphologic changes and an IgG staining pattern consistent with DX3 escape from the nucleus into the cytoplasm, indicative of dissolution of the nuclear membrane caused by cell death. Bars: 10 pm. (Fig. 7C) ANADC is toxic to U87 glioma cells. Cells were treated with titrated doses of IgG control, ADC control, DX3, and ANADC, and surviving fractions determined by colony formation assay. ***P<0.001, student’s t- test, N=3. (Fig. 7D) ANADC induces CatB and CatS nuclear translocation. Merged immunofluorescence confocal microscopy images of ANADC-treated U87 glioma cells stained for IgG and cathepsin with DAPI nuclear counterstain are shown. CatB and CatS signal co-localized with DX3 and DAPI signals in the nucleus, while CatK and CatL signal remained cytoplasmic / lysosomal. Bars: 10 pm.

[0074] Figures 8A-8B. Determination of ANADC DAR. (Fig. 8A) ANADC HIC profile shows mean DAR 4.8, with four linked MMAE molecules the predominant species. (Fig. 8B) ANADC LC-MS profile shows mean DAR 4.5, consistent with the HIC results.

[0075] Figures 9A-9B. ANADC penetrates cells and progressively impacts morphologies. (Fig. 9 A) Brightfield microscopy images of U87 cells treated with IgG control, ADC control, DX3, and ANADC corresponding with the immunofluorescence images from Fig. 7B. (Fig 9B) U87 cells treated with 4 pM ANADC imaged by brightfield and anti-lgG fluorescence microscopy at the indicated timepoints after treatment show progressive loss of cell architecture and subsequent escape of DX3 signal from the nucleus into the cytoplasm, consistent with MMAE- induced toxicity. Bars: 20 pm.

[0076] Figure 10. ANADC induces CatB and CatS nuclear translocation in NHAs. ANADC induces CatB and CatS nuclear translocation. Merged immunofluorescence confocal microscopy images of ANADC-treated NHAs stained for IgG and cathepsin with DAPI nuclear counterstain are shown. CatB and CatS signal co-localized with DX3 and DAPI signals in the nucleus, while CatK and CatL signal remained cytoplasmic / lysosomal. Bars: 10 pm.

[0077] Figures 11A-11D. DX3 localizes to orthotopic brain tumors and facilitates prolongation of survival by ANADC in mice bearing brain tumors. (Fig. 11 A, Fig. 11B) DX3 preferentially localizes to brain tumors. Brains were taken from mice bearing intracranial U87 glioma tumors six hours after treatment with radiolabeled IgG control or DX3. Tumor and normal brain cortex and other components were separately analyzed by gamma counter to quantify antibody uptake. Antibody uptake in glioma compared to normal brain cortex is shown in (Fig. 11 A) and remaining normal brain regions in (Fig. 11B). Results are expressed as %ID / g. N=2. *P<0.05, **P<0.01, Tukey’s multiple comparisons test. (Fig. 11C) Systemic distribution of DX3 in mice with brain tumors. Tissues were taken from mice bearing intracranial U87 glioma tumors 96-hours after treatment with radiolabeled IgG control or DX3 and analyzed by gamma counter to quantify antibody uptake. Compared to IgG control, DX3 content was increased in blood and decreased in livers. The remaining tissues showed no statistically significant differences between IgG control and DX3 content. Results are expressed as %ID / g. N=3, *P<0.05, one-tailed t-test. (Fig. 1 ID) ANADC prolongs survival in an orthotopic glioma model. Mice bearing intracranial U 87 glioma tumors were treated with four weekly tail vein injections of control buffer (N=6), DX3 (10 mg / kg, N=6), or ANADC (10 mg / kg, N=5). Mice were followed for endpoints of neurologic changes or weight loss. Kaplan- Meier plots show median survivals of 49.5, 47, and 68 days associated with control buffer, DX3, and ANADC, respectively (P<0.01, log -rank test).

[0078] Figures 12A-12D. ANADC suppresses extracranial breast cancer tumor growth. (Fig. 12A) ANADC penetrates MCF7 breast cancer cells. Representative images of MCF7 cells treated with ADC control or ANADC and immunostained for IgG with DAPI counterstain are shown. Bars: 20 pm. (Fig. 12B - Fig. 12D) ANADC suppresses MCF7 tumor growth. Mice bearing MCF7 subcutaneous xenograft flank tumors with mean volume -150 mm3 were randomized to treatment with four weekly IP injections of control buffer (N=6), ADC control (10 mg / kg, N=6), unconjugated DX3 (10 mg / kg, N=6), or ANADC (10 mg / kg, N=6) once a week for four weeks (treatments on days 0, 7, 14, and 21 in the figures). ANADC showed the greatest inhibition of tumors compared to all controls, with mean tumor volumes one week after completion of treatment (day 28) of 216+45 and 960+85 mm3 in mice treated with ANADC or ADC control, respectively (**P<0.01, one-tailed student’s t-test) (Fig. 12B). Median tumor tripling times associated with buffer, DX3, or ADC control were 7, 8.5, and 10 days, respectively, compared to 45 days with DX3 vedotin (P<0.01, log-rank test). Kaplan-Meier plots are shown in (Fig. 12C). ANADC was well tolerated based on stability of mouse body weights (Fig. 12D).

[0079] Figure 13. Schematic illustrating the implications of ANA-induced cathepsin nuclear translocation. ANADC localizes to environments enriched in extracellular nucleosides and crosses live cell membranes through ENT2-mediated nucleoside salvage pathways. Inside the cell ANADC localizes into the nucleus and induces nuclear translocation by lysosomal cathepsin proteases CatB and CatS. Activated CatS subsequently promotes degradation of BRCA1 thereby inhibiting DNA repair and causing accumulation of DNA damage that is synthetically lethal to cells with pre-existing defects in the DNA damage response. Concurrently, CatB cleaves the dipeptide VC linker on ANADC to release the cytotoxic cargo drug MMAE.

[0080] Figure 14A-14G. DX3 induces CatB nuclear translocation. (Fig. 14A) U87 glioma cells treated for 15 minutes with 4 pM TgG control or DX3 were immunostained for CatB, IgG, with DAPI nuclear counterstain. Representative confocal immunofluorescence microscopy images are shown, including single channel and merged images. IgG control did not penetrate cells, but DX3 was taken into nuclei. CatB signal localized in the cytoplasm of cells treated with IgG control and in the nucleus of DX3-treated cells. Bars: 10 pm. Images of cells treated with control buffer and immunostained for additional cathepsins are in Fig. 15. (Fig. 14B) Nuclear overlap coefficients for CatB in U87 glioma cells treated with control buffer, 4 pM IgG control, or 4 pM DX3 were determined by ImageJ Colocalization Finder. DX3 induced a significant increase in CatB nuclear overlap compared to control buffer or IgG control. ****P<0.0001, two-tailed student’s t-test, N=3. (Fig. 14C and Fig. 14D) Nuclear contents isolated from U87 glioma cells treated for 0-6 hours with 4 pM IgG control or DX3 were analyzed by western blot probed for CatB, with Lamin Bl (~66 kDa) for loading control. DX3 caused a significant increase in nuclear content of the active form of CatB (~29 kDa). Representative western blot on nuclear extracts is shown in (Fig. 14C), and ImageJ quantification of CatB nuclear content relative to content in cells treated with IgG control at 6 hours is shown in (Fig. 14D) respectively. *P<0.05, two-tailed student’s t-test, N=3. (Fig. 14E) CatB activity is increased in nuclear extracts from DX3 -treated cells. Cleavage of a CatB fluorogenic substrate by nuclear extracts from U87 cells treated with IgG control (IgG nuclei), DX3 (DX3 nuclei), or DX3 + CA-074Me CatB inhibitor (DX3 nuclei + inhibitor) was measured by fluorescence plate reader, and results expressed relative to 0.4 ng reference purified CatB protein activity (CatB ref). **P<0.0001, Tukey’s multiple comparisons test, N=3. (Fig. 14F and Fig. 14G) Nuclear extracts of U87 cells exposed to DX3 in the presence of DMSO control or the CatB inhibitor C A-074Me were probed by western blot for DX3 content one hour or two days after application of DX3. Representative western blots are shown in (Fig. 14E) and ImageJ quantification of relative DX3 content in (Fig. 14D) respectively. ****P<0.0001, two-tailed student’s t-test, N=3.

[0081] Figure 15A-15C. Cathepsin localization in cells treated with control buffer, IgG control, or DX3. (Fig. 15A and 15B) U87 glioma cells treated for 15 minutes with control buffer or 4 pM IgG control or DX3 were immunostained for CatB, K, L, or S, IgG, with DAPI nuclear counterstain. Representative confocal immunofluorescence microscopy images are shown, including cathepsin single channel (Fig. 15 A) and merged images of DAPI, anti-IgG, and anti-cathepsin (Fig. 15B). Bars: 10 pm. (Fig. 15C) Cathepsin nuclear overlap coefficients. Nuclear overlap coefficients for CatK, L, and S in U87 glioma cells treated with IgG control or DX3 were determined by ImageJ Colocalization Finder. DX3 did not cause any significant changes compared to IgG control in nuclear signal for CatK or L and had only a small effect on CatS. **P<0.01, Tukey’s multiple comparisons test, N=3.

[0082] Figure 16A-16B. Cathepsin nuclear overlap coefficients. (Fig. 16A and 16B) Cytoplasmic contents isolated from U87 glioma cells treated for 0-6 hours with 4 pM IgG control or DX3 were analyzed by western blot probed for CatB, with tubulin for loading control. As shown in the main manuscript figures, DX3 caused a significant increase in nuclear content, and here a corresponding decrease in cytoplasmic content of the active form of CatB (~29 kDa) is shown. Representative western blot on nuclear extracts is shown in (Fig. 16A), and ImageJ quantification of CatB cytoplasmic content relative to content in cells treated with IgG control at 6 hours is shown in (Fig. 16B). ****P<0.0001, two-tailed student’s t-test, N=3.

[0083] Figure 17A-17H. DX3 induces nuclear accumulation of CatB in a panel of cancer cells. (Figure 17A-H) Representative immunofluorescence confocal microscopy images of CatB, DAPI and anti-IgG in a panel of cancer cells treated with 4 pM IgG control or DX3 for 24 hours are shown, along with corresponding nuclear overlap coefficients for CatB and DAPI signals determined by ImageJ. (Fig. 17A and 17B) ER+ MCF7 breast cancer cells. (Fig. 17A and 17B) Triple negative MDA-MB-231 breast cancer cells. (Fig. 17E and 17F) DLD1 colon cancer cells. (Fig. 17G and 17H) A549 lung adenocarcinoma cells. Bars: 20 irn. **P<0.01, ***P<0.001, ***P<0.0001, two-tailed student’s test, N=3.

[0084] Figure 18A-18B. DX3 induces nuclear accumulation in primary NHAs. (Fig. 18A and 18B) Representative immunofluorescence confocal microscopy images of NHAs treated with control buffer or DX3 immunostained for CatB, DAPI, IgG, and merged images are shown in (Fig. 18A), and nuclear overlap coefficients measured by ImageJ Colocalization Finder to determine overlap coefficient for CatB and DAPI channels in (Fig. 18B). Bars: 10 pm. ***P<0.001, two-tailed student’s test, N=3.

[0085] Figure 19A-19E. ANADC causes CatB nuclear translocation and CatB -dependent cytotoxicity. (Fig. 19A-C) ANADC induces CatB nuclear translocation. Immunofluorescence confocal microscopy images of ANADC-treated U87 glioma cells stained for IgG and CatB or CatL with DAPI nuclear counterstain show CatB (Fig. 19A) but not CatL (Fig. 19B) co-localizing with DX3 and DAPI in the nucleus. Bars: 10 pm. Nuclear overlap coefficients quantified by ImageJ Colocalization Finder confirmed ANADC induced nuclear translocation by CatB but not CatL ****P<0.0001, two-tailed student’s t-test, N=3 (Fig. 19C). (Fig. 19D) The dependence of ANADC cytotoxicity on CatB-mediated cleavage of the VC linker to release cargo drug was tested. U87 glioma cells preincubated with control buffer (DMSO) or 10 pM protease inhibitors (E64 Cys protease inhibitor, CA-074Me CatB specific inhibitor, or LY3000328 CatS specific inhibitor) were treated with control media or 4 pM ANADC ± inhibitors. Cells were monitored by brightfield microscopy with quantification of morphologic changes by ImageJ. The Cys protease inhibitor (which inhibits CatB and other Cys proteases) and the CatB specific inhibitor provided protection to the cells compared to control buffer or the CatS specific inhibitor, consistent with CatB-dependent cytotoxicity. ***P<0.001, Tukey’s multiple comparisons test, N=3. (Fig. 19E) ANADC is toxic to U87 glioma cells. Cells were treated with titrated doses of IgG control, ADC control, DX3, and ANADC, and surviving fractions determined by colony formation assay. ****P<0.0001, Tukey’s multiple comparisons test, N=3.

[0086] Figure 20A-20B. ANADC cytotoxicity is suppressed by CatB inhibition. (Fig. 20A) Representative brightfield microscopy images of U87 glioma cells 45 minutes after treatment with control media or 4 pM ANADC in the presence of control buffer, E64 Cys protease inhibitor, CA- 074Me CatB specific inhibitor, or LY3000328 CatS specific inhibitor are shown. Cell morphologies were rapidly lost after ANADC treatment in control buffer or with CatS inhibitor but were maintained in the presence of the broad spectrum Cys protease inhibitor or the specific CatB inhibitor, consistent with CatB- dependent cytotoxicity. Bars: 40 pm. (Fig. 20B) Confirmation of CatS inhibition. Nuclear extracts from cells treated with IgG control or DX3 ± CatS inhibitor as described in (Fig. 20A) were tested for CatS activity by assaying the nuclear extracts for cleavage of a CatS Anorogenic substrate measured by fluorescence plate reader. ****P<0.0001, two-tailed student’s t-test, N=3.

[0087] Figure 21A-21E. ANADC penetrates and is toxic to a panel of cancer cells. (Fig. 21 A) Representative immunofluorescence confocal microscopy images of DAPI and anti-IgG in a panel of cancer cells (ER+ MCF7 and triple negative MDA-MB-231 breast cancer, DLD1 colon cancer, and A549 lung adenocarcinoma) treated with 4 pM ANADC are shown. (Fig. 21B-E) Cells were treated with titrated doses of DX3 or ANADC and surviving fractions determined by colony formation assay. ****P<0.0001, two-tailed student’s t-test, N=3.

[0088] Figure 22A-22F. ANADC suppresses tumor growth in multiple subcutaneous xenograft models. (Fig. 22A and Fig. 22B) ANADC suppresses ER+ breast cancer tumor growth. Tumor volumes in mice bearing MCF7 subcutaneous xenograft flank tumors treated with four weekly IP injections of control buffer, ADC control (10 mg / kg), unconjugated DX3 (10 mg / kg), or ANADC (10 mg / kg) (N=6 per group) once a week for four weeks (treatments on days 0, 7, 14, and 21 in the figure) are shown in (Fig. 22A) and body mass measurements over the study in (Fig. 22B). ANADC showed the greatest inhibition of tumors (****P<0.0001, Tukey’s multiple comparison test) and was well tolerated. (Fig. 22C and Fig. 22D) ANADC suppresses triple negative breast cancer tumor growth in a dose-dependent manner. Tumor volumes in mice bearing MDAMB- 231 subcutaneous xenograft Rank tumors treated with four weekly IP injections of control buffer or ANADC at 1, 5, or 10 mg / kg (N=8 per group) once a week for four weeks (treatments on days 0, 7, 14, and 21 in the figure) are shown in (Fig. 22C) and body mass measurements over the study in (Fig. 22D). ANADC showed dose-dependent suppression of tumor growth (****P<0.0001, Tukey’s multiple comparison test) and was well-tolerated. (Fig. 22E and 22F) ANADC suppresses colon cancer tumor growth. Tumor volumes in mice bearing HT29 subcutaneous xenograft Hank tumors treated with four weekly IP injections of control buffer, unconjugated DX3 (10 mg / kg), or ANADC (10 mg / kg) (N=6 per group) once a week for four weeks (treatments on days 0, 7, 14, and 21 in the 1'igure) are shown in (E) and body mass measurements over the study in (Fig. 22F). ANADC significantly suppressed tumor growth (**P<0.01, Tukey’s multiple comparison test) and was well-tolerated. Figure 23A-23C. DX3 localizes to brain tumors and ANADC prolongs survival in an orthotopic glioma model. Representative IVIS images at day 29 (one day after completion of treatment are shown in (Fig. 23A). All treatments were well-tolerated based on stable body masses (Fig. 23B). (Fig. 23C) Schematic showing design of parallel brain tumor localization and survival studies in an orthotopic GBM model.

[0089] KEY TO SEQUENCE LISTING

[0090] SEQ ID NO I - 3E10 VH CDR1 SEQ ID NO 2 - 3E10 VH CDR2 SEQ ID NO 3 - 3E10 VH CDR3 SEQ ID NO 4 - 3E10 VL CDR1 SEQ ID NO 5 - 3E10 VL CDR2 SEQ ID NO 6 - 3E10 VL CDR3 SEQ ID NO 7 - 3E10 VH SEQ ID NO 8 - 3E10 VL SEQ ID NO 9 - VH CDR1 of DX1 / DX3 SEQ ID NO 10 - VH CDR1 variant of DX3 SEQ ID NO I I - VH CDR2 of DX1 / DX3 SEQ ID NO 12 - VH CDR2 variant of DX3 SEQ ID NO 13 - VH CDR3 of DX1 / DX3 SEQ ID NO 14 - VL CDR1 of DXl / DX3 SEQ ID NO 15 - VL CDR1 variant of DX3 SEQ ID NO 16 - VL CDR2 of DX1 / DX3 SEQ ID NO 17 - VL CDR3 of DX1 / DX3 SEQ ID NO 18 - VH of DX1 / DX3 SEQ ID NO 19 - VH of DX3 variant 1 SEQ ID NO 20 - VH of DX3 variant 2 SEQ ID NO 21 - VH of DX3 variant 3 SEQ ID NO 22 - VL of DX1 / DX3 SEQ ID NO 23 - VL of DX3 variant 1 SEQ ID NO 24 - VL of DX3 variant 2 SEQ ID NO 25 - VL of DX3 variant 3 SEQ ID NO 26 - Linker sequence 1 separating scFv molecules SEQ ID NO 27 - Linker sequence 2 separating scFv molecules SEQ ID NO 28 - Hinge sequence SEQ ID NO 29 - Signal sequence SEQ ID NO 30 - VH chain of 5C6 SEQ ID NO 31 - VH CDR1 of 5C6 SEQ ID NO 32 - VH CDR2 of 5C6 SEQ ID NO 33 - VH CDR3 of 5C6 SEQ ID NO 34 - VL of 5C6 SEQ ID NO 35 - VL CDR1 of 5C6 SEQ ID NO 36 - VL CDR2 of 5C6 SEQ ID NO 37 - VL CDR3 of 5C6 SEQ ID NO 38 - DX1 sequence SEQ ID NO 39 - DX3 sequence SEQ ID NO 40 - VH CDR1 KABAT SEQ ID NO 41 - VH CDR2 of variants 2 - 4, 6 - 8, 10 - 12 KABAT SEQ ID NO 42 - VH CDR2 of variants 13 - 19 KABAT SEQ ID NO 43 - VH CDR3 KABAT SEQ ID NO 44 - VL CDR1 of variants 2 - 4, 6 - 8, 10 - 12 KABAT SEQ ID NO 45 - VL CDR1 of variants 13 - 19 KABAT SEQ ID NO 46 - VL CDR2 KABAT SEQ ID NO 47 - VL CDR3 KABAT SEQ ID NO 48 - VH CDR1 IMGT SEQ ID NO 49 - VH CDR2 of variants 2 - 4, 6 - 8, 10 - 12 IMGT SEQ ID NO 50 - VH CDR2 of variants 13 - 19 IMGT SEQ ID NO 51 - VH CDR3 IMGT SEQ ID NO 52 - VL CDR1 of variants 2 - 4, 6 - 8, 10 - 12) IMGT SEQ ID NO 53 - VL CDR1 of variants 13 - 19 IMGT SEQ ID NO 54 - VL CDR2 IMGT SEQ ID NO 55 - VL CDR3 IMGT SEQ ID NO 56 - VH of variants 2, 6 and 10 SEQ ID NO 57 - VH of variants 3, 7 and 11 SEQ ID NO 58 - VH of variants 4, 8 and 12 SEQ ID NO 59 - VH of variants 6 and 10 SEQ ID NO 60 - VH of variants 13, 16 and 19 SEQ ID NO 61 VH of variants 14 and 17 SEQ ID NO 62 - VH of variants 15 and 18 SEQ ID NO 63 - VL of variants 2, 3 and 4 SEQ ID NO 64 - VL of variants 6, 7 and 8 SEQ ID NO 65 - VL of variants 10, 11 and 12 SEQ ID NO 66 - VL of variants 13, 14 and 15 SEQ ID NO 67 - VL of variants 16, 17 and 18 SEQ ID NO 68 VL of variant 19 SEQ ID NO 69 - Variant 2 SEQ ID NO 70 - Variant 3 SEQ ID NO 71 - Variant 4 SEQ ID NO 72 - Variant 6 SEQ ID NO 73 - Variant 7 SEQ ID NO 74 - Variant 8 SEQ ID NO 75 - Variant 10 SEQ ID NO 76 - Variant 11 SEQ ID NO 77 - Variant 12 SEQ ID NO 78 - Variant 13 SEQ ID NO 79 - Variant 14 SEQ ID NO 80 - Variant 15 SEQ ID NO 81 - Variant 16 SEQ ID NO 82 - Variant 17 SEQ ID NO 83 - Variant 18 SEQ ID NO 84 - Variant 19 SEQ ID NO 85 - VH CDR2 variant A of 3E10 SEQ ID NO: 2 SEQ ID NO 86 - VL CDR1 variant A of 3E10 SEQ ID NO: 4 SEQ ID NO 87 - VL CDR2 variant A of 3E10 SEQ ID NO: 5 SEQ ID NO 88 - VH CDR1 variant A of 3E10 SEQ ID NO: 1 SEQ ID NO 89 - VH-CDR2 variant B of 3E10 SEQ ID NO: 2 SEQ ID NO 90 - VL-CDR1 variant B of 3E10 SEQ ID NO: 1 SEQ ID NO 91 - VL-CDR2 variant B of 3E10 SEQ ID NO: 5 SEQ ID NO 92 - VH-CDR1 variant C of 3E10 SEQ ID NO: 1 SEQ ID NO 93 - VH-CDR1 variant D of 3E10 SEQ ID NO: 1 SEQ ID NO 94 - VH-CDR1 variant E of 3E10 SEQ ID NO: 1 SEQ ID NO 95 - VH-CDR1 variant F of 3E10 SEQ ID NO: 1 SEQ ID NO 96 - VH-CDR1 variant G of 3E10 SEQ ID NO: 1 SEQ ID NO 97 - VH-CDR2 variant C of 3E10 SEQ ID NO: 2 SEQ ID NO 98 - VH-CDR2 variant D of 3E10 SEQ ID NO: 2 SEQ ID NO 99 - VH-CDR2 variant E of 3E10 SEQ ID NO: 2 SEQ ID NO 100 - VH-CDR3 variant A of 3E10 SEQ ID NO: 3 SEQ ID NO 101 - VH-CDR3 variant B of 3E10 SEQ ID NO: 3 SEQ ID NO 102 - VH-CDR3 variant C of 3E10 SEQ ID NO: 3 SEQ ID NO 103 - VL-CDR1 variant C of 3E10 SEQ ID NO: 4 SEQ ID NO 104 - VL-CDR1 variant D of 3E10 SEQ ID NO: 4 SEQ ID NO 105 - VL-CDR1 variant E of 3E10 SEQ ID NO: 4 SEQ ID NO 106 - VL-CDR1 variant F of 3E10 SEQ ID NO: 4 SEQ ID NO 107 - VL-CDR1 variant G of 3E10 SEQ ID NO: 4 SEQ ID NO 108 - VL-CDR1 variant F of 3E10 SEQ ID NO: 4 SEQ ID NO 109 - VL-CDR2 variant C of 3EI0 SEQ ID NO: 5 SEQ ID NO 110 - VL-CDR3 variant A of 3E10 SEQ ID NO: 6 SEQ ID NO 111 - VL-CDR3 variant B of 3E10 SEQ ID NO: 6 SEQ ID NO 112 - VL-CDR3 variant C of 3E10 SEQ ID NO: 6 SEQ ID NO 113 - VL-CDR3 variant D of 3E10 SEQ ID NO: 6 SEQ ID NO 114 - VL-CDR3 variant E of 3E10 SEQ ID NO: 6 SEQ ID NO 115 - VL-CDR3 variant F of 3E10 SEQ ID NO: 6 SEQ ID NO 116 - VH-CDR1 variant H of 3E10 SEQ ID NO: 1 SEQ ID NO 117 - VH-CDR2 variant F of 3E10 SEQ ID NO: 2 SEQ ID NO 118 - VH-CDR3 variant D of 3E10 SEQ ID NO: 3 SEQ ID NO 119 - VL-CDR1 variant G of 3E10 SEQ ID NO: 4 SEQ ID NO 120 - VL-CDR2 variant D of 3E10 SEQ ID NO: 5 SEQ ID NO 121 - VL-CDR3 variant G of 3E10 SEQ ID NO: 6

[0091] DETAILED DESCRIPTION OF THE INVENTION

[0092] General Techniques and Selected Definitions

[0093] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., molecular biology, biochemistry, antibodies, antibody fragments such as single chain fragment variable and clinical studies).

[0094] The term “culture conditions” is used in the context of the present disclosure to refer to an assessment of cells in culture. In certain examples, it may be necessary to confirm structure and / or function of a conjugate described herein under culture conditions. For example, a population of cells (i.e. a cell culture) may be contacted with a conjugate of the disclosure to determine whether the conjugate can penetrate cell nuclei and be processed by a nuclear cathepsin to release its payload.

[0095] The term “conjugate” is used in the context of the present disclosure to refer to a construct comprising a targeting molecule (e.g. a binding protein) that is attached to a payload via a linker. Such constructs are also known as “immunoconjugates”. Unless otherwise specified, the term “conjugate” as used herein can be understood to mean an “immunoconjugate” (i.e. comprising a binding protein). Accordingly, the conjugate is defined by a generic formula: P-L-B, wherein P = payload, L = linker, and B = binding protein (e.g., a nuclear penetration anti-DNA binding protein). Conjugates can also be defined by the generic formula in reverse order: B-L-P. In an example, the conjugate is an antibody-drug conjugate. Accordingly, the term “conjugated” is used in the context of the present disclosure to refer to binding proteins of the present disclosure that are conjugated to a payload, e.g., therapeutic agent or a diagnostic agent.

[0096] “Cathepsins” are endopeptidases found in most cells, which takes part in cell autolysis and self-digestion of tissues. Based on their structure and catalytic type, cathepsins are classified into serine (cathepsins A and G), aspartic (cathepsins D and E), and cysteinecathepsins (cathepsins B, C, F, H, K, L, O, S, V, W, andX). Some lysosomal cathepsin proteases are released into the cytoplasm and translocate into the nucleus and are implicated in mechanisms of nucleocytoplasmic shuttling and regulation of DNA repair proteins such as BRCA1. In the context of the present disclosure, the term “nuclear cathepsins” is used to refer to cathepsins that are present in the nucleus of cells. Such cathepsins are capable of cleaving linkers described herein. In an example, the nuclear cathepsin may have translocated to the nucleus from the cytoplasm (e.g. a lysosome). For example, a conjugate of the disclosure may be associated (or direct) translocation of a cathepsin to the nucleus, with the translocation leading to the eventual cleavage of the payload from the conjugate in the nucleus.

[0097] The term “linker”, as used in the context of nuclear penetrating conjugates disclosed herein, refers to a chemical moiety that attaches a payload to a binding protein. In an example, the “linker” refers to a bifunctional or multifunctional chemical moiety that can be used to covalently link one or more payloads to a binding protein (e.g. an antibody) to form a conjugate of the present disclosure. As would be appreciated by those of skill in the art, suitable linkers generally do not substantively interfere with the targeting function of the delivery vehicle they are attached to (e.g. a binding protein of the disclosure). Such linkers may be referred to as a “payload linker” because they link a payload to a conjugate disclosed herein.

[0098] In an example, suitable linkers allow the biological activity of a payload to be realised once they are cleaved. For example, in the context of the present disclosure, a suitable linker can be cleaved in the nucleus. Accordingly, in an example, the linker is a cleavable linker. Cleavable linkers are stable in circulation, but allow for the release of the payload following cleavage via proteases or other stimuli that are present in the target environment. In an example, the linker can be cleaved by a specific enzyme, in particular, a cathepsin such as a nuclear cathepsin and / or another cathepsin that has translocated to the nucleus from a lysosome.

[0099] In an example, the linker is capable of being cleaved by a cathepsin. The term “capable” is used in this context to define linkers that can be cleaved by a cathepsin as well as another mechanism such as an alternative protease. In another example, the linker is specifically cleaved by a cathepsin. “Specifically cleaved” in this context refers to a linker that is preferentially cleaved by a particular protease, e.g. a cathepsin. Accordingly, a linker that is specifically cleaved by a cathepsin, is preferentially cleaved by the cathepsin even when other proteases are present. In one example, the linker is cleaved more frequently, more rapidly, with greater duration and / or with greater affinity by a cathepsin than it does with alternative proteases. It is also understood by reading this definition that, for example, the linker that is specifically cleaved by cathepsin may or may not be specifically cleaved by another protease. As such, “specifically cleaved” does not necessarily require exclusive cleavage or non-detectable cleavage by another protease. Methods for determining specific cleavage will be apparent to the skilled person. For example, a linker of the disclosure may be contacted with a cathepsin or alternative protease. Cleavage of the linker by cathepsin or alternative protease is then determined and the linker that is preferentially cleaved by cathepsin rather than the alternative protease is considered to be specifically cleaved by the cathepsin. In another example, specific cleavage of a linker disclosed herein is determined by measuring whether a conjugate of the disclosure releases its payload in the nucleus of a cell.

[0100] Examples of cathepsin-cleavable linkers include, but are not limited to, peptide linkers (e.g. dipeptide linkers or tripeptide linkers) containing valine-citrulline (Val-Cit), phenylalanine-lysine (Phe-Lys), or valine-alanine (Vai-Ala) sequences. In an example, the linker is cleaved by cysteine cathepsins, in particular cathepsin B or cathepsin S. Further examples of suitable linkers are disclosed herein.

[0101] In certain examples describing binding proteins of the disclosure, the term “linker” is used to describe a molecule that attaches two associated polypeptide chains, for example a variable heavy (VH) and a variable light (VL) chain or two binding proteins such as a pair of scFVs. Examples of such constructs include polypeptide chains comprising the structure VL-X-VH, VH- -VL or scFv-X-scFv. In the context of structures VL-X-VH, VH-X-VL, X is a linker comprising insufficient residues to permit the VH and VL in a single polypeptide chain to associate (or form an Fv). Such linkers may be referred to as a “binding protein component linker” because they join (or form) structural components of a binding protein disclosed, such as a di-scFV.

[0102] The term "cell penetrating” is used in the context of the present disclosure to refer to an anti-DNA binding protein that is transported into the cytoplasm of living mammalian cells and, preferably, binds DNA (e.g., single-stranded and / or doublestranded DNA). In an example, cell penetrating binding proteins also penetrate cell nuclei. In an example, cell penetrating binding proteins enter the cell via a nucleoside transporter pathway, for example the ENT2 pathway. In an example, cell penetrating binding proteins enter the cell independently of receptor-mediated endocytosis. In an example, cell penetrating binding proteins enter the cell independently of lysosomes.

[0103] “Nuclear-penetrating” binding proteins and conjugates comprising the same are binding proteins and conjugates that localise in cell nuclei. Such binding proteins are distinguished from antibodies and other cell penetrating peptides that can penetrate into cells but remain sequestered in the cytoplasm.

[0104] The term “anti-DNA binding protein” is used in the context of the present disclosure to refer to binding proteins that are capable of binding DNA.

[0105] The term “binding protein” is used in the context of the present disclosure to refer to immunoglobulin molecules, preferably human or humanised immunoglobulin molecules, immunologically reactive with a particular antigen and includes both polyclonal and monoclonal antibodies. The term “binding protein” also includes antigen binding forms of antibodies, including fragments with antigen-binding capability (e.g., Fab', F(ab')2, Fab, Fv and rlgG as discussed in Pierce Catalogue and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3rdEd., W.H. Freeman & Co., New York (1998). The term is also used to refer to recombinant single chain Fv fragments (scFv) as well as divalent (di-scFv) and trivalent (tri-scFV) forms thereof. The term antibody also includes diabodies, triabodies, and tetrabodies.

[0106] The term binding protein as used herein encompasses binding proteins which comprise an antibody such as a bi-specific molecule. For example, a binding protein may comprise an above referenced immunoglobulin such as an antibody and an above referenced fragment such as an Fv.

[0107] An “antigen binding fragment” of an antibody comprises one or more variable regions of an intact antibody. Examples of antibody fragments include Fab, Fab’, F(ab’)2 and Fv fragments; diabodies; linear antibodies and single-chain antibody molecules formed from antibody fragments. For example, the term antigen binding fragment may be used to refer to recombinant single chain Fv fragments (scFv) as well as divalent (di- scFv) and trivalent (tri-scFV) forms thereof. In an example, the binding protein is an antigen binding fragment. Such fragments can be produced via various methods known in the art.

[0108] The term “immunoglobulin” will be understood to include binding proteins of the disclosure, such as anti-DNA binding proteins, which comprise an immunoglobulin domain. Exemplary immunoglobulins are antibodies. Additional proteins encompassed by the term “immunoglobulin” include domain antibodies, camelid antibodies and antibodies from cartilaginous fish (i.e., immunoglobulin new antigen receptors (IgNARs)). Generally, camelid antibodies and IgNARs comprise a VH, however lack a VL and are often referred to as heavy chain immunoglobulins.

[0109] The terms “full-length antibody”, “intact antibody” or “whole antibody” are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antigen binding fragment of an antibody. Specifically, whole antibodies include those with heavy and light chains. In an example, whole antibodies include an Fc region. The constant domains may be wild-type sequence constant domains (e.g., human wild-type sequence constant domains) or amino acid sequence variants thereof. In an example, the binding protein is an antibody. In an example, the antibody is an IgG.

[0110] As used herein, “variable region” refers to the portions of the light and / or heavy chains of an antibody as defined herein that specifically binds to an antigen and, for example, includes amino acid sequences of CDRs; i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). For example, the variable region comprises three or four FRs (e.g., FR1, FR2, FR3 and optionally FR4) together with three CDRs. VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.

[0111] As used herein, the term “complementarity determining regions” (syn. CDRs; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable region the presence of which are major contributors to specific antigen binding. Each variable region typically has three CDR regions identified as CDR1, CDR2 and CDR3. In one example, the amino acid positions assigned to CDRs and FRs are defined according to Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as “the Kabat numbering system” or “Kabat”.

[0112] Other conventions that include corrections or alternate numbering systems for variable domains include IMGT (Lefranc, el al. (2003), Dev Comp Immunol 27: 55- 77), Chothia (Chothia C, Lesk AM (1987), J Mai Biol 196: 901-917; Chothia, et al. (1989), Nature 342: 877-883) and AHo (Honegger A, Pliickthun A (2001) J Mol Biol 309: 657- 670). For convenience, examples of antibodies of the present disclosure may also be labelled according to IMGT.

[0113] “Framework regions” (Syn. FR) are those variable domain residues other than the CDR residues.

[0114] The term “constant region” as used herein, refers to a portion of heavy chain or light chain of an antibody other than the variable region. In a heavy chain, the constant region generally comprises a plurality of constant domains and a hinge region, e.g., a IgG constant region comprises the following linked components, a constant heavy CHI, a linker, a CH2 and a CH3. In a heavy chain, a constant region comprises a Fc. In a light chain, a constant region generally comprise one constant domain (a CL1). The term “fragment crystalizable” or “Fc” or “Fc region” or “Fc portion” (which can be used interchangeably herein) refers to a region of an antibody comprising at least one constant domain and which is generally (though not necessarily) glycosylated and which is capable of binding to one or more Fc receptors and / or components of the complement cascade. The heavy chain constant region can be selected from any of the five isotypes: a, 6, e, y, or p. Exemplary heavy chain constant regions are gamma 1 (IgGl), gamma 2 (IgG2) and gamma 3 (IgG3), or hybrids thereof.

[0115] A “constant domain” is a domain in an antibody the sequence of which is highly similar in antibodies / antibodies of the same type, e.g., IgG or IgM or IgE. A constant region of an antibody generally comprises a plurality of constant domains, e.g., the constant region of y, a or 5 heavy chain comprises two constant domains.

[0116] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill of those practicing in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0117] As used herein, the term “binds” in reference to the interaction of a binding protein and an antigen means that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen. For example, a binding protein recognizes and binds to a specific antigen structure rather than to antigens generally. For example, if a binding protein binds to epitope "A", the presence of a molecule containing epitope “A” (or free, unlabelled “A”), in a reaction containing labelled “A” and the binding protein, will reduce the amount of labelled “A” bound to the binding protein.

[0118] As used herein, the term “specifically binds” shall be taken to mean that the binding interaction between the binding protein and DNA is dependent on detection of the DNA by the binding protein. Accordingly, the binding protein preferentially binds or recognizes DNA even when present in a mixture of other molecules or organisms.

[0119] In one example, the binding protein reacts or associates more frequently, more rapidly, with greater duration and / or with greater affinity with DNA than it does with alternative antigens or cells. It is also understood by reading this definition that, for example, the binding protein that specifically binds to DNA may or may not specifically bind to a second antigen. As such, “specific binding” does not necessarily require exclusive binding or non-detectable binding of another antigen. The term “specifically binds” can be used interchangeably with “selectively binds” herein. Generally, reference herein to binding means specific binding, and each term shall be understood to provide explicit support for the other term. Methods for determining specific binding will be apparent to the skilled person. For example, a binding protein of the disclosure is contacted with DNA or an alternative antigen. Binding of the binding protein to DNA or alternative antigen is then determined and the binding protein that binds as set out above to the DNA rather than the alternative antigen is considered to specifically bind to DNA.

[0120] Binding proteins according to the present disclosure and compositions comprising the same can be administered to a subject to treat various indications such as inflammatory diseases. Terms such as “subject”, “patient” or “individual” are terms that can, in context, be used interchangeably in the present disclosure. In an example, the subject is a mammal. The mammal may be a companion animal such as a dog or cat, or a livestock animal such as a horse or cow. In one example, the subject is a human. For example, the subject can be an adult. In another example, the subject can be a child.

[0121] As used herein, the term “treatment” refers to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. An individual is successfully “treated”, for example, if one or more symptoms associated with a disease are mitigated or eliminated. In an example, treatment is characterised by a reduction of inflammatory markers in a subject. In addition, the term “treatment” includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; prophylactic treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0122] As used herein, the term “prevention” includes providing prophylaxis with respect to occurrence or recurrence of a disease in an individual. An individual may be predisposed to or at risk of developing the disease or disease relapse but has not yet been diagnosed with the disease or the relapse. In an example, methods of the present disclosure inhibit disease progression or disease complication in a subject.

[0123] An “effective amount” refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic, prophylactic, diagnostic or otherwise informative result. An effective amount can be provided in one or more administrations. In some examples of the present disclosure, the term “effective amount” is meant an amount necessary to effect treatment of a disease or condition described below. The effective amount may vary according to the disease or condition to be treated and also according to the weight, age, racial background, sex, health and / or physical condition and other factors relevant to the subject being treated. Typically, the effective amount will fall within a relatively broad range (e.g. a “dosage” range) that can be determined through routine trial and experimentation by a medical practitioner. The effective amount can be administered in a single dose or in a dose repeated once or several times over a treatment period. It is understood that the specific dose level for any particular patient depends upon a variety of factors including the activity of the specific antibody employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

[0124] A “therapeutically effective amount” is at least the minimum concentration required to effect a measurable improvement of a particular disorder (e.g. inflammatory disease). A therapeutically effective amount herein may also vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the binding protein are outweighed by the therapeutically beneficial effects. In the case of disease, the therapeutically effective amount of the conjugate or binding protein of the disclosure may inhibit (i.e., slow to some extent and, in some examples, stop) disease symptoms, disease progression; and / or relieve to some extent one or more of the symptoms associated with the disease being treated. For diseases disclosed herein, efficacy in vivo can, for example, be measured by assessing the duration of survival, time to disease progression (TTP), the response rates (RR), duration of response, and / or quality of life.

[0125] Conjugates

[0126] The present disclosure relates to nuclear penetrating conjugates, comprising a nuclear-penetrating anti-DNA binding protein attached to a payload via a cleavable linker, wherein the linker is capable of being cleaved by a cathepsin. In an example, the conjugate is an antibody-drug conjugate. In this instance, the binding protein is an antibody.

[0127] Methods of preparing conjugates according to the present disclosure are disclosed herein and are known in the art. Methods for preparing conjugates are described, for example, in Yao et al., Int J Mol Sci. 2016; 17(2): 194; Jackson. Org. Process Res. Dev. 2016, 20(5): 852-866; Hutchinson et al., Chapter 40 - Process Development and Manufacturing of Antibody-Drug Conjugates, Biopharmaceutical Processing, Elsevier, 2018, Pages 813-836, ISBN 9780081006238.

[0128] Binding proteins can be conjugated to payloads using various approaches. The most common methods utilize lysine or cysteine residues present in the binding protein. Other conjugating platforms are known in the art, e.g. THIOMAB drug conjugates, which utilize site-specific conjugation. These can be divided in two groups: those that utilize antibody engineering to introduce new conjugation sites, and those that conjugate via sites that are native to the antibody.

[0129] Lysine-directed ADCs are conjugated via native lysine residues on the antibody. The large number of lysines available for conjugation, typically upwards of 40 separate sites in a given antibody. A lysine-directed ADC with an average Drug-to-antibody ratio (DAR) between 3 and 4 is comprised of individual antibodies with a distribution of anywhere between 0-9 drugs per molecule (Ab). Lysine directed conjugations typically involve N-hydroxysuccinimide (NHS) esters or imidoesters.

[0130] Interchain-cysteine ADCs are conjugated via native antibody cysteines that are generated by partial reduction of interchain disulfide bonds. Typically, a predefined level reductant is used in order to reduce only a portion of the interchain disulfides present, which results in a heterogeneous conjugate comprised of 0, 2, 4, 6 and 8-DAR forms. A typical target average DAR is between 3 and 4. Conjugation reactions targeting antibody cysteines (i.e., free thiols) are most often performed using maleimide linker chemistry to form thio-ether bonds, but can alternatively be targeted using pyridyldisulfide, iodoacetamide, bromoacetamide or other thiol-reactive chemistries. Non-limiting examples of reducing agents include tris(2-carboxyethyl) phosphine (TCEP) and dithiothreitol (DTT).

[0131] Conjugates of the disclosure can be prepared by several routes employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of a nucleophilic group of an antibody with a bivalent linker reagent to form B-L via a covalent bond, followed by reaction with a payload moiety P; and (2) reaction of a nucleophilic group of a drug moiety with a bivalent linker reagent, to form P-L, via a covalent bond, followed by reaction with a nucleophilic group of an antibody or binding protein. Exemplary methods for preparing a conjugate via the latter route are described, for example, in US 7498298.

[0132] Nucleophilic groups on antibodies include: (i) N-terminal amine groups, (ii) side chain amine groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; and (iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain antibodies have reducible interchain disulfides, i.e. cysteine bridges. Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol) or tricarbonylethylphosphine (TCEP), such that the antibody is fully or partially reduced. Each cysteine bridge will thus form, theoretically, two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies through modification of lysine residues, e.g., by reacting lysine residues with 2-iminothiolane (Traut's reagent), resulting in conversion of an amine into a thiol. Reactive thiol groups may also be introduced into an antibody by introducing one, two, three, four, or more cysteine residues (e.g., by preparing variant antibodies comprising one or more non-native cysteine amino acid residues).

[0133] Conjugates according to the disclosure can also be produced by reaction between an electrophilic group on an antibody, such as an aldehyde or ketone carbonyl group, with a nucleophilic group on a linker reagent or payload. Useful nucleophilic groups on a linker reagent include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. In an example, an antibody is modified to introduce electrophilic moieties that are capable of reacting with nucleophilic substituents on the linker reagent or pay load. In another example, the sugars of glycosylated antibodies may be oxidized, e.g. with periodate oxidizing reagents, to form aldehyde or ketone groups which may react with the amine group of linker reagents or payload moieties. The resulting imine Schiff base groups may form a stable linkage, or may be reduced, e.g. by borohydride reagents to form stable amine linkages. In one example, reaction of the carbohydrate portion of a glycosylated antibody with either galactose oxidase or sodium meta-periodate may yield carbonyl (aldehyde and ketone) groups in the antibody that can react with appropriate groups on the payload (Hermanson, Bioconjugate Techniques). In another example, antibodies containing N-terminal serine or threonine residues can react with sodium meta-periodate, resulting in production of an aldehyde in place of the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146; US 5362852 ). Such an aldehyde can be reacted with a payload moiety or linker nucleophile.

[0134] Exemplary nucleophilic groups on a payload moiety include, amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (hi) aldehydes, ketones, carboxyl, and maleimide groups.

[0135] Methods for preparing conjugates can be broken down into three main steps: antibody functionalisation, conjugation reaction, and purification and formulation.

[0136] Antibody functionalization: The antibody is prepared for conjugation by generating the required number of reactive sites per antibody, most often by reduction, reduction / reoxidation or modification with a linker. Additional adjustment steps may also be performed in order to achieve the conditions required for downstream conjugation reaction, potentially including changes to the concentration, pH, temperature, solvent level, or buffer composition of the pool.

[0137] Conjugation reaction: The payload (e.g. a small -molecule drug) is solubilized in organic solvent and added to the antibody solution to form the conjugate. Typically, the payload is added in excess to the antibody reactive groups in order to ensure complete conjugation. Depending on the conjugation reaction chemistry, a quenching step may be performed following the conjugation reaction. Binding proteins can be conjugated to payloads by using thiols, amines, alcohols, aldehydes and azides.

[0138] Purification and formulation: The conjugate is purified to remove excess payload, process-related impurities, and product variants that may be present following conjugation. Ultrafiltration and diafiltration (UFDF) is commonly used to remove smallmolecule impurities following conjugation. Depending on the molecule characteristics of the antibody and drug, additional purification prior to UFDF may also be required. Other purification and analysis methods include hydrophobic interaction chromatography (HIC), size-exclusion chromatography (SEC), and liquid chromatography mass spectrometry (LC-MS).

[0139] In an example, conjugates according to the present disclosure can be conjugated via modification of native cysteine side chain thiols of a binding protein with a linkerpayload. Briefly, this method involves reduction of the solvent-exposed disulfides of a binding protein (e.g. antibody) with dithiothreitol (DTT) or tris(2-carboxyethyl) phosphine (TCEP) followed by modification of the resulting thiols with a linker-payload (e.g. maleimide-containing drugs). In an example, thiols can be modified with MC-VC- PAB-MMAE, which incorporates a cathepsin B protease cleavage site (VC: valine, citrulline) and a self-immolative linker (PAB: para-ami nobenzyloxycarbonyl; also abbreviated as PAB A) between the maleimide group (MC: maleimidocaproyl):

[0140] The binding protein can be reduced by treatment with tris(2-carboxyethyl) phosphine (TCEP) or DTT under suitable conditions. For example, the binding protein is incubated with TCEP at 37°C for two hours and conjugated to the cathepsin-cleavable linker by incubation in 50 mM PBS pH 7.0 for two hours at 4°C. In an example, the linker is prepared in a state in which it is connected to the payload, and the end of the linker has a reactive group that can be linked to the antibody. Further examples of linkers and payloads are provided herein. The skilled person will be able to determine suitable conditions for conjugating the nuclear-penetrating anti-DNA binding proteins and cathepsin-cleavable linkers of the disclosure with a payload of choice.

[0141] Payload Linkers

[0142] Conjugates according to the present disclosure comprise a cleavable linker that is capable of being cleaved by a cathepsin. Cathepsin-cleavable linkers are known in the art and are described, for example in Bargh et al. Chem. Soc. Rev., 2019, 48, 4361. The linker may be any suitable linker known in the art that is capable of being cleaved by a cathepsin, including the linkers described herein. The cathepsin capable of cleaving the linker may be a nuclear cathepsin. In another example, the cathepsin capable of cleaving the linker is a cathepsin that has translocated from a lysosome to the nucleus. In another example, the cathepsin capable of preferentially cleaving the linker may be a nuclear cathepsin. In another example, the cathepsin capable of preferentially cleaving the linker is a cathepsin that has translocated from a lysosome to the nucleus.

[0143] In an example, the cathepsin is cathepsin B (CatB) or cathepsin S (CatS). In an example, the cathepsin is cathepsin B. In an example, the linker is capable of being cleaved by cathepsin B (CatB) or Cathepsin S (CatS). In an example, the linker is capable of being cleaved by cathepsin B.

[0144] The conjugates of the present disclosure may be prepared using a linker having reactive functionalities for covalently attaching to the payload and to the nuclear- penetrating anti-DNA binding protein (e.g. antibody). In an example, a cysteine thiol and / or a lysine amine of an antibody (Ab) can form a bond with a reactive functional group of a linker or a payload-linker intermediate to make a conjugate of the present disclosure.

[0145] In an example, the linker has a functionality that is capable of reacting with a free cysteine and / or a free lysine present on a nuclear-penetrating anti-DNA binding protein (e.g. an antibody) to form a covalent bond. Exemplary reactive functionalities include maleimide, haloacetamides, a-haloacetyl such as bromoacetyl, thiols, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, isothiocyanates, amino acids (including natural and non-natural amino acids), or using any amine or thiol-modifying chemistry known in the art. See, e.g., the conjugation method at page 766 of Klussman, et al (2004), Bioconjugate Chemistry 15(4) :765-773, and the Examples herein.

[0146] The linker may comprise one or more linker components. Exemplary linker components include 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), maleimido- propylethyleneglycol (Mal-PEG), valine-citrulline (Val-Cit or VC), alaninephenylalanine (Ala-Phe), para-aminobenzyl (P AB), para-aminobenzyloxycarbonyl (PABA), / jara-aminobenzyloxycarbamate (PABC), N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), 4-(N-maleimidomethyl) cyclohexane- 1 carboxylate (MCC), N- succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl-4-(2-pyridyldithio)- 2-sulfo butanoate (sulfo-SPDB), and hydrazone. Various linker components are known in the art, some of which are described below.

[0147] In certain examples, the linker (L) has the following Formula II:

[0148] -Xx-Ww-Yy- wherein X is a stretcher unit, and x is 0 to 1 ; W is a peptide unit, and w is an integer from 1 to 12; and Y is a spacer unit, and y is 0, 1 or 2.

[0149] In an example, the linker comprises a peptide unit (W). Preferably, the peptide unit allows for cleavage of the linker by a protease, thereby facilitating release of the payload from the binding protein (e.g. antibody) upon exposure to intracellular proteases, in particular cathepsins. Exemplary peptide units include dipeptides, tripeptides, tetrapeptides, and pentapeptides. A peptide unit may comprise amino acid residues that occur naturally and / or minor amino acids and / or non-naturally occurring amino acid analogs, such as citrulline. Peptide units can be designed and optimized for enzymatic cleavage by a particular enzyme, for example a cathepsin. Typically, peptide-type linkers can be prepared by forming a peptide bond between two or more amino acids and / or peptide fragments. In an example, the linker comprises a dipeptide unit. Cleavable dipeptides were first developed as cathepsin B-cleavable linkers for Doxorubicin (Dox)-prodrugs in 1998. The previously described Phe-Arg sequence, a general substrate for cathepsin B and L, was used as a starting point for investigations of dipeptide-cleavage rates by cathepsin B. Two general trends were observed: (1) a hydrophilic residue at Pl is required and hydrolysis rates increase with basicity. Citrulline (Cit) is isoelectronic with Arg, and was preferred to Arg due to synthetic ease, despite the lower basicity. (2) The hydrophobic residues Phe, Vai and Ala at P2 enable cleavage by cathepsin B whilst imparting plasma stability. Examples of suitable dipeptide linkers include Phe-Lys, Phe- Arg, Vai-Ala, and Val-Cit. In an example, the dipeptide unit is valine-citrulline (Val- Cit). In an example, the dipeptide unit is valine- alanine (Val-Ala).

[0150] In an example, the linker comprises a tripeptide unit. Examples of tripeptide linkers include Gly- Val-Cit, Glu- Val-Cit, Ser- Val-Cit.

[0151] In an example, the linker comprises a peptidomimetic unit. In an example, the peptidomimetic unit comprises a di- or tri- peptide unit and a cyclobutane- 1 , 1 - dicarboxamide (cBu) moiety.

[0152] In an example, the linker comprises a stretcher unit (X) that links the nuclear- penetrating anti-DNA binding protein (e.g. antibody) to the peptide unit (W) of the linker. Exemplary stretcher units are shown below (wherein the wavy line indicates sites of covalent attachment to a binding protein, pay load, or additional linker component):

[0153] In an example, the linker comprises a spacer unit (Y) that links the payload to the peptide unit (W) of the linker. In an example, the spacer unit of the linker comprises a para-aminobenzyl (PAB) unit. In an example, the para-aminobenzyl alcohol is attached to the peptide unit via an amide bond, and a carbamate, methylcarbamate, or carbonate is made between the benzyl alcohol and the payload. In an example, the spacer unit comprises para-aminobenzyloxycarbonyl (PABA). In an example, the spacer unit comprises the spacer unit comprises para-aminobenzyloxycarbamate (PABC). Other exemplary spacer units include aromatic compounds that are electronically similar to the PAB group, such as 2-aminoimidazol-5-methanol derivatives and ortho- or para- aminobenzylacetals. In some embodiments, spacers can be used that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4- aminobutyric acid amides (Rodrigues et al (1995) Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al (1972) J. Amer. Chem. Soc. 94:5815) and 2- aminophenylpropionic acid amides (Amsberry, et al (1990) J. Org. Chem. 55:5867). Linkage of a drug to the a-carbon of a glycine residue is another example of a self-immolative spacer that may be useful in ADC (Kingsbury et al (1984) J. Med. Chem. 27: 1447).

[0154] In an example, the linker comprises Val-Cit-PABC. In an example, the linker comprises Phe-Lys-PABC.

[0155] In an example, the linker comprises Val-Cit-PABA. In an example, the linker comprises Phe-Lys-PABA.

[0156] In an example, the linker comprises a valine-citrulline unit and a pentafluorophenyl group. In an example, the linker comprises a valine-citrulline unit and a succinimide group. In an example, the linker comprises a valine-citrulline unit and a para aminobenzoic acid group. In an example, the linker comprises a valine-citrulline unit, a para aminobenzoic acid group and a pentafluorophenyl group. In an example, the linker comprises a valine-citrulline unit, a para aminobenzoic acid group and a succinimide group.

[0157] In an example, the linker comprises cBu-Cit-PABC.

[0158] In an example, the linker comprises cBu-Cit-PABA.

[0159] Exemplary linkers of the present disclosure are shown below (wherein the wavy line indicates sites of covalent attachment to a binding protein or pay load):

[0160]

[0161] The present disclosure contemplates conjugates prepared using the following linker reagents: bis-maleimido-trioxyethylene glycol (BMPEO), N-(f>- maleimidopropyloxy)-N-hydroxy succinimide ester (BMPS), N-(e- maleimidocaproyloxy) succinimide ester (EMCS), N-[y- maleimidobutyryloxy]succinimide ester (GMBS), 1,6-hexane-bis-vinylsulfone (HBVS), succinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxy-(6-amidocaproate) (LC- SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N- Maleimidophenyl)butyric acid hydrazide (MPBH), succinimidyl 3- (bromoacetamido)propionate (SBAP), succinimidyl iodoacetate (SIA), succinimidyl (4- iodoacetyljaminobenzoate (SIAB), N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), succinimidyl 4-(N- maleimidomethyl)cyclohexane- 1 -carboxylate (SMCC), succinimidyl 4-(p- maleimidophenyl)butyrate (SMPB), succinimidyl 6-[(beta- maleimidopropionamido)hexanoate] (SMPH), iminothiolane (IT), sulfo-EMCS, sulfo- GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and succinimidyl-(4-vinylsulfone)benzoate (SVSB), and including bis-maleimide reagents: dithiobismaleimidoethane (DTME), 1,4-Bismaleimidobutane (BMB), 1,4 Bismaleimidyl-2,3-dihydroxybutane (BMDB), bismaleimidohexane (BMH), bismaleimidoethane (BMOE), BMiPEGh (shown below), and BMiPEGh; bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6- diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4- dinitrobenzene). In some examples, bis-maleimide reagents allow the attachment of the thiol group of a cysteine in the binding protein (e.g. antibody) to a thiol-containing linker, or linker-drug intermediate. Other functional groups that are reactive with thiol groups include, iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyl disulfide, isocyanate, and isothiocyanate.

[0162] Nuclear Penetrating Anti-DNA Binding Proteins

[0163] The present disclosure relates to anti-DNA binding proteins and conjugates comprising the same.

[0164] In an example, the anti-DNA binding protein is also nuclear-penetrating. Accordingly, the present disclosure contemplates use of nuclear-penetrating, anti-DNA binding proteins. In other words, the binding protein can enter the nucleus of a cell and bind DNA rather than remaining sequestered in the cytoplasm of a cell. The skilled person will appreciate that nuclear penetrating anti-DNA binding proteins are also cell penetrating. Accordingly, the present disclosure contemplates use of cell penetrating, anti-DNA binding proteins.

[0165] In one example, the binding protein is an autoantibody derived from a subject or an animal with an autoimmune disease. In an example, the autoantibody is derived from a subject with systemic lupus erythematous, or an animal model thereof. The term “derived” as used herein encompasses recombinant forms of an antibody of the disclosure produced using recombinant techniques such as the methods discussed below. For example, a nucleic acid sequence encoding an autoantibody from a subject with systemic lupus erythematous, or an animal model thereof can be provided in a recombinant system to produce a recombinant form of the antibody. Examples of anti- DNA autoantibodies are known in the art (Hansen et al. (2012) Sci Transl Med., 4:157ral42; Noble et al. (2015) Cancer Research., 75:2285-2291 ; Noble et al. (2016) Nat Rev Rheumatol., 12:429-34). In an example, the autoantibody is 3E10 (i.e. antibody having a VH comprising SEQ ID NO: 7 and a VL comprising SEQ ID NO: 8) or a humanised form thereof.

[0166] In one example, an anti-DNA binding protein according to the present disclosure comprises a heavy chain variable region (VH) having a CDR 1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 2, a CDR3 as shown in SEQ ID NO: 3 and a light chain variable region (VL) having a CDR1 as shown in SEQ ID NO: 4, a CDR2 as shown in SEQ ID NO: 5 and a CDR3 as shown in SEQ ID NO: 6 or a humanized form thereof. Accordingly, in an example, the present disclosure encompasses an binding protein which comprises a heavy chain variable region (VH) having a CDR 1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 2, a CDR3 as shown in SEQ ID NO: 3 and a light chain variable region (VL) having a CDR1 as shown in SEQ ID NO: 4, a CDR2 as shown in SEQ ID NO: 5 and a CDR3 as shown in SEQ ID NO: 6 or a humanized form thereof. In an example, the binding protein competes for binding to DNA with an binding protein which comprises a heavy chain variable region (VH) having a CDR 1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 2, a CDR3 as shown in SEQ ID NO: 3 and a light chain variable region (VL) having a CDR1 as shown in SEQ ID NO: 4, a CDR2 as shown in SEQ ID NO: 5 and a CDR3 as shown in SEQ ID NO: 6 or a humanized form thereof. In these examples, the VH CDR 1 may rather comprise SEQ ID NO: 40.

[0167] In an example, the binding protein inhibits DNA repair. Inhibition of DNA repair can be assessed in-vitro by contacting cells with a DNA damaging agent and the binding protein before measuring the capacity of cells to repair DNA. In an example, the cells are cancer cells. In an example, inhibition of DNA repair is assessed based on the level of apoptosis in damaged cells.

[0168] Binding proteins of the disclosure such as antibodies or fragments thereof are particularly useful in that they can translocate cellular cathepsins into the nucleus. Accordingly, in an example, the present disclosure encompasses a binding protein which comprises a heavy chain variable region (VH) having a CDR 1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 2, a CDR3 as shown in SEQ ID NO: 3 and a light chain variable region (VL) having a CDR1 as shown in SEQ ID NO: 4, a CDR2 as shown in SEQ ID NO: 5 and a CDR3 as shown in SEQ ID NO: 6 or a humanized form thereof, wherein, the antibody or fragment thereof translocate cellular cathepsins into the nucleus under culture conditions. In an example, the VH CDR1 may rather comprise SEQ ID NO: 40.

[0169] The present disclosure also encompasses humanized forms and CDR variants of the above referenced example. Accordingly in another example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 9 or SEQ ID NO: 10, a CDR2 as shown in SEQ ID NO: 11 or SEQ ID NO: 12, a CDR3 as shown in SEQ ID NO: 13 and a VL having a CDR1 as shown in SEQ ID NO: 14 or SEQ ID NO: 15, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH having a CDR1 as shown in SEQ ID NO: 9 or SEQ ID NO: 10, a CDR2 as shown in SEQ ID NO: 11 or SEQ ID NO: 12, a CDR3 as shown in SEQ ID NO: 13 and a VL having a CDR1 as shown in SEQ ID NO: 14 or SEQ ID NO: 15, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17.

[0170] In another example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 9, a CDR2 as shown in SEQ ID NO: 11, a CDR3 as shown in SEQ ID NO: 13 and a VL having a CDR1 as shown in SEQ ID NO: 14, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH having a CDR1 as shown in SEQ ID NO: 9, a CDR2 as shown in SEQ ID NO: 11 , a CDR3 as shown in SEQ ID NO: 13 and a VL having a CDR1 as shown in SEQ ID NO: 14, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17. In another example, the humanized form of the antibody or fragment thereof comprises a VH having a CDR1 as shown in SEQ ID NO: 9, a CDR2 as shown in SEQ ID NO: 11 , a CDR3 as shown in SEQ ID NO: 13 and a VL having a CDR1 as shown in SEQ ID NO: 14, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17.

[0171] In another example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 10, a CDR2 as shown in SEQ ID NO: 12, a CDR3 as shown in SEQ ID NO: 13 and a VL having a CDR1 as shown in SEQ ID NO: 15, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH having a CDR1 as shown in SEQ ID NO: 10, a CDR2 as shown in SEQ ID NO: 12, a CDR3 as shown in SEQ ID NO: 13 and a VL having a CDR1 as shown in SEQ ID NO: 15, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17.

[0172] In the above referenced examples, the CDRs are subject to at least one amino acid substitution. In an example, the substitution(s) are in CDR1. In another example, the substitution(s) are in VH CDR1. In another example, the substitution(s) are in VL CDR2. In another example, the substitution(s) are in VH CDR2.

[0173] In another example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 6, a CDR2 as shown in SEQ ID NO: 7 and a CDR3 as shown in SEQ ID NO: 8 or a humanized form thereof.

[0174] In one example, the binding protein comprises a VH which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 18-21 and a VL as shown in any one of SEQ ID NOs: 22-25 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 18-21 and a VL as shown in any one of SEQ ID NOs: 22-25.

[0175] In one example, the binding protein comprises a Vn which comprises an amino acid sequence as shown in SEQ ID NO: 18 and a VL as shown in SEQ ID NO: 22 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 18 and a VL as shown in SEQ ID NO: 22. In an example, binding protein comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 18 and a VL as shown in SEQ ID NO: 22.

[0176] In one example, the binding protein comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 19 and a VL as shown in SEQ ID NO: 23 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 19 and a VL as shown in SEQ ID NO: 23.

[0177] In one example, the binding protein comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 20 and a VL as shown in SEQ ID NO: 24 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 20 and a VL as shown in SEQ ID NO: 24.

[0178] In one example, the binding protein comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 21 and a VL as shown in SEQ ID NO: 25 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 21 and a VL as shown in SEQ ID NO: 25.

[0179] In an example, the binding protein is a variant of a binding protein which comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 18 and a VL as shown in SEQ ID NO: 22. In an example, the variant has the CDRs set forth in SEQ ID NO: 18 and SEQ ID NO: 22. It will be appreciated that various inconsequential amino acid changes can be made outside of this region without substantially impacting on function. Accordingly, in an example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 9, a CDR2 as shown in SEQ ID NO: 11, a CDR3 as shown in SEQ ID NO: 13 and a VL having a CDR1 as shown in SEQ ID NO: 14, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17, wherein the Vuis 95% identical to SEQ ID NO: 18 and the Vris 95% identical to SEQ ID NO: 22. In another example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 9, a CDR2 as shown in SEQ ID NO: 11, a CDR3 as shown in SEQ ID NO: 13 and a VL having a CDR1 as shown in SEQ ID NO: 14, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17, wherein the VH is 97% identical to SEQ ID NO: 18 and the VL is 97% identical to SEQ ID NO: 22. In another example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 9, a CDR2 as shown in SEQ ID NO: 1 1, a CDR3 as shown in SEQ ID NO: 13 and a VL having a CDR1 as shown in SEQ ID NO: 14, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17, wherein the VH is 98% identical to SEQ ID NO: 18 and the VL IS 98% identical to SEQ ID NO: 22. In another example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 9, a CDR2 as shown in SEQ ID NO: 11, a CDR3 as shown in SEQ ID NO: 13 and a VL having a CDR1 as shown in SEQ ID NO: 14, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17, wherein the VH is 99% identical to SEQ ID NO: 18 and the VL is 99% identical to SEQ ID NO: 22. In these examples, % sequence identity is determined by aligning the complete amino acid sequence of the respective VH or VL with the complete amino acid sequence of a corresponding variant to determine sequence identity. In an example, the variant has the same number of amino acid residues as the respective VH or VL(i.e. SEQ ID NO: 18 or SEQ ID NO: 22).

[0180] In an example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 40, a CDR2 as shown in SEQ ID NO: 41 and a CDR3 as shown in SEQ ID NO: 43, and a VL having a CDR1 as shown in SEQ ID NO: 44, a CDR2 as shown in SEQ ID NO: 46 and a CDR3 as shown in SEQ ID NO: 47.

[0181] In another example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 40, a CDR2 as shown in SEQ ID NO: 42 and a CDR3 as shown in SEQ ID NO: 43, and a VL having a CDR1 as shown in SEQ ID NO: 45, a CDR2 as shown in SEQ ID NO: 46 and a CDR3 as shown in SEQ ID NO: 47.

[0182] In another example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 48, a CDR2 as shown in SEQ ID NO: 49 and a CDR3 as shown in SEQ ID NO: 51, and a VL having a CDR1 as shown in SEQ ID NO: 52, a CDR2 as shown in SEQ ID NO: 54 and a CDR3 as shown in SEQ ID NO: 55.

[0183] In another example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 48, a CDR2 as shown in SEQ ID NO: 50 and a CDR3 as shown in SEQ ID NO: 51, and a VL having a CDR1 as shown in SEQ ID NO: 53, a CDR2 as shown in SEQ ID NO: 54 and a CDR3 as shown in SEQ ID NO: 55.

[0184] In an example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 40, a CDR2 as shown in SEQ ID NO: 85 and a CDR3 as shown in SEQ ID NO: 3, and a VL having a CDR1 as shown in SEQ ID NO: 44, a CDR2 as shown in SEQ ID NO: 46 and a CDR3 as shown in SEQ ID NO: 47. In another example, the binding protein comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 56 and a VL as shown in SEQ ID NO: 63.

[0185] In another example, the binding protein comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 57 and a VL as shown in SEQ ID NO: 63.

[0186] In another example, the binding protein comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 58 and a VL as shown in SEQ ID NO: 63.

[0187] In another example, the binding protein comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 56 and a VL as shown in SEQ ID NO: 64.

[0188] In another example, the binding protein comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 57 and a VL as shown in SEQ ID NO: 64.

[0189] In another example, the binding protein comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 58 and a VL as shown in SEQ ID NO: 64.

[0190] In another example, the binding protein comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 56 and a VL as shown in SEQ ID NO: 65.

[0191] In another example, the binding protein comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 57 and a VL as shown in SEQ ID NO: 65.

[0192] In another example, the binding protein comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 58 and a VL as shown in SEQ ID NO: 65.

[0193] In another example, the binding protein comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 60 and a VL as shown in SEQ ID NO: 66.

[0194] In another example, the binding protein comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 61 and a VL as shown in SEQ ID NO: 66.

[0195] In another example, the binding protein comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 62 and a VL as shown in SEQ ID NO: 66.

[0196] In another example, the binding protein comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 60 and a VL as shown in SEQ ID NO: 67.

[0197] In another example, the binding protein comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 61 and a VL as shown in SEQ ID NO: 67.

[0198] In another example, the binding protein comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 62 and a VL as shown in SEQ ID NO: 67.

[0199] In another example, the binding protein comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 60 and a VL as shown in SEQ ID NO: 68.

[0200] In another example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 40, a CDR2 as shown in SEQ ID NO: 41 and a CDR3 as shown in SEQ ID NO: 43, and a VL having a CDR1 as shown in SEQ ID NO: 44, a CDR2 as shown in SEQ ID NO: 46 and a CDR3 as shown in SEQ ID NO: 47, wherein the VH comprises an amino acid sequence that is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 56 and the VL that is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 63.

[0201] In another example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 40, a CDR2 as shown in SEQ ID NO: 41 and a CDR3 as shown in SEQ ID NO: 43, and a VL having a CDR1 as shown in SEQ ID NO: 44, a CDR2 as shown in SEQ ID NO: 46 and a CDR3 as shown in SEQ ID NO: 47, wherein the VH comprises an amino acid sequence that is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 57 and the VL that is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 63.

[0202] In another example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 40, a CDR2 as shown in SEQ ID NO: 41 and a CDR3 as shown in SEQ ID NO: 43, and a VL having a CDR1 as shown in SEQ ID NO: 44, a CDR2 as shown in SEQ ID NO: 46 and a CDR3 as shown in SEQ ID NO: 47, wherein the VH comprises an amino acid sequence that is at least 95 % identical to SEQ ID NO: 58 and the VL is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 63.

[0203] In another example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 40, a CDR2 as shown in SEQ ID NO: 41 and a CDR3 as shown in SEQ ID NO: 43, and a VL having a CDR1 as shown in SEQ ID NO: 44, a CDR2 as shown in SEQ ID NO: 46 and a CDR3 as shown in SEQ ID NO: 47, wherein the VH comprises an amino acid sequence that is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO:

[0204] 56 and the VL is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 64.

[0205] In another example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 40, a CDR2 as shown in SEQ ID NO: 41 and a CDR3 as shown in SEQ ID NO: 43, and a VL having a CDR1 as shown in SEQ ID NO: 44, a CDR2 as shown in SEQ ID NO: 46 and a CDR3 as shown in SEQ ID NO: 47, wherein the VH comprises an amino acid sequence that is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO:

[0206] 57 and the VL is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 64.

[0207] In another example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 40, a CDR2 as shown in SEQ ID NO: 41 and a CDR3 as shown in SEQ ID NO: 43, and a VL having a CDR1 as shown in SEQ ID NO: 44, a CDR2 as shown in SEQ ID NO: 46 and a CDR3 as shown in SEQ ID NO: 47, wherein the VH comprises an amino acid sequence that is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO:

[0208] 58 and the VL is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 64.

[0209] In another example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 40, a CDR2 as shown in SEQ ID NO: 41 and a CDR3 as shown in SEQ ID NO: 43, and a VL having a CDR1 as shown in SEQ ID NO: 44, a CDR2 as shown in SEQ ID NO: 46 and a CDR3 as shown in SEQ ID NO: 47, wherein the VH comprises an amino acid sequence that is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO:

[0210] 56 and the VL is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 65.

[0211] In another example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 40, a CDR2 as shown in SEQ ID NO: 41 and a CDR3 as shown in SEQ ID NO: 43, and a VL having a CDR1 as shown in SEQ ID NO: 44, a CDR2 as shown in SEQ ID NO: 46 and a CDR3 as shown in SEQ ID NO: 47, wherein the VH comprises an amino acid sequence that is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO:

[0212] 57 and the VL is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 65.

[0213] In another example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 40, a CDR2 as shown in SEQ ID NO: 41 and a CDR3 as shown in SEQ ID NO: 43, and a VL having a CDR1 as shown in SEQ ID NO: 44, a CDR2 as shown in SEQ ID NO: 46 and a CDR3 as shown in SEQ ID NO: 47, wherein VH comprises an amino acid sequence that is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO:

[0214] 58 and a VL that is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 65.

[0215] In another example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 40, a CDR2 as shown in SEQ ID NO: 42 and a CDR3 as shown in SEQ ID NO: 43, and a VL having a CDR1 as shown in SEQ ID NO: 45, a CDR2 as shown in SEQ ID NO: 46 and a CDR3 as shown in SEQ ID NO: 47, wherein the VH comprises an amino acid sequence that is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO:

[0216] 60 and the VL is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 66.

[0217] In another example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 40, a CDR2 as shown in SEQ ID NO: 42 and a CDR3 as shown in SEQ ID NO: 43, and a VL having a CDR1 as shown in SEQ ID NO: 45, a CDR2 as shown in SEQ ID NO: 46 and a CDR3 as shown in SEQ ID NO: 47, wherein the VH comprises an amino acid sequence that is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO:

[0218] 61 and the VL is at least 95%, 97%, 98%, or 99% to SEQ ID NO: 66.

[0219] In another example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 40, a CDR2 as shown in SEQ ID NO: 42 and a CDR3 as shown in SEQ ID NO: 43, and a VL having a CDR1 as shown in SEQ ID NO: 45, a CDR2 as shown in SEQ ID NO: 46 and a CDR3 as shown in SEQ ID NO: 47, wherein the VH comprises an amino acid sequence that is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO:

[0220] 62 and the VL is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 66.

[0221] In another example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 40, a CDR2 as shown in SEQ ID NO: 42 and a CDR3 as shown in SEQ ID NO: 43, and a VL having a CDR1 as shown in SEQ ID NO: 45, a CDR2 as shown in SEQ ID NO: 46 and a CDR3 as shown in SEQ ID NO: 47, wherein VH comprises an amino acid sequence that is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO:

[0222] 60 and the VL is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 67.

[0223] In another example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 40, a CDR2 as shown in SEQ ID NO: 42 and a CDR3 as shown in SEQ ID NO: 43, and a VL having a CDR1 as shown in SEQ ID NO: 45, a CDR2 as shown in SEQ ID NO: 46 and a CDR3 as shown in SEQ ID NO: 47, wherein the VH comprises an amino acid sequence that is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO:

[0224] 61 and the VL is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 67.

[0225] In another example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 40, a CDR2 as shown in SEQ ID NO: 42 and a CDR3 as shown in SEQ ID NO: 43, and a VL having a CDR1 as shown in SEQ ID NO: 45, a CDR2 as shown in SEQ ID NO: 46 and a CDR3 as shown in SEQ ID NO: 47, wherein VH which comprises an amino acid sequence that is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO:

[0226] 62 and the VL is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 67.

[0227] In another example, the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 40, a CDR2 as shown in SEQ ID NO: 42 and a CDR3 as shown in SEQ ID NO: 43, and a VL having a CDR1 as shown in SEQ ID NO: 45, a CDR2 as shown in SEQ ID NO: 46 and a CDR3 as shown in SEQ ID NO: 47, wherein the VH comprises an amino acid sequence that is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 60 and the VL is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 68.

[0228] Other examples of suitable anti-DNA binding proteins are known in the art (Zack et al. (1995) J. Immunol., 154:1987-1994; Gu et al. (1998) J. Immunol., 161 :6999-7006; Noble et al. (2014) Sci Rep., 4:5958; ATCC Accession No. PTA 2439 hybridoma; WO2019 / 018426). In an example, the antibody is not an anti-guanosine antibody.

[0229] In an example, the anti-DNA binding protein can be a fragment such as a cell penetrating anti-DNA Fv. In an example, the Fv is a scFv. In an example, the fragment has an antigen binding domain, wherein the antigen binding domain binds to or specifically binds to DNA. For example, the Fv can bind the same epitope as an antibody having a VH comprising an amino acid sequence as shown in SEQ ID NO: 7 and a VL comprising an amino acid sequence as shown in SEQ ID NO: 8. In another example, the Fv can bind the same epitope as an antibody having a VH comprising an amino acid sequence as shown in SEQ ID NO: 18 and a VL comprising an amino acid sequence as shown in SEQ ID NO: 22. In another example, the Fv can bind the same epitope as an antibody having a VH comprising an amino acid sequence as shown in SEQ ID NO: 19 and a VL comprising an amino acid sequence as shown in SEQ ID NO: 23. In another example, the Fv can bind the same epitope as an antibody having a VH comprising an amino acid sequence as shown in SEQ ID NO: 20 and a VL comprising an amino acid sequence as shown in SEQ ID NO: 24. In another example, the Fv can bind the same epitope as an antibody having a VH comprising an amino acid sequence as shown in SEQ ID NO: 21 and a VL comprising an amino acid sequence as shown in SEQ ID NO: 25. In an example, the Fv is a scFv.

[0230] In an example, the Fv comprises a linker. Various suitable linkers and methods for their design have been described previously (e.g. U.S. Patent No. 4,946,778; WO 1994 / 012520; and U.S. Patent No. 4,704,692). In an example, the Fv comprises a glycine- serine (GS) linker. In one example, the Fv comprises a linker comprising the sequence as shown in SEQ ID NO: 26.

[0231] In an example, the VH and VL of the Fv can be in a single polypeptide chain. In another example, the Fv lacks an Fc region. For example, the Fv can be a single chain Fv fragment (scFv), a dimeric scFv (di-scFv), a trimeric scFv (tri-scFv). In an example, the Fv is an scFv. In another example, the Fv is a di-scFv. The scFvs may be separated by a linker. In one example, the linker comprises the sequence shown in SEQ ID NO: 27.

[0232] Thus, in an example, the binding protein may be a scFv comprising a Vn which comprises an amino acid sequence as shown in SEQ ID NO: 18 and a VL as shown in SEQ ID NO: 22 or a variant thereof that competes for binding to DNA with an antibody which comprises a Vn which comprises an amino acid sequence as shown in SEQ ID NO: 18 and a VL as shown in SEQ ID NO: 22 and a linker separating the scFvs comprising the sequence shown in SEQ ID NO: 27.

[0233] In another example, the binding protein may be a scFv comprising a Vn which comprises an amino acid sequence as shown in SEQ ID NO: 19 and a VL as shown in SEQ ID NO: 23 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 19 and a VL as shown in SEQ ID NO: 23 and a linker separating the scFvs comprising the sequence shown in SEQ ID NO: 27.

[0234] In another example, the binding protein may be a scFv comprising a VH which comprises an amino acid sequence as shown in SEQ ID NO: 20 and a VL as shown in SEQ ID NO: 24 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 20 and a VL as shown in SEQ ID NO: 24 and a linker separating the scFvs comprising the sequence shown in SEQ ID NO: 27.

[0235] In another example, the binding protein may be a scFv comprising a VH which comprises an amino acid sequence as shown in SEQ ID NO: 21 and a VL as shown in SEQ ID NO: 25 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 21 and a VL as shown in SEQ ID NO: 25 and a linker separating the scFvs comprising the sequence shown in SEQ ID NO: 27.

[0236] Thus, in another example, the binding protein may be a di-scFV comprising a VH which comprises an amino acid sequence as shown in SEQ ID NO: 18 and a VL as shown in SEQ ID NO: 22 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 18 and a VL as shown in SEQ ID NO: 22 and a linker separating the scFvs comprising the sequence shown in SEQ ID NO: 27.

[0237] In another example, the binding protein may be a di-scFv comprising a VH which comprises an amino acid sequence as shown in SEQ ID NO: 19 and a VL as shown in SEQ ID NO: 23 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 19 and a VL as shown in SEQ ID NO: 23 and a linker separating the scFvs comprising the sequence shown in SEQ ID NO: 27.

[0238] In another example, the binding protein may be a di-scFv comprising a VH which comprises an amino acid sequence as shown in SEQ ID NO: 20 and a VL as shown in SEQ ID NO: 24 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 20 and a VL as shown in SEQ ID NO: 24 and a linker separating the scFvs comprising the sequence shown in SEQ ID NO: 27.

[0239] In another example, the binding protein may be a di-scFv comprising a VH which comprises an amino acid sequence as shown in SEQ ID NO: 21 and a VL as shown in SEQ ID NO: 25 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 21 and a VL as shown in SEQ ID NO: 25 and a linker separating the scFvs comprising the sequence shown in SEQ ID NO: 27.

[0240] In another example, the binding protein is a di-scFv which comprises an amino acid sequence as shown in SEQ ID NO: 38.

[0241] In another example, the Fv is a tri-scFv.

[0242] In another example, the scFv, di-scFv or tri-scFv can be linked to a constant region of an antibody, Fc or a heavy chain constant domain Cn2 and / or Cu3. In one example, the scFv, di-scFv or tri-scFv is linked to the a constant region of an antibody, Fc or a heavy chain constant domain CH2 and / or CH3 by a hinge region. In one example, the hinge region comprises a sequence as shown in SEQ ID NO: 28.

[0243] In an example, the binding protein is a di-scFv having an antigen binding domain, wherein the antigen binding domain binds to or specifically binds to DNA. In another example, the Vn and VL of the binding protein are in a separate polypeptide chain. For example, the binding protein can be a diabody, triabody, tetrabody, Fab, F(ab’)2. In another example, the binding protein can be an Fv which comprises a Vn and VL in separate polypeptide chains. In these examples, the binding proteins may be linked to a constant region of an antibody, Fc or a heavy chain constant domain CH2 and / or CH3. In another example, the binding protein can be an intact antibody. Accordingly, in an example, the present disclosure encompasses an antibody having an antigen binding domain, wherein the antigen binding domain binds to or specifically binds to DNA. For example, the antibody comprises a VH comprising an amino acid sequence as shown in SEQ ID NO: 7 and a VL comprising an amino acid sequence as shown in SEQ ID NO: 8. In another example, the antibody comprises a VH comprising an amino acid sequence as shown in SEQ ID NO: 18 and a VL comprising an amino acid sequence as shown in SEQ ID NO: 22 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 18 and a VL as shown in SEQ ID NO: 22.

[0244] In another example, the antibody comprises a Vn comprising an amino acid sequence as shown in SEQ ID NO: 19 and a VL comprising an amino acid sequence as shown in SEQ ID NO: 23 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 19 and a VL as shown in SEQ ID NO: 23.

[0245] In another example, the antibody comprises a VH comprising an amino acid sequence as shown in SEQ ID NO: 20 and a VL comprising an amino acid sequence as shown in SEQ ID NO: 24 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 20 and a VL as shown in SEQ ID NO: 24.

[0246] In another example, the antibody comprises a VH comprising an amino acid sequence as shown in SEQ ID NO: 21 and a VL comprising an amino acid sequence as shown in SEQ ID NO: 25 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 21 and a VL as shown in SEQ ID NO: 25.

[0247] In another example, the antibody is an intact antibody comprising a VH having a CDR1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 2, a CDR3 as shown in SEQ ID NO: 3 and a VL having a CDR 1 as shown in SEQ ID NO: 4, a CDR2 as shown in SEQ ID NO: 5 and a CDR3 as shown in SEQ ID NO: 6 or a humanized form thereof. In an example, the antibody is a chimeric antibody. In an example, the VH CDR1 may rather comprise SEQ ID NO: 40. In another example, the antibody is an intact antibody comprising a Vn having a CDR1 as shown in SEQ ID NO: 9 or SEQ ID NO: 10, a CDR2 as shown in SEQ ID NO: 11 or SEQ ID NO: 12, a CDR3 as shown in SEQ ID NO: 13 and a VL having a CDR1 as shown in SEQ ID NO: 14 or SEQ ID NO: 15, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH having a CDR1 as shown in SEQ ID NO: 9 or SEQ ID NO: 10, a CDR2 as shown in SEQ ID NO: 11 or SEQ ID NO: 12, a CDR3 as shown in SEQ ID NO: 13 and a VL having a CDR1 as shown in SEQ ID NO: 14 or SEQ ID NO: 15, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17. The CHI and CH2 domains of the antibody may be linked by a hinge region. In one example, the hinge region comprises a sequence as shown in SEQ ID NO: 28.

[0248] In another example, the antibody is an intact antibody comprising a VH having a CDR1 as shown in SEQ ID NO: 9, a CDR2 as shown in SEQ ID NO: 11, a CDR3 as shown in SEQ ID NO: 13 and a VL having a CDR1 as shown in SEQ ID NO: 14, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH having a CDR1 as shown in SEQ ID NO: 9, a CDR2 as shown in SEQ ID NO: 11 , a CDR3 as shown in SEQ ID NO: 13 and a VL having a CDR1 as shown in SEQ ID NO: 14, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17.

[0249] In another example, the antibody is an intact antibody comprising a VH having a CDR1 as shown in SEQ ID NO: 10, a CDR2 as shown in SEQ ID NO: 12, a CDR3 as shown in SEQ ID NO: 13 and a Vt having aCDRl as shown in SEQ ID NO: 15, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH having a CDR1 as shown in SEQ ID NO: 10, a CDR2 as shown in SEQ ID NO: 12, a CDR3 as shown in SEQ ID NO: 13 and a Vr having aCDRl as shown in SEQ ID NO: 15, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17.

[0250] In one example, the antibody is an intact antibody comprising a VH which comprises an amino acid sequence as shown in SEQ ID NO: 18 and a VL as shown in SEQ ID NO: 22 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 18 and a VL as shown in SEQ ID NO: 22.

[0251] In one example, the antibody is an intact antibody comprising a VH which comprises an amino acid sequence as shown in SEQ ID NO: 19 and a VL as shown in SEQ ID NO: 23 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 19 and a VL as shown in SEQ ID NO: 23.

[0252] In one example, the antibody is an intact antibody comprising a VH which comprises an amino acid sequence as shown in SEQ ID NO: 20 and a VL as shown in SEQ ID NO: 24 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 20 and a VL as shown in SEQ ID NO: 24.

[0253] In one example, the antibody is an intact antibody comprising a VH which comprises an amino acid sequence as shown in SEQ ID NO: 21 and a VL as shown in SEQ ID NO: 25 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 21 and a VL as shown in SEQ ID NO: 25.

[0254] In one example, the antibody is an intact antibody comprising the amino acid sequence shown in SEQ ID NO: 39.

[0255] As known in the art, antibodies can come in different isotypes such as IgA, IgD, IgE, IgG, and IgM. In one example, antibodies encompassed by the present disclosure are IgG.

[0256] In an example, the binding protein is a "nucleolytic binding protein" such as an nucleolytic antibody. These binding proteins can bring about and catalyse cleavage of nucleic acids, such as RNA or DNA. Nucleolytic binding proteins can recognize and interact with DNA or RNA to bring about cleavage of nucleotide-nucleotide linkages at, or near to the region of contact with the DNA or RNA. Accordingly, in certain examples, binding proteins of the disclosure can have "nucleolytic" activity. The term “nucleolytic” is used in this context to refer to a binding protein that can cleave the nucleotidenucleotide linkages between nucleic acids, for example, by hydrolysis. In an example, the binding protein comprises a VH having an amino acid sequence as shown in SEQ ID NO: 30 and a VL having an amino acid sequence as shown in SEQ ID NO: 34 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH having an amino acid sequence as shown in SEQ ID NO: 30 and a VL having an amino acid sequence as shown in SEQ ID NO: 34. In an example, the antibody comprises a VH having a CDR1 as shown in SEQ ID NO: 31, a CDR2 as shown in SEQ ID NO: 32, a CDR3 as shown in SEQ ID NO: 33 and a VL having a CDR1 as shown in SEQ ID NO: 35, a CDR2 as shown in SEQ ID NO: 36 and a CDR3 as shown in SEQ ID NO: 37 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH having a CDR1 as shown in SEQ ID NO: 31, a CDR2 as shown in SEQ ID NO: 32, a CDR3 as shown in SEQ ID NO: 33 and a VL having a CDR1 as shown in SEQ ID NO: 35, a CDR2 as shown in SEQ ID NO: 36 and a CDR3 as shown in SEQ ID NO: 37. An example, of a nucleolytic antibody is disclosed in Noble et al. (2014) Sci Rep- Uk„ 4:5958.

[0257] In another example, binding proteins of the disclosure can bind DNA without facilitating degradation of the DNA.

[0258] In an example, the antibody is monoclonal. Monoclonal antibodies are one exemplary form of antibodies contemplated by the present disclosure. The term “monoclonal antibody" or “MAb” refers to a homogeneous antibody population capable of binding to the same antigen(s), for example, to the same epitope within the antigen. This term is not intended to be limited as regards to the source of the antibody or the manner in which it is made.

[0259] In an example, antibodies encompassed by the present disclosure may be “humanized”. In an example, the CDRs are humanized. A “humanized antibody” is an immunoglobulin molecule which contains minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin consensus sequence. In an example, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)). In an example, antibodies of the disclosure are used to deliver a payload to cell nuclei in the brain of a subject.

[0260] Binding proteins can be identified by their ability to compete with a reference binding protein for binding to DNA using various methods known in the art. For example, an anti-DNA binding protein of the disclosure is conjugated with biotin using established procedures (Hofmann K, et al. (1982) Biochemistry 21 : 978-84). Candidate binding proteins are then evaluated by their capacity to compete with the binding of the biotinylated antibody to DNA. The binding of biotinylated antibody to DNA may be assessed by the addition of fluorescein-labelled streptavidin which will bind to biotin on the labelled binding protein. Fluorescence staining is then quantified, and the competitive effect of binding protein(s) expressed as a percentage of the fluorescence levels obtained in the absence of the candidate competitor. In other examples, affinity measurements are used to determine the competitive effect of candidate binding proteins. Affinity measurements can be determined by standard methodology for antibody reactions, for example, immunoassays, surface plasmon resonance (SPR) (Rich and Myszka Curr. Opin. Biotechnol 11 :54, 2000; Englebienne Analyst. 123: 1599, 1998), isothermal titration calorimetry (ITC) or other kinetic interaction assays known in the art. In one example, the constants are measured by using surface plasmon resonance assays, e.g., using BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) with immobilized DNA. Exemplary SPR methods are described in U.S. Patent No. 7,229,619.

[0261] Single Chain Fv (scFv) Fragments

[0262] One of skill in the art will be aware that scFv’s comprise VH and VL regions in a single polypeptide chain and a polypeptide linker between the VH and VL which enables the scFv to form the desired structure for antigen binding (i.e., for the VH and VL of the single polypeptide chain to associate with one another to form a Fv). Single-chain variable fragments lack the constant Fc region found in complete antibody molecules and therefore can have reduced immunogenicity. Exemplary linkers used in this context comprise in excess of 12 amino acid residues with (Gly4Ser)3 being one of the more favoured linkers for a scFv. Another example of a suitable linker is provided in SEQ ID NO: 26.

[0263] The present disclosure also contemplates a disulfide stabilized Fv (or diFv or dsFv), in which a single cysteine residue is introduced into a FR of VH and a FR of VL and the cysteine residues linked by a disulfide bond to yield a stable Fv.

[0264] In another example, the present disclosure encompasses a dimeric scFv (di-scFV), i.e., a protein comprising two scFv molecules linked by a non-covalent or covalent linkage, e.g., by a leucine zipper domain (e.g., derived from Fos or Jun) or trimeric scFV (tri-scFv). In another example, two scFv’s are linked by a peptide linker of sufficient length to permit both scFv’s to form and to bind to an antigen, e.g., as described in U.S. Published Application No. 20060263367. An exemplary linker is provided in SEQ ID NO: 27.

[0265] Diabodies, Triabodies, Tetrabodies

[0266] In some examples, an antigen binding fragment of the disclosure is or comprises a diabody, triabody, tetrabody or higher order protein complex such as those described in W098 / 044001 and / or W094 / 007921. For example, a diabody is a protein comprising two associated polypeptide chains, each polypeptide chain comprising the structure VL-X-VH or VH-X-VL, wherein X is a linker comprising insufficient residues to permit the VH and VL in a single polypeptide chain to associate (or form an Fv) or is absent, and wherein the VH of one polypeptide chain binds to a VL of the other polypeptide chain to form an antigen binding site, i.e., to form a Fv molecule capable of specifically binding to one or more antigens. The VL and VH can be the same in each polypeptide chain or the VL and VH can be different in each polypeptide chain so as to form a bispecific diabody (i.e., comprising two Fv’s having different specificity).

[0267] Other Antibodies and Antibody Fragments

[0268] Other examples of antibodies encompassed by the present disclosure include:

[0269] (i) “key and hole” bispecific proteins as described in U.S. Patent No. 5,731,168;

[0270] (ii) heteroconjugate proteins, e.g., as described in U.S. Patent No. 4,676,980;

[0271] (iii) heteroconjugate proteins produced using a chemical cross-linker, e.g., as described in U.S. Patent No. 4,676,980; and

[0272] (iv) Faba (e.g., as described in EPl 9930302894).

[0273] Recombinant Expression

[0274] In an example, the binding protein is recombinant.

[0275] In the case of a recombinant binding protein such as an antibody or fragment thereof, a nucleic acid encoding the same can be cloned into expression vectors, which are then transfected into host cells, such as E. coli cells, yeast cells, insect cells, or mammalian cells, such as simian COS cells, Chinese Hamster Ovary (CHO) cells, human embryonic kidney (HEK) cells, or myeloma cells that do not otherwise produce immunoglobulin or antibody protein.

[0276] Suitable molecular cloning techniques are known in the art and described, for example in Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present) or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). A wide variety of cloning and in vitro amplification methods are suitable for the construction of recombinant nucleic acids. Methods of producing recombinant antibodies are also known in the art. See U.S. Patent No. 4,816,567 or U.S. Patent No. 5,530,101.

[0277] Following isolation, the nucleic acid is operably linked to a promoter in an expression construct or expression vector for further cloning (amplification of the DNA) or for expression in a cell-free system or in cells. Thus, another example of the disclosure provides an expression construct that comprises an isolated nucleic acid encoding a binding protein of the disclosure and one or more additional nucleotide sequences. Suitably, the expression construct is in the form of, or comprises genetic components of, a plasmid, bacteriophage, a cosmid, a yeast or bacterial artificial chromosome as are understood in the art. Expression constructs may be suitable for maintenance and propagation of the isolated nucleic acid in bacteria or other host cells, for manipulation by recombinant DNA technology and / or for expression of the nucleic acid encoding a binding protein of the disclosure.

[0278] Many vectors for expression in cells are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence (e.g. SEQ ID NO: 29), a sequence encoding the binding protein (e.g., derived from the amino acid sequence information provided herein), an enhancer element, a promoter, and a transcription termination sequence. Exemplary signal sequences include prokaryotic secretion signals (e.g., pelB, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretion signals (e.g., invertase leader, a factor leader, or acid phosphatase leader) or mammalian secretion signals (e.g., herpes simplex gD signal).

[0279] Exemplary promoters active in mammalian cells include cytomegalovirus immediate early promoter (CMV-IE), human elongation factor 1 -a promoter (EFl ), small nuclear RNA promoters (Ula and Ulb), a-myosin heavy chain promoter, Simian virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), Adenovirus major late promoter, [3-actin promoter; hybrid regulator}' element comprising a CMV enhancer / 0- actin promoter or an immunoglobulin or antibody promoter or active fragment thereof. Examples of useful mammalian host cell lines are monkey kidney CV 1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture; baby hamster kidney cells (BHK, ATCC CCL 10); or Chinese hamster ovary cells (CHO).

[0280] Typical promoters suitable for expression in yeast cells such as for example a yeast cell selected from the group comprising Pichia pastoris, Saccharomyces cerevisiae and S. pombe, include, but are not limited to, the ADH1 promoter, the GALI promoter, the GAIA promoter, the CUP1 promoter, the PHO5 promoter, the nmt promoter, the RPR1 promoter, or the TEF1 promoter.

[0281] Means for introducing the isolated nucleic acid or expression construct comprising same into a cell for expression are known to those skilled in the art. The technique used for a given cell depends on the known successful techniques. Means for introducing recombinant DNA into cells include microinjection, transfection mediated by DEAE-dextran, transfection mediated by liposomes such as by using lipofectamine (Gibco, MD, USA) and / or cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation and microparticle bombardment such as by using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA) amongst others.

[0282] The host cells used to produce the binding protein may be cultured in a variety of media, depending on the cell type used. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPM1-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing mammalian cells. Media for culturing other cell types discussed herein are known in the art.

[0283] The skilled artisan will understand from the foregoing description that the present disclosure also provides an isolated nucleic acid encoding a binding protein of the present disclosure.

[0284] The present disclosure also provides an expression construct comprising an isolated nucleic acid of the disclosure operably linked to a promoter. In one example, the expression construct is an expression vector.

[0285] In one example, the expression construct of the disclosure comprises a nucleic acid encoding a polypeptide (e.g., comprising a VH) operably linked to a promoter and a nucleic acid encoding another polypeptide (e.g., comprising a VL) operably linked to a promoter.

[0286] The disclosure also provides a host cell comprising an expression construct according to the present disclosure.

[0287] The present disclosure also provides an isolated cell expressing a binding protein of the disclosure or a recombinant cell genetically-modified to express the binding protein.

[0288] Methods for purifying antibodies according to the present disclosure are known in the art and / or described in publications such as WO2019 / 018426.

[0289] Payloads

[0290] Conjugates of the disclosure provide a platform for transporting various payloads to the nucleus so that they can be released by a cathepsin. This platform allows suitable payloads to be selected based on desired application. Exemplary payloads are discussed in more detail below.

[0291] Gene editing agents

[0292] The nuclear penetrating capability of conjugates disclosed herein opens the door to delivering gene editing agents and / or molecular machinery used in the same. In an example, the payload is a gene editing agent. Examples of gene editing agents are well known in the art and are described, for example, in Porteus et al. Annu. Rev. Pharmacol. Toxicol. 2016. 56:163-90.

[0293] Non-limiting examples of suitable gene editing agents include, for example, a triplex-forming molecules, pseudocomplementary oligonucleotides, a CRISPR system, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and intron encoded meganucleases.

[0294] In an example, the payload is a triplex-forming molecules, Examples of triplexforming molecules include triplex forming oligonucleotides (TFOs), peptide nucleic acids (PNAs), Tail clamp peptide nucleic acids (tcPNA), and pseudocomplementary oligonucleotides / PN As .

[0295] In an example, the payload is a CRISPR system, such as a CRISPR / Cas system. CRISPR / Cas systems are well known in the art and are described, for example, in Xu Y. and Li Z. Comput Struct Biotechnol J. 2020. 18:2401-2415. In an example, the CRISPR / Cas system is a CRISPR / Cas9 nickase (e.g., D10A, H840A, N854A, and N863A nickase). In an example, the CRISPR / Cas system is a CRISPR / Cas9 D10A nickase.

[0296] In an example, the payload is a donor oligonucleotide. In an example, the donor oligonucleotide contains a sequence that can correct a mutation in a cell's genome.

[0297] Nucleic acids

[0298] In an example, the payload is a nucleic acid. In an example, the nucleic acid is a DNA molecule (e.g. a single stranded (ss) DNA, or double stranded (ds) DNA). Unless specified, it is to be understood that such DNA molecules are isolated, i.e. it exists as a separate molecule independent of other sequences (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease treatment), as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote.

[0299] In an example, the nucleic acid is a RNA molecule. For example, the nucleic acid is a messenger RNA (mRNA). In another example, the nucleic acid is a micro RNA (miRNA). In an example, the nucleic acid is an RNA interference molecule, for example a short Interfering RNA (siRNA).

[0300] In an example, the nucleic acid is antisense molecule. For example, the nucleic acid is an antisense oligonucleotide.

[0301] Other non-limiting examples of suitable nucleic acids include aptamers, external guide sequences (ESGs), ribozymes, a cyclic dinucleotides. Modulators of DNA replication and repair

[0302] In an example, the payload is a modulator of DNA methylation, a modulator of chromatin, or a modulator of DNA replication and repair.

[0303] Non-limiting examples of modulators of DNA methylation, chromatin, and / or DNA replication and repair include Lysine-specific histone demethylase 1A (ESDI), Enhancer of zeste homolog 2 (EZH2) histone-lysine-N-methyltransferase, DNA methyltransferase 1 (DNMT1), or a histone deacetylase (HDAC).

[0304] Cathepsin inhibitors

[0305] In an example, the pay load is a cathepsin inhibitor. In an example, the payload is a cathepsin B and / or cathepsin S inhibitor.

[0306] In an example, the inhibitor is a cathepsin B inhibitor. Cathepsin B inhibitors are known in the art and are described, for example, in Xie et al. Cell Death Dis. 2023. 14(4):255. Non-limiting examples of cathepsin B inhibitors include, a cystatin (e.g. Stefin A, Stefin B, Cystatin C, Cystatin SN) , peptidyl aldehyde (e.g. Leupeptin, Tokaramide, YM-51084), an aziridinyl peptide (e.g. Miraziridine), an epoxysuccinyl peptide (e.g. E-64), an epoxysuccinyl inhibitor (e.g. E-64d, CA074, CA074Me), an aziridine (e.g. Aziridine-2,3- dicarboxylate containing inhibitor), a nitrile (e.g. Azapeptide nitriles KI 1777, Z-Phe-Ala-CH2F (Z-FA-fmk), or a monoclonal antibody (e.g. ProCatB -CDR3H of Herceptin, mAb 2A2).

[0307] Nucleoside transporter inhibitors

[0308] In an example, the payload is an inhibitor of a nucleoside transporter. As used herein, “inhibitor of a nucleoside transporter” and “nucleoside transporter inhibitor” are used interchangeably.

[0309] Examples of inhibitors of nucleoside transporters are described, for example, in W02021077070. In an example, the inhibitor of the nucleoside transporter is a purine nucleoside analogue, a pyrimidopyrimidine or pteridine derivative, or a flazine calcium channel blocker. In an example, the inhibitor of the nucleoside transporter is dipyridamole or a tautomer, geometrical isomer, optically active form, enantiomeric mixture thereof, pharmaceutically acceptable salt or pharmaceutically active analogue thereof.

[0310] In an example, the inhibitor of the nucleoside transporter is dipyridamole or pharmaceutically acceptable salt thereof. In an example, the inhibitor of the nucleoside transporter is a dipyridamole analogue or pharmaceutically acceptable salt thereof.

[0311] In an example, the inhibitor of the nucleoside transporter is a peptide.

[0312] In an example, the inhibitor of the nucleoside transporter is a binding protein. In an example, the inhibitor of the nucleoside transporter is an antibody or fragment thereof.

[0313] In an example, the inhibitor of the nucleoside transporter is an oligonucleotide inhibitor. In an example, the oligonucleotide inhibitor is an antisense RNA or DNA, siRNA or siDNA, miRNA, miRNA mimic, shRNA or DNA and Chimeric Antisense DNA or RNA. In an example, the inhibitor of the nucleoside transporter is an siRNA, shRNA, or miRNA.

[0314] Other payloads

[0315] In an example, the payload is a drug. The skilled person will appreciate that suitable drugs can be selected based on the disease or disorder to be treated (i.e. by administering a conjugate of the disclosure). For example, when treating cancer, the drug may be a chemotherapeutic agent, cytotoxic agent, radiosensitizer or a combination thereof. For treating cardiovascular disease or ischemia, the drug may increase blood flow, reduce coagulation, induce arterial dilation, induce or increase thrombolysis, protect and prolong survival of cells in the area of ischemia, or a combination thereof. For example, the drug may be a beta-blocker or a thrombolytic. For treating a neurological condition, the drug may he an opioid, an anticonvulsant, dopamine or a dopamine analogue, or a dopamine agonist, or a cholinesterase inhibitor.

[0316] In an example, the payload is an anti-cancer drug. In an example, the drug is chemotherapeutic agent, a cytotoxic agent, and / or a radiosensitizer. Non-limiting examples of drugs that are chemotherapeutic agents, cytotoxic agents, and / or radiosensitizers include methotrexate, vinblastine, doxorubicin, monomethyl auristatin E, camptothecin topoisomerase inhibitors, noncamptothecin topoisomerase inhibitors, a radioisotope, and monomethyl auristatin F. In an example, the drug is a PARP inhibitor, for example, Olaparib. In an example, the drug is niraparib. In an example, the drug is rucaparib. In an example, the drug is monomethyl auristatin E (MMAE). In an example, the drug is doxorubicin. In an example, the drug is a camptothecin or noncamptothecin topoisomerase inhibitor such as irinotecan or topotecan. In an example, the payload is a radioisotope.

[0317] In an example, the payload is a recombinant viral expression vector. In an example, the recombinant viral expression vector is a recombinant adeno-associated virus (AAV) vector, a recombinant adenovirus (AdV) vector, or a recombinant lentivirus (LV) vector.

[0318] Compositions The present disclosure includes pharmaceutical compositions for administration to subjects. Exemplary compositions comprise one or more of the above referenced conjugates.

[0319] The compositions can also contain a pharmaceutically acceptable carrier or adjuvant for administration of the conjugate of the disclosure. In some embodiments, the carrier is pharmaceutically acceptable for use in humans. The carrier or adjuvant should not itself induce the production of antibodies harmful to the individual receiving the composition and should not be toxic. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, poly lactic acids, polyglycolic acids, polymeric amino acids, ammo acid copolymers and inactive virus particles.

[0320] In an example, compositions can also contain DNA. For example, a composition may comprise a conjugate described herein and DNA. In an example, the binding protein component of the composition may be bound to the DNA. In an example, the composition comprises a saturating concentration of DNA so that substantially all binding protein in the composition is bound to DNA. In this example, the composition may facilitate delivery of DNA as well as a conjugated payload to the nucleus of a cell.

[0321] Pharmaceutically acceptable salts can be used, for example mineral acid salts, such as hydrochlorides, hydrobromides, phosphates and sulphates, or salts of organic acids, such as acetates, propionates, malonate and benzoates.

[0322] Pharmaceutically acceptable carriers in therapeutic compositions can additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents or pH buffering substances, can be present in such compositions.

[0323] The compositions of the presently disclosed subject matter can further comprise a carrier to facilitate composition preparation and administration. Any suitable delivery vehicle or carrier can be used, including but not limited to a microcapsule, for example a microsphere or a nanosphere (Manome et al. (1994) Cancer Res 54:5408-5413; Saltzman & Fung (1997) Adv Drug Deliv Rev 26:209-230), a glycosaminoglycan (U.S. Pat. No.6, 106,866), a fatty acid (U.S. Pat. No.5,994,392), a fatty emulsion (U.S. Pat. No.5,651,991), a lipid or lipid derivative (U.S. Pat. No.5, 786, 387), collagen (U.S. Pat. No.5,922,356), a polysaccharide or derivative thereof (U.S. Pat. No.5, 688, 931), a nanosuspension (U.S. Pat. No.5,858,410), a polymeric micelle or conjugate (Goldman et al. (1997) Cancer Res 57:1447-1451 and U.S. Pat. Nos.4, 551,482, 5,714,166, 5,510,103, 5,490,840, and 5,855,900), and a polysome (U.S. Pat. No.5,922,545).

[0324] A composition of the present invention may comprise a pharmaceutical composition that includes a pharmaceutically acceptable carrier. Suitable formulations include aqueous and non-aqueous sterile injection solutions which can contain antioxidants, buffers, bacteriostats, bactericidal antibiotics and solutes which render the formulation isotonic with the bodily fluids of the intended recipient; and aqueous and non- aqueous sterile suspensions which can include suspending agents and thickening agents. The formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a frozen or freeze-dried (lyophilized) condition requiring only the addition of sterile liquid carrier, for example water for injections, immediately prior to use. Some exemplary ingredients are SDS in the range of 0.1 to 10 mg / ml, about 2.0 mg / ml; and / or mannitol or another sugar in the range of 10 to 100 mg / ml, in some embodiments about 30 mg / ml; and / or phosphate- buffered saline (PBS). Any other agents conventional in the art having regard to the type of formulation in question can be used. In some examples, the carrier is pharmaceutically acceptable. In some examples, the carrier is pharmaceutically acceptable for use in humans.

[0325] Compositions of the present disclosure can have a pH between 5.5 and 8.5, preferably between 6 and 8, and more preferably about 7. The pH can be maintained by the use of a buffer. The composition can be sterile and / or pyrogen free. The composition can be isotonic with respect to humans. Compositions of the presently disclosed subject matter can be supplied in hermetically-sealed containers.

[0326] Conjugates of the disclosure and compositions comprising the same can be administered in a variety of unit dosage forms depending upon the method of administration. Dosages for typical binding protein such as antibody or fragment pharmaceutical compositions are well known to those of skill in the art. Such dosages are typically advisory in nature and are adjusted depending on the particular therapeutic context or patient tolerance. The amount binding protein adequate to accomplish this is defined as a “therapeutically effective dose.” The dosage schedule and amounts effective for this use, i.e., the “dosing regimen,” will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient’s health, the patient’s physical status, age, pharmaceutical formulation and concentration of active agent, and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration. The dosage regimen must also take into consideration the pharmacokinetics, i.e., the pharmaceutical composition’s rate of absorption, bioavailability, metabolism, clearance, and the like. See, e.g., the latest Remington’s; Egleton, Peptides 18: 1431-1439, 1997; Langer, Science 249: 1527-1533, 1990.

[0327] Routes of administration include, but are not limited to, injection, subcutaneous, intramuscular, intravenous, intraarterial. In one example, the conjugate or binding protein or composition comprising the same is delivered through intravenous administration. In another example, the conjugate or binding protein or composition comprising the same is delivered through injection. In another example, the conjugate or binding protein or composition comprising the same is delivered through infusion. In another example, the conjugate or binding protein or composition comprising the same is delivered through intraarticular injection into a joint space. In another example, the conjugate or binding protein or composition comprising the same is delivered through intraarticular injection into an arthritic joint.

[0328] The compositions can be administered in a single dose treatment or in multiple dose treatments on a schedule and over a time period appropriate to the age, weight and condition of the subject, the particular binding protein formulation used, and the route of administration.

[0329] Methods of treatment

[0330] The disclosed conjugates can be used to deliver payloads to the nucleus. Accordingly, in an example, the disclosure provides a method of delivering a payload to the nucleus of a cell, comprising contacting the cell with an effective amount of a nuclear penetrating conjugate according to the disclosure. This delivery platform of the disclosure allows for various conditions to be treated by selecting relevant payload(s).

[0331] In an example, the conjugate is administered directly to the subject (e.g. in vivo).

[0332] In an example, the conjugate is administered indirectly to the cell / s of a subject (e.g. ex vivo). In this example, a population of cells is obtained from a subject, the population of cells is contacted with the conjugate, and the population of cells is then administered back into the subject. In an example, the population of cells are autologous. In another example, the population of cells are allogenic (i.e. are obtained from a donor). Suitable populations of cells include, for example, a stem population, an immune cell population (e.g. a T cell population), or a blood cell population.

[0333] Typically an effective amount of nuclear penetrating conjugate is administered to a subject in need thereof. As used herein, the term "effective amount" or "therapeutically effective amount" means a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of the disorder being treated or to otherwise provide a desired pharmacologic and / or physiologic effect. The precise dosage will vary according to the selected payload and a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the disease, and the treatment being effected.

[0334] Gene editing In an example, the disclosure provides a method of gene editing comprising, administering to a subject in need thereof an effective amount of a nuclear penetrating conjugate of the disclosure comprising a gene editing agent payload. Examples of suitable gene editing agents are disclosed above and are generally known in the art (see, e.g. Porteus. Anna. Rev. Pharmacol. Toxicol. 2016. 56: 163-90).

[0335] Gene editing, also known as genome editing, refers to a group of technologies that allow genetic material to be added, removed, or altered at particular locations in the genome. The person skilled in the art would appreciate that gene editing can be used to treat a wide range of diseases. For example, methods of gene editing according to the disclosure can be used to treat genetic disorders. Non-limiting examples of genetic disorders include hemophilia, muscular dystrophy, globinopathies (e.g. sickle cell anemia or beta-thalassemia), cystic fibrosis, xeroderma pigmentosum, lysosomal storage diseases (e.g. Gaucher's disease, Fabry disease, or Hurler syndrome), immune deficiency syndromes such as X-linked severe combined immunodeficiency and ADA deficiency, tyrosinemia, Fanconi anemia, the red cell disorder spherocytosis, alpha- 1- anti-trypsin deficiency, Wilson’s disease, Leber’s hereditary optic neuropathy, or chronic granulomatous disorder. The globinopathy can be sickle cell anemia or beta-thalassemia.

[0336] Tn an example, gene editing can be used to treat a HIV infection. For example, gene editing can be used to induce a mutation that reduces HIV infection, for example, by reducing an activity of a cell surface receptor that facilitates entry of HIV into the cell.

[0337] In an example, gene editing can be used in cell therapy, such as CAR-T cell therapy or stem cell therapy. In this example, the cells (e.g. hematopoietic stem cells or immune cells) are contacted with the conjugate ex vivo and then administered to a subject in need thereof.

[0338] Cathepsin-related diseases

[0339] The findings reported herein provide basis for treating diseases associated with cellular accumulation of cathepsin, comprising administering to a subject in need thereof an effective amount of a nuclear penetrating conjugate of the disclosure. The conjugate can comprise a cathepsin inhibitor payload, preferably a cathepsin B inhibitor payload, and / or an additional therapeutic agent suitable for treating the disease. In an example, the conjugate comprises a a cathepsin B inhibitor payload. In an example, the conjugate comprises a a cathepsin B inhibitor payload and an additional therapeutic agent. In an example, the conjugate comprises a therapeutic agent suitable for treating the disease.

[0340] Diseases associated with cellular accumulation of cathepsin are known in the art and are disclosed, for example, in Xie et al. Cell Death Dis. 2023. 14(4):255. Examples of diseases associated with cellular accumulation of cathepsin Alzheimer’s disease, Parkinson’s disease, traumatic brain injury, cerebral ischemia, diabetes, myocardial infarction, diabetic cardiomyopathy, stroke, pancreatitis, liver disease, lysosomal storage disorders (LSDs), retinitis pigmentosa, Kawaski disease, or Sjogren syndrome. In an example, the disease is (or is associated with) cardiovascular disease such as myocardial infarction, diabetes, diabetic cardiomyopathy, and stroke.

[0341] Neurological and neuro generative diseases hi an example, the disclosure provides a method of neurodegeneration or a neurological disease, comprising administering to a subject in need thereof an effective amount of a nuclear penetrating conjugate of the disclosure.

[0342] Neurodegeneration is a process that results in the loss or damage of neurons in the brain. This process is characteristic of several debilitating, incurable conditions known as neurodegenerative diseases. Accordingly, “neurodegenerative disease” refers to conditions which primarily affect the neurons in the human brain. “Neurological disease” refers to conditions that affect the nervous system, which includes the brain, spinal cord, and peripheral nerve. As the skilled person would appreciate, a neurodegenerative disease can also be a neurological disease, and vice-versa.

[0343] Tn an example, the neurological disease is Alzheimer’s disease, Parkinson’s disease, traumatic brain injury, cerebral ischemia, retinitis pigmentosa, lyosomal storage disease, amyotrophic lateral ischemia, spinocerebral ataxia, or HIV-inf ection.

[0344] In an example, the conjugate comprises a cathepsin inhibitor payload, preferably a cathepsin B inhibitor. In an example, the payload further comprises an additional therapeutic agent suitable for treating the disease. For example, the conjugate comprises a payload comprising a cathepsin B inhibitor and a therapeutic agent for treating Alzheimer’s disease (e.g. a acetylocholinesterase inhibitor such as galantamine, rivastigmine, or donepezil). The person skilled in the art would be able to determine therapeutic agents that are suitable for treating the above referenced diseases.

[0345] The findings reported herein also provide basis for a method of treating neurodegeneration or neurological disease, comprising administering to subject a nuclear penetrating anti-DNA binding protein in an amount effective to attenuate nuclear cathepsin activity. As disclosed herein, results show that nuclear penetrating anti-DNA binding proteins translocate cathepsin’s into the nucleus without resulting in cell-death. This supports the conclusion that anti-DNA binding proteins have the ability to attenuate nuclear cathepsin activity.

[0346] In this example, an amount effective to attenuate nuclear cathepsin activity refers to amount of nuclear penetrating anti-DNA binding protein effective to prevent cell death under culture conditions. This can be determined by treating a population of cells with an anti-DNA binding protein in cell culture and measuring whether cell death is prevented using routine cell death assays known in the art.

[0347] Examples of suitable neurodegenerative or neurological diseases are disclosed above. In an example, the neurodegenerative or neurodegeneration or neurological disease is Alzheimer’s disease, Parkinson’s disease, or traumatic brain injury, or cerebral ischemia. In an example, the neurodegenerative or neurological disease is aging.

[0348] Autoimmune diseases

[0349] In an example, the disclosure provides a method of treating an autoimmune disease, comprising, administering to a subject in need thereof an effective amount of a nuclear penetrating conjugate according to the disclosure. In an example, the payload is an inhibitor of a nucleoside transporter. Suitable nucleoside transporters are disclosed above. In an example, the payload further comprises an additional therapeutic agent suitable for treating the autoimmune disease. Examples, of suitable therapeutic agents include a steroid, colchicine, hydroxychloroquine, sulfasalazine, dapsone, methotrexate, mycophenolate mofetil, or azathioprine.

[0350] Examples of autoimmune diseases include systemic lupus erythematosus (lupus or SLE), CNS, lupus, systemic sclerosis (scleroderma), Graves’ disease, myasthenia gravis, autoimmune hemolytic anemia, and pemphigus vulgaris, and additionally may contribute to the severity of disease in other autoimmune diseases such as rheumatoid arthritis, autoimmune thrombocytopenia, autoimmune atrophic gastritis of pernicious anemia, myasthenia gravis, Goodpasture’s syndrome, diabetes mellitus, multiple sclerosis, Hashimoto’s thyroiditis, Crohn’s disease, and Sjogren’s syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, POEMS syndrome, dermatomyositis, inclusion body myositis, inflammatory myopathies, vasculitis syndromes including but not limited to Churg-Strauss Syndrome, Wegener granulomatosis, Behcet’s disease, Buerger’s disease, Kawasaki disease, Takayasu’s arteritis, Henoch-Schonlein purpura, Giant cell arteritis, or polyarteritis nodosa.

[0351] In an example, the autoimmune disease is SLE.

[0352] Cancer

[0353] In an example, the disclosure provides a method of treating cancer, comprising, administering to a subject in need thereof an effective amount of a nuclear penetrating conjugate according to the disclosure. In an example, the payload is a chemotherapeutic agent, a cytotoxic agent, or a radiosensitiser. Examples of suitable chemotherapeutic agents, cytotoxic agents, and radiosensitiser are disclosed above. In an example, the payload is Olaparib. The types of cancer that can be treated with the conjugates of the disclosure include, but are not limited to, vascular cancer such as multiple myeloma; adenocarcinomas and sarcomas of bone, bladder, brain, breast, cervical, colo-rectal, esophageal, kidney, liver, lung, nasopharangeal, pancreatic, prostate, skin, stomach, and uterine. In an example, the disclosed conjugates are used to treat multiple cancer types concurrently. The conjugates can also be used to treat metastases or tumors at multiple locations. In an example, the cancer is brain cancer. For example, the brain cancer may be glioblastoma.

[0354] Conjugates according to the disclosure are particularly usefully for treating cells with impair DNA repair, and can be used alone or in combination with DNA damaging agents and radiotherapy.

[0355] In an example, the conjugate is lethal to cells with impaired DNA repair. The cells can be defective in the expression of a gene or in the function of a protein involved in DNA repair, DNA synthesis, or homologous recombination. Exemplary genes and associated products include XRCC1, ADPRT (PARP-1), ADPRTL2, (P ARP-2), POLYMERASE BETA, CTPS, MLH1, MSH2, FANCD2, PMS2, p53, p21, PTEN, RPA, RPA1, RPA2, RPA3, XPD, ERCC1, XPF, MMS19, RAD51, RAD51B, RAD51C, RAD51D, DMC1 , XRCCR, XRCC3, BRCA1 , BRCA2,PALB2, RAD52, RAD54, RAD50, MRE11, NB51 , WRN, BLM, KU70, KU80, ATM, ATR CHK1, CHK2, FANC family of genes, FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCL, FANCM, RADI, and RAD9. If required, genetic mutation(s) in one or more of these genes can easily be determined prior to therapy via standard genomic testing.

[0356] In an example, the defective gene is a tumor suppressor gene.

[0357] In an example, the gene is associated with maintenance of chromosomal integrity and / or protection from genotoxic stress. In some embodiments, the cells are deficient in single and / or double strand break repair.

[0358] In an example, the cells have one or more mutations in BRCA1, BRCA2, and / or PTEN. Gene mutations, such as BRCA1, BRCA2, PTEN mutations, can be identified using standard PCR, hybridization, or sequencing techniques.

[0359] In an example, the conjugates can be used to treat cancers arising from DNA repair deficient familial syndromes, such as breast, ovarian, and pancreatic cancers. For example, the conjugates can be used to treat cancers that are linked to mutations in BRCA1, BRCA2, PALB2, or RAD51B, RAD51C, RAD 5 ID, or related genes. The conjugates can also be used to treat colon cancers, endometrial tumors, or brain tumors linked to mutations in genes associated with DNA mismatch repair, such as MSH2, MLH1, PMS2, and related genes. The conjugates can also be used to treat cancers with silenced DNA repair genes, such as BRCA1, MLH1, OR RAD51B, RAD51C, orRAD51D. The conjugates can also be used to treat cancers associated with chromosomal maintenance or genotoxic stress, for example, cancers in which PTEN is mutated or silences. PTEN is frequently inactivated in many cancers including breast, prostate, glioma, melanoma, and lung cancers.

[0360] A representative but non-limiting list of cancers that can be treating using the disclosed conjugates include cancers of the blood and lymphatic system (including leukemias, Hodgkin's lymphomas, non-Hodgkin’s lymphomas, solitary plasmacytoma, multiple myeloma), cancers of the genitourinary system (including prostate cancer, bladder cancer, renal cancer, urethral cancer, penile cancer, testicular cancer,), cancers of the nervous system (including mengiomas, gliomas, glioblastomas, ependymomas) cancers of the head and neck (including squamous cell carcinomas of the oral cavity, nasal cavity, nasopharyngeal cavity, oropharyngeal cavity, larynx, and paranasal sinuses), lung cancers (including small cell and non-small cell lung cancer), gynecologic cancers (including cervical cancer, endometrial cancer, vaginal cancer, vulvar cancer ovarian and fallopian tube cancer), gastrointestinal cancers (including gastric, small bowel, colorectal, liver, hepatobiliary, and pancreatic cancers), skin cancers (including melanoma, squamous cell carcinomas, and basal cell carcinomas), breast cancer (including ductal and lobular cancer and triple negative breast cancers), and pediatric cancers (including neuroblastoma, Ewing's sarcoma, Wilms tumor, medulloblastoma).

[0361] In an example, the cancer is brain cancer. In an example, the brain cancer is glioma. In an example, the brain cancer is glioblastoma.

[0362] In another example, the cancer is breast cancer. In an example, the breast cancer is triple negative breast cancer. In an example, the breast cancer is estrogen receptorpositive (ER+) breast cancer.

[0363] In another example, the cancer is lung cancer. In an example, the lung cancer is an adenocarcinoma.

[0364] In another example, the cancer is colon cancer. In an example, the colon cancer is colorectal cancer. In an example, the colon cancer is an adenocarcinoma.

[0365] Infectious diseases

[0366] Cathepsins, in particular cysteine cathepsins, are known to be implicated in viral entry and unloading. Accordingly, it is envisioned that a nuclear penetrating conjugate according to the disclosure may be used for impairing cathepsin-mediated viral activity. Results also show that nuclear penetrating conjugates of the disclosure increase translocation of cathepsins into the nucleus. In certain examples, it is also envisioned that this mechanism can be exploited in the context of infectious diseases by enhancing cathepsin activity in the nucleus to promote immune cell activity against pathogens.

[0367] Accordingly, in an example, the disclosure provides a method of treating an infectious disease, comprising, administering to a subject in need thereof an effective amount of a nuclear penetrating conjugate according to the disclosure.

[0368] In an example, the infectious disease is a virus. A representative but non-limiting list of viruses that can be treating using the disclosed conjugates include hepatitis virus, herpes simplex virus, rhinovirus, respiratory syncytial virus, influenza virus, cytomegalovirus, coronavirus, human immunodeficiency virus (HIV), and ebolavirus. In an example, the virus is a coronavirus. In an example, the virus is HIV. In an example, the virus is Ebola.

[0369] In an example, the nuclear penetrating conjugate comprises a cathepsin inhibitor payload. In an example, the nuclear penetrating conjugate comprises an anti-viral agent.

[0370] Dose control

[0371] Results show that nuclear penetrating anti-DNA binding proteins, and nuclear- penetrating conjugates comprising the same, penetrate cells via the nucleoside transporter ENT2 pathway. Transcellular transport of the conjugate can therefore be prevented by blocking the ENT2 pathway. Accordingly, the findings reported herein underpin methods of delivery / treatment which further comprise a step of administering to the subject an effective amount of an inhibitor of a nucleoside transporter to reduce or block nuclear delivery of the payload.

[0372] It has also been shown that conjugates comprising anti-DNA binding proteins and a cathepsin labile linker (e.g. a cathepsin B labile linker) do not release their payload into the cell when a cathepsin inhibitor is also present. The findings reported herein also underpin methods of delivery / treatment which further comprise a step of administering to the subject an effect amount of a cathepsin B inhibitor.

[0373] Such methods are useful in the context of titrating or controlling dose of the conjugate and / or preventing the activity of the conjugate (i.e. a safety-switch which blocks activity in the nucleus). Clinical situations where this might be desirable include where the subject has an adverse reaction, where the subject achieves a clinical response and no further treatment is required, or where a clinician is required to optimise or titrate the dose of the conjugate.

[0374] In an example, the inhibitor of the nucleoside transporter inhibits activity or expression of one or more of ENT1, ENT2, ENT3, or ENT4, optionally at least or only ENT2. Examples of suitable inhibitors of the nucleoside transporters are disclosed above and are described, for example, in W02021077070. In an example, the inhibitor of the nucleoside transporter is dipyridamole or pharmaceutically acceptable salt thereof. In an example, the inhibitor of the nucleoside transporter is a dipyridamole analogue or pharmaceutically acceptable salt thereof.

[0375] The person skilled in the art will appreciate that any inhibitor that inhibits the particular cathepsin responsible for linker cleavage can be used. In an example, the cathepsin inhibitor is a cathepsin B inhibitor. Non-limiting examples of cathepsin B inhibitors include, a cystatin (e.g. Stefin A, Stefin B, Cystatin C, Cystatin SN) , peptidyl aldehyde (e.g. Leupeptin, Tokaramide, YM-51084), an aziridinyl peptide (e.g. Miraziridine), an epoxysuccinyl peptide (e.g. E-64), an epoxysuccinyl inhibitor (e.g. E- 64d, CA074, CA074Me), an aziridine (e.g. Aziridine-2,3- dicarboxylate containing inhibitor), a nitrile (e.g. Azapeptide nitriles KI 1777, Z-Phe-Ala-CH2F (Z-FA-fmk), or a monoclonal antibody (e.g. ProCatB -CDR3H of Herceptin, mAb 2A2). In an example, the cathepsin B inhibitor is E64. In an example, the cathepsin B inhibitor is CA074Me.

[0376] The conjugate and nucleoside transport inhibitor or cathepsin B inhibitor can be administered to a subject in need thereof by a parenteral, enteral, transdermal, or transmucosal route of administration.

[0377] In an example, the subject is administered the conjugate separately or together with the inhibitor of the nucleoside transporter. In an example, the subject is administered the conjugate separately or together with the cathepsin B inhibitor.

[0378] Kits

[0379] Another example of the disclosure provides kits containing a conjugate of the present disclosure.

[0380] In one example, the kit comprises (a) a container comprising a conjugate optionally in a pharmaceutically acceptable carrier or diluent, and (b) at least one further therapeutically active compound or drug. In an example, the therapeutically active compound or drug is an inhibitor of a nucleoside transporter, e.g. dipyridamole or pharmaceutically acceptable salt thereof.

[0381] Such kits may be useful for methods of treatment disclosed herein. In an example, the kit further comprises a package insert with instructions for titrating or controlling dose of the conjugate and / or preventing the activity of the conjugate.

[0382] In an example, the kit comprises (a) at least one conjugate optionally in a pharmaceutically acceptable carrier or diluent; (b) at least one further therapeutically active compound or drug; e.g. an inhibitor of a nucleoside transporter; and (c), optionally, a package insert with instructions for using the kit in a method of treating a subject in need thereof. In accordance with this example of the disclosure, the package insert is on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds or contains a composition that is effective for inhibiting complement activity or for treating or preventing or delaying progression of a complement-mediated disorder and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is the sCRl variant. The label or package insert indicates that the composition is used for treating a subject eligible for treatment, e.g., one having or predisposed to developing a complement-mediated disorder with specific guidance regarding dosing amounts and intervals of the sCRl variant and any other medicament being provided. The kit may further comprise an additional container comprising a pharmaceutically acceptable diluent buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and / or dextrose solution. The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0383] SEQUENCES OF THE DISCLOSURE

[0384]

[0385] EXAMPLES

[0386] Example 1 : DX3 is a nuclear-penetrating ANA

[0387] The autoantibody 3E10 isolated from the MRL / lpr lupus mouse model is an IgG2a kappa ANA that binds DNA, localizes to tumors, crosses outer cell membranes through the nucleoside transporter ENT2, and penetrates the nucleus where it inhibits DNA repair. In the present work humanized 3E10 complementarity-determining regions (CDRs) were grafted onto a human IgGl framework bearing the Asn297Asp (N297D) mutation that reduces FcyR interactions. The activity of the resulting antibody, hereafter referred to as DX3, was characterized.

[0388] DX3 bound a test 30-mer single-stranded DNA oligonucleotide ligand with KD 113 nM determined by surface plasmon resonance (SPR) (Fig. 1A) and penetrated the nuclei of live colon cancer and glioma cells and normal human astrocytes (NHAs) (Fig. IB, 1C). Promotion and inhibition of nucleoside transport by addition of the nucleoside adenosine or the nucleoside transport inhibitor dipyridamole enhanced and suppressed DX3 uptake, respectively (Fig. 2A, 2B). Confocal fluorescence microscopy of DX3- treated cells immunostained for IgG and the lysosomal marker LAMP1 demonstrated DX3 nuclear penetration and lysosomal avoidance (Fig. 2C).

[0389] Example 2: DX3 induces cathepsin nuclear translocation.

[0390] As noted, antibodies that target cell surface antigens are internalized through endocytosis and subsequently degraded in lysosomes, but mechanisms responsible for catabolism of antibodies that penetrate nuclei and avoid lysosomes are unknown. Some lysosomal cathepsin proteases are released into the cytoplasm and translocate into the nucleus and are implicated in mechanisms of nucleocytoplasmic shuttling and regulation of DNA repair proteins such as BRCA1. Cathepsins involved in nuclear import and export (CatB, CatK, CatL, and CatS) were examined in U87 glioma cells (Figs. 3A-3B) and NHAs (Figs. 4A-4B) 15 minutes after treatment with buffer control, IgG control, or DX3 by confocal fluorescence microscopy. IgG control did not impact cathepsin localization in glioma or NHAs, but DX3 caused a marked shift of CatB and CatS signal into the nucleus in both cell types. In contrast, DX3 had no effect on CatK or CatL (Figs. 3A-3B, 4A-4B). DX3 induction of nuclear accumulation of pro-CatB and pro-CatS (as distinguished by MW 50 kDa) was orthogonally verified by western blots of nuclear extracts from IgG control or DX3-treated U87 glioma cells (Fig. 5A, 5B). Taken together, these results demonstrate that CatB and CatS rapidly accumulate in the nucleus of DX3-treated cells, which indicates they are involved in mechanisms of DX3 nuclear import and / or catabolism.

[0391] Example 3: DX3 promotes loss of BRCA1 and is synthetically lethal to BRCA2-deficient tumors.

[0392] 3E10, the prototype autoantibody on which DX3 is based, inhibits homologous recombination (HR) and causes DNA damage to accumulate in and kill tumor cells with pre-existing defects in the DNA damage response (DDR). Previous theories on the mechanism by which 3E10 inhibits HR center on antibody steric hindrance of repair factor access to damaged DNA, but the present work raises the possibility that nuclear accumulation of cathepsins provoked by antibody penetration into nuclei causes loss of important HR proteins such as BRCA1. Notably, CatS regulates BRCA 1 protein stability and is recognized as a prognostic marker in some malignancies. Total cell lysates of U87 glioma cells treated with TgG control or DX3 were analyzed by western blots probed for CatS and BRCA1. Compared to IgG control, DX3-treated cells exhibited increased mature activated CatS (based on size 24 kDa) and correspondingly reduced BRCA1 content (Fig. 5C-5F). This finding indicates cathepsin- mediated degradation of BRCA1 is a likely mechanism of HR inhibition by 3E10 / DX3.

[0393] The impact of DX3 on HR-deficient tumors was examined. 3E10 has previously been shown to be synthetically lethal to cancer cells with HR-defects, but the sensitivity of matched HR-proficient and deficient tumors to 3E10 has not been directly compared in vivo. Matched BRCA2-deficient and WT DLD1 tumors were simultaneously established in the flanks of athymic nude mice, with BRCA2-deficient tumors in the right flank and WT in the left. Mice were then randomized to treatment with single agent DX3 (50 mg / kg) (N=8) or vehicle control (N=8) by tail vein injection twice a week. DX3 did not yield any significant suppression of BRCA2-WT tumors compared to vehicle control (Fig. 6A, 6B). In contrast, DX3 significantly suppressed growth of the BRCA2-deficient tumors (Fig. 6C, 6D). These findings confirm differential sensitivity of BRC A2-deficient and WT tumors to DX3 in vivo and are consistent with the present finding of BRCA1 loss promoted by DX3 and previous reports of inhibition of DNA repair and 3E10 selective toxicity to BRCA2- deficient cancer cells in cell culture work. Example 4: Leveraging cathepsin nuclear translocation to design an antinuclear antibody-drug conjugate (ANADC).

[0394] In addition to pointing to BRCA1 loss as a likely mechanism underlying HR inhibition by DX3, the present discovery of DX3 induction of cathepsin nuclear translocation offered a path towards design of a first-in-class ANADC based on a cathepsin-labile drug linker strategy. The dipeptide valine-citrulline (VC) linker, which is already in use in clinical stage conventional ADCs such as brentuximab vedotin, was originally designed to be specifically cleavable by CatB. Here the VC linker strategy to develop a DX3-based ADC (hereafter referred to as ANADC) that leverages CatB nuclear accumulation in DX3-treated cells to facilitate drug release from the CatB- labile linker (Fig. 7 A).

[0395] ANADC was generated by maleimide-based linkage of DX3 to the anti-mitotic agent monomethyl auristatin E (MMAE) with incorporation of the CatB-labile VC dipeptide and self-immolative PABA spacer. IgG control linked to MMAE was prepared using the same protocol and is hereafter called ADC control. Purity and absence of free drug was confirmed by size exclusion high performance liquid chromatography (SEC- HPLC) and liquid chromatography mass spectrometry (LC-MS). Both ADCs were >98% pure with undetectable free drug, and drug-antibody-ratios (DARs) were closely matched between ANADC and ADC control at 4.5-4.8 and 4.7-4.9 as determined by LC-MS and hydrophobic interaction chromatography (HIC), respectively (Figs. 8A-8B).

[0396] ANADC activity in cell culture was characterized. U87 glioma cells treated with IgG control, ADC control, unconjugated DX3, or ANADC for 60 minutes were washed, fixed, and immunostained to detect antibody uptake. IgG control and ADC control showed minimal uptake into cells. Unconjugated DX3 localized into nuclei, while ANADC signal was detected in both nuclear and cytoplasmic compartments including some punctate foci of staining in the cytoplasm. Brightfield examination of cell morphologies showed loss of normal contours in ANADC-treated cells compared to cells treated with IgG control, ADC control, or unconjugated DX3. These findings indicate that cell death caused by release of MMAE from ANADC allowed some escape of DX3 from the nucleus into the cytoplasm (Fig. 7B, Fig. 9A). To further probe this effect, ANADC localization in U87 cells was examined at earlier time points by immunofluorescence. At 15 and 30 minutes after treatment ANADC signal was primarily in the nucleus, with progressive escape into the cytoplasm at 45-60 minutes (Fig. 9B) consistent with cell death and dissolution of nuclear membranes secondary to MMAE release. ANADC toxicity to the U87 cells was subsequently evaluated in colony formation assays. Cells tolerated IgG control, ADC control, and unconjugated DX3 over a dose range of 0.5 to 31.25 nM but were significantly killed by ANADC at concentrations above 7 nM (Fig. 7C). Lastly, the ability of ANADC to induce CatB and CatS nuclear accumulation was confirmed by fluorescence confocal microscopy. ANADC-treated U87 glioma cells and NHAs showed nuclear localization by CatB and CatS, but not CatK and CatL, consistent with the effects of unconjugated DX3 (Fig. 7D, Fig. 10).

[0397] Example 5: ANADC prolongs survival in a U87 orthotopic glioma model.

[0398] The nucleoside transporter ENT2 manages nucleoside flux at the blood-brain barrier (BBB) and facilitates BBB crossing and brain tumor localization by a 3E10 fragment. Based on this, experiments were designed to test if DX3 would localize to orthotopic brain tumors and that ANADC would deliver cargo drug to tumors to prolong survival in an orthotopic brain tumor model. The 3E10 di-scFv fragment that was previously shown to localize to orthotopic glioma is a smaller than the full IgGl design in DX3, which may be advantageous in crossing the BBB. Biodistribution and trafficking to brain tumors by the full IgGl DX3 and IgG control was evaluated in NMRI-Foxnlnumice. Mice bearing orthotopic U87 brain tumors confirmed by MRI were randomized to treatment with125I-radiolabeled IgG control (N=5) or DX3 (N=5). Brains were taken six hours after treatment from two mice in each group, and tumors and normal brain regions separated for analysis of antibody uptake by gamma counter. Results expressed as percentage of injected dose / gram of tissue (%ID / g) showed increased DX3 localization into brain tumor compared to IgG control (%lD / g 2.7+0.3 versus 1. 1+0.2, P<0.05) but not into normal brain cortex or other regions of normal brain (Fig. 11 A, 1 IB). Brains and additional normal tissues (blood, kidneys, liver, lungs, spleen) from the remaining three mice in each group were taken for gamma counting at 96 hours post-treatment. DX3 was present at higher concentrations than IgG control in blood (1.02+0.31 versus 0.14+0.01, P<0.05), but hepatic uptake of DX3 was lower than IgG control (0.39+0.09 versus 0.95+0.14, P<0.05). The remaining tissues did not show any statistically significant differences between IgG control and DX3 content (Fig. 11C). These data indicate DX3 bioavailability to orthotopic brain tumors without significant deposition in normal brain or other tissues relative to IgG control and supported testing of ANADC efficacy in a brain tumor model.

[0399] Athymic nude mice bearing orthotopic U87 brain tumors confirmed by I VIS were randomized to treatment with four cycles of weekly tail vein injections of control buffer (N=6), DX3 (10 mg / kg) (N=6), or ANADC (10 mg / kg) (N=5). Mice were monitored and euthanized for weight loss or neurologic changes. ANADC was well-tolerated and significantly prolonged survival compared to buffer or unconjugated DX3, with median survivals of 68, 49.5, and 47 days, respectively (P<0.01, log-rank test) (Fig. 1 ID). All treatments were well-tolerated based on stability of behaviours and body mass (Fig. 23A). IVIS images taken on day 29 (one day after the last treatment) showed an apparent suppression of tumor growth associated with ANADC (Fig. 23B). A schematic for the brain tumour localisation and survival studies in the U87 glioma model is shown in Fig. 23C.

[0400] Example 6: ANADC suppresses extracranial breast cancer tumors.

[0401] An extracranial breast cancer model was subsequently used both to confirm tumor agnostic behavior of ANADC and to compare the efficacy of ANADC and ADC control against tumors not sequestered behind the BBB. ANADC penetration into MCF7 breast cancer cells in culture was confirmed (Fig. 12A), and then mice bearing MCF7 subcutaneous xenograft tumors with mean volume -150 mm3 were randomized to treatment with four weekly intraperitoneal (IP) injections of control buffer, unconjugated DX3 (10 mg / kg) (N=6 mice per group), ADC control (10 mg / kg), or ANADC (10 mg / kg). Study endpoints were tumor volume or changes in behavior or weights. ANADC significantly suppressed tumor growth compared to all controls. Mean tumor volumes one week after completion of treatment were 216+45 and 960+85 mm3 in mice treated with ANADC or ADC control, respectively (P<0.01 , two- tailed Student’s t-test) (Fig. 9B). Median tumor tripling times in mice treated with buffer, DX3, or ADC control were 7, 8.5, and 10 days, respectively, compared to 45 days in mice treated with ANADC (P<0.01, log-rank test) (Fig. 12C). Because the MCF7 tumors are estrogen responsive, the study was stopped at day 60 based on the expected lifespan of the implanted 17-3- estradiol pellets. All treatments were well-tolerated, with no significant differences in body mass observed throughout treatment (Fig 12D). Taken together, the finding of ANADC activity against both brain and breast cancer tumors in vivo is consistent with tumor agnostic targeting. ANADC appeared to have a greater magnitude of effect in the breast cancer model, which is likely due to absence of the BBB.

[0402] Example 7: Model of ANA-cathepsin interactions and implications.

[0403] A central finding of this study is the discovery of cathepsin protease nuclear translocation induced by a cell-penetrating ANA. Corollary findings include recognition of ANA-provoked activation of CatS with associated loss of BRCA1. The findings unlock the opportunity to harness nuclear cathepsins in the design of therapeutic agents such as ANADC that use nucleoside salvage pathways to cross live cell membranes and localize into the nucleus. A schematic illustrating these concepts is shown in Fig. 13.

[0404] Example 8: Methods Antibodies and cell lines.

[0405] DX3 (PAT-DX3, Patrys Ltd., Melbourne, Australia) was designed as detailed in the results and generated in CHO cells and purified. Matched BRCA2-WT and BRCA2- deficient DLD1 cells, U87 glioma and MCF7 breast cancer cells, and NHA were obtained from Horizon Discovery (Cambridge, UK), ATCC (Manassas, VA), and Lonza (Morristown, NJ), respectively.

[0406] DNA binding assay.

[0407] KD for DX3-DNA binding was determined by surface plasmon resonance using the Biacore T200 (Cytiva, Marlborough, MA, USA). The CM5 sensor chip in flow cells (FC) 1 and 2 were coated with neutravidin using the Amine Coupling Kit (Cytiva, BR100050). FC1 was used as reference cell. A 30-mer biotinylated single- stranded DNA ligand (0.025 pM, Sigma-Aldrich) was then immobilized onto the MC5 chip in FC2 through interaction with neutravidin. FC2 was used as the active flow cell. After three conditioning cycles with 5 M NaCl and six startup cycles with PBS pH 7.4, the analyte DX3 in PBS was added with flow rate 30 pl / min, contact time 120 seconds, and dissociation time 400 seconds. Results were analyzed using BIA Evaluation Software version 3.1 using a 1 : 1 fit kinetic binding analysis. The experiment was performed twice, with KDs of 113 nM and 112 nM determined in each experiment.

[0408] Cell penetration assays.

[0409] DLD1, U87, MCF7, or NHA cells were treated with 4 pM IgG control, ADC control, DX3, or ANADC for the indicated times after which cells were washed, fixed in chilled 100% ethanol, and immunostained to detect antibody with Alexa Fluor 488 (AF488) conjugated secondary antibody detection and DAPI counterstain. Images were obtained using an EVOS fl digital fluorescence microscope (Advanced Microscopy Group, Bothell, WA) and a Zeiss Axioimager Ml Microscope (Zeiss, Germany).

[0410] Lysosomal and cathepsin localization assays.

[0411] Cells treated with 4 pM buffer control, IgG control, or DX3 for 15 minutes were washed, fixed, immunostained for LAMP1 (Invitrogen, 14-1079-80), CatB (Invitrogen, MA5-32651), CatK (Proteintech, 11239-1- AP), CatL (Invitrogen, BMS1032), or CatS (Invitrogen, PA5-82049), co-immunostained for IgG and subjected to DAPI nuclear counterstain and visualized under fluorescence confocal microscopy. Nuclear extracts of U87 glioma cells treated with DX3 were probed for CatB and CatS antibodies described above. BRCA2-WT and deficient tumor xenograft study.

[0412] Female a thymic nude mice were injected subcutaneously with matched DLD1 BRCA2-WT and BRCA2-deficient cells to generate matched tumors in the same mouse. 2x106 DLD1 BRCA2-WT cells were injected in the left flank and 2x106 DLD1 BRCA2- deficient cells in the right flank. Once tumors reached mean volume of -100 mm3 mice were randomized to groups for tail vein injection of vehicle control (N=8) or DX3 (50 mg / kg) (N=8) twice per week. Tumor volumes were tracked by caliper measurements and mouse weights and behaviors monitored through the study. Kaplan-Meier plots of tumor tripling time (relative to tumor volumes on day 0 of treatment) were generated in Prism 9.5.1 (GraphPad Software, San Diego, CA, USA). Studies were conducted under an IACUC approved protocol, and mice were humanely euthanized for endpoints of tumor size, weight loss, or behavior changes.

[0413] Generation ofANADC and ADC control.

[0414] Isotype control IgG or DX3 at 5 mg / mL were incubated with TCEP at 37oC for two hours for reduction, followed by conjugation to VC- MMAE by incubation in 50 mM PBS pH 7.0 for two hours at 4°C. DX3 was conjugated to the cytotoxic drug monomethylauristatin E (MMAE) via modification of native cysteine side chain thiols. This method involves reduction of the solvent-exposed disulfides with dithiothreitol (DTT) or tris(2-carboxyethyl) phosphine (TCEP) followed by modification of the resulting thiols with maleimide-containing drugs. The thiols were modified with MC- VC-PAB-MMAE, which incorporates a cathepsin B protease cleavage site (VC: valine, citrulline) and a self-immolative linker (PAB: para-aminobenzyloxycarbonyl) between the maleimide group (MC: maleimidocaproyl) and the cytotoxic drug (MMAE). The DX3-MMAE was purified to generate 4 drug molecules per Ab (DAR4). DAR, purity, and percentage of free drug was determined by HIC-HPLC, SEC-HPLC, and LC-MS. ADCs were >98% pure and had undetectable free drug. Minimal levels of endotoxin were confirmed by PTS Cartridge.

[0415] Cytotoxicity assays.

[0416] U87 cells cultured in 12- well plates were treated with titrated doses of IgG control, ADC control, unconjugated DX3, or AN ADC and cell survival was evaluated by colony formation assay and plotted in Prism version 9.5.1.

[0417] Biodistribution study.

[0418] Female NMRl-Foxnlnu mice (Charles River) aged 6 weeks were subjected to stereotactic intracranial inoculation of 50,000 U87 glioma cells in 2 L PBS as previously described. Tumors were confirmed by volumetric MRI two weeks after inoculation. Mean volumes were 4.3 ± 0.1 mm3 at the time of the study. Mice were treated with tail vein injections of ~35 pg (corresponding to an activity of ~16 MBq) radiolabeled IgG control (N=5) or DX3 (N=5), and tissues were taken and analyzed for antibody uptake by gamma counter (Wizard 2, PerkinElmer, Finland) at 6 and 96 hours (N=2 and 3 / group, respectively). Results are expressed as percentage of injected dose per gram of tissue analyzed (%ID / g).

[0419] U87 orthotopic glioma survival study.

[0420] Orthotopic U87 glioma tumors were established in female athymic nude mice aged 6 weeks by intracranial inoculation of 50,000 luciferase-expressing cells. One week after inoculation mice were evaluated for tumors by IVIS and then randomized into groups for four weekly treatments by tail vein injection of control buffer (N=6), unconjugated DX3 (N=6), and ANADC (N=5). Mice were closely observed and euthanized for humane endpoints of neurologic or behavior changes or weight loss. Kaplan-Meier survival curves were plotted in Prism 9.5.1.

[0421] MCF7 breast cancer xenograft study.

[0422] Female Balb / c nude mice were administered 60- day release 17-P-estradiol pellets, and three days later injected subcutaneously with 5xl06MCF7 cells in Matrigel to generate flank tumors. Once tumors reached mean volume of -150 mm3 mice were randomized to groups for intraperitoneal treatment with vehicle control (N=6), ADC control (10 mg / kg) (N=6), unconjugated DX3 (10 mg / kg) (N=6), or ANADC (10 mg / kg) (N=6) once a week for four weeks. Tumor volumes were tracked by caliper measurements and mouse weights and behaviors monitored through the study. Kaplan- Meier plots of tumor tripling time (relative to tumor volumes on day 0 of treatment) were generated in Prism 9.5.1 (GraphPad Software, San Diego, CA, USA). One mouse in the ANADC group was lost (unrelated to tumor or treatment) prior to reaching tumor tripling time and was excluded from the Kaplan-Meier analysis. Studies were conducted under an lACUC-approved protocol, and mice were humanely euthanized for endpoints of tumor size, weight loss, or behavior changes. Statistics. P values were determined as indicated by student’s t-test, Tukey’s multiple comparisons test, or log -rank test in Prism 9.5.1. P<0.05 was considered significant.

[0423] Western blots

[0424] Nuclear and cytoplasmic extracts of U87 glioma cells treated with 4 pM IgG control or DX3 were prepared using the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) and probed for CatB using the CatB primary antibody described above, and signal detection facilitated by HRP-conjugated secondary antibody. DX3 was probed by HRP-conjugated anti-human IgG Fc (abeam, Ab97225). Antibodies against Lamin Bl (Invitrogen, PA5- 19468) or a-tubulin (Invitrogen, PA5- 19489) were used for loading controls. Band intensities normalized to loading control were quantified by ImageJ.

[0425] CatB and CatS activity assays

[0426] CatB and CatS enzymatic activity was measured using the Cathepsin B Inhibitor Screening Assay Kit (BPS Bioscience, #79590) and Cathepsin S Inhibitor Screening Assay Kit (BPS Bioscience, #79588), respectively, which uses a fluorogenic cathepsin substrate to report enzymatic activity. Activity signal was quantified by fluorescence plate reader (Synergy HT, BioTek) and expressed relative to reference purified CatB and CatS protein activity.

[0427] Protease inhibitor assays

[0428] DX3 stability assays. U87 cells cultured in 12-well plates were treated with 4 pM DX3 in the presence of 10 pM CA-074Me CatB inhibitor (HY-100350, MCE) or an equivalent volume of DMSO for one hour, after which media was removed and replaced with fresh media lacking DX3 but still containing 10 pM CA-074Me CatB inhibitor or an equivalent volume of DMSO. Nuclear extracts were prepared from the cells at the time of media replacement (one hour after treatment with DX3) or 2 days after treatment and were probed for the presence of DX3 by western blot. Relative DX3 content was determined by ImageJ analysis of band intensities.

[0429] Morphology change assays

[0430] U87 cells cultured in 10 cm plates were treated with 10 pM protease inhibitors (E64 Cys protease inhibitor from the Cathepsin B Inhibitor Screening Assay Kit, BPS Bioscience, #79590), CA-074Me CatB inhibitor (HY-100350, MCE), or LY3000328 CatS inhibitor (HY- 15533, MCE) or media containing an equivalent volume of DMSO control for three hours, followed by addition of control media or media containing 4 pM ANADC. Cell morphologies were monitored by brightfield microscopy for 60 minutes, and changes quantified by ImageJ.

[0431] Clonogenic assays

[0432] U87, MCF7, MDA-MB-231, DLD1, or A549 cells seeded at 1,000 cells per well in 6-well plates were treated with titrated doses of IgG control, ADC control, unconjugated DX3, or ANADC and cell survival was evaluated by colony formation assay as previously described (Hansen et al. Sci Transl Med. 2012; 4(157):157ra42) and plotted in Prism version 9.5.1.

[0433] Triple negative MDA-MB-231 breast cancer xenograft study

[0434] Female NOG mice were injected subcutaneously with 5x106 MDA-MB-231 cells in Matrigel to generate flank tumors. Once tumors reached mean volume of -250 mm3 mice were randomized to groups for IP treatment with vehicle control (N=8) or ANADC at 1, 5, or 10 mg / kg) (N=8 / group) once a week for four weeks. Tumor volumes were tracked by caliper measurements and mouse weights and behaviors monitored through the study.

[0435] HT29 colon cancer xenograft study

[0436] Female Balb / c nude mice were injected subcutaneously with 5x106 HT29 cells in Matrigel to generate flank tumors. Once tumors reached mean volume of -150 mm3 mice were randomized to groups for IP treatment with vehicle control (N=6), unconjugated DX3 (10 mg / kg) (N=6), or ANADC (10 mg / kg) (N=6) once a week for four weeks. Tumor volumes were tracked by caliper measurements and mouse weights and behaviors monitored through the study.

[0437] Example 9: Characterisation of DX3-mediated nuclear accumulation of cathepsin B

[0438] Following the experiments described in Example 2, additional studies were conducted to examine the mechanisms responsible for catabolism of ANAs that penetrate nucleic and avoid lysosomes.

[0439] Some lysosomal cathepsin proteases are released into the cytoplasm and translocate into the nucleus and are implicated in mechanisms of nucleocytoplasmic shuttling. Intracellular localization of cathepsins involved in nuclear import and export (CatB, CatK, CatL, and CatS) were examined in U87 glioma cells 15 minutes after treatment with buffer control, IgG control, or DX3 by confocal fluorescence microscopy. IgG control did not impact localization of these cathepsins, but DX3 caused a marked shift of CatB signal into the nucleus (Fig. 14A, Fig. 15 A, 15B). To quantitatively evaluate this effect nuclear overlap coefficients were determined for each of the cathepsins in IgG control and DX3-treated cells using Imagel Colocalization Finder. CatB nuclear overlap coefficient was increased to 0.95 ± 0.01 in DX3-treated cells compared to 0.53 ± 0.02 in cells treated with IgG control (P<0.0001), while coefficients were unchanged by DX3 for CatK and CatL and only minimally increased for CatS (Fig. 14B, Fig. 15C). Western blots with ImageJ quantification of bands on nuclear and cytoplasmic extracts from IgG control or DX3 -treated U87 cells confirmed DX3 promotes nuclear accumulation of the active isoform of CatB (molecular weight ~29 kDa) with a corresponding decrease in cytoplasmic content of active CatB (Fig. 14C, 14D, Fig. 16). Consistent with this, CatB enzymatic activity was increased in nuclear extracts from DX3 compared to IgG control- treated U87 cells, and this activity was suppressed by pre-treatment of cells with the specific CatB inhibitor CA-074Me (Fig. 14E). The impact of DX3 on CatB nuclear flux was consistent over a panel of cancer cell lines (including glioma, breast, colon, and lung cancer cells) and in primary cells (Fig. 17, Fig. 18).

[0440] The effect of CatB inhibition on DX3 stability in the nucleus was probed to determine if CatB is involved in ANA nuclear catabolism. U87 cells were treated with DX3 in the presence of DMSO control or the CatB inhibitor CA-074Me. One hour after DX3 application, media was removed and replaced with fresh media lacking DX3 but containing DMSO or CA-074Me. Intranuclear DX3 content was then measured by western blot on nuclear extracts taken at timepoints of one hour or two days after initial exposure to DX3 or two days later. CatB inhibition significantly delayed the intranuclear degradation of DX3 (Fig. 14F, 14G). Taken together, these data demonstrate that active CatB rapidly accumulates in the nucleus of DX3-treated cells and degrades DX3, revealing a CatB role in intranuclear catabolism of ANAs.

[0441] Example 10: ANADC characterisation

[0442] Following the experiments in Example 3, ANADC activity in cell culture was further characterized. U87 glioma cells treated with IgG control, ADC control, unconjugated DX3, or ANADC for 60 minutes were washed, fixed, and immunostained to detect antibody uptake. IgG control and ADC control showed minimal uptake into cells. Unconjugated DX3 localized into nuclei, while ANADC signal was detected in both nuclear and cytoplasmic compartments, including some punctate foci of staining in the cytoplasm. Brightfield examination of cell morphologies showed loss of normal contours in ANADC-treated cells compared to cells treated with IgG control, ADC control, or unconjugated DX3. These findings indicated cell death caused by MMAE released from ANADC allowed some DX3 to leak out of the nucleus into the cytoplasm that was detectable by immunostaining (Fig. 9A). To further probe this effect, ANADC localization in U87 cells was examined at earlier time points. At 15 and 30 minutes after treatment ANADC signal was primarily in the nucleus, with progressive leakage into the cytoplasm at 45-60 minutes (Fig. 9B) consistent with cell death and dissolution of nuclear membranes secondary to MMAE release. The ability of ANADC to induce CatB nuclear accumulation in glioma cells was confirmed by fluorescence confocal microscopy (Fig. 19A-C). To test whether CatB-mediated cleavage of the VC linker in ANADC is required for release of the cytotoxic MMAE cargo drug, the ability of CatB inhibition to protect cells against the cytotoxicity of subsequent ANADC treatment was examined. U87 cells pre-treated with control buffer or E64 Cys protease inhibitor, CA-074Me CatB specific inhibitor, or LY3000328 CatS specific inhibitor were incubated in control media or media containing ANADC ± the inhibitors listed above. Toxicity was followed by brightfield microscopy and quantification of morphologic changes by ImageJ. ANADC rapidly induced loss of cell morphologies in the presence of control buffer or the CatS inhibitor. In contrast, the broad spectrum Cys protease inhibitor (which has inhibitory effect on CatB and other Cys proteases) and the specific CatB inhibitor significantly suppressed these morphologic changes, consistent with CatB -dependent cytotoxicity (Fig. 19D, Fig. 20). The effects of IgG control, ADC control, unconjugated DX3, and ANADC on the survival of U87 glioma cells were then evaluated in colony formation assays. Cells tolerated IgG control, ADC control, and unconjugated DX3 over a dose range of 0.5 to 31.25 nM, but ANADC was highly toxic to the cells at concentrations above 7 nM (Fig. 19E). ANADC penetration and cytotoxicity was consistent in additional cancer cells including MCF7 and MDA-MB-231 breast, DLD1 colon, and A549 lung cancer cells (Fig. 21).

[0443] Example 11 : ANADC suppresses subcutaneous xenograft tumors

[0444] The impact of ANADC on estrogen receptor-positive (ER+) and triple negative breast cancer and on colon cancer tumors was examined in subcutaneous xenograft models. For the ER+ positive breast cancer model, mice with subcutaneous MCF7 tumors were randomized to treatment with four weekly intraperitoneal (IP) injections of control buffer, unconjugated DX3 (10 mg / kg), ADC control (10 mg / kg), or ANADC (10 mg / kg) (N=6 mice per group (as discussed in Example 6). ANADC dose-dependency was examined in the triple negative breast cancer model by randomization of mice with MDA-MB-231 subcutaneous tumors to treatment with four weekly IP injections of control buffer or ANADC at 1, 5, or 10 mg / kg (N=8 mice per group). ANADC efficacy against tumors from colon cancer was tested in the HT29 model, with mice randomized to four weekly IP injections of control buffer, unconjugated DX3 (10 mg / kg), or ANADC (10 mg / kg) (N=6 / group). Endpoints in each study were tumor volume or changes in behavior or weights. ANADC significantly suppressed tumor growth compared to all controls in each model, exhibited dose-dependent activity, and was well tolerated with no significant differences in body mass observed throughout treatment (Fig 22A-22F).

[0445] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the disclosure as shown in the specific embodiments without departing from the spirit or scope of the disclosure as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0446] All publications discussed above are incorporated herein in their entirety. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present disclosure. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

[0447] This application claims priority from United States of America Provisional Patent Application 63 / 593,747 filed on 27 October 2023, the disclosure of which is incorporated herein in its entirety.

Claims

CLAIMS1. A nuclear penetrating conjugate comprising a nuclear penetrating, anti-DNA binding protein attached to a payload via a cleavable linker, wherein the linker is capable of being cleaved by a cathepsin.

2. The conjugate according to claim 1 , wherein the cathepsin is a nuclear cathepsin.

3. The conjugate according to claim 1 or 2, wherein the cathepsin is cathepsin B (CatB) or cathepsin S (CatS).

4. The conjugate according to any one of claims 1 to 3, wherein the linker comprises a peptide unit.

5. The conjugate according to any one of claims 1 to 4, wherein the linker comprises a dipeptide unit selected from the group consisting of valine-citrulline; valine-alanine; and phenylalanine-arginine.

6. The conjugate according to any one of claims 1 to 5 wherein the linker comprises a valine-citrulline unit.

7. The conjugate according to claim 6, wherein the linker comprises: a) a valine-citrulline unit and a pentafluorophenyl group; b) a valine-citrulline unit and a succinimide group; c) a valine-citrulline unit and a para aminobenzoic acid group; d) a valine-citrulline unit and a para aminobenzoic acid group and a pentafluorophenyl group; or e) a valine-citrulline unit, a para aminobenzoic acid group and a succinimide group.

8. The conjugate according to any one of claims 1 to 7, wherein the binding protein is an autoantibody derived from a subject or an animal with an autoimmune disease, preferably wherein the autoantibody is derived from a subject with systemic lupus erythematous, or an animal model thereof.

9. The conjugate according to claim 8, wherein the autoantibody is 3E10 or a humanised form thereof.

10. The conjugate according to any one of claims 1 to 9, wherein the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 2, a CDR3 as shown in SEQ ID NO: 3 and a VL having a CDR 1 as shown in SEQ ID NO: 4, a CDR2 as shown in SEQ ID NO: 5 and a CDR3 as shown in SEQ ID NO: 6 or a humanized form thereof.

11. The conjugate according to any one of claims 1 to 10, wherein the binding protein comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 7 and a VL as shown in SEQ ID NO: 8 or a humanized form thereof.

12. The conjugate according to any one of claims 1 to 9, wherein the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 9 or SEQ ID NO: 10, a CDR2 as shown in SEQ ID NO: 11 or SEQ ID NO: 12, a CDR3 as shown in SEQ ID NO: 13 and a VL having a CDR1 as shown in SEQ ID NO: 14 or SEQ ID NO: 15, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH having a CDR1 as shown in SEQ ID NO: 9 or SEQ ID NO: 10, a CDR2 as shown in SEQ ID NO: 11 or SEQ ID NO: 12, a CDR3 as shown in SEQ ID NO: 13 and a VL having a CDR1 as shown in SEQ ID NO: 14 or SEQ ID NO: 15, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17.

13. The conjugate of claim 12, wherein the binding protein comprises a VH which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 18-21 and a VL as shown in any one of SEQ ID NOs: 22-25 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 18-21 and a VL as shown in any one of SEQ ID NOs: 22-25.

14. The conjugate of any one of claims 1 to 13, wherein the binding protein is an intact IgG or a di-scFv.

15. The conjugate according to any one of claims 1 to 14, wherein the payload is a gene editing agent.

16. The conjugate according to claim 15, wherein the payload is a gene editing agent selected from the group consisting of triplex-forming molecules, pseudocomplementary oligonucleotides, a CRISPR system, zinc finger nucleases (ZFN), transcription activatorlike effector nucleases (TALEN), and intron encoded meganucleases.

17. The conjugate according to any one of claims 1 to 16, wherein the payload is an oligonucleotide.

18. The conjugate of claim 17, wherein the oligonucleotide is a siRNA, a mRNA, an aptamer, or an antisense oligonucleotide.

19. The conjugate according to any one of claims 1 to 14, wherein the payload is a drug.

20. The conjugate according to claim 19, wherein the drug is an anti-cancer drug, preferably a chemotherapeutic agent, a cytotoxic agent, and / or a radiosensitizer.

21. The conjugate according to any one of claims 1 to 14, wherein the pay load is a recombinant viral expression vector.

22. The conjugate of claim 21, wherein the recombinant viral expression vector is a recombinant adeno-associated virus (AAV) vector, a recombinant adenovirus (AdV) vector, or a recombinant lentivirus (LV) vector.

23. The conjugate according to any one of claim 1 to 14, wherein the payload is a modulator of DNA methylation, a modulator of chromatin, or a modulator of DNA replication and repair, preferably wherein the payload is Lysine-specific histone demethylase 1A (LSD1), Enhancer of zeste homolog 2 (EZH2) histone-lysine-N- methyltransferase, DNA methyltransferase 1 (DNMT1), or a histone deacetylase (HDAC).

24. The conjugate according to any one of claims 1 to 14, wherein the pay load is a cathepsin inhibitor, preferably a cathepsin B and / or cathepsin S inhibitor.

25. The conjugate according to any one of claims 1 to 14, wherein the payload is an inhibitor of a nucleoside transporter, preferably dipyridamole.

26. A composition comprising a conjugate according to claim 17 or 18 and DNA.

27. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 25, or the composition according to claim 26, and a pharmaceutically acceptable carrier.

28. A method of delivering a payload to the nucleus of a cell, comprising contacting the cell with an effective amount of a nuclear penetrating conjugate comprising a nuclear penetrating, anti-DNA binding protein attached to a payload via a linker, wherein the linker is capable of being cleaved via cathepsin.

29. The method of claim 28, wherein the contacting step comprises administering the conjugate to a subject.

30. A method of gene editing comprising, administering to a subject in need thereof an effective amount of a nuclear penetrating conjugate comprising a nuclear penetrating, anti-DNA binding protein attached to a payload via a linker, wherein the linker is capable of being cleaved via cathepsin, and wherein the payload is a gene editing agent selected from the group consisting of triplex-forming molecules, pseudocomplementary oligonucleotides, a CRISPR system, zinc finger nucleases (ZFN), transcription activatorlike effector nucleases (TALEN), and intron encoded meganucleases.

31. The method according to claim 30, comprising administering to the subject the composition according to claim 26.

32. A method of treating a disease associated with cellular accumulation of cathepsin, comprising administering to a subject in need thereof an effective amount of a nuclear penetrating conjugate comprising a nuclear penetrating, anti-DNA binding protein attached to a payload via a linker, wherein the linker is capable of being cleaved via cathepsin, and wherein the payload is a cathepsin inhibitor.

33. The method according to claim 32, wherein the cathepsin is cathepsin B.

34. The method according to claim 32 or 33, wherein the cathepsin inhibitor is a cathepsin B inhibitor.

35. The method according to claim 34, wherein the cathepsin B inhibitor is a cystatin, a peptidyl aldehyde, an aziridinyl peptide, an epoxysuccinyl peptide, an epoxysuccinyl inhibitor, an aziridine, a nitrile, or a monoclonal antibody.

36. The method according to any one of claims 32 to 35, wherein the disease is a neurological disease.

37. The method according to any one of claims 32 to 36, wherein the disease is Alzheimer’s disease, Parkinson’s disease, traumatic brain injury, or cerebral ischemia.

38. The method according to any one of claims 32 to 35, wherein the disease is a cardiovascular disease.

39. The method according to claim 38, wherein the cardiovascular disease is myocardial infarction, diabetes, diabetic cardiomyopathy, or stroke.

40. The method according to any one of claims 32 to 35, wherein the disease is pancreatitis, liver disease, lysosomal storage disorders (LSDs), retinitis pigmentosa, Kawaski disease, or Sjogren syndrome.

41. A method of treating an autoimmune disease, comprising, administering to a subject in need thereof an effective amount of a nuclear penetrating conjugate comprising a nuclear penetrating, anti-DNA binding attached linked to a payload via a linker, wherein the linker is capable of being cleaved via cathepsin, and wherein the payload is an inhibitor of a nucleoside transporter.

42. The method of claim 41, wherein the autoimmune disease is one or more of systemic lupus erythematosus (lupus or SLE), CNS, lupus, systemic sclerosis (scleroderma), Graves’ disease, myasthenia gravis, autoimmune hemolytic anemia, and pemphigus vulgaris, and additionally may contribute to the severity of disease in other autoimmune diseases such as rheumatoid arthritis, autoimmune thrombocytopenia, autoimmune atrophic gastritis of pernicious anemia, myasthenia gravis, Goodpasture’s syndrome, diabetes mellitus, multiple sclerosis, Hashimoto’s thyroiditis, Crohn’s disease, and Sjogren’s syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, POEMS syndrome, dermatomyositis, inclusion body myositis, inflammatory myopathies, vasculitis syndromes including but not limited to Churg-Strauss Syndrome, Wegener granulomatosis, Behcet’s disease, Buerger’s disease, Kawasaki disease, Takayasu’s arteritis, Henoch-Schonlein purpura, Giant cell arteritis, or polyarteritis nodosa.

43. A method of treating cancer, comprising, administering to a subject in need thereof an effective amount of a nuclear penetrating conjugate comprising a nuclear penetrating, anti-DNA binding protein attached to a payload via a linker, wherein the linker is capable of being cleaved via cathepsin, and wherein the payload is a chemotherapeutic agent, a cytotoxic agent, or a radiosensitiser.

44. The method of claim 43, wherein the payload is olaparib, niraparib, or rucaparib.

45. The method according to any one of claims 28 to 44, wherein the method further comprises the step of administering to the subject an effective amount of an inhibitor of a nucleoside transporter to reduce transcellular transport of the conjugate thus reducing nuclear delivery of the payload.

46. The method of claim 45, wherein the inhibitor of the nucleoside transporter inhibits activity or expression of one or more of ENT1, ENT2, ENT3, or ENT4, optionally at least or only ENT2.

47. The method of claim 46, wherein the inhibitor of the nucleoside transporter is dipyridamole or pharmaceutically acceptable salt thereof.

48. The method according to any one of claims 45 to 47, wherein the conjugate and inhibitor of the nucleoside transporter are administered to a subject in need thereof by a parenteral, enteral, transdermal, or transmucosal route of administration.

49. The method according to any one claims 45 to 48, wherein the subject is administered the conjugate separately or together with the inhibitor of the nucleoside transporter.

50. Use of a nuclear penetrating conjugate comprising a nuclear penetrating, anti-DNA binding protein attached to a payload via a linker in the manufacture of a medicament for gene editing, wherein the linker is capable of being cleaved via cathepsin, and wherein the payload is a gene editing agent selected from the group consisting of triplex-forming molecules, pseudocomplementary oligonucleotides, a CR1SPR system, zinc fingernucleases (ZFN), transcription activator-like effector nucleases (TALEN), and intron encoded meganucleases.

51. Use of a nuclear penetrating conjugate comprising a nuclear penetrating, anti-DNA binding protein attached to a payload via a linker in the manufacture of a medicament for treating a disease characterized by cellular accumulation of cathepsin, wherein the linker is capable of being cleaved via cathepsin, and wherein the payload is a cathepsin inhibitor.

52. Use of a nuclear penetrating conjugate comprising a nuclear penetrating, anti-DNA binding protein attached to a payload via a linker in the manufacture of a medicament for treating an autoimmune disease, wherein the linker is capable of being cleaved via cathepsin, and wherein the payload is an inhibitor of a nucleoside transporter.

53. Use of a nuclear penetrating conjugate comprising a nuclear penetrating, anti-DNA binding protein attached to a payload via a linker in the manufacture of a medicament for treating cancer, wherein the linker is capable of being cleaved via cathepsin, and wherein the payload is a chemotherapeutic agent, a cytotoxic agent, or an radiosensitiser.

54. The method according to any one of claims 28 to 49, or use according to any one of claims 51 to 53, wherein the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 9 or SEQ ID NO: 10, a CDR2 as shown in SEQ ID NO: 11 or SEQ ID NO: 12, a CDR3 as shown in SEQ ID NO: 13 and a VL having a CDR1 as shown in SEQ ID NO: 14 or SEQ ID NO: 15, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH having a CDR1 as shown in SEQ ID NO: 9 or SEQ ID NO: 10, a CDR2 as shown in SEQ ID NO: 11 or SEQ ID NO: 12, a CDR3 as shown in SEQ ID NO: 13 and a VL having a CDR1 as shown in SEQ ID NO: 14 or SEQ ID NO: 15, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17.

55. The method according to any one of claims 28 to 49, or use according to any one of claims 51 to 53, wherein the binding protein comprises a VH which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 18-21 and a VL as shown in any one of SEQ ID NOs: 22-25 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 18-21 and a VL as shown in any one of SEQ ID NOs: 22-25.

56. A kit comprising the conjugate according to any one of claims 1 to 25, the composition according to claim 26, or the pharmaceutical composition according to claim 27, and an inhibitor of a nucleoside transporter, preferably wherein the inhibitor of the nucleoside transporter is dipyridamole or pharmaceutically acceptable salt thereof.

57. A method of treating neurodegeneration or neurological disease, comprising administering to subject a nuclear penetrating anti-DNA binding protein in an amount effective to attenuate nuclear cathepsin activity.

58. The method according to claim 57, wherein the cathepsin is cathepsin B.

59. The method according to claim 57 or 58, wherein the neurodegeneration is Alzheimer’s disease, Parkinson’s disease, traumatic brain injury, cerebral ischemia, or aging.

60. The method according to any one of claims 57 to 59, wherein the nuclear penetrating anti-DNA binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 2, a CDR3 as shown in SEQ ID NO: 3 and a VL having a CDR 1 as shown in SEQ ID NO: 4, a CDR2 as shown in SEQ ID NO: 5 and a CDR3 as shown in SEQ ID NO: 6 or a humanized form thereof.

61. The method according to any one of any one of claims 57 to 60, wherein the binding protein comprises a VH which comprises an amino acid sequence as shown in SEQ ID NO: 7 and a VL as shown in SEQ ID NO: 8 or a humanized form thereof.

62. The method according to any one of claims 57 to 59, wherein the binding protein comprises a VH having a CDR1 as shown in SEQ ID NO: 9 or SEQ ID NO: 10, a CDR2 as shown in SEQ ID NO: 11 or SEQ ID NO: 12, a CDR3 as shown in SEQ ID NO: 13 and a VL having a CDR1 as shown in SEQ ID NO: 14 or SEQ ID NO: 15, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH having a CDR1 as shown in SEQ ID NO: 9 or SEQ ID NO: 10, a CDR2 as shown in SEQ ID NO: 11 or SEQ ID NO: 12, a CDR3 as shown in SEQ ID NO: 13 and a VL having a CDR1as shown in SEQ ID NO: 14 or SEQ ID NO: 15, a CDR2 as shown in SEQ ID NO: 16 and a CDR3 as shown in SEQ ID NO: 17.

63. The method according to any one of claims 57 to 59, wherein the binding protein comprises a VH which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 18-21 and a VL as shown in any one of SEQ ID NOs: 22-25 or a variant thereof that competes for binding to DNA with an antibody which comprises a VH which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 18-21 and a VL as shown in any one of SEQ ID NOs: 22-25.

64. The method according to any one of claims 57 to 63, wherein the binding protein is a di-scFv.

65. Use of a nuclear penetrating anti-DNA binding protein in the manufacture of a medicament for treating neurodegeneration or a neurological disease.

66. The method according to any one of claims 28 to 44, wherein the method further comprises the step of administering to the subject an effective amount of a cathepsin inhibitor to reduce cleavage of the linker thus reducing release of the payload.

67. The method according to claim 66, wherein the conjugate and cathepsin inhibitor are administered to a subject in need thereof by a parenteral, enteral, transdermal, or transmucosal route of administration.

68. The method according to claim 66 or 67, wherein the subject is administered the conjugate separately or together with the cathepsin inhibitor.

69. The method according to any one of claims 66 to 68, wherein the cathepsin inhibitor is a cathepsin B inhibitor.

Images