Composition comprising a cell penetrating, Anti-DNA binding protein and methods of treatment

Abstract

The present disclosure relates to therapeutic and prophylactic applications of cell penetrating, anti-DNA binding proteins, that inhibit Neutrophil Extracellular Trap (NET) formation, and preserve neutrophil function. The cell penetrating, anti-DNA binding proteins are used in the context of immunosuppression and conditions associated with the same.

Description

[0001] COMPOSITION COMPRISING A CELL PENETRATING, ANTI-DNA BINDING

[0002] PROTEIN AND METHODS OF TREATMENT

[0003] This application claims priority from United States provisional application 63 / 612,079, filed on 19 December 2023, the disclosures of which are incorporated herein by reference in their entirety.

[0004] INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A SEQUENCE LISTING XML FILE

[0005] A Sequence Listing is provided herewith as a Sequence Listing XML, “RICE- 235PCT_Sequence_listing_ST26.xml”, created on December 19, 2024 and having a size of 131,831 bytes. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety.

[0006] FIELD OF THE INVENTION

[0007] The present disclosure relates to therapeutic and prophylactic applications of cell penetrating, anti-DNA binding proteins, particularly in the context of immunosuppression and conditions associated with the same.

[0008] BACKGROUND OF THE INVENTION

[0009] Organ transplantation is a lifesaving procedure for many individuals with endstage organ disease. However, the need for lifelong maintenance immunosuppression (M-IMS) post transplantation is nearly universal in this patient group. Non-adherence to M-IMS is a contributing cause of poor post-transplant outcomes.

[0010] Standard immunosuppression generally consists of triple therapy: a calcineurin inhibitor, an anti-proliferative agent, and a corticosteroid. One of the most commonly used regimes consists of cyclosporine / tacrolimus, mycophenolate mofetil, and prednisone.

[0011] M-IMS is problematic because, among other things, it can result in neutropenia (low numbers of neutrophils (white blood cells)). Neutropenia is a common and problematic secondary occurrence, because it leaves patients at high risk of infections, which in turn can result in death.

[0012] Accordingly, there remains a need in the art for improved methods of immunosuppression. SUMMARY OF THE INVENTION

[0013] The present inventors have surprisingly found that cell penetrating, anti-DNA binding proteins prevent neutrophil activation in response to a variety of inflammatory stimuli, whilst also preventing neutrophil cell death and preserving the ability of neutrophils to fight infection. This finding is particularly advantageous in the context of immunosuppression therapy, where there is a need for agents that preserve neutrophil function.

[0014] Accordingly, the findings of the present inventors provides basis for the use of cell penetrating, anti-DNA binding proteins in immunosuppression therapy, and for the treatment of solid organ transplant rejection and Ischemia-reperfusion injury where there is a need for agents that can prevent neutrophil activation whilst preserving neutrophil function.

[0015] Standard of care immunosuppressive agents, including calcineurin inhibitors and corticosteroids, are associated with a variety of negative side effects including neutropenia. Accordingly, the findings of the present inventors also provide basis for using anti-DNA binding proteins of the disclosure in calcineurin inhibitor and / or steroid- sparing immunosuppressive regimens.

[0016] Finally, the findings of the present inventors also provide basis for the use of anti- DNA binding proteins of the disclosure to treat neutropenia, as they are able to impair neutrophil activation without killing remaining neutrophils.

[0017] The present disclosure provides a method of immunosuppression, the method comprising administering to a subject in need thereof, a composition comprising a cell penetrating, anti-DNA binding protein, wherein the cell penetrating, anti-DNA binding protein (i) inhibits Neutrophil Extracellular Trap (NET) formation, and (ii) preserves neutrophil function.

[0018] The present disclosure also provides a method of treating solid organ transplant rejection, the method comprising administering to a subject in need thereof, a cell penetrating, anti-DNA binding protein, wherein the cell penetrating, anti-DNA binding protein (i) inhibits Neutrophil Extracellular Trap (NET) formation, and (ii) preserves neutrophil function.

[0019] The present disclosure also provides a method of treating or preventing neutropenia in a subject requiring immunosuppression, the method comprising administering to the subject a cell penetrating, anti-DNA binding protein, wherein the cell penetrating, anti-DNA binding protein (i) inhibits Neutrophil Extracellular Trap (NET) formation, and (ii) preserves neutrophil function. In an example, the cell penetrating, anti-DNA binding protein reduces one or more of the following in a subject: autoantibody levels, preferably wherein the autoantibodies are anti-neutrophil cytoplasmic autoantibodies (ANCA); and / or IL-ip levels. In an example, the cell penetrating, anti-DNA binding protein reduces autoantibody levels and IL-ip levels in a subject. In an example, the cell penetrating, anti-DNA binding protein reduces autoantibody levels or IL-ip levels in a subject.

[0020] In an example, the cell penetrating, anti-DNA binding protein reduces autoantibody levels in the subject. In an example, the cell penetrating, anti-DNA binding protein reduces anti-neutrophil cytoplasmic autoantibody (ANCA) levels in the subject. In an example, the ANCA is myeloperoxidase (MPO)-ANCA. In an example, the ANCA is proteinase 3 (PRT3)-ANCA.

[0021] In an example, the cell penetrating, anti-DNA binding protein reduces IL-ip levels. in a subject:

[0022] In an example, the subject has been treated with a steroid. In an example, the steroid is a corticosteroid. In an example, the steroid is prednisolone. In an example, the steroid is prednisone. In an example, the steroid is a corticosteroid, preferably, wherein the steroid is prednisolone.

[0023] In an example, the subject is also being treated with a calcineurin inhibitor and an anti-proliferative agent. In an example, the subject is also being treated with a calcineurin inhibitor or an anti -proliferative agent. In an example, the subject is also being treated with a calcineurin inhibitor and / or an anti-proliferative agent. In an example, the subject is also being treated with a calcineurin inhibitor. In an example, the subject is also being treated with an anti-proliferative agent. In an example, the calcineurin inhibitor is cyclosporine or tacrolimus. In an example, the calcineurin inhibitor is cyclosporine. In an example, the calcineurin inhibitor is tacrolimus. In an example, the anti-proliferative agent is mycophenolate mofetil.

[0024] In an example, the method comprises reducing or discontinuing the steroid component of the subject’s treatment. In an example, the method comprises reducing or discontinuing the calcineurin inhibitor component of the subject’s treatment. In an example, the method comprises reducing or discontinuing the anti-proliferative agent component of the subject’s treatment.

[0025] In an example, treatment preserves neutrophil function in the subject. In an example, the preservation of neutrophil function in the subject is determined by assessing one or more of the following parameter(s) in a sample obtained from the patient: level of neutrophil viability; level of neutrophil cytotoxicity; level of neutrophil apoptosis; level of release of pro-inflammatory neutrophil enzymes; and / or level of neutrophil phagocytosis. In an example, the preservation of neutrophil function in the subject is determined by assessing the level of neutrophil viability, the level of neutrophil cytotoxicity, or the level of neutrophil apoptosis in a sample obtained from the patient. In an example, the preservation of neutrophil function in the subject is determined by assessing the level of release of pro-inflammatory neutrophil enzymes or the level of neutrophil phagocytosis in a sample obtained from the patient. In an example, the preservation of neutrophil function in the subject is determined by assessing the level of one of (i) neutrophil viability, (ii) neutrophil cytotoxicity, or (iii) neutrophil apoptosis; and one of (i) release of pro-inflammatory neutrophil enzymes, or (ii) neutrophil phagocytosis, in a sample obtained from the patient.

[0026] In an example, the determination is based on a comparison between the level of the relevant parameter(s) in the sample and a corresponding level(s) in a control sample. In an example, the control sample is a sample obtained from the subject prior to receiving treatment with the cell penetrating, anti-DNA binding protein. In an example, the determination is based on a comparison between the level of the relevant parameter(s) in the sample and a corresponding level(s) in a control sample, preferably wherein the control sample is a sample obtained from the subject prior to receiving treatment with the cell penetrating, anti-DNA binding protein.

[0027] In an example, the immunosuppression is maintenance immunosuppression. In an example, the immunosuppression is induction immunosuppression. In an example, the immunosuppression is anti -rejection immunosuppression.

[0028] In an example, the solid organ is selected from the group consisting of a heart, a kidney, a liver, and a lung. In an example, the solid organ is a heart or a kidney.

[0029] In an example, the subject has Ischaemia-Reperfusion injury (IRI). In an example, the IRI is in one or more of the subject’s kidney, heart, liver or brain.

[0030] The present disclosure also provides a method of treating Ischaemia-Reperfusion injury (IRI) in a subject, wherein the method comprises administering to the subject a composition comprising a cell penetrating, anti-DNA binding protein in an amount effective to (i) inhibit Neutrophil Extracellular Trap (NET) formation, and (ii) preserve neutrophil function.

[0031] In an example, the subject has received a solid organ transplant. In an example, the solid organ transplant is a heart transplant. In an example, the solid organ transplant is a kidney transplant. In an example, the solid organ transplant is a liver transplant. In an example, the solid organ transplant is a lung transplant.

[0032] In an example, the subject has received a cardiopulmonary bypass or is about to undergo a cardiopulmonary bypass. In an example, the subject has received a cardiopulmonary bypass. In an example, the subject is about to undergo a cardiopulmonary bypass.

[0033] In an example, the composition is administered before or during the cardiopulmonary bypass. In an example, the composition is administered before, during, or after the cardiopulmonary bypass. In an example, the composition is administered before, during, and after the cardiopulmonary bypass. In an example, the composition is administered before the cardiopulmonary bypass. In an example, the composition is administered during the cardiopulmonary bypass. In an example, the composition is administered after the cardiopulmonary bypass.

[0034] In an example, the cardiopulmonary bypass occurs during coronary artery bypass graft surgery.

[0035] In an example, the IRI is in the heart or kidney. In an example, the IRI is in the heart. In an example, the IRI is in the kidney. In an example, the IRI is in the heart and kidney. In an example, the IRI is Acute kidney injury (AKI). In an example, the AKI is caused by surgical revascularisation. In an example, the surgical revascularisation as part of a solid organ transplantation.

[0036] In an example, the subject has neutropenia.

[0037] In an example, the present disclosure relates to a method of treating acute kidney injury, wherein the method comprises administering a binding protein disclosed herein.

[0038] The present disclosure provides a method of reducing calcineurin inhibitor use or steroid use in a subject in need thereof, the method comprising administering to the subject an effective amount of a cell penetrating, anti-DNA binding protein, wherein the cell penetrating, anti-DNA binding protein (i) inhibits Neutrophil Extracellular Trap (NET) formation, and (ii) preserves neutrophil function. In an example, the steroid is a corticosteroid, preferably prednisolone, or the calcineurin inhibitor is cyclosporine or tacrolimus.

[0039] In an example, the disclosure provides a method of reducing calcineurin inhibitor use in a subject in need thereof, the method comprising administering to the subject an effective amount of a cell penetrating, anti-DNA binding protein, wherein the cell penetrating, anti-DNA binding protein (i) inhibits Neutrophil Extracellular Trap (NET) formation, and (ii) preserves neutrophil function. In an example, the calcineurin inhibitor is cyclosporine or tacrolimus. In an example, the calcineurin inhibitor is cyclosporine. In an example, the calcineurin inhibitor is tacrolimus.

[0040] In an example, the disclosure provides a method of reducing steroid use in a subject in need thereof, the method comprising administering to the subject an effective amount of a cell penetrating, anti-DNA binding protein, wherein the cell penetrating, anti-DNA binding protein (i) inhibits Neutrophil Extracellular Trap (NET) formation, and (ii) preserves neutrophil function. In an example, the steroid is a corticosteroid. In an example, the steroid is prednisone. In an example, the steroid is prednisolone. In an example, the steroid is a corticosteroid, preferably prednisolone.

[0041] In an example, the subject requires immunosuppression. In an example, the immunosuppression is maintenance immunosuppression. In an example, the immunosuppression is induction immunosuppression. In an example, the immunosuppression is anti-rejection immunosuppression

[0042] In an example, the subject has neutropenia.

[0043] In an example, the subject’s calcineurin inhibitor or steroid use is reduced or discontinued after treatment. In an example, the subject’s calcineurin inhibitor use is reduced or discontinued after treatment. In an example, the subject’s steroid use is reduced or discontinued after treatment.

[0044] In an example, the subject is using the calcineurin inhibitor or the steroid for immunosuppression. In an example, the subject is using the calcineurin inhibitor for immunosuppression. In an example, the subject is using the steroid for immunosuppression.

[0045] The present disclosure also provides the use of a cell penetrating, anti-DNA binding protein in the manufacture of a medicament for: treating solid organ transplant rejection; treating or preventing neutropenia; treating Ischaemia-Reperfusion injury (IRI); or, reducing steroid use, wherein the cell penetrating, anti-DNA binding protein (i) inhibit Neutrophil Extracellular Trap (NET) formation, and (ii) preserve neutrophil function.

[0046] In an example, the medicament further comprises at least one additional immunosuppressant agent.

[0047] In an example, the additional immunosuppressant agent is a calcineurin inhibitor, an antiproliferative agent, or a corticosteroid. In an example, the additional immunosuppressant agent is a calcineurin inhibitor. In an example, the additional immunosuppressant agent is an antiproliferative agent. In an example, the additional immunosuppressant agent is a corticosteroid.

[0048] In an example, the additional immunosuppressant agent is cyclosporine, tacrolimus, mycophenolate mofetil, or prednisolone. In an example, the additional immunosuppressant agent is cyclosporine. In an example, the additional immunosuppressant agent is tacrolimus. In an example, the additional immunosuppressant agent is mycophenolate mofetil. In an example, the additional immunosuppressant agent is prednisolone.

[0049] In an example, the medicament is not formulated for use with a steroid, or the medicament is not formulated for use with a calcineurin inhibitor. In an example, the medicament is not formulated for use with a steroid. In an example, the medicament is not formulated for use with a calcineurin inhibitor. In an example, the medicament is not formulated for use with a steroid and a calcineurin inhibitor.

[0050] In an example, the anti-DNA 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 anti-DNA 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. In an example, the anti-DNA binding protein 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.

[0051] In an example, the anti-DNA 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. In an example, the anti-DNA 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. In an example, the anti-DNA binding protein 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. In an example, the anti-DNA binding protein comprises a VH which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 18 and a VL as shown in any one of SEQ ID NOs: 22. In an example, the anti-DNA binding protein comprises a VH which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 19 and a VL as shown in any one of SEQ ID NOs: 23. In an example, the anti- DNA binding protein comprises a VH which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 20 and a VL as shown in any one of SEQ ID NOs: 24. In an example, the anti-DNA binding protein comprises a VH which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 21 and a VL as shown in any one of SEQ ID NOs: 25.

[0052] In an example, the cell penetrating anti-DNA 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. In an example, the cell penetrating anti-DNA binding protein 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 an example, the cell penetrating anti-DNA 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.

[0053] In an example, the cell penetrating anti-DNA binding protein is an intact antibody, an scFv or a di-scFv. In an example, the cell penetrating anti-DNA binding protein is an intact antibody. In an example, the cell penetrating anti-DNA binding protein is an scFv. In an example, the cell penetrating anti-DNA binding protein is a di-scFv.

[0054] In an example, the anti-DNA binding protein is nuclear penetrating.

[0055] In an example, the binding protein comprises a linker which comprises an amino acid sequence shown in any one of SEQ ID NO: 17; SEQ ID NO: 26 or SEQ ID NO: 27.

[0056] In an example, inhibition of NET formation and preservation of neutrophil function is determined under culture conditions, wherein the culture conditions comprise culturing neutrophil-like PLB-985 cells or neutrophils in culture medium which comprises an inflammatory stimulus. In an example, inhibition of NET formation and preservation of neutrophil function is determined under culture conditions. In an example, the culture conditions comprise culturing neutrophil-like PLB-985 cells or neutrophils in culture medium which comprises an inflammatory stimulus.

[0057] In an example, preservation of neutrophil function under culture conditions is determined by:

[0058] (i) treating a population of neutrophils with the anti-DNA binding protein;

[0059] (ii) stimulating the population of neutrophils with an inflammatory stimulus; and

[0060] (iii) determining the level of one or more markers of neutrophil function in the population of neutrophils with the anti-DNA binding protein and a control population of neutrophils that do not comprise an anti-DNA binding protein; wherein an equivalent level of the one or more markers of neutrophil function between the control neutrophil population and the anti-DNA binding protein population indicates that neutrophil function is preserved.

[0061] In an example, the marker(s) used to determine preservation of neutrophil function under culture conditions are selected from the group consisting of:

[0062] - neutrophil viability;

[0063] - neutrophil cytotoxicity;

[0064] - neutrophil apoptosis;

[0065] - release of pro-inflammatory neutrophil enzymes; and

[0066] - neutrophil phagocytosis.

[0067] In an example, the inflammatory stimulus is phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS), a calcium ionophore ionomycin (IM), or tumour necrosis factor alpha (TNFa).

[0068] In an example, NET formation under culture conditions is determined by one or more markers selected from the group consisting of:

[0069] 1) level of cell death and extracellular DNA in NETs; and / or,

[0070] 2) reduced DNA release from neutrophil-like cells after culture with the inflammatory stimulus.

[0071] BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0072] Figure 1: DX-1 and DX-3 reduces NET formation regardless of stimulus.

[0073] Figure 2: NET formation in PMA or ionomycin-stimulated neutrophils treated with DX- 1.

[0074] Figure 3: Viability of neutrophils treated with DX-1 and DX-3. Figure 4: Cytoxicity neutrophils treated with DX-1 and DX-3

[0075] Figure 5: Apoptosis of neutrophils treated with DX-1 and DX-3.

[0076] Figure 6: MPO release from neutrophils treated with DX-1 and DX-3.

[0077] Figure 7: Neutrophil elastase (NE) release from neutrophils treated with DX-1 and DX- 3.

[0078] Figure 8: PRT3 release from neutrophils treated with DX-1 and DX-3.

[0079] Figure 9: Phagocytosis of neutrophils treated with DX-1 and DX-3.

[0080] Figure 10: Schematic of murine experimental model of MPO-AAV.

[0081] Figure 11: DX-1 and DX-3 reduce systemic responses to autoimmunity to MPO.

[0082] Figure 12: Urine analysis (functional injury).

[0083] Figure 13: Kidney Inflammatory mRNA transcription unaltered with DX-1 and DX-3.

[0084] Figure 14: DX-1 and DX-3 attenuates glomerular injury.

[0085] Figure 15: DX-1 and DX-3 attenuates neutrophil recruitment to the kidney.

[0086] Figure 16: DX-1 and DX-3 attenuates macrophage recruitment to the kidney.

[0087] Figure 17: DX-1 and DX-3 attenuates CD4 T cell recruitment to the kidney.

[0088] Figure 18: DX-1 and DX-3 reduces NETosis in the kidney.

[0089] Figure 19: Schematic of murine experimental model of MPO-AAV (preventative or maintenance dosing schedule).

[0090] Figure 20: Effect of DX-1 and DX-3 on systemic responses to autoimmunity to MPO.

[0091] Figure 21: DX-1 and DX-3 attenuates glomerular injury.

[0092] Figure 22: DX-1 and DX-3 attenuates neutrophil recruitment to the kidney.

[0093] Figure 23: DX-1 and DX-3 attenuates macrophage recruitment to the kidney.

[0094] Figure 24: DX-1 and DX-3 attenuates CD4 T cell recruitment to the kidney.

[0095] Figure 25: Kidney Inflammatory mRNA transcription unaltered with DX-1 and DX-3.

[0096] KEY TO SEQUENCE LISTING

[0097] SEQ ID NO: 1 - 3E10 VH CDR1

[0098] SEQ ID NO: 2 - 3E10 VH CDR2

[0099] SEQ ID NO: 3 - 3E10 VH CDR3

[0100] SEQ ID NO: 4 - 3E10 VL CDR1

[0101] SEQ ID NO: 5 - 3E10 VL CDR2

[0102] SEQ ID NO: 6 - 3E10 VL CDR3

[0103] SEQ ID NO: 7 - 3E10 VH

[0104] SEQ ID NO: 8 - 3E10 VL

[0105] SEQ ID NO: 9 - VH CDR1 of DX1 / DX3

[0106] SEQ ID NO: 10 - VH CDR1 variant of DX3 SEQ ID NO: 11 - VH CDR2 of DX1 / DX3

[0107] SEQ ID NO: 12 - VH CDR2 variant of DX3

[0108] SEQ ID NO: 13 - VH CDR3 of DX1 / DX3

[0109] SEQ ID NO: 14 - VL CDR1 of DX1 / DX3

[0110] SEQ ID NO: 15 - VL CDR1 variant of DX3

[0111] SEQ ID NO: 16 - VL CDR2 of DX1 / DX3

[0112] SEQ ID NO: 17 - VL CDR3 of DX1 / DX3

[0113] SEQ ID NO: 18 - VH of DX1 / DX3

[0114] SEQ ID NO: 19 - VH of DX3 variant 1

[0115] SEQ ID NO: 20 - VH of DX3 variant 2

[0116] SEQ ID NO: 21 - VH of DX3 variant 3

[0117] SEQ ID NO: 22 - VL of DX1 / DX3

[0118] SEQ ID NO: 23 - VL of DX3 variant 1

[0119] SEQ ID NO: 24 - VL of DX3 variant 2

[0120] SEQ ID NO: 25 - VL of DX3 variant 3

[0121] SEQ ID NO: 26 - Linker sequence 1 separating scFv molecules

[0122] SEQ ID NO: 27 - Linker sequence 2 separating scFv molecules

[0123] SEQ ID NO: 28 - Hinge sequence

[0124] SEQ ID NO: 29 - Signal sequence

[0125] SEQ ID NO: 30 - VH chain of 5C6

[0126] SEQ ID NO: 31 - VH CDR1 of 5C6

[0127] SEQ ID NO: 32 - VH CDR2 of 5C6

[0128] SEQ ID NO: 33 - VH CDR3 of 5C6

[0129] SEQ ID NO: 34 - VL of 5C6

[0130] SEQ ID NO: 35 - VL CDR1 of 5C6

[0131] SEQ ID NO: 36 - VL CDR2 of 5C6

[0132] SEQ ID NO: 37 - VL CDR3 of 5C6

[0133] SEQ ID NO: 38 - DX1 sequence

[0134] SEQ ID NO: 39 - DX3 sequence

[0135] SEQ ID NO: 40 - VH CDR1 KABAT

[0136] SEQ ID NO: 41 - VH CDR2 of variants 2 - 4, 6 - 8, 10 - 12 KABAT

[0137] SEQ ID NO: 42 - VH CDR2 of variants 13 - 19 KABAT

[0138] SEQ ID NO: 43 - VH CDR3 KABAT

[0139] SEQ ID NO: 44 - VL CDR1 of variants 2 - 4, 6 - 8, 10 - 12 KABAT

[0140] SEQ ID NO: 45 - VL CDR1 of variants 13 - 19 KABAT

[0141] SEQ ID NO: 46 - VL CDR2 KABAT SEQ ID NO: 47 - VL CDR3 KABAT

[0142] SEQ ID NO: 48 - VH CDR1 IMGT

[0143] SEQ ID NO: 49 - VH CDR2 of variants 2 - 4, 6 - 8, 10 - 12 IMGT

[0144] SEQ ID NO: 50 - VH CDR2 of variants 13 - 19 IMGT

[0145] SEQ ID NO: 51 - VH CDR3 IMGT

[0146] SEQ ID NO: 52 - VL CDR1 of variants 2 - 4, 6 - 8, 10 - 12) IMGT

[0147] SEQ ID NO: 53 - VL CDR1 of variants 13 - 19 IMGT

[0148] SEQ ID NO: 54 - VL CDR2 IMGT

[0149] SEQ ID NO: 55 - VL CDR3 IMGT

[0150] SEQ ID NO: 56 - VH of variants 2, 6 and 10

[0151] SEQ ID NO: 57 - VH of variants 3, 7 and 11

[0152] SEQ ID NO: 58 - VH of variants 4, 8 and 12

[0153] SEQ ID NO: 59 - VH of variants 6 and 10

[0154] SEQ ID NO: 60 - VH of variants 13, 16 and 19

[0155] SEQ ID NO: 61 VH of variants 14 and 17

[0156] SEQ ID NO: 62 - VH of variants 15 and 18

[0157] SEQ ID NO: 63 - VL of variants 2, 3 and 4

[0158] SEQ ID NO: 64 - VL of variants 6, 7 and 8

[0159] SEQ ID NO: 65 - VL of variants 10, 11 and 12

[0160] SEQ ID NO: 66 - VL of variants 13, 14 and 15

[0161] SEQ ID NO: 67 - VL of variants 16, 17 and 18

[0162] SEQ ID NO: 68 VL of variant 19

[0163] SEQ ID NO: 69 - Variant 2

[0164] SEQ ID NO: 70 - Variant 3

[0165] SEQ ID NO: 71 - Variant 4

[0166] SEQ ID NO: 72 - Variant 6

[0167] SEQ ID NO: 73 - Variant 7

[0168] SEQ ID NO: 74 - Variant 8

[0169] SEQ ID NO: 75 - Variant 10

[0170] SEQ ID NO: 76 - Variant 11

[0171] SEQ ID NO: 77 - Variant 12

[0172] SEQ ID NO: 78 - Variant 13

[0173] SEQ ID NO: 79 - Variant 14

[0174] SEQ ID NO: 80 - Variant 15

[0175] SEQ ID NO: 81 - Variant 16

[0176] SEQ ID NO: 82 - Variant 17 SEQ ID NO: 83 - Variant 18

[0177] SEQ ID NO: 84 - Variant 19

[0178] 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 3E10 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

[0179] SEQ ID NO: 120 - VL-CDR2 variant D of 3E10 SEQ ID NO: 5

[0180] SEQ ID NO: 121 - VL-CDR3 variant G of 3E10 SEQ ID NO: 6

[0181] DETAILED DESCRIPTION OF THE INVENTION

[0182] General Techniques and Selected Definitions

[0183] 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, cell culture, immunology, and clinical studies).

[0184] Unless otherwise indicated, cell culture techniques and assays utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0185] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0186] As used herein, the term “about”, unless stated to the contrary, refers to + / - 10%, more preferably + / - 5%, of the designated value.

[0187] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0188] The terms “level” and “amount” are used to define the amount of a particular substance or cell in a sample from a subject or in a cell culture media (or sample therefrom). For example, a particular concentration, weight, percentage (e.g. v / v%) or ratio can be used to define the level of a particular substance / cell in a sample. In an example, the level is expressed in terms of how much of a particular marker is expressed by a population of cells under culture conditions. In an example, the level of the marker is representative of a certain state such as viability, cytotoxicity, apoptosis. In this example, the marker can be representative of the level of viability / cytotoxicity / apoptosis in a particular sample, in particular a certain cell population in a sample such as, for example, neutrophils. Other exemplary levels of interest in the context of the present disclosure include level of release of pro-inflammatory neutrophil enzymes and level of neutrophil phagocytosis. In an example, the level is determined under culture conditions. For example, the level can be determined based on how much of a particular marker is expressed / released from cells described herein under culture conditions. In an example, the sample is obtained from a patient or subject (e.g. a blood sample) and the level of a substance or cell is measured in the sample to determine the level of the substance in the sample. For example, neutrophils can be assessed in a sample obtained from a subject. In an example, one or more of the following may be assessed in a sample obtained from a subject: level of neutrophil viability; level of neutrophil cytotoxicity; level of neutrophil apoptosis; level of release of pro-inflammatory neutrophil enzymes; level of neutrophil phagocytosis.

[0189] In other examples, levels of interest of the present disclosure refer to the level of autoantibody in a sample obtained from a subject. For example, the level of antineutrophil cytoplasmic antibodies (ANCA) may be assessed in a sample obtained from a subject. In another example, levels of interest of the present disclosure refer to the level of interleukin 1 beta (IL-ip) in a sample obtained from a subject. For example, the level of IL- Ip may be assessed in a sample obtained from a subject.

[0190] “Immunosuppression” as used herein refers to the suppression of a subject’s immune system, in particular the impairment of immune cell recruitment, proliferation and / or function. For example, immunosuppression can be characterised by the impairment of lymphocyte proliferation, lymphocyte depletion, and / or inhibition of lymphocyte activation. Lymphocytes include T cells and B cells. Other immune cells who’s recruitment / function / proliferation can be impaired in the context of immunosuppression include other white blood cells such as Natural Killer (NK) cells, monocytes, macrophages, dendritic cells, and neutrophils. In an example, immunosuppression can be characterised by the impairment of immune cell recruitment. For example, immunosuppression may impair recruitment of cells such as neutrophils, macrophages and / or certain types of T-cells such as CD4+ T-cells. Conditions requiring immunosuppression, such as solid organ transplant rejection, are complex and involve processes such as antibody-mediated rejection (AMR) and acute cellular rejection (ACR). Such processes are generally targeted with classical immunosuppressive treatments. In an example, compositions of the disclosure can supress AMR. For example, in organ transplantation, the role of neutrophils is commonly related to antibody-mediated rejection. In this context, immunosuppression can involve impairment of neutrophil activity and / or function. In another example, immunosuppression can involve suppression of autoantibodies. In an example, immunosuppression reduces autoantibody levels. In other examples compositions of the disclosure can supress ACR. In yet other examples, compositions of the disclosure can suppress both AMR and ACR. In another example, compositions of the disclosure can reduce IL-ip levels.

[0191] The term “immunosuppressant” is used in the context of the present disclosure to refer to an agent capable of mediating immunosuppression. It will be appreciated that immunosuppression is often mediated by a combination of active agents rather than a single therapy. Accordingly, in certain examples, an immunosuppressant of the disclosure mediates immunosuppression in conjunction with other actives. In other words, an immunosuppressant of the disclosure may impair some but not all aspects of immune system function while still be considered an immunosuppressant. For example, one standard approach to immunosuppression consists of triple therapy with a calcineurin inhibitor, an anti-proliferative agent, and a corticosteroid. In an example, an immunosuppressant of the disclosure is added to this combination. In an example, such an addition allows one or more of the calcineurin inhibitor, the anti-proliferative agent or, the corticosteroid to be tapered. In an example, an immunosuppressant of the disclosure inhibits the recruitment, proliferation and / or function of immune cells. For example, an immunosuppressant may inhibit T-cell recruitment, activation and / or proliferation, or cause T-cell depletion. Immunosuppressants may also impair the function of other immune cells, such as B cells, NK cells, neutrophils, monocytes, and / or macrophages. In certain examples, immunosuppressant compositions of the disclosure impair immune cell recruitment and / or function without depletion. For example, immunosuppressant compositions of the disclosure can impair neutrophil activity without substantially depleting neutrophil levels. For example, an immunosuppressant composition may impair neutrophil activity without resulting neutropenia. In an example, impaired neutrophil activity is impaired NETosis. In another example, an immunosuppressant composition may reduce autoantibody levels, in particular antineutrophil cytoplasmic antibody (ANCA) levels. In another example, an immunosuppressant composition may reduce IL-ip levels. In other examples, immunosuppressant compositions of the disclosure impair recruitment and / or function of other immune cells such as T-cells (e.g. CD4+ T-cells) and / or macrophages. Impairment of such cells is particularly important in the context of certain diseases requiring immunosuppression as macrophages interact with T cells to bring about T cell activation. Accordingly, in certain examples, an immunosuppressant disclosed herein attenuates recruitment and / or function of these cell types. In an example, an immunosuppressant of the disclosure is a binding protein. In an example, such binding proteins are combined with other immunosuppressants to impart immunosuppression. In an example, the binding protein impairs recruitment of neutrophils, CD4+ T cells and / or macrophages. In another example, the binding protein impairs recruitment of neutrophils, CD4+ T cells and macrophages.

[0192] Immunosuppressants are a clearly recognised class of actives in that they mediate immunosuppression, as outlined above. While some immunosuppressants can also act as anti-inflammatories, when they act as an anti-inflammatory, they do so by targeting inflammation directly rather than the activity of the immune system generally. As would be appreciated by those of skill in the art, the term “inflammation” can be used to generically describe inflammatory pathways and cascades that are mediated by cytokines, acute phase proteins, and chemokines in response to an inflammatory stimuli. “Anti-inflammatory agent” refers to an agent that interferes with one or more of the pathways that contribute to inflammation (e.g. the activity of soluble mediators, their signalling pathways, or cellular receptors), but do not directly affect immune cell function. Examples of anti-inflammatory agents include pro-inflammatory cytokine inhibitions, such as Tumor Necrosis Factor-alpha (TNFa) inhibitors, and IL-6 receptor antagonists (e.g. tocilizumab).

[0193] Accordingly, immunosuppressants and methods of using the same disclosed herein are distinguished from anti-inflammatories because immunosuppression involves the inhibition of immune cell proliferation and / or function (e.g. autoantibody production), whereas anti-inflammatories target mediators of inflammatory pathways, such as cytokines or chemokines and their signalling pathways. Indeed, those of skill in the art will appreciate that “immunosuppressants” and “anti-inflammatories” are distinct classes of actives.

[0194] Although some agents can mediate both anti-inflammatory and immunosuppressive functions, these agents can be appropriately characterised as one or the other based on their activity in vivo. Corticosteroids are an example of agents that can mediate either anti-inflammatory or immunosuppressive function. For example, corticosteroids, such as prednisone, have anti-inflammatory properties such as inhibition of cytokine release and inhibition of cellular responses to pathogen-associated molecular patterns (PAMPS) and, damage-associated molecular patterns (DAMPS). Corticosteroids are therefore administered to treat certain anti-inflammatory diseases and, when used in this context, are appropriately described as an anti-inflammatory. However, corticosteroids can also act as an immunosuppressant by impairing both T cell and B cell proliferation and activation. Corticosteroids are therefore also administered to treat certain diseases which require immunosuppression, such as solid organ transplant rejection. When used in this context, the corticosteroids are appropriately described as an immunosuppressant. In certain examples, actives with dual anti- inflammatory / immunosuppressive activity can be distinguished as either an antiinflammatory or an immunosuppressant depending on dose administered. For example, example, low-dose corticosteroids are typically administered to treat inflammation, whereas higher-doses of corticosteroids are typically administered when immunosuppression is required. For the avoidance of doubt, where the term “immunosuppressant” is used to refer to agents herein that have dual anti- inflammatory / immunosuppressive functions, it is to be understood that the term “immunosuppressant” encompasses the agent when in an amount or dose effective to impart the immunosuppressive properties of said agent.

[0195] 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 nucleus of living mammalian cells and, preferably, binds DNA (e.g., single-stranded and / or double-stranded DNA). In an example, cell penetrating antibodies also penetrate cell nuclei. Accordingly, binding proteins of the disclosure can localise in cell nuclei and bind DNA (i.e. they are nuclear penetrating). Such binding proteins are distinguished from antibodies that can penetrate into cells but remain sequestered in the cytoplasm.

[0196] The term “anti-DNA binding protein” is used in the context of the present disclosure to refer to antibodies capable of binding DNA.

[0197] The term “binding protein” is used in the context of the present disclosure to refer to 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.

[0198] 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.

[0199] 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.

[0200] 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.

[0201] 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 antibody is an IgG.

[0202] 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. 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”.

[0203] Other conventions that include corrections or alternate numbering systems for variable domains include IMGT (Lefranc, et 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 labeled according to IMGT.

[0204] “Framework regions” (Syn. FR) are those variable domain residues other than the CDR residues.

[0205] 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).

[0206] 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, a, y, or p. Exemplary heavy chain constant regions are gamma 1 (IgGl), gamma 2 (IgG2) and gamma 3 (IgG3), or hybrids thereof.

[0207] 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 6 heavy chain comprises two constant domains.

[0208] 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 another compound, e.g., therapeutic compound or a diagnostic compound. Accordingly, in one example, the binding protein of the present disclosure are “conjugated”. The nature of the conjugation is not particularly limited so long as it maintains the capacity of the binding protein to inhibit Neutrophil Extracellular Trap (NET) formation and preserve neutrophil function.

[0209] "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.

[0210] 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, unlabeled “A”), in a reaction containing labeled “A” and the binding protein, will reduce the amount of labeled “A” bound to the binding protein.

[0211] 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.

[0212] 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.

[0213] 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. 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. In another example, the subject can be an adolescent. In one example, the subject has received a solid organ transplant. In another example, the subject has neutropenia. In another example, the subject has IRI.

[0214] 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.

[0215] 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. For example, compositions of the disclosure may be administered prophylactically to reduce the risk of neutropenia.

[0216] 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, for example, immunosuppression. An effective amount can be provided in one or more administrations. In some examples of the present disclosure, the term “effective amount” means an amount necessary to effect treatment of a disease or condition described below. In other example, the term “effective amount” means an amount necessary to supress the immune system. 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.

[0217] A “therapeutically effective amount” is at least the minimum concentration required to effect a measurable improvement of a particular disorder (e.g. solid organ transplant rejection). 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.

[0218] In the context of immunosuppression, the therapeutically effective amount of the binding protein may reduce autoantibody levels. In the case of solid organ transplant rejection, the therapeutically effective amount of the binding protein 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 solid organ transplant rejection, 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. In an example, the therapeutically effective amount is sufficient to reduce or discontinue calcineurin inhibitor use in a subject. In another example, the therapeutically effective amount is sufficient to reduce or discontinue antiproliferative agent use in a subject. In another example, the therapeutically effective amount is sufficient to reduce or discontinue steroid use in a subject. In another example, the therapeutically effective amount is sufficient to taper a subject’s immunosuppression. In this example, the immunosuppression may comprise administration of a calcineurin inhibitor, an antiproliferative agent, and a corticosteroid and, the therapeutically effective amount is sufficient to taper one or more or all of these actives. In another example, the therapeutically effective amount is sufficient to reduce a subject’s IL-ip levels.

[0219] Binding proteins of the disclosure preserve neutrophil function. In an example, preservation of neutrophil function is determined under culture conditions. The term “culture conditions” is used to refer to cells growing in culture. In an example, culture conditions refers to a population of cells in cell culture such as neutrophils or neutrophillike cells such as PLB-985, HL-60 cells (e.g. ATCC CCL-240 or a sub clone thereof), NB4. In an example, the population of cells are neutrophils that have been obtained from a subject. In an example, the subject has been administered a binding protein of the disclosure. In an example, the neutrophils are obtained from the subject prior to administering a binding protein of the disclosure. In an example, culture conditions refers to a culture expanded population of cells. In an example, culture conditions refers to an actively dividing population of cells. In an example, culture conditions comprises culturing cells with an inflammatory stimulus such as phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS), a calcium ionophore ionomycin (IM), or tumour necrosis factor alpha (TNFa). In these examples, cells will be assessed in culture to determine whether NET formation is inhibited. In another example, cells will be assessed in culture to determine whether neutrophil function is preserved. In an example, cells are assessed in culture to determine that NET formation is inhibited and that the remaining neutrophil function is preserved.

[0220] Preservation of neutrophil function can be measured under culture conditions using various approaches. In an example, the level of a particular marker that is representative of neutrophil function can be determined by taking a sample measuring the level of marker in the sample. In another example, the level of a particular marker can be determined by taking a sample of cells and measuring the level of the marker in the cell lysate. In another example, the level of a particular marker can be determined by taking a sample of cells and measuring the level of marker in the cell sample. Those of skill in the art will appreciate that secreted markers can be measured by sampling the culture media while markers expressed on the surface or inside cultured cell(s) may be measured by assessing a sample of cells or cell lysate. In an example, the sample is taken after at least 1 hour to two days in culture. In another example, the level of a particular marker can be determined by visually assessing cells under culture conditions. In an example, the marker of neutrophil function is neutrophil viability, neutrophil cytotoxicity, neutrophil apoptosis, neutrophil phagocytosis, or release of pro- inflammatory neutrophil enzymes relative to a control. In this example, preservation of neutrophil function is characterised by equivalent levels of neutrophil viability, neutrophil cytotoxicity, neutrophil apoptosis, neutrophil phagocytosis, or release of pro- inflammatory neutrophil enzymes relative to a control.

[0221] In certain examples, binding proteins of the disclosure preserve neutrophil function in a subject who has been administered said binding protein. In an example, preservation of neutrophil function is assessed in vivo. For example, preservation of neutrophil function is assessed by determining circulating neutrophil levels in a subject. In an example, the level of circulating neutrophils can be determined by taking a sample from a subject (e.g. a blood sample) and measuring the number of neutrophils in the blood sample. In another example, preservation of neutrophil function is assessed in a sample obtained from a subject who has been administered a binding protein of the disclosure. In this example, perseveration of neutrophil function is assessed by measuring the level of a particular marker of neutrophil function under culture conditions as described above.

[0222] Binding proteins of the disclosure also inhibit Neutrophil Extracellular Trap (NET) formation. In an example, NET formation is determined under culture conditions. In this example, culture conditions comprises culturing cells with a stimulus of NET formation such as phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide (LPS). Other exemplary stimulus of NET formation include calcium ionophores such as A23187 (A23) or inomycin. In an example, culture conditions comprises culturing cells with 300 nM PMA for 4.5 hours.

[0223] In an example, NET formation is measured in a population of neutrophils that have been obtained from a subject. In an example, the subject has been administered a binding protein of the disclosure. In an example, the neutrophils are obtained from the subject prior to administering a binding protein of the disclosure. In an example, the level of a particular marker that is representative of NET formation can be determined by taking a sample of cell culture media and measuring the level of marker in the sample. In another example, the level of a particular marker can be determined by taking a sample of cells and measuring the level of the marker in the cell lysate. Those of skill in the art will appreciate that secreted markers can be measured by sampling the culture media while markers expressed on the surface or inside cultured cell(s) may be measured by assessing a sample of cell lysate. In an example, the sample is taken after 2 hours to two days in culture. In another example, the level of a particular marker can be determined by visually assessing cells under culture conditions. In an example, the marker of NET formation is level of cell death and extracellular DNA in NETs. In another example, the marker of NET formation is DNA release from neutrophil-like cells after culture with the inflammatory stimulus. In this example, inhibition of NET formation is characterised by reduced DNA release from neutrophil-like cells after culture with the inflammatory stimulus. Various markers of NET formation are known in the art. Examples include circulating cell free DNA, myeloperoxidase (MPO), Citrullinated histone 3 (H3Cit), and neutrophil elastase (NE).

[0224] In certain examples, binding proteins of the disclosure inhibit NET formation in a subject who has been administered said binding protein. In an example, NET formation is assessed in vivo. In this example, NET formation is assessed by determining NET formation in a subject. In an example, NET formation can be determined by taking a sample from a subject (e.g. a plasma sample) and measuring NET formation using an immunoassay (e.g. an Enzyme-Linked Immunosorbent Assay (ELISA)). In this example, an ELISA is used to detect markers of in vivo NET formation, including citrullinated histone 3 (H3Cit), nucleosomes, cell-free DNA, or neutrophil-derived enzymes such as MPO and NE, in a sample. Other methods for measuring NET formation in vivo are described, for example, in Stoimenou et al. International Journal of Molecular Sciences. 2022; 23(24): 15823.

[0225] In some examples, binding proteins of the disclosure reduce anti-neutrophil cytoplasmic autoantibody (ANCA) levels. For example, ANCA levels are reduced in a subject that has been administered a binding protein of the disclosure. In an example, the ANCA is myeloperoxidase (MPO)-ANCA. In an example, the ANCA is myeloperoxidase (MPO)-ANCA. In an example, the ANCA is proteinase 3 (PRT3)- ANCA. In an example, ANCA levels can be determined by taking a sample from a subject (e.g. a serum sample or a plasma sample) and measuring the level of ANCA using an immunoassay (e.g. an Enzyme-Linked Immunosorbent Assay (ELISA)).

[0226] In certain examples, binding proteins of the disclosure can be used to taper a subject’s dose of an immunosuppressant. The term “taper” and “tapering” as used herein means gradually decreasing the total dose of a medication, typically with the goal of discontinuing the medication. For example, binding proteins of the disclosure can be used as sparing agents, and / or in methods of reducing or discontinuing a subject’s use of an immunosuppressant agent.

[0227] The term “sparing” or “sparing agent” is used in the context of the present disclosure to refer to a composition that can be administered to a subject using an alternative immunosuppressant agent in order to facilitate reduction of the subject’s intake of that particular immunosuppressant agent. In other words, subj ects administered a sparing agent (e.g. a binding protein of the disclosure) are able to take a lower dose of an alternative immunosuppressant agent over time. In an example, binding proteins of the disclosure can be used as a corticosteroid sparing agent. In an example, the corticosteroid sparing agent supresses a subject’s immune response and / or reduces the subject’s risk of neutropenia. In an example, the subject’s risk of neutropenia is reduced relative to a subject who has not been administered a corticosteroid sparing agent (e.g. a binding protein of the disclosure). In an example, corticosteroid sparring agents of the disclosure are administered to a subject with neutropenia.

[0228] In another example, binding proteins of the disclosure can be used as a calcineurin inhibitor sparing agent. In an example, the calcineurin inhibitor sparing agent supresses a subject’s immune response and / or reduces the subject’s risk of neutropenia. In an example, the subject’s risk of neutropenia is reduced relative to a subject who has not been administered a calcineurin inhibitor sparing agent (e.g. a binding protein of the disclosure). In an example, calcineurin inhibitor sparring agents of the disclosure are administered to a subject with neutropenia.

[0229] Immunosuppressant Anti-DNA Binding Proteins

[0230] The present inventors surprisingly identified immunosuppressant anti-DNA binding proteins that impair immune cell recruitment and / or activation. In certain examples, these binding proteins can also impair immune cell recruitment and / or activation whilst preserving immune cell function. For example, binding proteins of the disclosure can impair recruitment of cells such as neutrophils, macrophages and certain types of T-cells, such as CD4+ T-cells. In certain examples, binding proteins of the disclosure can also direct immunosuppression by reducing autoantibody levels, in particular ANCAs. In an example, binding proteins of the disclosure can impair recruitment of cells such as neutrophils whilst preserving neutrophil function.

[0231] In particular, binding proteins of the disclosure preserve neutrophil viability, and do not cause neutrophil cytotoxicity or neutrophil apoptosis. Accordingly, binding proteins of the disclosure maintain preserve neutrophil viability and do not cause neutrophil cell death. Binding proteins of the disclosure also preserve release of pro- inflammatory neutrophil enzymes and neutrophil phagocytosis. Phagocytosis and release of pro-inflammatory enzymes, such as MPO and NE, are indicators that the immune function of neutrophils is preserved.

[0232] Neutrophils represent the most abundant innate immune cells. Activated neutrophils generate neutrophil extracellular traps (NETs), which are extracellular fibers composed of chromatin, histones and granular proteins. Neutrophil activation, and subsequent NETosis, can occur in a range of disease states, such as transplant rejection and reperfusion injury, and, in response to inflammatory stimuli. ANCAs are autoantibodies that react to pro-inflammatory neutrophil enzymes. ANCAs occur prominently in patients that require immunosuppression, for example patients having vasculitis or glomerulonephritis. Two main forms of ANCA have been distinguished on the basis of the target antigens: PR3-ANCA (C-ANCA), which reacts with proteinase-3 (PR3), and MPO-ANCA (P-ANCA), which reacts with myeloperoxidase (MPO).

[0233] Accordingly, anti-DNA binding proteins of the disclosure can supress immune responses by targeting key cell types, such as neutrophils, that are also targeted by classical immunosuppression regimens, however they have the added advantage of not depleting neutrophil levels.

[0234] In an example, prevention of neutrophil activation by anti-DNA binding proteins of the disclosure is characterised by the inhibition of NETosis. In an example, anti-DNA binding proteins of the disclosure reduce autoantibody levels, such as ANCA levels. The reduction of ANCA levels in this example is a measure of both perseveration of neutrophils and inhibition of neutrophil activation. In an example, the anti-DNA binding protein impairs recruitment of neutrophils, CD4+ T cells and / or macrophages. In another example, the anti-DNA binding protein impairs recruitment of neutrophils, CD4+ T cells and macrophages.

[0235] 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. In an example, the anti-DNA binding protein is cell penetrating.

[0236] 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. 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. 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 CDR1 may rather comprise SEQ ID NO: 40.

[0237] 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.

[0238] 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.

[0239] 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 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.

[0240] In an example, the binding protein 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.

[0241] 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.

[0242] 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.

[0243] In an 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.

[0244] In an example, the binding protein 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.

[0245] 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. 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.

[0246] 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.

[0247] In one example, the 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 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.

[0248] 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.

[0249] 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.

[0250] 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.

[0251] 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 Vn is 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: 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 Vnis 98% identical to SEQ ID NO: 18 and the V 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 Vnis 99% identical to SEQ ID NO: 18 and the Vris 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).

[0252] 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.

[0253] 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.

[0254] 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. 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.

[0255] 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.

[0256] 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.

[0257] 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.

[0258] 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.

[0259] 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.

[0260] 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.

[0261] 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.

[0262] 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.

[0263] 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.

[0264] 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.

[0265] 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.

[0266] 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.

[0267] 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.

[0268] 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.

[0269] 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.

[0270] 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. 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.

[0271] 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.

[0272] 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.

[0273] 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:

[0274] 56 and the VL is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 64.

[0275] 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:

[0276] 57 and the VL is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 64.

[0277] 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:

[0278] 58 and the VL is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 64. 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:

[0279] 56 and the VL is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 65.

[0280] 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:

[0281] 57 and the VL is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 65.

[0282] 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:

[0283] 58 and a VL that is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 65.

[0284] 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:

[0285] 60 and the VL is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 66.

[0286] 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:

[0287] 61 and the VL is at least 95%, 97%, 98%, or 99% to SEQ ID NO: 66.

[0288] 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:

[0289] 62 and the VL is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 66. 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:

[0290] 60 and the VL is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 67.

[0291] 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:

[0292] 61 and the VL is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 67.

[0293] 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:

[0294] 62 and the VL is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 67.

[0295] 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.

[0296] 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; WO20 19 / 018426). In an example, the antibody is not an anti-guanosine antibody.

[0297] 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.

[0298] 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.

[0299] 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.

[0300] Thus, in an example, the binding protein may be a 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.

[0301] 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: 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.

[0302] 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.

[0303] 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.

[0304] 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.

[0305] 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.

[0306] 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.

[0307] 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.

[0308] In another example, the binding protein is a di-scFv which comprises an amino acid sequence as shown in SEQ ID NO: 38.

[0309] In another example, the Fv is a tri-scFv. 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 CH2 and / or CH3. In one example, the scFv, di-scFv or tri-scFv is linked to the 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.

[0310] 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.

[0311] In another example, the VH 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 VH 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.

[0312] In another example, the antibody comprises 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 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.

[0313] 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.

[0314] 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.

[0315] 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.

[0316] In another example, the antibody is an intact antibody comprising 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. 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.

[0317] 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.

[0318] 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 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. 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.

[0319] 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.

[0320] 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.

[0321] 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.

[0322] In one example, the antibody is an intact antibody comprising the amino acid sequence shown in SEQ ID NO: 39.

[0323] 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.

[0324] 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 nucleoti de-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.

[0325] An example, of a nucleolytic antibody is disclosed in Noble et al. (2014) Sci Rep- Uk., 4:5958.

[0326] In another example, binding proteins of the disclosure can bind DNA without facilitating degradation of the DNA.

[0327] 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.

[0328] 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.

[0329] 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.

[0330] Single Chain Fv (scFv) Fragments

[0331] 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.

[0332] 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.

[0333] 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.

[0334] Diabodies, Triabodies, Tetrabodies

[0335] 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.

[0336] 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).

[0337] Other Antibodies and Antibody Fragments

[0338] Other examples of antibodies encompassed by the present disclosure include:

[0339] (i) “key and hole” bispecific proteins as described in U.S. Patent No. 5,731,168;

[0340] (ii) heteroconjugate proteins, e.g., as described in U.S. Patent No. 4,676,980;

[0341] (iii) heteroconjugate proteins produced using a chemical cross-linker, e.g., as described in U.S. Patent No. 4,676,980; and

[0342] (iv) Fabs (e.g., as described in EP19930302894).

[0343] Recombinant Expression

[0344] In an example, the binding protein is recombinant.

[0345] 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. 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.

[0346] 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.

[0347] 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).

[0348] 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, P-actin promoter; hybrid regulatory element comprising a CMV enhancer / P- actin promoter or an immunoglobulin or antibody promoter or active fragment thereof. Examples of useful mammalian host cell lines are monkey kidney CV1 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).

[0349] Typical promoters suitable for expression in yeast cells such as for example a yeast cell selected from the group comprising Pichiapastoris, Saccharomyces cerevisiae and S. pombe. include, but are not limited to, the ADH1 promoter, the GALI promoter, the GAL4 promoter, the CUP 1 promoter, the PHO 5 promoter, the nmt promoter, the RPR1 promoter, or the TEF1 promoter.

[0350] 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.

[0351] 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.

[0352] 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.

[0353] 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.

[0354] 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.

[0355] The disclosure also provides a host cell comprising an expression construct according to the present disclosure.

[0356] 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. Methods for purifying antibodies according to the present disclosure are known in the art and / or described in publications such as WO2019 / 018426.

[0357] Methods of measuring NET formation and neutrophil function

[0358] Immunosuppressant binding proteins of the disclosure inhibit neutrophil activation (e.g., NET formation) and preserve neutrophil function. Methods for determining inhibition of NET formation and preservation of neutrophil function are known in the art and can be performed using routine techniques.

[0359] In an example, inhibition of NET formation and / or preservation of neutrophil function are determined under culture conditions. In an example, the culture conditions comprise culturing neutrophils or a neutrophil-like cell line in culture medium which comprises an inflammatory stimulus. The person skilled in the art will appreciate that any appropriate neutrophil or neutrophil-like cell line can be used. In an example, the cells are primary neutrophils, for example human neutrophils. In an example, the cells are PLB-985 cells. Non-limiting examples of suitable inflammatory stimuli include phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS), a calcium ionophore ionomycin (IM), or tumour necrosis factor alpha (TNFa).

[0360] In some examples, inhibition of NET formation and / or preservation of neutrophil function are determined ex vivo. In this example, neutrophils are obtained from a subject. In an example, neutrophils are obtained from a subject who has been administered an immunosuppressant binding protein of the disclosure.

[0361] In an example, preservation of neutrophil function under culture conditions is determined by:

[0362] (i) treating a population of neutrophils with the anti-DNA binding protein;

[0363] (ii) stimulating the population of neutrophils with an inflammatory stimulus; and

[0364] (iii) determining the level of one or more markers of neutrophil function in the population of neutrophils with the anti-DNA binding protein and a control population of neutrophils that do not comprise an anti-DNA binding protein; wherein an equivalent level of the one or more markers of neutrophil function between the control neutrophil population and the anti-DNA binding protein population indicates that neutrophil function is preserved.

[0365] Various markers of neutrophil function known in the art and include, for example, neutrophil viability; neutrophil cytotoxicity; neutrophil apoptosis; release of pro- inflammatory neutrophil enzymes; and neutrophil phagocytosis.

[0366] There are various reagents and assays available for determining neutrophil viability, cytotoxicity, and apoptosis. For example, neutrophil viability can be determined by routine assays such as tetrazolium reduction, resazurin reduction, and ATP detection. Briefly, tetrazolium compounds such as MTT, MTS, XTT, and WST-1 are incubated with the cells. Viable cells with active metabolism convert the compound into a coloured product with an absorbance maximum near a particular wavelength (e.g. 570 nm), which can be detected using spectrophotometry.

[0367] Cytotoxicity assays measure parameters associated with loss of membrane integrity upon cell death. Cytotoxicity assays may also detect cytosolic proteins that should not be released by the cell, unless the cell has lost membrane integrity and the proteins have leaked out of the cell. For example, lactate dehydrogenase (LDH) release is a well-accepted measure of cell death that can be detected with the bioluminescent or fluorescent reagents that bind LHD. Alternatively, cytotoxicity assays may detect the ability of cell impermeant dyes to enter cells upon loss of membrane activity, for example fluorescent DNA binding dyes can be used that binds DNA upon cell death and generates a stable signal that can be measured using techniques such as spectrophotometry or flow cytometry.

[0368] Apoptosis assays detect and quantify the cellular events associated with programmed cell death, including caspase activation, cell surface exposure of phosphatidyl serine (PS) and DNA fragmentation. Reagents such as annexin V, which bind PS, can be used to measure apoptosis.

[0369] Release of pro-inflammatory neutrophil enzymes into culture supernatants can be measured using standard techniques, such as ELISA, flow cytometry, western blot, or multiplex assays. Pro-inflammatory neutrophil enzymes that can be measured include myeloperoxidase (MPO), neutrophil elastase (NE), and proteinase 3 (PRT3).

[0370] Neutrophils mainly kill pathogens through phagocytosis, a process in which bacteria are engulfed into intracellular vesicles called phagosomes. Neutrophil phagocytosis function can be measured by incubating neutrophils with fluorescent bacteria and measuring the number of bacteria-associated neutrophils by methods such as spectrophotometry, flow cytometry, or fluorescence microscopy.

[0371] Neutrophil function can also be assessed in vivo. For example, preservation of neutrophil function can assessed by determining circulating neutrophil levels in a subject. In an example, the level of circulating neutrophils can be determined by taking a sample from a subject (e.g. a blood sample) and measuring the number of neutrophils in the blood sample. This is determined by performing a white blood cell count (WBC) or complete blood count (CBC) on a blood sample. Neutropenia is characterised by an absolute neutrophil count (ANC) of less than 1.5* 109 / L in circulating blood. Moderate to severe neutropenia is characterised by an ANC of less than 1 * 109 / L in circulating blood. Severe neutropenia is characterised by an ANC of less than 0.5* 109 / L in circulating blood. Neutropenia is characterised as chronic when the low neutrophil counts persist over at least a 3 month period. For example, neutropenia is deemed “severe” and “chronic” when the counts are less than 0.5 * 109 / L on at least 3 occasions in a 3 month period.

[0372] Immunosuppressant binding proteins of the disclosure, and compositions comprising the same, inhibit neutrophil activation without substantially depleting neutrophil levels. For example, an immunosuppressant binding protein may inhibit neutrophil activation without resulting neutropenia.

[0373] Methods of measuring inhibition of NET formation under culture conditions are known in the art and are described herein. In an example, NET formation can determined based on one or both of: 1) level of cell death and extracellular DNA in NETs; and / or, 2) reduced DNA release from neutrophil-like cells after culture with the inflammatory stimulus. NET formation and extracellular DNA can be visualised by the addition of a DNA stain, such as DAPI, to cells, and counting the cells with visualized extrusion of DNA using fluorescence microscopy. Extracellular DNA can also be measured using SYTOX green and A260. An example of NET morphology is provided in Figure 2. Another indicator of NETosis is chromatin decondensation. Chromatin decondensation can also be visualized by DAPI staining and imaged using fluorescence microscopy. In this example, a diffuse DAPI stain is suggestive of chromatin decondensation.

[0374] Extracellular DNA can also be measured by spectrophotometry. In this example, cells are stimulated in assay plates in under sufficient conditions to allow adherence of DNA-protein complexes to plate wells. Supernatants are then aspirated and residual contents adherent to the wells can be extracted. The DNA content of the adherent contents can then determined by spectrophotometry.

[0375] NET formation can also be assessed in vivo. In an example, NET formation can be determined by taking a sample from a subject (e.g. a plasma sample) and measuring NET formation using an immunoassay (e.g. an Enzyme-Linked Immunosorbent Assay (ELISA)). In this example, an ELISA is used to detect markers of in vivo NET formation, including citrullinated histone 3 (H3Cit), nucleosomes, cell-free DNA, or neutrophil- derived enzymes such as MPO and NE, in a sample. Other methods for measuring NET formation in vivo are described, for example, in Stoimenou et al. International Journal of Molecular Sciences. 2022; 23(24): 15823. In an example, compositions comprising anti-DNA binding proteins of the disclosure are selected for use in a method of the disclosure based on their ability to (i) inhibit NET formation, and (ii) preserve neutrophil function. In this example, inhibition of NET formation and preservation of neutrophil function is measured according to the methods described above.

[0376] Immunosuppressant Compositions

[0377] Anti-DNA binding proteins of the disclosure can be used in combination with other immunosuppressive agents. For example, immunosuppressive therapy can comprise an anti-DNA binding protein, and one or more additional immunosuppressive agents.

[0378] Immunosuppressive therapy regimens typically comprise a combination of systemic immunosuppressants, including calcineurin inhibitors, mechanistic target of rapamycin (mTOR) inhibitors, anti-proliferative agents, corticosteroids and antibodies (Mahmud et al., mAbs. 2010. 2(2): 148-156).

[0379] Antibody Therapies

[0380] Various antibody therapies are used in immunosuppression regimens, Typically, antibody therapies are used in induction or anti -rejection regimens. These treatments are also used in the treatment of acute rejection in organ transplantation and often used in conjunction with other immunosuppressants, including corticosteroids, to allow the administration of lower and less toxic doses.

[0381] Examples of antibody therapies include Muromonab-CD3 (also known as Orthoclone OKT3), anti -lymphocyte globulin (ALG), anti-thymocyte globulin (ATG), daclizumab and basiliximab. Muromonab-CD3 blocks T-cell function by binding to the epsilon chain of CD3 -T cell-receptor complex found on the surface of T cells, which is involved in antigen recognition (Klipa et al. mAbs. 2010. 2(6): 607-612). Antilymphocyte globulin (ALG) and anti-thymocyte globulin (ATG) are infusions of animal antibodies (from hyperimmunized mammals such as horses and rabbits) against human T cells. Side effects include anaphylactic reactions, serum sickness, and glomerulonephritis. ALG and ATG modulate the T cell reaction by binding to T-cell- specific antigens, leading to T-cell death. Daclizumab and basiliximab are chimeric or humanized murine antibodies that are directed against the IL-2R-alpha-chain and target activated T cells. They prevent binding of IL-2, which in turn inhibits T cell proliferation. Calcineurin Inhibitors

[0382] Calcineurin inhibitors (tacrolimus and cyclosporine) prevent the synthesis of IL- 2 and other cytokines produced by T cells. Both tacrolimus and cyclosporine have the ability to inhibit calcineurin, an intracellular calcium / calmodulin-dependent phosphatase that plays a key role in the translocation of nuclear factor of activated T cells from the T cell receptor to the nucleus. By inhibiting calcineurin, tacrolimus and cyclosporine block the transcription and synthesis of IL-2, IL-3, interferon-gamma and TNF-alpha, thereby interfering with activated CD4+ Th cell function. Consequently, T-cell proliferation and differentiation of precursor cytotoxic lymphocytes are inhibited. In addition, inhibition of the calcineurin pathway is known to subsequently inhibit NET formation (Mutua and Gershwin. Clin Rev Allergy Immunol. 2021. 61(2): 194-211).

[0383] Calcineurin inhibitors have been associated with many side effects, including susceptibility to infection, refractory hypertension, and liver and kidney disease, an increased risk of malignancy, headache, seizure, confusion, weakness, tremors, diffuse encephalopathy and posterior reversible encephalopathy syndrome.

[0384] Anti-Proliferative Agents

[0385] Anti-proliferative agents are often used concomitantly with other immunosuppressive agents for the prevention of rejection in organ transplantation. It is usually used as part of a three-compound regimen of immunosuppressants, including a calcineurin inhibitor and corticosteroids.

[0386] Examples of anti-proliferative agents include mycophenolate mofetil (MMF), my cophenolate sodium and azathioprine. These agents block the production of DNA in B and T cells, which prevents these cells from multiplying.

[0387] MMF is a prodrug of mycophenolic acid (MPA) that is rapidly converted to mycophenolic acid after oral administration. Mycophenolate sodium is another formulation of mycophenolic acid. MPA acts via a series of downstream signalling pathways that ultimately inhibits the attachment of leukocytes to endothelial cells and prevents the recruitment of lymphocytes and monocytes to sites of inflammation.

[0388] Azathioprine is an immunosuppressant antimetabolite that inhibits purine nucleotide synthesis, thus interfering with synthesis and metabolism of RNA. The most common adverse events associated with anti-proliferative agents include diarrhea, nausea, abdominal pain, anemia and leukopenia. mTOR Inhibitors mTOR inhibitors (sirolimus and everolimus) are mainly used in combination with other immunosuppressive drugs as part of maintenance therapy. mTOR inhibitors block IL-2 signals to T cells, resulting into a reduction in T cell proliferation. mTOR inhibition also affects the activity of T cells, as well as other populations of immune cells, including dendritic cells (Stallone, et al. Journal of Translational Medicine. 2016. 14: 152 2016). Common adverse effects of mTOR inhibitors include abdominal pain, diarrhoea, high blood pressure, low levels of platelets or red blood cells, hypokalemia and hypophosphatemia.

[0389] Corticosteroids

[0390] Examples of corticosteroids include prednisone, prednisolone and methylprednisolone. Corticosteroids bind to the glucocorticoid receptor to form a complex, and this steroid-receptor complex translocates into the cell nucleus where it binds DNA, altering the transcription of numerous genes in leukocytes. This leads to an anti-inflammatory and immunosuppressive response. Corticosteroids are also referred to as “glucocorticoids”. Unless otherwise specified, these terms are used interchangeably throughout.

[0391] Corticosteroids effect on peripheral blood leukocyte trafficking, proliferation and differentiation, as well as leukocyte infiltration into inflamed sites. Lymphocyte, monocyte and basophil counts decrease considerably in response to corticosteroid administration. High doses of intravenous corticosteroid form are often administered as part of an induction immunosuppression regimen, and can also be taken as daily for maintenance therapy along with other immunosuppressive medications.

[0392] Mechanisms of action, immune cell effects, and side effects of common immunosuppressive agents are summarized in Tables 1-3.

[0393] Table 1 : Mechanisms of Action of Common Immunosuppressive Therapies (Roberts and Fishman. Clin Infect Dis. 2021, 73(7):el302-el317),

[0394] Abbreviations: ATG, Anti -thymocyte globulin; IL, interleukin; IMPDH, inosine-5'- monophosphate dehydrogenase; MMF, mycophenolate mofetil; mTOR, mechanistic target of rapamycin. Table 2: Effects of Common Immunosuppressive Agents on Immune Function (Roberts and Fishman. Clin Infect Dis. 2021, 73(7):el302-el317)

[0395]

[0396] Abbreviations: ATG, Anti -thymocyte globulin; CNI, calcineurin inhibitor; Ig, immunoglobulin; IL, interleukin; LPS, lipopolysaccharide; MMF, mycophenolate mofetil; mTOR, mechanistic target of rapamycin; Treg, T-regulatory cell; VEGF, vascular endothelial growth factor.

[0397] Table 3: Side effects of common immunosuppressive drugs (Claeys et al. Journal of Immunological Sciences. 2019,3(4): 14-21),

[0398] The present disclosure includes compositions for administration to subjects. Exemplary compositions comprise one or more of the above referenced immunosuppressant binding proteins. In other examples, compositions of the disclosure comprise an immunosuppressant binding protein, and one or more additional immunosuppressive agents.

[0399] In an example, the immunosuppressant composition comprises an anti-DNA binding protein and a calcineurin inhibitor, - an anti -proliferative agent, and / or

[0400] - a corticosteroid. In an example, the immunosuppressant composition comprises an anti-DNA binding protein and a calcineurin inhibitor. In an example, the calcineurin inhibitor is cyclosporine. In an example, the calcineurin inhibitor is tacrolimus.

[0401] In an example, the immunosuppressant composition comprises an anti-DNA binding protein and an anti-proliferative agent. In an example, the anti-proliferative agent is MMF. In an example, the anti-proliferative agent is Azathioprine.

[0402] In an example, the immunosuppressant composition comprises an anti-DNA binding protein and a corticosteroid. In an example, the corticosteroid is prednisolone. In an example, the corticosteroid is prednisone.

[0403] In an example, the immunosuppressant composition comprises an anti-DNA binding protein, a calcineurin inhibitor, and an anti-proliferative agent. In an example, the immunosuppressant composition comprises an anti-DNA binding protein, a calcineurin inhibitor, and a corticosteroid. In an example, the immunosuppressant composition comprises an anti-DNA binding protein, an anti-proliferative agent, and a corticosteroid. In an example, the immunosuppressant composition comprises an anti- DNA binding protein, a calcineurin inhibitor, an anti-proliferative agent, and a corticosteroid.

[0404] Immunosuppressant compositions of the disclosure can also comprise an anti- DNA binding protein and an immunosuppressant antibody. In an example, the immunosuppressant antibody is ATG. In an example, the immunosuppressant antibody is an anti-CD20 antibody (e.g. rituximab).

[0405] In an example, the immunosuppressant composition comprises an anti-DNA binding protein and an mTOR inhibitor. In an example, the mTOR inhibitor is sirolimus. In an example, the mTOR inhibitor is everolimus.

[0406] Compositions can also contain a pharmaceutically acceptable carrier or adjuvant for administration of the binding protein. 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, polylactic acids, polyglycolic acids, polymeric amino acids, ammo acid copolymers and inactive virus particles.

[0407] 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. 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.

[0408] 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).

[0409] 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.

[0410] 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. Compositions can include an effective amount of one or more binding proteins as described herein. In some embodiments, a pharmaceutical composition can comprise an amount that is sufficient to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic effect. For example, the composition includes an effective amount of binding protein to preserve neutrophil function under culture conditions or in a subject in need thereof, e.g. relative to a control. In some examples, the composition includes an effective amount of one or more binding proteins to preserve neutrophil function in an effective amount to reduce or prevent one or more symptoms of disease or disorder (e.g. neutropenia). In some examples, the composition includes an effective amount of one or more binding proteins to reduce Neutrophil Extracellular Trap (NET) formation or NETosis under culture conditions or in a subject in need thereof, e.g., relative to a control. In some examples, the composition includes an effective amount of one or more binding proteins to reduce Neutrophil Extracellular Trap (NET) formation or NETosis in an effective amount to reduce or prevent one or more symptoms of disease or disorder. In some examples, the composition includes an effective amount of one or more binding proteins to reduce autoantibody levels, in particular ANCA levels, in an effective amount to reduce or prevent one or more symptoms of disease or disorder.

[0411] Binding proteins 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.

[0412] In one example, the binding protein is delivered through intravenous administration. In another example, the binding protein is delivered through subcutaneous administration. In another example, the binding protein is delivered through injection. In another example, the binding protein is delivered through infusion.

[0413] 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.

[0414] Tapering

[0415] Binding proteins of the disclosure can be used in immunosuppression therapy, in particular to reduce the use of standard immunosuppressive agents and thus minimise the harmful side effects of these agents. In this example, binding proteins of the disclosure can be used as a substitute for one or more standard immunosuppressive agents, such as a calcineurin inhibitor, an anti-proliferative agent, and / or a steroid. Accordingly, in an example, the disclosure provides a steroid-free immunosuppression regimen, comprising administering a binding protein of the disclosure. In another example, the disclosure provides a calcineurin inhibitor-free immunosuppression regimen, comprising administering a binding protein of the disclosure. In another example, the disclosure provides an anti-proliferative-free immunosuppression regimen, comprising administering a binding protein of the disclosure.

[0416] In an example, the disclosure provides a method of reducing calcineurin inhibitor use in a subject in need thereof, the method comprising administering to the subject an effective amount of a cell penetrating, anti-DNA binding protein in an amount effective to (i) inhibit Neutrophil Extracellular Trap (NET) formation, and (ii) preserve neutrophil function, wherein the subject is using a calcineurin inhibitor to supress their immune response.

[0417] Calcineurin inhibitor sparing regimens are known in the art and are described, for example, in Mathis et al. World J Transplant. 2014. 4(2):57-80. Current strategies include use of agents such as mycophenolate mofetil (MMF), mycophenolate sodium (MPS), sirolimus, everolimus or belatacept to facilitate late calcineurin inhibitor withdrawal (e.g. beyond 6 months post-transplant), or using these agents to plan early withdrawal within 6 months; or to avoid the calcineurin inhibitor all together using calcineurin inhibitor- free regimens. Anti-DNA binding proteins of the disclosure can be used in any calcineurin inhibitor sparing regimen, for example as a substitute for mycophenolate mofetil (MMF), mycophenolate sodium (MPS), sirolimus, everolimus or belatacept.

[0418] In an example, the subject has been using can be using a calcineurin inhibitor to supress their immune response for greater than 1, 2, 3, 4, 5, 6, 12, 24 months or more. In an example, the subject has been using a calcineurin inhibitor for at least a month. In another example, the subject has been using a calcineurin inhibitor for at least 3 months. In another example, the subject has been using a calcineurin inhibitor for at least 6 months. In another example, the subject has been using a calcineurin inhibitor for at least 12 months. In another example, the subject has been using a calcineurin inhibitor for at least 24 months. In another example, the subject has been using a calcineurin inhibitor for at least 36 months. In another example, the subject has been using a calcineurin inhibitor for at least 48 months. In another example, the subject has been using a calcineurin inhibitor for at least 60 months. In another example, the subject has been using a calcineurin inhibitor for between 6 and 78 months. In another example, the subject has been using a calcineurin inhibitor for between 6 and 70 months. In another example, the subject has been using a calcineurin inhibitor for between 6 and 68 months. In an example, the subject has been using a calcineurin inhibitor for between 0 and 6 months. In an example, the subject has been using a calcineurin inhibitor for between 2 and 6 months. In an example, the subject has been using a calcineurin inhibitor for between 3 and 6 months. In an example, the subject has been using a calcineurin inhibitor for between 4 and 6 months.

[0419] In an example, the calcineurin inhibitor is a cyclosporine. In another example, the calcineurin inhibitor is tacrolimus.

[0420] The initial dose of calcineurin inhibitor administered to the subject depends on a variety of factors, including age, weight, sex, disease, risk factors, and concomitant medication, including other immunosuppressants. General guidelines for calcineurin inhibitor dosage are described, for example, in Safarini et al. In: StatPearls [Internet], Treasure Island (FL): StatPearls Publishing; 2023 (Available from: https: / / www.ncbi.nlm.nih.gov / books / NBK558995 / ). In an example, the subject is using a calcineurin inhibitor in an amount of between about 1 mg and about 50 mg per day. In an example, the subject is using a calcineurin inhibitor in an amount of between about 10 mg and about 50 mg per day. In an example, the subject is using a calcineurin inhibitor in an amount of between about 1 mg and about 10 mg per day.

[0421] In an example, calcineurin inhibitor use is reduced 1 month after administration of the composition. In another example, calcineurin inhibitor use is reduced 3 month after administration of the composition. In another example, calcineurin inhibitor use is reduced 6 month after administration of the composition. In another example, calcineurin inhibitor use is reduced 12 month after administration of the composition. In another example, calcineurin inhibitor use is reduced 18 month after administration of the composition. In another example, calcineurin inhibitor use is reduced 24 month after administration of the composition. In another example, calcineurin inhibitor use is reduced 36 month after administration of the composition. In an example, the subjects calcineurin inhibitor use is reduced relative to their baseline calcineurin inhibitor use prior to administering a composition of the disclosure. For example, the subject’s calcineurin inhibitor use can be reduced relative to the subject’s average baseline calcineurin inhibitor equivalent dose prior to administering the composition.

[0422] In an example, the subject’s calcineurin inhibitor use is reduced by about 20% relative to their baseline calcineurin inhibitor use prior to administering the composition. In another example, the subject’s calcineurin inhibitor use is reduced by about 30% relative to their baseline calcineurin inhibitor use prior to administering the composition. In another example, the subject’s calcineurin inhibitor use is reduced by about 40% relative to their baseline calcineurin inhibitor use prior to administering the composition.

[0423] In an example, the subject discontinues calcineurin inhibitor use after administering a composition of the disclosure. In an example, the subject discontinues calcineurin inhibitor use 12 months after administering a composition of the disclosure. In another example, the subject discontinues calcineurin inhibitor use 18 months after administering a composition of the disclosure. In another example, the subject discontinues calcineurin inhibitor use 24 months after administering a composition of the disclosure. In another example, the subject discontinues calcineurin inhibitor use 36 months after administering a composition of the disclosure. In another example, the subject discontinues calcineurin inhibitor use between 18 and 36 months after administering a composition of the disclosure.

[0424] In an example, the disclosure provides a method of reducing steroid use in a subject in need thereof, the method comprising administering to the subject an effective amount of a cell penetrating, anti-DNA binding protein in an amount effective to (i) inhibit Neutrophil Extracellular Trap (NET) formation, and (ii) preserve neutrophil function, wherein the subject is using a steroid to supress their immune response.

[0425] In an example, the subject has been using can be using a steroid to supress their immune response for greater than 1, 2, 3, 4, 5, 6, 12, 24 months or more. In an example, the subject has been using a steroid for at least a month. In another example, the subject has been using a steroid for at least 3 months. In another example, the subject has been using a steroid for at least 6 months. In another example, the subject has been using a steroid for at least 12 months. In another example, the subject has been using a steroid for at least 24 months. In another example, the subject has been using a steroid for at least 36 months. In another example, the subject has been using a steroid for at least 48 months. In another example, the subject has been using a steroid for at least 60 months. In another example, the subject has been using a steroid for between 6 and 78 months. In another example, the subject has been using a steroid for between 6 and 70 months. In another example, the subject has been using a steroid for between 6 and 68 months.

[0426] In an example, the steroid is a corticosteroid. In an example, the steroid is prednisone.

[0427] In an example, the subject is using a steroid in an amount between about 5 mg / day and about 60 mg / day. In an example, the subject is using a steroid in an amount between about 20 mg / day and about 60 mg / day. In an example, the subject is using a steroid in an amount between about 30 mg / day and about 60 mg / day. In an example, the subject is using a steroid in an amount between about 40 mg / day and about 60 mg / day. In an example, the subject is using a steroid in an amount of about 60 mg / day. In an example, the subject is using a steroid in an amount between about 5 mg / day and about 10 mg / day. In an example, the subject is using a steroid in an amount between about 5 mg / day and about 15 mg / day. In an example, the subject is using a steroid in an amount between about 5 mg / day and about 20 mg / day.

[0428] In the above examples, “high-dose immunosuppression” is considered use of steroids in an amount of between about 20 mg / day and about 60 mg / day, or higher. “Low- dose immunosuppression” is considered use of steroids in an amount of less than 20 mg / day.

[0429] In an example, steroid use is reduced 1 month after administration of the composition. In another example, steroid use is reduced 3 month after administration of the composition. In another example, steroid use is reduced 6 month after administration of the composition. In another example, steroid use is reduced 12 month after administration of the composition. In another example, steroid use is reduced 18 month after administration of the composition. In another example, steroid use is reduced 24 month after administration of the composition. In another example, steroid use is reduced 36 month after administration of the composition. In an example, the subjects steroid use is reduced relative to their baseline steroid use prior to administering a composition of the disclosure. For example, the subject’s steroid use can be reduced relative to the subject’s average baseline steroid equivalent dose prior to administering the composition.

[0430] In an example, the subject’s steroid use is reduced by about 20% relative to their baseline steroid use prior to administering the composition. In another example, the subject’s steroid use is reduced by about 30% relative to their baseline steroid use prior to administering the composition. In another example, the subject’s steroid use is reduced by about 40% relative to their baseline steroid use prior to administering the composition. In an example, the steroid is prednisone and the reduction is relative to the subjects average baseline prednisone equivalent dose prior to administering the composition.

[0431] In an example, the subject discontinues steroid use after administering a composition of the disclosure. In an example, the subject discontinues steroid use 12 months after administering a composition of the disclosure. In another example, the subject discontinues steroid use 18 months after administering a composition of the disclosure. In another example, the subject discontinues steroid use 24 months after administering a composition of the disclosure. In another example, the subject discontinues steroid use 36 months after administering a composition of the disclosure. In another example, the subject discontinues steroid use between 18 and 36 months after administering a composition of the disclosure.

[0432] Methods of the disclosure relate to subjects who are using a steroid, antiproliferative agent, and / or a calcineurin inhibitor to supress their immune response. In an example, the subject is suffering from a disease or disorder that is being treated by a steroid and / or a calcineurin inhibitor to supress their immune response.

[0433] In an example, the inflammatory disease is an autoimmune disease. Examples of autoimmune diseases include lupus, rheumatoid arthritis and ANCA associated vasculitis (AAV). In an example, the subject has ANCA associated vasculitis (AAV).

[0434] In an example, the inflammatory disease is graft versus host disease. In an example, the disease is solid organ transplant rejection.

[0435] Methods of treatment

[0436] Immunosuppression

[0437] The present disclosure provides a method of immunosuppression, the method comprising administering to a subject in need thereof, a composition comprising an anti- DNA binding protein of the disclosure.

[0438] In an example, the subject is administered a composition comprising an anti-DNA binding protein of the disclosure and one or more additional immunosuppressive agents.

[0439] In an example, the subject is administered an anti-DNA binding protein and a calcineurin inhibitor,

[0440] - an anti-proliferative agent, and / or

[0441] - a corticosteroid.

[0442] In an example, the subject is administered an anti-DNA binding protein and a calcineurin inhibitor. In an example, the calcineurin inhibitor is cyclosporine. In an example, the calcineurin inhibitor is tacrolimus. In an example, the subject is not administered an anti -proliferative agent. In an example, the subject is not administered a corticosteroid.

[0443] In an example, the subject is administered an anti-DNA binding protein and an anti-proliferative agent. In an example, the anti-proliferative agent is MMF. In an example, the anti-proliferative agent is Azathioprine. In an example, the subject is not administered a calcineurin inhibitor. In an example, the subject is not administered a corticosteroid.

[0444] In an example, the subject is administered an anti-DNA binding protein and a corticosteroid. In an example, the corticosteroid is prednisolone. In an example, the corticosteroid is prednisone. In an example, the subject is not administered a calcineurin inhibitor. In an example, the subject is not administered an anti-proliferative agent.

[0445] In an example, the subject is administered an anti-DNA binding protein, a calcineurin inhibitor, and an anti-proliferative agent. In an example, the subject is not administered a corticosteroid.

[0446] In an example, the subject is administered an anti-DNA binding protein, a calcineurin inhibitor, and a corticosteroid. In an example, the subject is not administered an anti-proliferative agent.

[0447] In an example, the subject is administered an anti-DNA binding protein, an antiproliferative agent, and a corticosteroid. In an example, the subject is not administered a calcineurin inhibitor.

[0448] In an example, the subject is administered an anti-DNA binding protein, a calcineurin inhibitor, an anti-proliferative agent, and a corticosteroid.

[0449] In an example, the subject is administered an anti-DNA binding protein and an immunosuppressant antibody. In an example, the immunosuppressant antibody is ATG. In an example, the immunosuppressant antibody is an anti-CD20 antibody (e.g. rituximab). In an example, the subject is not also administered a calcineurin inhibitor, an anti-proliferative agent, or a corticosteroid.

[0450] In an example, the subject is administered an anti-DNA binding protein and an mTOR inhibitor. In an example, the mTOR inhibitor is sirolimus. In an example, the mTOR inhibitor is everolimus. In an example, the subject is not also administered a calcineurin inhibitor, an anti-proliferative agent, or a corticosteroid.

[0451] The immunosuppression regimen administered to a subject is tailored to the individual and can depend on a number of factors and will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The immunosuppression regimen can be adjusted by the individual physician in the event of any complication. Exemplary immunosuppression regimens are described, for example, in Pilch et al. Pharmacotherapy. 2021. 41(1): 119-131.

[0452] Immunosuppressive regimens can generally be classified as induction or maintenance. Induction regimens provide intense early postoperative immune suppression while maintenance regimens are used throughout the patient's life to prevent both acute and chronic immune responses. Other immunosuppressive regimens include anti -rejection regimens, which are an acute-type of regimen used in response to an acute rejection reaction or other acute flare-up.

[0453] In an example, the immunosuppression is maintenance immunosuppression. In an example, the maintenance immunosuppression is life-long. In another example, the immunosuppression is induction immunosuppression. In another example, the immunosuppression is anti-rejection immunosuppression.

[0454] In an example, the binding protein is administered at an initial (or induction) dose which is higher than subsequent (maintenance doses). For example, the binding protein is administered at an initial dose of between about lOmg / kg to about 30mg / kg. The binding protein is then administered at a maintenance dose of between about O.OOOlmg / kg to about 30mg / kg. The maintenance doses may be administered every 2- 30 days, such as, every 2 or 3 or 6 or 9 or 12 or 15 or 18 or 21 or 24 or 27 or 30 days.

[0455] The maintenance dose may be continued for up to 1 year post-induction immunosuppression (e.g. post-transplantation). In an example, the maintenance dose is continued for at least 1 year post-induction immunosuppression. In an example, the maintenance is continued for at least 1 year, 2 years, 3, years, 5 years, 10 years, 15 years, 20 years, 30 years, or greater than 30 years post-induction immunosuppression. In an example, the maintenance dose is continued for the duration of the subject’s life.

[0456] Immunosuppression can be monitored by measuring functional and molecular correlates of immune reactivity and responses. Appropriate monitoring methods are dependent on the underlying reason for immunosuppression and can be easily determined by the person skilled in the art (see, e.g. Ashton-Chess et al. European Society for Organ Transplantation. 2009. 22(1): 110-119).

[0457] In the context of transplant rejection, conventional techniques routinely used include: the measurement of blood creatinine, creatinine clearance and proteinuria to evaluate renal function;

[0458] - protocol biopsy to evaluate histological lesions;

[0459] - detection of anti-HLA antibodies;

[0460] - soluble CD30 ELISA; and / or - AlloMap quantitative PCR.

[0461] In an example, the subject being immunosuppressed according to the methods disclosed herein has solid organ transplant rejection. In an example, the subject being immunosuppressed according to the methods disclosed herein has ischemia reperfusion injury.

[0462] In an example, the subject’s risk of neutropenia is reduced after treatment according to the methods disclosed herein. In an example, the subject’s risk of neutropenia is reduced relative to a subject who has not been administered an anti-DNA binding protein of the disclosure.

[0463] In another example, a binding protein of the disclosure can be administered to a subject prophylactically to reduce the risk of neutropenia.

[0464] In an example, the binding protein reduces the level of IL-ip in a subject. In the context of the present disclosure, the reduction of IL-ip in a subject administered a binding protein of the disclosure is indicative of effective immunosuppression. In this example, the subject’s level of IL-ip is reduced relative to baseline (i.e. the level of IL- ip present in the subject before administration of the binding protein). In an example, the subject’s level of IL-ip is reduced by about 5% relative to baseline. In an example, the subject’s level of IL-ip is reduced by about 10% relative to baseline. In an example, the subject’s level of IL-ip is reduced by about 20% relative to baseline. In an example, the subject’s level of IL-ip is reduced by about 30% relative to baseline. In an example, the subject’s level of IL-ip is reduced by about 40% relative to baseline. In an example, the subject’s level of IL-ip is reduced by about 50% relative to baseline. In an example, the subject’s level of IL-ip is reduced by about 75% relative to baseline. In an example, the subject’s level of IL-ip is reduced by greater than 50% relative to baseline. In an example, the subject’s level of IL-ip is reduced by greater than 75% relative to baseline.

[0465] Methods for measuring IL-ip are known in the art. IL-ip levels can be measured in plasma or serum samples obtained from subject using standard immunoassays, such as ELISAs and multiplex assays. In other examples, IL-ip levels can be determined by measuring mRNA expression in plasma or serum samples using standardised assays, such as quantitative reverse transcription polymerase chain reactions (RT-PCR).

[0466] In an example, the binding protein reduces the level of autoantibodies in a subject. In the context of the present disclosure, a reduction of autoantibodies in a subject administered a binding protein of the disclosure is indicative of immunosuppression. In some examples, the binding protein reduces the level of ANCA in a subject. In this example, the subject’s level of ANCA is reduced relative to baseline (i.e. the level of ANCA present in the subject before administration of the binding protein). In an example, the subject’s level of ANCA is reduced by about 5% relative to baseline. In an example, the subject’s level of ANCA is reduced by about 10% relative to baseline. In an example, the subject’s level of ANCA is reduced by about 20% relative to baseline. In an example, the subject’s level of ANCA is reduced by about 30% relative to baseline. In an example, the subject’s level of ANCA is reduced by about 40% relative to baseline. In an example, the subject’s level of ANCA is reduced by about 50% relative to baseline. In an example, the subject’s level of ANCA is reduced by greater than 50% relative to baseline.

[0467] ANCA are routinely tested for in clinical settings. Accordingly, there are several standardized diagnostic assays for detecting ANCA known in the art. ANCA levels can be measured by indirect immunofluorescence (IIF) and ELISA. IIF assays involve incubating patient sera with a substrate containing ethanol-fixed neutrophils. The substrate is then stained with fluorescently-labelled antihuman antibodies and observed under a fluorescence microscope for fluorescence patterning. Immunoassays such as ELISAs, or multiplex assays, addressable laser bead immunoassays (ALBIA) or lineimmunoassays (LIA), can also be used. Detailed methods for determining the level of ANCA autoantibodies are described in Deka et al. J Lab Physicians. 2021. 13(3):286- 290.

[0468] In an example, the subject with ANCA has vasculitis. In an example, the subject has ANCA associated vasculitis (AAV).

[0469] Organ transplant rejection

[0470] Organ transplantation represents the preferred treatment option for many patients in terminal organ failure. However, the vast majority of the organs fail within the first two decades following transplantation due to immune-mediated rejection of the organ. Organ transplantation almost always requires lifelong maintenance immunosuppression (M-IMS) post transplantation. Accordingly, these subjects are at severe risk of developing neutropenia due to M-IMS. Morbidity due to infection is also incredibly high in these chronically immunosuppressed patients.

[0471] Accordingly, the disclosure provides a method of treating solid organ transplant rejection, the method comprising administering to a subject in need thereof, a cell penetrating, anti-DNA binding protein, in an amount effective to (i) inhibit Neutrophil Extracellular Trap (NET) formation, and (ii) preserve neutrophil function.

[0472] In an example, the subject has had a heart transplant. In an example, the subject has had a liver transplant. In an example, the subject has had a kidney transplant. In an example, the subject has had a lung transplant. In an example, an additional immunosuppressant agent is also administered to the subject. For example, the immunosuppressant agent is a calcineurin inhibitor, an antiproliferative agent, or a corticosteroid.

[0473] In another example, an additional immunosuppressant agent is not administered to the subject.

[0474] In an example, the subject’s risk of neutropenia is reduced. In an example, the subject’s risk of neutropenia is reduced relative to a subject who has not been administered a cell penetrating, anti-DNA binding protein of the disclosure.

[0475] Neutropenia

[0476] The disclosure provides a method of treating or preventing neutropenia in a subject requiring immunosuppression, the method comprising administering to the subject an anti-DNA binding protein as disclosed herein.

[0477] Neutropenia is characterised by an abnormally low number of circulating neutrophils in the blood. Neutropenia is characterised by an absolute neutrophil count (ANC) of less than 1.5>< 109 / L in circulating blood. Moderate to severe neutropenia is characterised by an ANC of less than 1 >< 1O9 / L in circulating blood. Severe neutropenia is characterised by an ANC of less than 0.5x l09 / L in circulating blood. Neutropenia is characterised as chronic when the low neutrophil counts persist over at least a 3 month period. For example, neutropenia is deemed “severe” and “chronic” when the counts are less than 0.5 x 109 / L on at least 3 occasions in a 3 month period. Methods for measuring and monitoring neutropenia are described above. Briefly, the absolute neutrophil count is determined by a blood test, typically a whole blood count (WBC) or complete blood count (CBC), which are routine assays known in the art.

[0478] In an example, the subject has a baseline absolute neutrophil count of less than 1.5X 109 / L. In an example, the subject has a baseline absolute neutrophil count of less than 1 x 109 / L. In an example, the subject has a baseline absolute neutrophil count of less than 0.5X 109 / L.

[0479] In an example, the subject has a baseline absolute neutrophil count of less than 1.5X 109 / L for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or more than 6 months. In an example, the subject has a baseline absolute neutrophil count of less than 1.5x l09 / L for at least 3 months.

[0480] In an example, the subject has a baseline absolute neutrophil count of less than 1 x 109 / L for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or more than 6 months. In an example, the subject has a baseline absolute neutrophil count of less than 1 * 109 / L for at least 3 months.

[0481] In an example, the subject has a baseline absolute neutrophil count of less than 0.5* 109 / L for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or more than 6 months. In an example, the subject has a baseline absolute neutrophil count of less than 0.5>< 109 / L for at least 3 months.

[0482] In an example, the subject’s baseline absolute neutrophil count increases following administration of an anti-DNA binding protein of the disclosure. For example, the subject’s baseline absolute neutrophil count can increase by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100%.

[0483] In an example, the subject’s absolute neutrophil count increases to greater than about 0.5* 109 / L following administration of an anti-DNA binding protein of the disclosure. In an example, the subject’s absolute neutrophil count increases to greater than about l * 109 / L following administration of an anti-DNA binding protein of the disclosure. In an example, the subject’s absolute neutrophil count increases to greater than about 1.5>< 109 / L following administration of an anti-DNA binding protein of the disclosure.

[0484] In an example, the subject’s baseline absolute neutrophil count increases within about 1 year following administration of a binding protein of the disclosure. In an example, the subject’s baseline absolute neutrophil count increases within about 6 months following administration of a binding protein of the disclosure. In an example, the subject’s baseline absolute neutrophil count increases within about 3 months following administration of a binding protein of the disclosure.

[0485] In another example, a binding protein of the disclosure can be administered to a subject prophylactically to reduce the risk of neutropenia. In this example, the subject’s risk of neutropenia is reduced relative to a subject who has not been administered an anti- DNA binding protein of the disclosure.

[0486] Ischemia reperfusion injury (IRI)

[0487] Ischemia-Reperfusion injury (IRI) is the paradoxical exacerbation of cellular dysfunction and death, following restoration of blood flow to previously ischaemic tissues. IRI occurs in a wide range of organs including the heart, lung, kidney, gut, skeletal muscle and brain and may involve not only the ischaemic organ itself but may also induce systemic damage to distant organs, potentially leading to multi-system organ failure. For example, IRI resulting from reperfusion into an ischemic heart can also result in injury in the kidney, such as acute kidney injury (AKI). IRI can also occur as a result of revascularisation. The term “revascularisation” as used herein refers to the restoration of vascularity and blood flow to a tissue or organ. Revascularisation can occur via a surgical procedure, such as during solid organ transplantation, or in some examples can occur organically.

[0488] Cardiopulmonary bypass (CPB) is a form of extracorporeal circulation used to facilitate surgery on the heart and great vessels, such as coronary artery bypass graft surgery. It incorporates an extracorporeal circuit to provide physiological support in which venous blood is drained to a reservoir, oxygenated and sent back to the body using a pump. ,A major complication associated CPB is IRI.

[0489] A hallmark of IRI is the increased expression of numerous cytokines, including tumour necrosis factor-alpha (TNF-a,) interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8) and platelet activating factor (PAF). These cytokines are released systemically and are thus important in the development of systemic inflammatory response syndrome and ultimately multi-system organ failure. These cytokines are also potent neutrophil chemoattractants and play a role in endothelial cell regulation and neutrophil recruitment into injured tissues following ischaemia-reperfusion.

[0490] Reperfusion of a large amount of ischaemic tissue results in neutropenia. Both local and systemic damage are associated with neutrophil activation, enhanced neutrophil adhesion to endothelium, and consequent neutrophil accumulation in damaged tissue (see, e.g. Grace. British Journal of Surgery. 1994. 81 :637-647; Welbourn et al. British Journal of Surgery. 1991. 78:651 -655).

[0491] It is envisioned that binding proteins of the disclosure will be particularly useful in the context of treating IRI, due to their ability to inhibit neutrophil activation whilst preserving neutrophil function.

[0492] Accordingly, the disclosure provides a method of treating Ischaemia-Reperfusion injury (IRI) in a subject, wherein the method comprises administering to the subject a composition comprising a cell penetrating, anti-DNA binding protein in an amount effective to (i) inhibit Neutrophil Extracellular Trap (NET) formation, and (ii) preserve neutrophil function.

[0493] In an example, the subject has received a solid organ transplant. In an example, the solid organ is a heart, kidney, liver, or lung. In an example, the subject has received a heart transplant. In an example, the subject has received a kidney transplant. In an example, the subject has received a liver transplant. In an example, the subject has received a lung transplant.

[0494] In an example, the subject has received a cardiopulmonary bypass or is about to undergo a cardiopulmonary bypass. The composition can be administered before, during, and / or after cardiopulmonary bypass. In an example, the composition can be administered before, during, and after cardiopulmonary bypass. In an example, the composition can be administered before, during, or after cardiopulmonary bypass. In an example, the composition can be administered before or during the cardiopulmonary bypass. In an example, the composition is administered before the cardiopulmonary bypass. In an example, the composition is administered during the cardiopulmonary bypass. In an example, the composition is administered after the cardiopulmonary bypass.

[0495] In an example, the cardiopulmonary bypass occurs during coronary artery bypass graft surgery. In an example, the IRI is in the heart or kidney. In an example, the IRI is in the heart. In an example, the IRI is in the kidney. In an example, the IRI is in the heart and the kidney.

[0496] In an example, the IRI is Acute kidney injury (AKI).

[0497] In an example, the subject with IRI has neutropenia. In an example, the subject’s risk of neutropenia is reduced. In an example, the subject’s risk of neutropenia is reduced relative to a subject with IRI who has not been administered an anti-DNA binding protein of the disclosure.

[0498] SEQUENCES OF THE DISCLOSURE

[0499]

[0500]

[0501] EXAMPLES

[0502] EXAMPLE 1 - DX-1 and DX-3 reduces NET formation regardless of stimulus.

[0503] Human neutrophils were treated with 10 pM DX-1 or 10 pM DX-3 and stimulated with PMA, ionomycin, LPS, or TNFa. One sample was kept as an unstimulated control. Cells were then stained with DAPI and immunostained for markers of NETs including myeloperoxidase (MPO), citrullinated Histone (H3Cit), and peptidylarginine deiminase 4 (PAD4) and imaged via fluorescence microscopy to determine % NETosis. Cells with a diffuse DAPI stain suggestive of chromatin decondensation were counted as undergoing NETosis. The percentage of NET structures was determined by directly counting the number of total cells and cells with visualized extrusion of DNA colocalised with H3Cit in a minimum of 150 cells per treatment condition.

[0504] As shown in Fig. 1, both DX-1 and DX-3 reduced NET formation in response to all stimuli tested (PMA, ionomycin, LPS, and TNFa). Figure 2 shows NETosis morphology in PMA and ionomycin stimulated neutrophils that were not treated with DX-1, as compared to stimulated neutrophils that were treated with DX-1. DX-l-treated neutrophils have a similar morphology to the unstimulated neutrophils.

[0505] These data suggest that DX-1 and DX-3 are effective inhibitors of neutrophil activation in response to a variety of pro-inflammatory stimuli.

[0506] EXAMPLE 2 - DX-1 and DX-3 has no effect on viability, cytotoxicity or apoptosis of neutrophils.

[0507] To evaluate the effect of DX-1 and DX-3 on viability of stimulated neutrophils, cells were again treated with 10 M DX1 or 10 pM DX-3 and stimulated with PMA, ionomycin, LPS, or TNFa. One sample was kept as an unstimulated control. Cell viability, cytotoxicity, and apoptosis was then measured by incubating samples with a fluorescent indicator of viability, cytotoxicity, or apoptosis, respectively. Neither DX-1 nor DX-3 had any effect on neutrophil viability (Fig. 3), cytotoxicity (Fig. 4), or apoptosis (Fig. 5).

[0508] Accordingly, DX-1 and DX-3 do not cause neutrophil cell death. This is a surprising finding in light of the data above showing that DX-1 and DX-3 effectively inhibit NETosis in response to inflammatory stimuli. It also suggests that DX-1 and DX- 3 will not cause neutropenia in patients, which can be triggered by standard immunosuppression agents (e.g. calcineurin inhibitors, steroids). Furthermore, DX-1 and DX-3 are useful for treating neutropenia, as they are able to be impair neutrophil activation and NETosis, without killing remaining neutrophils. This is an important finding as it suggests that DX-1 and DX-3 can be used to allow neutrophil levels to recover while contributing to immunosuppression.

[0509] EXAMPLE 3 - DX-1 and DX-3 do not activate neutrophil enzyme release or neutrophil phagocytosis.

[0510] To determine whether DX-1 and DX-3 activate pro-inflammatory neutrophil enzyme release in response to inflammatory stimuli, cells were again treated with 10 pM DX1 or 10 pM DX3 and stimulated with PMA, ionomycin, LPS, or TNFa. One sample was kept as an unstimulated control. Levels of myoperioxidase (MPO), neutrophil elastase (NE) and proteinase 3 (PRT3) were then measured. Briefly. NE, MPO, and PRT3 were all measured by using DuoSET (R&D), a solid phase Sandwich ELISA where the level of enzyme is determined from a standard curve and measured via an absorbance plate reader. Neither DX1 nor DX3 had any effect on MPO (Fig. 6), NE (Fig. 7), or PRT3 (Fig. 8) release.

[0511] Phagocytosis of neutrophils was also measured to determine whether DX1 / DX3 have any detrimental effect on neutrophil host defence. A phagocytosis assay (ABCAM) was used, which utilises zymosan particles that have been pre-labeled to react with specific substrate that produces a colorimetric signal. The signal can be detected by absorbance at 405nm. As shown in Fig. 9, DX-1 and DX-3 have no effect on neutrophil phagocytosis in cells treated with PMA, ionomycin, LPS, or TNFa.

[0512] This data provides further evidence that DX-1 and DX-3 preserve immunosuppressive functions of neutrophils. In addition to preventing neutrophil cell death, DX-1 and DX-3 preserve the phagocytic function of neutrophils which is critical for fighting infection.

[0513] EXAMPLE 4 - Assessment of DX-1 and DX-3 efficacy in attenuation of inflammation in intervention studies replicating sudden onset or acute relapse treatment.

[0514] Efficacy of DX-1 and DX-3 in the attenuation of inflammation was examined in an animal model of MPO-induced ANCA-associated vasculitis (AAV) (summarized in Fig. 10). Firstly, systemic responses to autoimmunity to MPO were measured. Briefly, IFN-y in lymph nodes (LN) and the spleen were measured using IFN-y- and IL-17A- enzyme-linked immunospot (ELISPOT) assays. Serum levels of MPO autoantibodies (MPO-ANCA) were also measured. As shown in Fig. 11, both DX-1 and DX-3 reduced systemic responses to autoimmunity to MPO in this model. Treatment with DX-1 and DX-3 also reduced both leukocytes and protein levels in urine, indicative of a reduction in functional kidney injury (Fig. 12).

[0515] Kidney inflammatory mRNA transcription was also found to be unaltered with DX-1 and DX-3 treatment (Fig. 13).

[0516] As shown in Fig. 14 - Fig. 17, DX-1 and DX-3 attenuates kidney injury. DX-1 and DX-3 reduced the number of abnormal glomeruli (Fig. 14), and reduced neutrophil (Fig. 15), macrophage (Fig. 16), and CD4 T cell (Fig. 17) recruitment to the kidney. Fig. 18 shows that NETosis is also reduced in the kidneys of DX-1 and DX-3 treated mice.

[0517] These data indicate that DX-1 and DX-3 direct immunosuppression in vivo by supressing systemic autoimmunity (in particular the suppression of autoantibodies), preventing neutrophil activation (NETosis), as well as neutrophil, macrophage, and CD4- T cell recruitment to the kidney. This finding supports the use of DX-1 or DX-3 in induction immunosuppression therapy regimens, as well as anti -rejection therapy regimens.

[0518] EXAMPLE 5 - Assessment of DX-1 and DX-3 efficacy in attenuation of inflammation in prevention studies replicating treatment to prevent relapse (maintenance).

[0519] The treatment regimen in Example 4 (i.e. single dose of DX-l / DX-3) is representative of treatment given in acute / sudden onset settings (e.g. as part of an induction therapy regimen or anti-rejection regimen). To examine dosing in the context of preventive or maintenance therapy, DX-1 and DX-3 were administered across multiple doses at different time points following MPO challenge. The model and dosing schedule is summarized in Fig. 19.

[0520] Multiple doses of DX-1 and DX-3 at day 8, day 12, and day 18 significantly reduced production of the autoantibodies to MPO, MPO-ANCA. (Fig. 20). DX-1 and DX-3 treatment also reduced the number of abnormal glomeruli (Fig. 21), and neutrophil (Fig. 22), macrophage (Fig. 23), and CD4 T cell (Fig. 24) recruitment to the kidney. DX- 1 and DX-3 also significantly reduced IL-ip, which is produced by the inflammasome (Fig. 25).

[0521] This suggests that DX-1 and DX-3, when used in a maintenance therapy context, are sufficient to attenuate the most severe manifestations of an ongoing immune reaction (e.g. suppression of autoantibody production, reduction of abnormal glomeruli, and neutrophil, macrophage, and CD4 T cell recruitment)..

[0522] Together, the findings of the present inventors show that DX-1 and DX-3 are effective immunosuppressants. Advantageously, DX-1 and DX-3 impair neutrophil activation in response to a variety of inflammatory stimuli, whilst also avoiding neutrophil cell death, preserving neutrophils to fight infection. DX-1 and DX-3 are also able to reduce autoantibody levels via a mechanism that is distinct from an antiinflammatory effect on cytokines. This supports the use of DX-1 and DX-3 in immunosuppression therapy, as well as, or instead of, standard of care drugs, which have many adverse side effects, including neutropenia.

[0523] 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.

[0524] All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0525] 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.

Claims

CLAIMS1. A method of immunosuppression, the method comprising administering to a subject in need thereof, a composition comprising a cell penetrating, anti-DNA binding protein, wherein the cell penetrating, anti-DNA binding protein (i) inhibits Neutrophil Extracellular Trap (NET) formation, and (ii) preserves neutrophil function.

2. A method of treating solid organ transplant rejection, the method comprising administering to a subject in need thereof, a cell penetrating, anti-DNA binding protein, wherein the cell penetrating, anti-DNA binding protein (i) inhibits Neutrophil Extracellular Trap (NET) formation, and (ii) preserves neutrophil function.

3. A method of treating or preventing neutropenia in a subject requiring immunosuppression, the method comprising administering to the subject a cell penetrating, anti-DNA binding protein, wherein the cell penetrating, anti-DNA binding protein (i) inhibits Neutrophil Extracellular Trap (NET) formation, and (ii) preserves neutrophil function.

4. The method according to any one of claims 1 to 3, wherein the cell penetrating, anti-DNA binding protein reduces one or more of the following in a subject: autoantibody levels, preferably wherein the autoantibodies are antineutrophil cytoplasmic autoantibodies (ANCA); and / orIL-ip levels.

5. The method according to any one of claims 1 to 4, wherein the subject has been treated with a steroid.

6. The method of claim 5, wherein the steroid is a corticosteroid, preferably, wherein the steroid is prednisolone.

7. The method according to any one of claims 1 to 6, wherein the subject is also being treated with a calcineurin inhibitor and an anti-proliferative agent.

8. The method according to any one of claims 1 to 7, wherein treatment preserves neutrophil function in the subject.

9. The method according to any one of claims 5 to 8, wherein method comprises reducing or discontinuing the steroid component of the subject’s treatment.

10. The method of claim 8 or claim 9, wherein the preservation of neutrophil function in the subject is determined by assessing one or more of the following parameter(s) in a sample obtained from the patient: level of neutrophil viability; level of neutrophil cytotoxicity; level of neutrophil apoptosis; level of release of pro-inflammatory neutrophil enzymes; level of neutrophil phagocytosis.

11. The method of claim 10, wherein the determination is based on a comparison between the level of the relevant parameter(s) in the sample and a corresponding level(s) in a control sample, preferably wherein the control sample is a sample obtained from the subject prior to receiving treatment with the cell penetrating, anti-DNA binding protein.

12. The method according to any one of claims 1 to 11, wherein the immunosuppression is maintenance immunosuppression.

13. The method according to any one of claims 2 or 4 to 12, wherein the solid organ is selected from the group consisting of a heart, a kidney, a liver, and a lung.

14. The method of claim 13, wherein the solid organ is a heart or a kidney.

15. The method according to any one of claims 1 to 14, wherein the subject has Ischaemia-Reperfusion injury (IRI).

16. The method according to claim 15, wherein the IRI is in one or more of the subject’s kidney, heart, liver or brain.

17. A method of treating Ischaemia-Reperfusion injury (IRI) in a subj ect, wherein the method comprises administering to the subject a composition comprising a cell penetrating, anti-DNA binding protein in an amount effective to (i) inhibit Neutrophil Extracellular Trap (NET) formation, and (ii) preserve neutrophil function.

18. The method according to any one of claims 1 to 17, wherein the cell penetrating anti-DNA 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.

19. The method according to any one of claims 1 to 18, wherein the cell penetrating anti-DNA binding protein is an intact antibody, an scFv or a di-scFv.

20. The method according to any one of claims 17 to 19, wherein the subject has received a solid organ transplant.

21. The method according to any one of claims 17 to 19, wherein the subject has received a cardiopulmonary bypass, or is about to undergo a cardiopulmonary bypass.

22. The method according to claim 21, wherein the composition is administered before or during the cardiopulmonary bypass.

23. The method according to claim 21, wherein the composition is administered after the cardiopulmonary bypass.

24. The method according to any one of claims 21 to23, wherein the cardiopulmonary bypass occurs during coronary artery bypass graft surgery.

25. The method according to any one of claims 17 to 24, wherein the IRI is in the heart or kidney.

26. The method of claim 25, wherein the IRI is Acute kidney injury (AKI).

27. The method according to any one of claims 1, 2 or 4 to 26, wherein the subject has neutropenia.

28. A method of reducing calcineurin inhibitor use or steroid use in a subject in need thereof, the method comprising administering to the subject an effective amount of a cell penetrating, anti-DNA binding protein, wherein the cell penetrating, anti-DNA binding protein (i) inhibits Neutrophil Extracellular Trap (NET) formation, and (ii) preserves neutrophil function.

29. The method according to claim 28, wherein the steroid is a corticosteroid, preferably prednisolone, or wherein the calcineurin inhibitor is cyclosporine or tacrolimus.

30. The method of claim 28 or 29, wherein the subject requires immunosuppression.

31. The method of claim 30, wherein the immunosuppression is maintenance immunosuppression, or induction immunosuppression.

32. The method according to any one of claims 28 to 31, wherein the subject has neutropenia.

33. The method according to any one of claims 28 to 32, wherein the subject’s calcineurin inhibitor or steroid use is reduced or discontinued after treatment.

34. The method according to any one of claims 28 to 32, wherein the subject is using the calcineurin inhibitor or the steroid for immunosuppression.

35. Use of a cell penetrating, anti-DNA binding protein in the manufacture of a medicament for: treating solid organ transplant rejection; treating or preventing neutropenia; treating Ischaemia-Reperfusion injury (IRI); or, reducing steroid use, wherein the cell penetrating, anti-DNA binding protein (i) inhibit Neutrophil Extracellular Trap (NET) formation, and (ii) preserve neutrophil function.

36. Use of claim 35, wherein the medicament further comprises at least one additional immunosuppressant agent.

37. Use of claim 36, wherein additional immunosuppressant agent is a calcineurin inhibitor, an anti-proliferative agent, or a corticosteroid.

38. Use of claim 36, wherein additional immunosuppressant agent is cyclosporine, tacrolimus, mycophenolate mofetil, or prednisolone.

39. Use according to claim 35 or claim 36, wherein the medicament is not formulated for use with a steroid, or wherein the medicament is not formulated for use with a calcineurin inhibitor.

40. The method according to any one of claims 1 to 34 or the use according to any one of claims 35 to 39, wherein the anti-DNA 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.

41. The method according to any one of claims 1 to 34 or the use according to any one of claims 35 to 39, wherein the anti-DNA 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.

42. The method according to any one of claims 1 to 34, 40 or 41, or the use according to any one of claims 35 to 41, wherein the anti-DNA binding protein is nuclear penetrating.

43. The method according to any one of claims 1 to 34, or 40 to 42, or the use according to any one of claims 35 to 42, wherein the binding protein is a di-scFv.

44. The method according to any one of claims 1 to 34 or 40 to 43, or the use according to any one of claims 35 to 43, wherein the binding protein comprises a linker which comprises an amino acid sequence shown in any one of SEQ ID NO: 17; SEQ ID NO: 26 or SEQ ID NO: 27.

45. The method according to any one of claims 1 to 34 or 40 to 42, or the use according to any one of claims 35 to 42, wherein the binding protein is an intact antibody.

46. The method according to any one of claims 1 to 34 or 40 to 44, or the use according to any one of claims 35 to 44, wherein inhibition of NET formation and preservation of neutrophil function is determined under culture conditions, wherein the culture conditions comprise culturing neutrophil-like PLB-985 cells or neutrophils in culture medium which comprises an inflammatory stimulus.

47. The method or use according to claim 46, wherein preservation of neutrophil function under culture conditions is determined by:(i) treating a population of neutrophils with the anti-DNA binding protein;(ii) stimulating the population of neutrophils with an inflammatory stimulus; and(iii) determining the level of one or more markers of neutrophil function in the population of neutrophils with the anti-DNA binding protein and a control population of neutrophils that do not comprise an anti-DNA binding protein; wherein an equivalent level of the one or more markers of neutrophil function between the control neutrophil population and the anti-DNA binding protein population indicates that neutrophil function is preserved.

48. The method or use according to claim 47, wherein the marker(s) used to determine preservation of neutrophil function under culture conditions are selected from the group consisting of: neutrophil viability; neutrophil cytotoxicity; neutrophil apoptosis; release of pro-inflammatory neutrophil enzymes; and neutrophil phagocytosis.

49. The method or use according to any one of claims 46 to 48, wherein the inflammatory stimulus is phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS), a calcium ionophore ionomycin (IM), or tumour necrosis factor alpha (TNFa).

50. The method or use according to any one of claims 46 to 48, wherein NET formation under culture conditions is determined by one or more markers selected from the group consisting of:1) level of cell death and extracellular DNA in NETs; and / or,2) reduced DNA release from neutrophil-like cells after culture with the inflammatory stimulus.

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