Methods of treating or preventing ischemia-reperfusion injury

Abstract

The disclosure relates to a method for preventing or treating ischemia-reperfusion injury in a subject, the method comprising administering a compound that inhibits granulocyte colony stimulating factor (G-CSF) signaling. In some examples, ischemia-reperfusion injury is due to or associated with tissue or organ transplantation. In some examples, the compound is an antibody.

Description

[0001] Relevant application materials

[0002] This application claims priority to Australian Patent Application No. 2017904822, entitled “Methods for treating or preventing ischemia-reperfusion injury”, filed on 29 November 2017. The entire contents of this application are incorporated herein by reference.

[0003] Sequence Listing

[0004] This application and the sequence listing are submitted in electronic form. The entire contents of the sequence listing are incorporated herein by reference. Technical Field

[0005] The present disclosure relates to methods of treating or preventing ischemia-reperfusion injury in a subject by antagonizing granulocyte colony stimulating factor (G-CSF) signaling and uses thereof, for example, in organ transplantation. Background Art

[0006] Ischemia-reperfusion injury (IRI) is a pathological condition caused by ischemia, i.e., limiting or reducing the blood supply to a tissue or organ (e.g., a dead donor organ for transplantation), followed by reperfusion and reoxygenation. Ischemia causes cellular hypoxia and nutrient deficiency, insufficient removal of metabolic waste, and can rapidly lead to necrosis and inflammation. Reperfusion of the tissue or organ after a period of ischemia returns blood supply and oxygen, but reperfusion itself can aggravate the oxidation and inflammation caused by the initial ischemia and cause further damage. Reoxygenation can cause oxidative damage to cellular proteins, DNA, and plasma membranes. This oxidative damage can in turn lead to the release of free radicals, leading to further cell damage. The characteristics of reperfusion injury are particularly the production of reactive oxygen species, complement activation, cellular inflammation, and endothelial cell damage.

[0007] In organ transplantation, ischemia-reperfusion injury can be defined as either "hot" IRI or "cold" IRI. Hot IRI occurs in situ during organ transplant surgery or during various forms of shock or trauma. Cold IRI occurs during ex vivo storage and is often coupled to hot IRI during organ transplant surgery.

[0008] The innate immune system and the adaptive immune system play a key role in the pathogenesis of ischemia-reperfusion injury. In terms of innate immunity, the danger signals released by dead cells activate Toll-like receptors, resulting in the activation and / or generation of cells and soluble factors, which regulate the generation of inflammatory cell recruitment and inflammatory mediators (such as chemokines, cytokines and free radicals). Neutrophils are the main inflammatory cell responders after ischemia and reperfusion. In an inflammatory environment, dendritic cells absorb and process antigens from dead cells, migrate to lymph nodes and activate antigen-specific cells of the adaptive immune system. Therefore, the pathogenesis of ischemia-reperfusion injury is complex, and tissue damage may occur through a variety of mechanisms, such as cell death, microvascular dysfunction, transcriptional reprogramming, activation of complement and innate and adaptive immune systems.

[0009] Ischemia-reperfusion injury is a common event in transplantation, including renal transplantation, and is a relevant factor in determining short-term and long-term transplant outcomes. In renal transplantation, IRI can affect endothelial cells and tubular epithelial cells and can lead to acute kidney injury and delayed graft function (DGF), thereby compromising graft survival. DGF is one of the more frequent early complications after deceased donor or extended criteria donor kidney transplantation and is primarily caused by tubular epithelial cell necrosis caused by IRI ( B et al., Kidney Int. 2014).

[0010] Compounds currently being tested in clinical trials for the treatment of ischemia-reperfusion injury include eculizumab, a humanized monoclonal antibody directed against the C5 component of the complement cascade (Rother R, et al. Nat Biotechnol. 2007). Granulocyte colony stimulating factor (G-CSF) has also been studied as a therapeutic agent for the treatment of reperfusion injury, see Nogueira et al. Cell Phys Biochem (2006) and Lu B et al. Nephrology (2008). These studies suggest that G-CSF treatment has a protective effect in renal ischemia.

[0011] However, effective therapies to prevent or treat ischemia-reperfusion injury are elusive.Thus, as will be apparent to those skilled in the art from the foregoing, strategies and methods to prevent or treat damage caused by ischemia-reperfusion injury are needed. Summary of the invention

[0012] In the preparation of the present invention, the inventors conducted a teaching contrary to the previous teaching that G-CSF may have a protective effect in ischemia-reperfusion injury, and instead studied the effect of inhibiting G-CSF signaling on ischemia-reperfusion injury. The inventors found that by administering a compound that inhibits G-CSF signaling, the effects of ischemia-reperfusion injury were mitigated. In addition, the inventors found that by inhibiting G-CSF signaling, they can prevent or treat ischemia-reperfusion injury to a similar extent as they inhibit complement activation at the C5 level. These findings provide a basis for a method of preventing or treating ischemia-reperfusion injury by inhibiting G-CSF signaling.

[0013] Thus, in one example, the present disclosure provides a method for preventing or treating ischemia-reperfusion injury in a subject, the method comprising administering a compound that inhibits granulocyte colony stimulating factor (G-CSF) signaling.

[0014] The present disclosure also provides compounds that inhibit G-CSF signaling for use in preventing or treating ischemia-reperfusion injury.

[0015] The present disclosure also provides use of a compound that inhibits G-CSF signaling in the preparation of a medicament for preventing or treating ischemia-reperfusion injury.

[0016] In some examples, the ischemia and / or reperfusion injury is due to or is associated with organ transplantation, cold organ storage, brain death, atherosclerosis, thrombosis, thromboembolism, lipid embolism, trauma, hemorrhage, stents, surgery, angioplasty, bypass surgery, global ischemia, myocardial infarction, stroke, peripheral vascular disease, sepsis, tumors, or a combination thereof.

[0017] In some examples, the ischemia-reperfusion injury is warm ischemia-reperfusion injury. In some examples, the ischemia-reperfusion injury is cold ischemia-reperfusion injury.

[0018] In some examples, a compound that inhibits G-CSF signaling is administered to a subject.

[0019] In one example, the ischemia-reperfusion injury is due to or is associated with one or more of the following:

[0020] (i) Organ transplantation;

[0021] (ii) surgical procedures;

[0022] (iii) trauma;

[0023] (iv) sepsis;

[0024] (v) tumors; and

[0025] (vi) Stroke.

[0026] In one example, the ischemia-reperfusion injury is due to surgery or is associated with surgery. In one example, the ischemia-reperfusion injury is due to coronary artery bypass surgery, such as a double bypass (wherein two coronary arteries are bypassed (e.g., the left anterior descending coronary artery (LAD) and the right coronary artery (RCA)); a triple bypass (wherein three vessels are bypassed, such as the LAD, RCA, and left circumflex artery (LCX)); a quad bypass (wherein four vessels are bypassed (e.g., the LAD, RCA, LCX, and the first diagonal artery of the LAD)); or a penta bypass (bypassing five arteries).

[0027] In one example, the ischemia-reperfusion injury is due to or is associated with an organ transplant, such as a solid organ transplant. In one example, the organ transplant is a kidney transplant. In one example, the organ transplant is a liver transplant. In one example, the organ transplant is a heart transplant. In one example, the organ transplant is a pancreas transplant. In one example, the organ transplant is a lung transplant. In one example, the organ transplant is a stomach transplant. In one example, the organ transplant is an intestinal transplant. In one example, the organ transplant is a testicular transplant.

[0028] In a further example, the organ transplant is a skin transplant, such as a full-thickness skin transplant.

[0029] In one example, the ischemia-reperfusion injury is due to or associated with a tissue transplant. In one example, the tissue transplant is a vascular transplant. In one example, the tissue transplant is a skin transplant, such as vascularized skin. In one example, the tissue transplant is a pancreatic islet transplant. In one example, the tissue transplant is a corneal transplant. In one example, the tissue transplant is a musculoskeletal transplant.

[0030] When the ischemia-reperfusion injury is due to or associated with a transplant (e.g., an organ transplant), a compound that inhibits G-CSF signaling can be administered before, during, and / or after the transplant. In some examples, a compound that inhibits G-CSF signaling is administered to a subject, wherein the subject is a tissue or organ transplant recipient. In some examples, a compound that inhibits G-CSF signaling is administered to a tissue or organ transplant donor.

[0031] In some examples, the tissue or organ transplant donor is a living donor. In some examples, the tissue or organ transplant donor is a deceased donor. In some examples, the tissue or organ transplant donor is a brain dead donation (DBD) donor. In some examples, the tissue or organ transplant donor is a circulatory death donation (DCD) donor. In some examples, the tissue or organ transplant donor is an extended criteria donor (ECD). In some examples, the tissue or organ transplant donor is a standard donor (SCD).

[0032] In some examples, a compound that inhibits G-CSF signaling is administered in vitro to a harvested organ prior to organ transplantation. For example, a harvested organ can be perfused or infused with a solution containing a compound that inhibits G-CSF signaling prior to transplantation.

[0033] The present disclosure also provides a method of tissue or organ transplantation or a method for improving the outcome of tissue or organ transplantation or improving the function of transplanted tissue or organ or for preventing delayed transplant function, the method comprising administering a compound that inhibits G-CSF signaling to harvested tissue or organ in vitro, and transplanting the harvested tissue or organ into a tissue or organ transplant recipient.

[0034] In some examples, compounds that inhibit G-CSF signaling are administered to organ transplant donors and / or organ transplant recipients and / or harvested organs. In some examples, compounds that inhibit G-CSF signaling are administered to organ transplant donors and organ transplant recipients. In some examples, compounds that inhibit G-CSF signaling are administered to organ transplant donors and harvested organs. In some examples, compounds that inhibit G-CSF signaling are administered to organ transplant recipients and harvested organs.

[0035] In one example of the present disclosure, a compound that inhibits G-CSF signaling is administered prior to reperfusion, for example, in the case of a transplant (e.g., an organ transplant), a compound that inhibits G-CSF signaling is administered to an organ transplant recipient prior to reperfusion of the transplanted organ (e.g., an organ) (e.g., a compound that inhibits G-CSF signaling is administered prior to the transplant or during the transplant but prior to reperfusion, e.g., prior to release of a clamp restricting blood flow).

[0036] In some examples, the compound that inhibits G-CSF signaling is administered before ischemia. In one example, the compound that inhibits G-CSF signaling is administered between 0 days (e.g., immediately before ischemia or reperfusion) to 7 days before ischemia or reperfusion. For example, the compound that inhibits G-CSF signaling is administered between 0 days to 6 days, 5 days, or 4 days before reperfusion or ischemia. For example, the compound that inhibits G-CSF signaling is administered between 0 and 72 hours before reperfusion or ischemia. In one example, the compound that inhibits G-CSF signaling is administered between 6 and 48 hours before reperfusion or ischemia. In one example, the compound that inhibits G-CSF signaling is administered between 12 and 36 hours before reperfusion or ischemia. In one example, the compound that inhibits G-CSF signaling is administered about 24 hours before reperfusion or ischemia.

[0037] In some examples, the compound that inhibits G-CSF signaling is administered at least 1 hour prior to reperfusion or ischemia (and up to 7 days prior to reperfusion or ischemia). In some examples, the compound that inhibits G-CSF signaling is administered at least 2 hours, or at least 4 hours, or at least 6 hours, or at least 8 hours, or at least 10 hours, or at least 12 hours, or at least 14 hours, or at least 16 hours, or at least 18 hours, or at least 20 hours, or at least 22 hours, or at least 24 hours prior to reperfusion or ischemia. In some examples, the compound that inhibits G-CSF signaling is administered at least 24 hours prior to reperfusion or ischemia.

[0038] When discussing the administration time of the compounds herein, the discussion shall relate to multiple administrations of the compounds. For example, when stating that the compounds are administered between 0 and 7 days before ischemia or reperfusion, the disclosure encompasses multiple administrations of the compounds (e.g., 2 or 3 or 4 or 5, etc.) between 0 and 7 days before ischemia or reperfusion.

[0039] Additionally, in the case of transplantation, the compound may be administered multiple times following transplantation.

[0040] When discussing the timing of administration of a compound herein, this disclosure will also be used to provide clear support for a single administration of the compound.

[0041] In light of the foregoing, the present disclosure provides a method of transplantation (e.g., organ transplantation) or for improving transplantation outcomes (e.g., organ transplantation) or improving transplant function (e.g., transplanted organ) or for preventing delayed transplant function, which will be apparent to one skilled in the art, comprising administering a compound that inhibits G-CSF signaling to a transplant donor prior to harvesting the tissue or organ; harvesting the transplant (e.g., tissue / organ) and transplanting the tissue or organ into an organ transplant recipient.

[0042] The present disclosure also provides a method for preparing a transplant tissue or organ from a tissue or organ donor to improve tissue or organ function in a tissue or organ transplant recipient, the method comprising administering a compound that inhibits G-CSF signaling to the tissue or organ donor before collecting the tissue or organ.

[0043] The present disclosure also provides a method for preventing tissue or organ transplant rejection, the method comprising administering a compound that inhibits G-CSF signaling to a tissue or organ donor before collecting the tissue or organ, collecting the tissue or organ, and transplanting the tissue or organ into a tissue or organ transplant recipient.

[0044] In some examples, the method further comprises administering to the transplant recipient a compound that inhibits G-CSF signaling. For example, the compound that inhibits G-CSF signaling is administered to the transplant recipient prior to transplantation or at or around the time of transplantation of the tissue or organ. In another example, the compound that inhibits G-CSF signaling is administered to the transplant recipient during the tissue or organ transplantation procedure.

[0045] The present disclosure also provides an organ transplant or a method for improving the outcome of tissue or organ transplantation or improving the function of a transplanted tissue or organ or for preventing delayed transplant function, the method comprising administering a compound that inhibits G-CSF signaling to a transplant recipient before transplanting the tissue or organ, and then transplanting the organ into the transplant recipient.

[0046] In one example, the organ transplant donor is brain dead. For example, the organ donor is alive and on life support, but brain dead. Additional donors are described herein and used for this example of the present disclosure.

[0047] In the case of administration to an organ donor, the compound that inhibits G-CSF signaling can be administered prior to organ harvesting, for example, between 0 and 72 hours prior to organ harvesting. In one example, the compound that inhibits G-CSF signaling is administered between 6 and 48 hours prior to organ harvesting. In one example, the compound that inhibits G-CSF signaling is administered between 12 and 36 hours prior to organ harvesting. In one example, the compound that inhibits G-CSF signaling is administered about 24 hours prior to organ collection.

[0048] In the case of administration to a brain-dead donor, the compound that inhibits G-CSF signaling can be administered at a time between brain death and organ harvesting. In some examples, the compound that inhibits G-CSF signaling can be administered within 48 hours of declaring brain death, such as within 24 hours, 12 hours, or 6 hours of declaring brain death.

[0049] In some examples, the compound that inhibits G-CSF signaling is administered in a single dose.

[0050] In some examples, the compound that inhibits G-CSF signaling is administered in multiple doses. For example, the compound is administered to a transplant recipient before or during a transplant, and then one or more additional doses are administered to the recipient after the transplant. In another example, the compound that inhibits G-CSF signaling is administered to a transplant donor or donor tissue or organ, and then one or more additional doses are administered to the transplant recipient after the transplant.

[0051] In some examples, the compound that inhibits G-CSF signaling is administered in a preventive or therapeutically effective amount. In some examples, the compound that inhibits G-CSF signaling is administered at a dose of about 0.01 mg / kg to about 50 mg / kg, for example, about 0.05 mg / kg to about 30 mg / kg, for example, about 0.1 mg / kg to about 20 mg / kg, for example, about 1 mg / kg to about 10 mg / kg. In some examples, the compound that inhibits G-CSF signaling is administered at a dose of about 0.1 mg / kg to about 1 mg / kg. In some examples, the compound that inhibits G-CSF signaling is administered at a dose of about 0.4 mg / kg to about 0.5 mg / kg. In one example, the compound that inhibits G-CSF signaling is administered at a dose of about 0.1 mg / kg. In one example, the compound that inhibits G-CSF signaling is administered at a dose of about 0.2 mg / kg. In one example, the compound that inhibits G-CSF signaling is administered at a dose of about 0.3 mg / kg. In one example, the compound that inhibits G-CSF signaling is administered at a dose of about 0.4 mg / kg. In one example, the compound that inhibits G-CSF signaling is administered at a dose of about 0.5 mg / kg. In one example, the compound that inhibits G-CSF signaling is administered at a dose of about 0.6 mg / kg. In one example, the compound that inhibits G-CSF signaling is administered at a dose of about 0.7 mg / kg. In one example, the compound that inhibits G-CSF signaling is administered at a dose of about 0.8 mg / kg. In one example, the compound that inhibits G-CSF signaling is administered at a dose of about 1 mg / kg. In some examples, the compound that inhibits G-CSF signaling is administered at a dose of about 2 mg / kg to about 8 mg / kg. In one example, the compound that inhibits G-CSF signaling is administered at a dose of about 4 mg / kg to about 6 mg / kg. In one example, the compound that inhibits G-CSF signaling is administered at a dose of about 5 mg / kg.

[0052] In some examples, the compound that inhibits G-CSF signaling is administered in an amount that causes neutropenia. For example, the compound that inhibits G-CSF signaling is administered in an amount that causes transient neutropenia, such as for a period of less than a week, or less than 5 days, or less than 3 days, or less than 1 day.

[0053] In some examples, the compound that inhibits G-CSF signaling is administered in an amount that does not cause neutropenia.

[0054] In one example, the compound that inhibits G-CSF signaling is administered in an amount sufficient to reduce or prevent inflammation. In one example, the compound that inhibits G-CSF signaling is administered in an amount sufficient to reduce or prevent oxidative damage.

[0055] In one example, the compound that inhibits G-CSF signaling is administered in an amount sufficient to have one or more of the following effects:

[0056] (i) reducing or preventing neutrophil infiltration, for example into the transplanted organ in the case of organ transplantation; and

[0057] (ii) Reducing or preventing macrophage infiltration, for example into the transplanted organ in the case of organ transplantation.

[0058] In one example, the compound that inhibits G-CSF signaling is administered in an amount sufficient to reduce or prevent neutrophil infiltration. In one example, the compound that inhibits G-CSF signaling is administered in an amount sufficient to reduce or prevent macrophage infiltration. In one example, the compound that inhibits G-CSF signaling is administered in an amount sufficient to reduce or inhibit the expression of interleukin 8 receptor beta (IL-8Rβ). In one example, the compound that inhibits G-CSF signaling is administered in an amount sufficient to reduce or inhibit the expression of monocyte chemoattractant protein 1 (MCP-1).

[0059] In one example, the compound that inhibits G-CSF signaling is administered in an amount sufficient to have one or more of the following effects:

[0060] (i) reducing or preventing increases in serum or plasma creatinine levels; and

[0061] (ii) reducing or preventing an increase in serum or plasma urea levels.

[0062] As described herein, serum or plasma creatinine levels and serum or plasma urea levels are measures of renal function and can be used, for example, to assess delayed graft function in the context of a renal transplant as described herein.

[0063] In one example, the compound that inhibits G-CSF signaling is administered in an amount sufficient to reduce or prevent an increase in serum or plasma creatinine levels. In one example, the compound that inhibits G-CSF signaling is administered in an amount sufficient to reduce or prevent an increase in serum or plasma urea levels.

[0064] Increased albumin in urine and / or blood levels can also indicate impaired renal function or renal injury. In one example, the compound that inhibits G-CSF signaling is administered in an amount sufficient to reduce or prevent elevated urinary albumin levels. In one example, the compound that inhibits G-CSF signaling is administered in an amount sufficient to reduce or prevent elevated blood levels in the urine of a subject.

[0065] In one example, a compound that inhibits G-CSF signaling is administered, for example, in the case of an organ transplant, via cells of the transplanted organ, in an amount sufficient to reduce or inhibit the expression of one or more of:

[0066] (i) Kidney injury molecule 1 (KIM-1);

[0067] (ii) neutrophil gelatinase-associated lipocalin (NGAL);

[0068] (iii) interleukin 1β (IL-1β);

[0069] (iv) interleukin-6 (IL-6);

[0070] (v) tumor necrosis factor α (TNFα);

[0071] (vi) complement component 5a receptor 1 (C5AR1);

[0072] (vii) macrophage inflammatory protein 2-α (MIP2-α);

[0073] (viii) intercellular adhesion molecule 1 (ICAM-1);

[0074] (ix) E-selectin;

[0075] (x) CXC motif chemokine ligand 1 (CXCL1);

[0076] (xi) interleukin 8 receptor beta (IL-8Rβ); and

[0077] (xii) Monocyte chemoattractant protein 1 (MCP-1).

[0078] In some examples, the compound that inhibits G-CSF signaling is administered in an amount sufficient to reduce or prevent complement C5 activation.

[0079] In some examples, the compound that inhibits G-CSF signaling is administered in an amount sufficient to reduce or prevent deposition of C5b-9, for example, in the case of organ transplantation, on the surface of cells of the transplanted organ.

[0080] Methods for assessing each of the foregoing are known in the art and / or described herein. In addition, one skilled in the art will appreciate that the term "reduced" is used herein to refer to a lower amount of any of the items listed above relative to the amount in a subject prior to administration of a compound that inhibits G-CSF signaling, or relative to the amount in a corresponding control subject.

[0081] In one example, a compound that inhibits G-CSF signaling is administered in combination with another compound. In some examples, the other compound is hydrogen sulfide. In some examples, the other compound is an anti-inflammatory compound and / or specifically inhibits or reduces the expression or activity of one or more molecules listed in (i)-(x) above. In some examples, the other compound is an immunosuppressant, such as cyclosporin. Suitable immunosuppressants will be known in the art and include those described in James & Mannon "Current Transplantation Report" (2015) (James & Mannon, Curr Transplant Rep (2015)), which is incorporated herein by reference. Alternatively or additionally, the other compound is a corticosteroid, such as prednisone and / or prednisolone. Alternatively or additionally, the other compound is methotrexate. Alternatively or additionally, the other compound is cyclophosphamide. Alternatively or additionally, the other compound is mycophenolate mofetil. In some examples, the other compound is TRO40303, as described in Le Lamer et al., J Transl Med (2014). In some examples, the other compound is superoxide dismutase. In some examples, the other compound is metformin. In some examples, the other compound is a cannabinoid or a synthetic analog thereof. In some examples, the other compound is a compound commonly used in transplant surgery.

[0082] In some examples, a compound that inhibits G-CSF signaling is administered in combination with a cell. In some examples, the cell is a stem cell, such as a mesenchymal stem cell.

[0083] In some examples, compounds that inhibit G-CSF signaling are administered in combination with gene therapy.

[0084] In one example, the compound that inhibits G-CSF signaling is administered simultaneously with another compound. In one example, the compound that inhibits G-CSF signaling is administered before the other compound. In one example, the compound that inhibits G-CSF signaling is administered after the other compound.

[0085] In one example, a compound that inhibits G-CSF signaling and another compound are both administered to an organ transplant recipient. Suitable additional compounds are described above.

[0086] In one example, a compound that inhibits G-CSF signaling is administered to an organ transplant donor prior to harvesting the organ for transplantation, and another compound is administered to an organ transplant recipient, optionally in combination with the compound that inhibits G-CSF signaling. Suitable additional compounds are described above.

[0087] In one example, the compound that inhibits G-CSF signaling binds to G-CSF or a G-CSF receptor (G-CSFR). In one example, the compound that inhibits G-CSF signaling binds to G-CSF. In one example, the compound that inhibits G-CSF signaling binds to a G-CSF receptor (G-CSFR).

[0088] In one example, the compound that inhibits G-CSF signaling is a protein.

[0089] In one example, a compound that inhibits G-CSF signaling is a protein that includes an antibody variable region that binds to G-CSFR or specifically binds to and neutralizes G-CSF signaling. Reference herein to a protein or antibody that "binds" to G-CSFR provides literal support for a protein or antibody that "specifically binds" to G-CSFR.

[0090] In one example, a compound that inhibits G-CSF signaling is a protein that includes an antibody variable region that binds to G-CSF or specifically binds to and neutralizes G-CSF signaling. Reference herein to a protein or antibody that "binds" G-CSF provides literal support for a protein or antibody that "specifically binds" G-CSF.

[0091] In some examples, the compound that inhibits G-CSF signaling is a protein comprising Fv. In some examples, the protein is selected from the group consisting of:

[0092] (i) single-chain Fv fragment (scFv);

[0093] (ii) dimeric scFv (di-scFv); or

[0094] (iv) diabodies;

[0095] (v) tri-antibody;

[0096] (vi) tetrabodies;

[0097] (vii) Fab;

[0098] (viii) F(ab')2;

[0099] (ix) Fv;

[0100] (x) (i) to (ix) which is linked to an antibody, Fc or heavy chain constant domain (C H )2 and / or C H 3 constant region;

[0101] (xi) one of (i) to (ix) which is linked to albumin, a functional fragment or variant thereof, or a protein that binds to albumin (e.g., an antibody or an antigen-binding fragment thereof); or

[0102] (xii) Antibodies.

[0103] In one example, the protein is an antibody. In one example, the antibody is a naked antibody. Exemplary antibodies are described in WO2012171057, which is incorporated herein by reference.

[0104] In one example, the protein is an antibody that binds to hG-CSFR expressed on the surface of a cell with an affinity of at least about 5 nM. In one example, the protein is an antibody that binds to hG-CSFR expressed on the surface of a cell with an affinity of at least about 4 nM. In one example, the protein is an antibody that binds to hG-CSFR expressed on the surface of a cell with an affinity of at least about 3 nM. In one example, the protein is an antibody that binds to hG-CSFR expressed on the surface of a cell with an affinity of at least about 2 nM. In one example, the protein is an antibody that binds to hG-CSFR expressed on the surface of a cell with an affinity of at least about 1 nM.

[0105] In one example, the protein is an antibody that inhibits G-CSF-induced proliferation of BaF3 cells expressing hG-CSFR with an IC50 of at least about 5 nM. In one example, the protein is an antibody that inhibits G-CSF-induced proliferation of BaF3 cells expressing hG-CSFR with an IC50 of at least about 4 nM. In one example, the protein is an antibody that inhibits G-CSF-induced proliferation of BaF3 cells expressing hG-CSFR with an IC50 of at least about 3 nM. In one example, the protein is an antibody that inhibits G-CSF-induced proliferation of BaF3 cells expressing hG-CSFR with an IC50 of at least about 2 nM. In one example, the protein is an antibody that inhibits G-CSF-induced proliferation of BaF3 cells expressing hG-CSFR with an IC50 of at least about 1 nM. In one example, the protein is an antibody that inhibits G-CSF-induced proliferation of BaF3 cells expressing hG-CSFR with an IC50 of at least about 0.5 nM.

[0106] In one example, the protein is chimeric, deimmunized, humanized, human or primatized. In one example, the protein or antibody is human.

[0107] In one example, the protein includes an antibody variable region that binds to G-CSFR or specifically binds to and competitively inhibits the binding of antibody C1.2G to G-CSFR, wherein antibody C1.2G includes a heavy chain variable region (V H ) and light chain variable region (VL ), the heavy chain variable region (V H ) comprises the sequence shown in SEQ ID NO: 4, the light chain variable region (V L ) includes the sequence shown in SEQ ID NO:5.

[0108] In one example, the protein binds to an epitope comprising residues within one or two or three or four regions selected from 111-115, 170-176, 218-234 and / or 286-300 of SEQ ID NO:1.

[0109] In one example, the protein is an antibody comprising a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO:4 and a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO:5.

[0110] In one example, the protein is an antibody comprising a VH comprising the amino acid sequence shown in SEQ ID NO:2 and a VL comprising the amino acid sequence shown in SEQ ID NO:3.

[0111] In one example, the protein is an antibody comprising VH and VL, wherein the VH comprises three CDRs of VH, the VH comprises the amino acid sequence shown in SEQ ID NO:4, and the VL comprises three CDRs of VL, the VL comprises the amino acid sequence shown in SEQ ID NO:5.

[0112] In one example, the protein is an antibody comprising VH and VL, wherein the VH comprises three CDRs of VH, the VH comprises the amino acid sequence shown in SEQ ID NO:2, and the VL comprises three CDRs of VL, the VL comprises the amino acid sequence shown in SEQ ID NO:3.

[0113] In one example, the protein is an antibody comprising:

[0114] (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or 18 and a light chain comprising the amino acid sequence of SEQ ID NO: 15; or

[0115] (ii) one heavy chain comprising the amino acid sequence shown in SEQ ID NO:14, one heavy chain comprising the amino acid sequence shown in SEQ ID NO:18, and two light chains comprising the amino acid sequence shown in SEQ ID NO:15.

[0116] Sequence Listing Keywords

[0117] SEQ ID NO: 1 - Amino acids 25-335 of Homo sapiens G-CSFR (hG-CSFR) with a C-terminal polyhistidine tag

[0118] SEQ ID NO:2-V of C1.2 H

[0119] SEQ ID NO:3-V of C1.2 L

[0120] SEQ ID NO:4-C1.2G V H

[0121] SEQ ID NO:5-C1.2G V L

[0122] SEQ ID NO:6-HCDR1 of C1.2

[0123] SEQ ID NO:7-HCDR2 of C1.2

[0124] SEQ ID NO:8-HCDR3 of C1.2

[0125] SEQ ID NO:9-LCDR1 of C1.2

[0126] SEQ ID NO:10-LCDR2 of C1.2

[0127] LCDR3 of SEQ ID NO:11-C1.2

[0128] SEQ ID NO: 12 - Consensus sequence of HCDR3 of C1.2

[0129] SEQ ID NO: 13 - Consensus sequence of LCDR3 of C1.2

[0130] SEQ ID NO: 14—C1.2G heavy chain with stabilized IgG4 constant region

[0131] SEQ ID NO: 15—C1.2G light chain with kappa constant region

[0132] SEQ ID NO: 16 - Sequence of an exemplary hG-CSFR

[0133] SEQ ID NO: 17—Polypeptide comprising the Ig and CRH domains of cynomolgus monkey G-CSFR (cynoG-CSFR) with a C-terminal polyhistidine tag

[0134] SEQ ID NO: 18—C1.2G heavy chain with stabilized IgG4 constant region and lacking the C-terminal lysine. BRIEF DESCRIPTION OF THE DRAWINGS

[0135] Figure 1 is a graphical representation of serum creatinine concentrations 24 hours after reperfusion in a renal warm ischemia-reperfusion injury model. Mice treated with 100 μg of anti-G-CSFR antibody VR81 had significantly reduced serum creatinine concentrations compared to mice treated with 100 μg of isotype control antibody. One-way ANOVA (with Bonferroni correction), *p<0.05, ***p<0.005 (n=8 mice / group). Results are representative of two independent experiments - see also FIG7 .

[0136] Figure 2 is a graphic representation of plasma urea concentrations 24 hours after reperfusion in a renal warm ischemia-reperfusion injury model. Mice treated with 100 μg of anti-G-CSFR antibody VR81 had significantly reduced plasma urea concentrations compared to mice treated with 100 μg of isotype control antibody. Unpaired T-test *p<0.05 (n=8 mice / group). Results are representative of two independent experiments - see also Figure 8 .

[0137] Figure 3 Is a graphic representation of neutrophil counts per high power field (HPF) in renal tissue. Compared to the neutrophil infiltration observed in mice treated with 100 μg of isotype control antibody assessed 24 hours after reperfusion, mice treated with 100 μg of anti-G-CSFR antibody VR81 significantly reduced neutrophil renal infiltration in the hot renal IRI model. One-way ANOVA (with Bonferroni correction), *p<0.05, **p<0.01, ***p<0.005 (n=5-6 mice / group).

[0138] Figure 4 is a graphic representation of macrophage counts per high power field (HPF) in kidney tissue. Mice treated with 100 μg of anti-G-CSFR antibody VR81 had significantly reduced macrophage renal infiltration in the hot renal IRI model compared to macrophage infiltration observed in mice treated with 100 μg of isotype control antibody assessed 24 hours after reperfusion. One-way ANOVA (with Bonferroni correction), *p<0.05, **p<0.01 (n=5-6 mice / group).

[0139] Figure 5Is a graphic representation of (A) neutrophil and (B) macrophage counts per high power field (HPF) in renal tissue. Mice treated with 500 μg of anti-G-CSFR antibody VR81 had significantly reduced neutrophil and macrophage renal infiltration in the hot renal IRI model compared to the neutrophil infiltration observed in mice treated with 500 μg of isotype control antibody assessed 24 hours after reperfusion. One-way ANOVA (with Bonferroni correction), ****p<0.0001 (n=5-6 mice / group).

[0140] Figure 6 is a graphic representation of tubular injury in the thermal IRI model, as determined by the extent of tubular necrosis observed in the renal cortex of treated mice (sham; 100 μg anti-G-CSFR antibody VR81; 100 μg of isotype control antibody). Necrosis was assessed by a semiquantitative method using fluorescence microscopy (Lu B et al. Nephrology 2008). The extent of tubular necrosis was graded and a modified scoring system was used (see Table 3). One-way ANOVA Kruskal-Wallis test, Dunn's multiple comparison test, *p<0.05, **p<0.01, ***p<0.005 (n=8 mice / group, not sham, where n=3).

[0141] Figure 7 is a graphical representation of KIM-1, NGAL, IL-6, IL-8Rβ / CXCR2, MCP-1 / CCL2, CXCL1, MIP-2 / CXCL2, ICAM-1, and C5aR mRNA expression in renal tissue in a hot renal IRI model assessed by qRT-PCR. VR81 treatment at a dose of 500 μg / mouse significantly reduced the expression of (A) KIM-1, NGAL, IL-6, IL-8Rβ / CXCR2, MCP-1 / CCL2, CXCL1, and (B) MIP-2 / CXCL2, ICAM-1, and C5aR compared to treatment with an isotype control antibody. One-way ANOVA Kruskal-Wallis test for VR81 vs. Ab control, Dunn's multiple comparison test *p<0.05, **p<0.01, ***p<0.005.

[0142] Figure 8 In the warm renal IRI model, neutrophils (CD11b + Gr1 +) as assessed by flow cytometry in (A) kidneys and (B) peripheral blood of mice treated with 100 μg of anti-G-CSFR antibody VR81 or 100 μg of anti-C5 antibody (BB5.1) compared to mice treated with 100 μg of isotype control antibody and sham-operated mice. Neutrophils (CD11b + Gr1 + ) levels were significantly reduced. Neutrophil populations in the blood were similar in all groups and were not affected by treatment. One-way ANOVA Kruskal-Wallis test, Dunn's multiple comparison test, *p<0.05, **p<0.01 (n=7-8 mice / group instead of sham, where n=4).

[0143] Fig. 9 is a graphical representation of serum creatinine concentrations in the hot renal IRI model. Treatment with 100 μg of anti-G-CSFR antibody VR81 or 100 μg of anti-C5 antibody BB5.1 significantly reduced creatinine concentrations compared to mice treated with 100 μg of isotype control antibody. There was no significant difference in the reduction observed with VR81 and BB5.1. One-way ANOVA Kruskal-Wallis test, Dunn's multiple comparison test, *p<0.05, **p<0.01, ***p<0.005 (n=7-8 mice / group).

[0144] Fig.10 is a graphical representation of plasma urea concentrations in the warm renal IRI model. Treatment with 100 μg of anti-G-CSFR antibody VR81 or 100 μg of anti-C5 antibody BB5.1 significantly reduced plasma urea concentrations compared to mice treated with 100 μg of isotype control antibody. There was no significant difference in the reduction observed with VR81 and BB5.1. One-way ANOVA Kruskal-Wallis test, Dunn's multiple comparison test, *p<0.05, **p<0.01 (n=7-8 mice / group).

[0145] Fig.11 is a graphical representation of serum urea concentration in the warm renal IRI model. Mice treated with 500 μg of anti-G-CSFR antibody VR81 had significantly reduced serum urea concentrations compared to mice treated with 500 μg of isotype control antibody. One-way ANOVA Kruskal-Wallis test, Dunn's multiple comparison test *p<0.05, **p<0.01 (n=6-8 mice / group).

[0146] Fig.12is a graphical representation of serum creatinine concentrations in the warm renal IRI model (mean ± SEM). Mice treated with 500 μg of anti-G-CSFR antibody (VR81) had significantly reduced serum creatinine concentrations compared to mice treated with 500 μg of isotype control antibody. One-way ANOVA Kruskal-Wallis test, Dunn's multiple comparison test *p<0.05 (n=6-8 mice / group).

[0147] Fig.13 Neutrophils (CD11b) in (A) blood and (B) kidney in the hot renal IRI model + Gr1 + 6G + ). Treatment with 200 μg / mouse and 500 μg / mouse of anti-G-CSFR antibody VR81 significantly reduced the frequency of neutrophils in the kidney compared to treatment with 500 μg / mouse of isotype control antibody. Statistics: One-way ANOVA (with Bonferroni correction), *p<0.05, **p<0.01 (n=6-7 mice / group).

[0148] Fig.14 Are graphical representations of plasma C5a (A) and renal C5b-9 deposition (B).

[0149] Fig.15 is a graphical representation of the frequency of neutrophils (CD11b+Grl+Ly6G+) in the blood and kidney in the hot renal IRI model. Treatment with 500 μg / mouse of the anti-G-CSFR antibody VR81 1 h before ischemia significantly reduced the frequency of neutrophils in the kidney compared to treatment with the isotype control antibody. Treatment with 500 μg / mouse of the control antibody BB5.1 did not significantly reduce the frequency of neutrophils in the kidney compared to treatment with the isotype control antibody. Statistics: One-way ANOVA Kruskal-Wallis test, Dunn's multiple comparison test *p<0.05, **p<0.01 (n=5-8 mice / group). DETAILED DESCRIPTION

[0150] generally

[0151] Throughout the specification, unless specifically stated otherwise or the context requires otherwise, references to individual steps, compositions of matter, groups of steps or groups of compositions of matter shall be considered to include both one and multiple (ie one or more) those steps, compositions of matter, groups of steps or groups of compositions of matter.

[0152] Those skilled in the art will appreciate that, in addition to those specifically described, the present disclosure is susceptible to variation and modification. It should be understood that the present disclosure includes all such variations and modifications. The present disclosure also includes all steps, features, compositions and compounds mentioned or indicated in this specification, either individually or in combination, and any and all combinations of any two or more of the steps or features.

[0153] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for illustrative purposes only. As described herein, functionally equivalent products, compositions and methods are clearly within the scope of the present disclosure.

[0154] Unless expressly stated otherwise, any example of the present disclosure herein shall apply mutatis mutandis to any other example of the present disclosure.

[0155] Unless specifically defined otherwise, all technical and scientific terms used herein shall have the same meanings as commonly understood by one of ordinary skill in the art (eg, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0156] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques used in this disclosure are standard procedures and are well known to those skilled in the art.These techniques are described and explained in, for example, 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); DM Glover and BD Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1992). Approach, Volumes 1-4, IRL Press (1995 and 1996); FM Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date); Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988); and JE Coligan et al. (editor), Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0157] The description and definition of variable regions and portions thereof, immunoglobulins, antibodies and fragments thereof herein can be further clarified by the discussion in Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991, Bork et al., J Mol. Biol. 242, 309-320, 1994, Chothia and Lesk J. Mol Biol. 196:901-917, 1987, Chothia et al., Nature 342, 877-883, 1989. et al. Nature 342, 877-883, 1989), and / or Al-Lazikani et al., J Mol Biol 273, 927-948, 1997.

[0158] The term "and / or", such as "X and / or Y", should be understood as "X and Y" or "X or Y", and should be regarded as providing clear support for both meanings or just one of them.

[0159] Throughout the specification, the word "comprise" or words such as "comprises or comprising" will be understood to imply the inclusion of a stated element, integer or step, or a group of elements, integers or steps, but not the exclusion of any other element, integer or step, or a group of elements, integers or steps.

[0160] As used herein, the term "derived from" should be understood to mean that the specified integer may be obtained from a particular source, although not necessarily directly from that source.

[0161] Selected Definitions

[0162] As contemplated by the present disclosure, a "compound" may take any of a variety of forms, including natural compounds, chemical small molecule compounds, or biological compounds or macromolecules. Exemplary compounds include antibodies or antigen-binding fragments of antibodies, nucleic acids, polypeptides, peptides, and small molecules.

[0163] "Granulocyte colony stimulating factor" (G-CSF) mentioned herein includes the natural form of G-CSF, its mutant forms, such as filgrastim and pegylated forms of G-CSF or filgrastim. The term also includes mutant forms of G-CSF that retain the activity of binding to G-CSFR (e.g., human G-CSFR) and inducing signal transduction.

[0164] G-CSF is the main regulator of granulocyte production. G-CSF is produced by bone marrow stromal cells, endothelial cells, macrophages and fibroblasts, and is induced by inflammatory stimuli. G-CSF works through G-CSF receptor (G-CSFR), which is expressed on early bone marrow progenitor cells, mature neutrophils, monocytes / macrophages, T and B lymphocytes and endothelial cells.

[0165] For purposes of nomenclature only and not limitation, an exemplary sequence of human G-CSFR is listed in NCBI Reference Sequence: NP_000751.1 (and in SEQ ID NO: 16). The sequences of G-CSFR from other species can be determined using the sequences provided herein and / or in public databases and / or using standard techniques (e.g., as described in Ausubel et al. (eds.), Current Protocols in Molecular Biology (1988, including all updates to date), Green Publishing Associates, Inc. and John Wiley & Sons, Inc., or Molecular Cloning: A Laboratory Manual, J. Sambrook et al., Cold Spring Harbor Laboratory Press). References to human G-CSFR may be abbreviated as hG-CSFR, while references to cynomolgus monkey G-CSFR may be abbreviated as cynoG-CSFR. References to soluble G-CSFR refer to polypeptides that include the ligand binding region of G-CSFR. The Ig and CRH domains of G-CSFR are involved in ligand binding and receptor dimerization (Layton et al., J. Biol Chem., 272:29735-29741, 1997 and Fukunaga et al., EMBO J. 10:2855-2865, 1991). Soluble forms of G-CSFR including these portions of the receptor have been used in various studies of the receptor, and mutation of the free cysteines at positions 78, 163, and 228 of the receptor facilitates the expression and isolation of soluble receptor polypeptides without affecting ligand binding (Mine et al., Biochem., 43:2458-2464 2004).

[0166] As used herein, the term "ischemia-reperfusion injury" refers to tissue or organ damage caused when blood supply returns to a tissue or organ after a period of ischemia or lack of oxygen (after hypoxia or hypoxia). The lack of oxygen and nutrients in the blood during ischemia causes a condition in which the restoration of circulation leads to inflammation and oxidative damage by the induction of oxidative stress rather than the restoration of normal function. Ischemia-reperfusion injury (IRI) is also referred to as "reperfusion injury", "reperfusion injury" and "reoxygenation injury". As used herein, "ischemia-reperfusion injury" includes warm ischemia-reperfusion injury and cold ischemia-reperfusion injury.

[0167] As used herein, the term "ischemia" (also referred to as "anemia") refers to a restriction in the blood supply to a tissue, resulting in a lack of oxygen and glucose required for cellular metabolism, and causing the accumulation and reduced removal of metabolic waste products.

[0168] As used herein, the term "reperfusion" relates to the restoration of blood flow or oxygen to a tissue.

[0169] As used herein, the term "condition" refers to a disruption or interference with normal function, and is not limited to any particular condition, and would include diseases or disorders.

[0170] As used herein, the terms "preventing," "prevent," or "prevention" include administering a compound of the present disclosure to at least partially halt or prevent the development of at least one symptom of a condition. The terms also include treating a subject in remission to prevent or prevent relapse.

[0171] As used herein, the terms "treating," "treat," or "treatment" include the administration of a compound described herein such that at least one symptom of a particular disease or condition is reduced or eliminated.

[0172] As used herein, the term "neutropenia" is used to refer to an absolute neutrophil count (ANC) below the lower limit of the normal range, such as less than 2000 cells / μL blood, or less than 1500 cells / μL blood, or less than 1000 cells / μL blood ANC, such as less than 500 cells / μL blood (see Sibille et al. 2010 Br J Clin Pharmacol 70 (5): 736-748). In some examples, the compound that inhibits G-CSF signaling is administered in an amount that does not cause severe neutropenia. As used herein, the term "severe neutropenia" is used to refer to an absolute neutrophil count (ANC) of less than 1000 cells / μL blood.

[0173] As used herein, the term "subject" should be understood to mean any animal (including humans), such as a mammal. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

[0174] As will be apparent to one skilled in the art from the preceding paragraphs, a donor (eg, an organ donor) or a recipient (eg, an organ recipient) includes a mammal, such as a human.

[0175] The term "protein" shall include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains covalently or non-covalently linked to each other (i.e., a polypeptide complex). For example, a series of polypeptide chains may be covalently linked using suitable chemical bonds or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0176] The term "polypeptide" or "polypeptide chain" will be understood from the preceding paragraphs to mean a series of consecutive amino acids linked by peptide bonds.

[0177] The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that, by virtue of its origin or source, is not associated with naturally associated components that accompany it in its native state; it is substantially free of other proteins from the same source. Proteins can be made substantially free of naturally associated components or substantially purified by isolation using protein purification techniques known in the art. "Substantially purified" means that the protein is substantially free of contaminating agents, for example, at least about 70% or 75% or 80% or 85% or 90% or 95% or 96% or 97% or 98% or 99% free of contaminating agents.

[0178] The term "recombinant" is understood to refer to the product of artificial genetic recombination. Therefore, in the context of recombinant proteins including antibody antigen binding domains, the term does not include naturally occurring antibodies in the subject, which are the product of natural recombination that occurs during B cell maturation. However, if such an antibody is separated, it is considered to be a separated protein including antibody antigen binding domains. Similarly, if a nucleic acid encoding a protein is separated and expressed using a recombinant method, the resulting protein is a recombinant protein including antibody antigen binding domains. Recombinant protein also includes proteins expressed by artificial recombination when it is in a cell, tissue or subject expressing it.

[0179] As used herein, the term "antigen binding site" shall refer to a structure formed by a protein that is capable of binding or specifically binding to an antigen. An antigen binding site need not be a series of consecutive amino acids, or even amino acids in a single polypeptide chain. For example, in an Fv produced by two different polypeptide chains, the antigen binding site consists of V L and V HThe antigen binding site is composed of a series of amino acids that interact with the antigen and is usually, but not always, in one or more CDRs in each variable region. H or V L or Fv.

[0180] Those skilled in the art will appreciate that an "antibody" is generally considered to include a polypeptide chain (e.g., comprising V L Peptides and including V H Antibodies also typically include constant domains, some of which, in the case of heavy chains, can be arranged as constant regions, including constant fragments or crystallizable fragments (Fc). H and V L Interaction forms Fv, and the Fv includes an antigen binding region that can specifically bind to one or more closely related antigens. Typically, the light chain from a mammal is a kappa light chain or a lambda light chain and the heavy chain from a mammal is alpha, delta, epsilon, gamma or mu. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), category (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. The term "antibody" also includes humanized antibodies, primatized antibodies, human antibodies and chimeric antibodies.

[0181] The terms "full-length antibody", "whole antibody" or "intact antibody" are used interchangeably and refer to an antibody in substantially intact form, relative to an antigen-binding fragment of an antibody. Specifically, intact antibodies include those having heavy and light chains that include an Fc region. The constant region may be a wild-type sequence constant region (e.g., a human wild-type sequence constant region) or an amino acid sequence variant thereof.

[0182] As used herein, "variable region" refers to the portion of the light chain and / or heavy chain of an antibody as defined herein that is capable of specific binding to an antigen and includes the amino acid sequence of the complementarity determining region (CDR); i.e., CDR1, CDR2, and CDR3 and framework region (FR). Exemplary variable regions include three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4) and three CDRs. In the case of a protein derived from an IgNAR, the protein may lack a CDR2. H Refers to the variable region of the heavy chain. L Refers to the variable region of the light chain.

[0183] The term "complementarity determining region" (synthetic CDR; i.e., CDR1, CDR2, and CDR3) as used herein refers to the amino acid residues in the variable region of an antibody, the presence of which is necessary for antigen binding. Each variable region typically has three CDR regions identified as CDR1, CDR2, and CDR3. The amino acid positions assigned to CDRs and FRs can be according to Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991, or other numbering systems in the present disclosure, e.g., Chothia and Lesk J. Mol Biol. 196:901-917, 1987; Chothia et al. Nature 342, 877-883, 1989; and / or Al-Lazikani et al. J. Biomolecules 273:927-948, 1997. al., J Mol Biol 273:927-948, 1997); the IMGT numbering system of Lefranc et al., Devel. And Compar. Immunol., 27:55-77, 2003; or the AHO numbering system of Honnegher and Plükthun, J. Mol. Biol., 309:657-670, 2001. For example, according to Kabat's numbering system, V H The framework regions (FRs) and CDRs are located as follows: residues 1-30 (FR1), 31-35 (CDR1), 36-49 (FR2), 50-65 (CDR2), 66-94 (FR3), 95-102 (CDR3), and 103-113 (FR4). According to the numbering system of Kabat, V LFRs and CDRs are located as follows: residues 1-23 (FR1), 24-34 (CDR1), 35-49 (FR2), 50-56 (CDR2), 57-88 (FR3), 89-97 (CDR3), and 98-107 (FR4). The present disclosure is not limited to FRs and CDRs as defined by the Kabat numbering system, but includes all numbering systems, including those discussed above. In one example, CDRs (or FRs) referred to herein are with respect to those regions according to the Kabat numbering system.

[0184] "Framework regions" (FR) are those variable region residues other than the CDR residues.

[0185] As used herein, the term "Fv" should be understood to mean any protein, whether composed of multiple polypeptides or a single polypeptide, in which V L and V H The V that forms the antigen binding site H and V L The fragments may be in a single polypeptide chain or in different polypeptide chains. In addition, the Fv of the present disclosure (and any protein of the present disclosure) may have multiple antigen binding sites, which may or may not bind to the same antigen. The term should be understood to include fragments directly derived from antibodies as well as proteins corresponding to such fragments produced using recombinant methods. In some examples, V H Not associated with the heavy chain constant domain (C H )1 Connect and / or V L Not associated with the light chain constant domain (C L ) connection. Exemplary Fv-containing polypeptides or proteins include Fab fragments, Fab' fragments, F(ab') fragments, scFv, diabodies, triabodies, tetrabodies or higher-order complexes, or any of the above linked to a constant region or a domain thereof, such as C H 2 or C H 3 domains, such as small antibodies. "Fab fragments" consist of monovalent antigen-binding fragments of immunoglobulins and can be produced by digesting whole antibodies with papain to obtain fragments consisting of the entire light chain and a portion of the heavy chain, or can be produced using recombinant methods. "Fab' fragments" of antibodies can be produced by treating whole antibodies with pepsin and then reducing to obtain fragments consisting of the entire light chain and a portion of the heavy chain. HThe Fab' fragment is obtained by treating each antibody in this way and obtaining a portion of the heavy chain of the antibody. Two Fab' fragments are obtained for each antibody treated in this way. Fab' fragments can also be produced by recombinant methods. The "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds and is obtained by treating the whole antibody molecule with pepsin without subsequent reduction. The "Fab2" fragment is a fragment that includes the use of, for example, leucine zippers or C H A recombinant fragment of two Fab fragments connected by 3 domains. "Single-chain Fv" or "scFv" is a recombinant molecule containing the variable region fragment (Fv) of an antibody, in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable flexible polypeptide linker.

[0186] As used herein, the term "binding" with respect to the interaction of a compound or its antigen binding site with an antigen means that the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the antigen. For example, an antibody recognizes and binds to a specific protein structure, rather than binding to a protein in general. If an antibody binds to epitope "A", then in a reaction comprising labeled "A" and a protein, the presence of a molecule comprising epitope "A" (or free, unlabeled "A") will reduce the amount of labeled "A" bound to the antibody.

[0187] As used herein, the term "specifically bind" or "bind specifically" should be understood to mean that the compounds of the present disclosure react or associate with a specific antigen or cell expressed by it more frequently, more rapidly, for a greater duration and / or with greater affinity than alternative antigens or cells. For example, the affinity of a compound that binds to G-CSFR (e.g., hG-CSFR) is greater (e.g., 20-fold or 40-fold or 60-fold or 80-fold to 100-fold or 150-fold or 200-fold) than it is to other cytokine receptors or antigens typically recognized by polyreactive natural antibodies (i.e., naturally occurring antibodies known to bind to multiple antigens naturally occurring in humans). Generally, but not necessarily, references to binding are intended to mean specific binding, and each term should be understood to provide explicit support for the other term.

[0188] If the dissociation constant (K D ) is less than the K of another polypeptide protein or antibody D, the protein or antibody can be considered to "preferentially bind" to the polypeptide. In one example, if a protein or antibody binds to a polypeptide with an affinity (i.e., KD) that is at least about 20 times, 40 times, 60 times, 80 times, 100 times, 120 times, 140 times, or 160 times higher than the KD of the protein or antibody for another polypeptide, then the protein or antibody is considered to preferentially bind to the polypeptide.

[0189] For purposes of clarity and as will be apparent to one of skill in the art based on the subject matter exemplified herein, references to "affinity" in this specification refer to the K of a protein or antibody. D .

[0190] For purposes of clarification and as will be apparent to one of skill in the art based on the description herein, reference to "an affinity of at least about ..." will be understood to mean an affinity (or K D ) is equal to the stated value or higher (i.e., the value stated when the affinity is lower), i.e., an affinity of 2 nM is greater than an affinity of 3 nM. In other words, the term can be "an affinity of X or less," where X is a value stated herein.

[0191] "At least about... 50 " shall be understood to mean IC 50 equal to the value or greater (i.e., when IC 50 lower), i.e., an IC of 2 nM 50 IC > 3nM 50 ). In other words, the term could be “X or smaller IC 50 ”, where X is a value described herein.

[0192] As used herein, the term "epitope" (synonymous with "antigenic determinant") is understood to mean a region of hG-CSFR to which a protein that includes an antigen binding site to which an antibody binds binds. The term is not necessarily limited to a specific residue or structure that contacts the protein. For example, the term includes a region spanning the amino acids that contact the protein and / or 5-10 or 2-5 or 1-3 amino acids outside the region. In some examples, the epitope includes a series of discontinuous amino acids that are positioned close to each other when the hG-CSFR is folded, i.e., a "conformational epitope". For example, a conformational epitope includes amino acids in one or more or two or more or all of the regions corresponding to 111-115, 170-176, 218-234, and / or 286-300 of SEQ ID NO: 1. Those skilled in the art will also know that the term "epitope" is not limited to peptides or polypeptides. For example, the term "epitope" includes chemically active surface groupings of a molecule (such as sugar side chains, phosphoryl side chains, or sulfonyl side chains), and in certain instances may have specific three dimensional structural characteristics and / or specific charge characteristics.

[0193] The term "competitive inhibition" is understood to mean that the protein (or its antigen binding site) of the present disclosure reduces or prevents the binding of the antibody or protein to G-CSFR, for example, to hG-CSFR. This may be due to the protein (or antigen binding site) and the antibody binding to the same or overlapping epitope. From the foregoing, it is obvious that the protein does not need to completely inhibit the binding of the antibody, but only needs to reduce the binding by a statistically significant amount, such as at least about 10% or 20% or 30% or 40% or 50% or 60% or 70% or 80% or 90% or 95%. Preferably, the protein reduces the binding of the antibody by at least about 30%, more preferably at least about 50%, more preferably at least about 70%, still more preferably at least about 75%, even more preferably at least about 80% or 85%, even more preferably at least about 90%. Methods for determining competitive inhibition of binding are known in the art and / or described herein. For example, the antibody is exposed to G-CSFR in the presence or absence of the protein. If the antibody bound in the presence of the protein is less than the antibody bound in the absence of the protein, the protein is considered to competitively inhibit the binding of the antibody. In one example, the competitive inhibition is not due to steric hindrance.

[0194] In the context of two epitopes, "overlap" should be taken to mean that the two epitopes share a sufficient number of amino acid residues to allow a protein (or antigen binding site thereof) that binds one epitope to competitively inhibit the binding of a protein (or antigen binding site) that binds the other epitope. For example, "overlapping" epitopes share at least 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 20 amino acids.

[0195] As used herein, the term "neutralizes" is understood to mean that the compound is able to block, reduce or prevent G-CSF-mediated signaling through G-CSFR in a cell. Methods for determining neutralization are known in the art and / or described herein.

[0196] Prevent or treat ischemia-reperfusion injury

[0197] The present disclosure provides, for example, a method for preventing or treating ischemia-reperfusion injury in a subject, comprising administering a compound that inhibits granulocyte colony stimulating factor G-CSF signaling. For example, the compound binds to G-CSF or G-CSFR.

[0198] Ischemia-reperfusion injury can be caused by natural events (e.g., restoration of blood flow after a myocardial infarction or stroke), trauma, a tumor, or by one or more surgical procedures or other therapeutic interventions that restore blood flow or oxygen to a tissue or organ that has been subjected to a reduced blood or oxygen supply.

[0199] In one example, the ischemia-reperfusion injury is caused by a surgical procedure. Such surgical procedures include, for example, coronary artery bypass surgery, transplant surgery, coronary angioplasty, organ transplant surgery, etc. (eg, cardiopulmonary bypass surgery).

[0200] In some examples, the ischemia and / or reperfusion injury is due to or is associated with organ transplantation, cold organ storage, brain death, atherosclerosis, thrombosis, thromboembolism, lipid embolism, trauma, hemorrhage, stents, surgery, angioplasty, bypass surgery, global ischemia, myocardial infarction, stroke, peripheral vascular disease, surgery, sepsis, or a combination thereof.

[0201] In some examples, the ischemia-reperfusion injury is due to or is associated with an organ transplant. In some examples, the organ is a solid organ. In some examples, the organ transplant is a kidney transplant. In some examples, the organ transplant is a liver transplant. In some examples, the organ transplant is a heart transplant. In some examples, the organ transplant is a pancreas transplant. In one example, the organ transplant is a lung transplant. In one example, the organ transplant is a stomach transplant. In one example, the organ transplant is an intestinal transplant. In one example, the organ transplant is a testicular transplant.

[0202] In one example, the ischemia-reperfusion injury is due to or associated with a tissue transplant. In one example, the tissue transplant is a vascular transplant. In one example, the tissue transplant is a skin transplant, such as vascularized skin. In one example, the tissue transplant is a pancreatic islet transplant.

[0203] The effects of ischemia-reperfusion injury include organ dysfunction, infarction, inflammation, oxidative damage, mitochondrial membrane potential damage, apoptosis, reperfusion-related arrhythmias, myocardial stunning, cardiac lipotoxicity, ischemia-derived scar formation, and combinations thereof. For ischemia-reperfusion injury caused by or associated with renal transplantation, the effects also include acute kidney injury and delayed graft function (DGF).

[0204] In some examples, the methods of the present disclosure prevent or reduce the likelihood of acute kidney injury after transplantation. For example, the methods of the present disclosure prevent or reduce the risk of one or more of the following:

[0205] Plasma creatinine level increased by at least ≥0.3 mg / dL (26.52 μmol / L) or ≥1.5- to 2-fold from baseline;

[0206] Less than about 0.5 mL of urine per kilogram per hour for at least 6 hours; and / or

[0207] Requires renal replacement therapy.

[0208] In some examples, the methods of the present disclosure prevent or reduce the possibility of delayed graft function after transplantation (e.g., renal transplantation). As will be known to those skilled in the art, delayed graft function is associated with poor long-term transplant outcomes, chronic transplant rejection, and / or organ survival. For example, the methods of the present disclosure prevent or reduce the risk of one or more of the following:

[0209] Requires one or more hemodialysis treatments within approximately 7 days before the onset of graft function;

[0210] Creatinine reduction between Day 0 and Day 7 was less than 70%.

[0211] In some examples, the methods of the present disclosure prevent or reduce the likelihood of one or more of the following:

[0212] Primary graft nonfunction: a condition in which the transplanted kidney never fully functions and the patient requires dialysis despite the transplant; and / or

[0213] Slow graft function: defined as serum creatinine greater than 3 milligrams (mg) / dL and no need for dialysis on day 5 after transplantation.

[0214] In some examples, compounds that inhibit G-CSF signaling are administered in an amount sufficient to reduce or prevent inflammation, for example, in a transplanted organ. As used herein, the term "inflammation" or "inflammatory response" refers to a group of changes that occur in tissues that undergo inflammation. In particular, inflammation involves a biological response to a noxious stimulus, including a pathogen, damaged cells, or an irritating substance. Methods for determining inflammation are known in the art and include, but are not limited to, measuring erythrocyte sedimentation rate (ESR), where a higher ESR indicates inflammation, measuring C-reactive protein (CRP), where a higher level of CRP indicates inflammation, and white blood cell count (increased in inflammation).

[0215] In some examples, the compound that inhibits G-CSF signaling is administered in an amount sufficient to reduce or prevent, for example, oxidative damage in a transplanted organ. As used herein, the term "oxidative damage" refers to biomolecular damage that can be caused by reactive species during oxygen recovery. Oxidative damage can involve lipid peroxidation, oxidative DNA damage, and oxidative damage to proteins. Methods for determining lipid peroxidation include, but are not limited to, determining MDA (malondialdehyde)-TBA (thiobarbituric acid) by HPLC, and quantifying isoprostanes (which are specific end products of polyunsaturated fatty acid peroxidation) by mass spectrometry. Methods for determining DNA oxidative damage include, but are not limited to, measuring 8-hydroxy-2'-deoxyguanosine (80HdG). Methods for determining oxidative damage to proteins include, but are not limited to, quantification of individual amino acid oxidation products, including kynurenine (from tryptophan), dityrosine (which appears to be metabolically stable and can be detected in urine), valine and leucine hydroxides, L-dihydroxyphenylalanine (L-DOPA), o-tyrosine, 2-oxo-histidine, glutamate semialdehyde and adipic acid semialdehyde, and carbonyl assays (including assays of protein carbonyls). Mitochondrial membrane potential (Δψιη) relates to the membrane potential in the form of a proton gradient across the inner mitochondrial membrane. Methods for assessing mitochondrial membrane potential damage are known to those skilled in the art, and include the use of fluorescent probes for monitoring membrane potential, including JC1 dyes (Cell Technology) and measurements of total fluorescence at excitation and emission wavelengths that allow quantification of green (485nm and 535nm) and red fluorescence (550nm and 600nm). Long-term ischemia of any tissue or organ is known to induce mitochondrial membrane potential damage.

[0216] In some examples, compounds that inhibit G-CSF signaling are administered in an amount sufficient to reduce or prevent neutrophil and / or macrophage infiltration. As used herein, the terms "neutrophil infiltration" and "macrophage infiltration" refer to the recruitment or accumulation of neutrophils and macrophages in tissues or cells in response to the release of various substances at the site of the inflammatory response caused by ischemia-reperfusion injury. For example, infiltration of neutrophils and / or macrophages may occur in donor organ tissue after organ transplantation. Methods for measuring neutrophil and / or macrophage infiltration are known to those skilled in the art. For example, neutrophils or macrophages can be measured directly, such as by visualization by fluorescence microscopy, or indirectly by measuring the abundance or activity of neutrophil / macrophage-specific proteins or enzymes in the affected tissue. Suitable methods for measuring neutrophil and / or macrophage infiltration are described in Soo-Jeong Yu et al. Korean J Intern Med (2008) and Pulli et al. PloS One (2013).

[0217] In some examples, the compound that inhibits G-CSF signaling is administered in an amount sufficient to reduce or prevent complement C5 activation. Methods for measuring complement C5a activation include, but are not limited to, assessing plasma C5a levels by enzyme-linked immunosorbent assay (ELISA).

[0218] In some examples, the compound that inhibits G-CSF signaling is administered in an amount sufficient to reduce or prevent C5b-9 deposition. Methods for measuring C5b-9 deposition include, but are not limited to, analysis by immunofluorescence staining of kidney sections.

[0219] In some examples, the compound that inhibits G-CSF signaling is administered in an amount sufficient to reduce the expression of one or more of:

[0220] Interleukin 8 receptor beta (IL-8Rβ);

[0221] Monocyte chemoattractant protein 1 (MCP-1);

[0222] Kidney injury molecule 1 (KIM-1);

[0223] Neutrophil gelatinase-associated lipocalin (NGAL);

[0224] Interleukin 1β (IL-1β);

[0225] Interleukin 6 (IL-6);

[0226] Tumor necrosis factor alpha (TNFα);

[0227] Complement component 5a receptor 1 (C5AR1);

[0228] Macrophage inflammatory protein 2-alpha (MIP2-alpha);

[0229] Intercellular adhesion molecule 1 (ICAM-1);

[0230] E-selectin; and

[0231] CXC motif chemokine ligand 1 (CXCL1).

[0232] Methods for measuring gene expression levels are known in the art. For example, the expression level of a gene can be measured by the amount of quantitative mRNA, such as by northern blotting or by quantitative reverse transcription PCR (qRT-PCR), as described in Riedy et al. Biotechniques (1995) (Riedy et al Biotechniques (1995)). Alternatively, or in addition, the expression level of a gene can be measured quantitatively by, for example, enzyme-linked immunosorbent assay (ELISA), fluorescence-linked immunosorbent assay (FLISA), immunofluorescence or immunoblotting (see J. Sambrook et al., Molecular Cloning: Laboratory Manual, published by Cold Spring Harbor Laboratory Press) to the level of a gene-encoded protein.

[0233] In some examples, the compound that inhibits G-CSF signaling is administered in an amount sufficient to reduce or prevent delayed transplantation function, such as that associated with renal transplantation. Delayed transplantation function is a manifestation of acute renal injury, resulting in oliguria after transplantation, increasing the risk of immunogenicity and acute rejection episodes of transplantation, and reducing long-term survival. Suitable biomarkers for detecting delayed transplantation function include NGAL, KIM-1, liver-type fatty acid binding protein (L-FABP), interleukin 18 (IL-18), YKL-40 (40kDa heparin binding glycoprotein and chitin binding glycoprotein, see, for example, Hakala et al. Journal of Biological Chemistry (1993) (Hakala et al., J Biol Chem 1993), clusterin, and serum cystatin C. Such biomarkers for detecting delayed transplantation function are described in Malyszko et al. Scientific Reports (2015) (Malyszko et al., Sci Rep (2015)), which are incorporated herein by reference.

[0234] In some examples, the compound that inhibits G-CSF signaling is administered in an amount sufficient to reduce or prevent an increase in serum or plasma creatinine levels. It will be appreciated by those skilled in the art that increased serum (or plasma) creatinine levels are indicators of impaired renal function. When the kidney is damaged for any reason, such as due to ischemia-reperfusion injury, the creatinine level in the blood will increase due to poor clearance of creatinine by the kidney. Therefore, abnormally high levels of creatinine warn of possible dysfunction or failure of the kidney. Suitable methods for measuring serum (or plasma) creatinine levels are known in the art, such as the Jaffe reaction using alkaline picrates, and are described in Peake and Whiting, Clin Biochem Rev (2006).

[0235] In some examples, the compound that inhibits G-CSF signaling is administered in an amount sufficient to reduce or prevent an increase in serum or plasma urea levels. Elevated serum or plasma urea levels are indicators of impaired renal function and may be caused by ischemia-reperfusion injury. Serum or plasma urea may be measured by any suitable method known in the art. For example, serum or plasma urea may be measured by chemical methods, such as diacetyl (produced by diacetyl monoxime and acid) reacting with urea to form diazines, or by enzymatic methods, such as using urease to produce and measure ammonia or glutamate dehydrogenase, wherein the consumption of NADH is measured. For example, Cell Biolabs' urea assay kit is based on the Berthelot reaction, in which urea is first degraded into ammonia and carbon dioxide, which further reacts with an alkaline developer to produce a blue-green product that can be measured with a standard spectrophotometric plate reader at an optical density between 580-630nm. Suitable methods for measuring serum or plasma urea levels include those described in Lamb E et al., Kidney Function Tests (Chapter 25): Tietz Textbook of Clinical Chemistry and Molecular Diagnostics (2012).

[0236] In some examples, the compound that inhibits G-CSF signaling is administered in an amount sufficient to reduce or prevent an increase in urine albumin levels. Increased urine albumin levels are an indicator of impaired renal function and may be caused by ischemia-reperfusion injury. Urine albumin levels can be determined by any suitable method known in the art. For example, urine albumin levels can be measured using a microalbuminuria test.

[0237] In one example, the compound that inhibits G-CSF signaling is administered in an amount sufficient to reduce or prevent an increase in blood levels in the subject's urine. Blood in the urine (also known as "hematuria") is an indicator of impaired renal function and can be caused by ischemia-reperfusion injury. The blood level in the subject's urine can be measured by any suitable method known in the art. For example, the blood level in the subject's urine can be measured using a urine dipstick, or by microscopic examination of a urine sample to detect the presence of red blood cells.

[0238] Antibody

[0239] In one example, the compound described herein according to any example is a protein comprising an antigen binding site of an antibody. In some examples, the compound that inhibits G-CSF signaling is an antibody. In some examples, the antibody binds to G-CSFR. In some examples, the antibody binds to G-CSF.

[0240] Methods for producing antibodies are known in the art and / or described in Harlow and Lane (editor) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988). Generally, in such methods, G-CSFR or G-CSF (e.g., hG-CSFR or hG-CSF) or a region (e.g., an extracellular domain) thereof or an immunogenic fragment or epitope thereof or a cell expressing and displaying the same (i.e., an immunogen), optionally formulated with any suitable or desired carrier, adjuvant or pharmaceutically acceptable excipient, is administered to a non-human animal, such as a mouse, chicken, rat, rabbit, guinea pig, dog, horse, cow, goat or pig. The immunogen can be administered intranasally, intramuscularly, subcutaneously, intravenously, intradermally, intraperitoneally or by other known routes.

[0241] Monoclonal antibodies are an exemplary form of antibodies contemplated by the present disclosure. The term "monoclonal antibody" or "mAb" refers to a homogeneous population of antibodies capable of binding to the same antigen (e.g., binding to the same epitope within an antigen). The term is not intended to limit the source of the antibody or how it is prepared.

[0242] To prepare mAbs, any of a variety of known techniques may be used, for example, as exemplified in US Pat. No. 4,196,265 or in Harlow and Lane (1988), supra.

[0243] Alternatively, MAb-secreting cell lines are generated using ABL-MYC technology (NeoClone, Madison WI 53713, USA) (e.g., as described in Largaespada et al, J. Immunol. Methods. 197:85-95, 1996).

[0244] Antibodies can also be produced or isolated by screening display libraries, such as phage display libraries, for example, as described in US6300064 and / or US5885793. For example, the inventors have isolated fully human antibodies from phage display libraries.

[0245] The antibodies of the present disclosure can be synthetic antibodies. For example, the antibodies are chimeric antibodies, humanized antibodies, human antibodies, or deimmunized antibodies.

[0246] In one example, the antibodies described herein are chimeric antibodies. The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species (e.g., mouse, such as a mouse) or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species (e.g., primates, such as humans) or belonging to another antibody class or subclass. Methods for producing chimeric antibodies are described in, for example, US4816567; and US5807715.

[0247] The antibodies of the disclosure may be humanized or human.

[0248] The term "humanized antibody" should be understood to refer to a chimeric antibody subclass having an antigen binding site or variable region derived from an antibody of a non-human species and a remaining antibody structure based on the structure and / or sequence of a human antibody. In a humanized antibody, the antigen binding site generally comprises a complementarity determining region (CDR) of a non-human antibody on an appropriate FR transplanted into the variable region of a human antibody and the remaining region from a human antibody. The antigen binding site can be wild-type (i.e., the same as those of a non-human antibody) or modified by one or more amino acid substitutions. In some cases, the FR residues of a human antibody are replaced by corresponding non-human residues.

[0249] Methods for humanizing non-human antibodies or parts thereof (e.g., variable regions) are known in the art. Humanization can be performed according to the methods of US5225539 or US5585089. Other methods for humanizing antibodies are not excluded.

[0250] The term "human antibody" as used herein refers to an antibody having variable regions (e.g., V H 、V L ) and, optionally, a constant region derived from or corresponding to a sequence found in humans, e.g., in human germline or somatic cells.

[0251] Exemplary human antibodies are described herein and include C1.2 and C1.2G and / or variable regions thereof. These human antibodies provide the advantage of reduced immunogenicity in humans compared to non-human antibodies. Exemplary antibodies are described in WO2012171057, which is incorporated herein by reference.

[0252] Antibody binding domain containing protein

[0253] Single domain antibodies

[0254] In some examples, the compounds of the present disclosure are or include proteins that are single domain antibodies (which can be used interchangeably with the terms "domain antibodies" or "dAbs"). Single domain antibodies are single polypeptide chains that include all or part of the variable region of an antibody heavy chain. In certain examples, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., US6248516).

[0255] Double antibody, triple antibody, quadruple antibody

[0256] In some examples, the proteins of the present disclosure are or include diabodies, triabodies, tetrabodies, or higher order protein complexes, such as those described in WO98 / 044001 and / or WO94 / 007921.

[0257] Single-chain Fv (scFv)

[0258] Those skilled in the art will appreciate that scFv comprises V H and V L Area and V H and V L The polypeptide linker between the scFv and the polypeptide linker enables the scFv to form the desired structure for antigen binding (i.e., to enable the V H and V L For example, the linker includes more than 12 amino acid residues, among which (Gly4Ser)3 is one of the more favorable linkers for scFv.

[0259] Heavy chain antibody

[0260] Heavy chain antibodies are structurally different from many other forms of antibodies in that they include heavy chains but no light chains. Therefore, these antibodies are also called "heavy chain-only antibodies." Heavy chain antibodies are found in, for example, camels and cartilaginous fish (also called IgNARs).

[0261] A general description of heavy chain antibodies and variable regions thereof from camelids and methods for their production and / or isolation and / or use is found in particular in the following references WO 94 / 04678, WO 97 / 49805 and WO 97 / 49805.

[0262] A general description of heavy chain antibodies from cartilaginous fish and their variable regions and methods for their production and / or isolation and / or use is found, inter alia, in WO 2005 / 118629.

[0263] Other antibodies and antibody fragments

[0264] The present disclosure also contemplates other antibodies and antibody fragments, such as:

[0265] (i) a "key and hole" bispecific protein as described in US5,731,168;

[0266] (ii) heteroconjugate proteins, e.g., as described in US 4,676,980;

[0267] (iii) heteroconjugated proteins produced using chemical cross-linkers (e.g., Neod) as described in US 4,676,980; and

[0268] (iv) Fab3 (eg as described in EP19930302894).

[0269] V-like proteins

[0270] The example of the compound of the present disclosure is T cell receptor. T cell receptor has two V-domains combined into a structure similar to antibody Fv module. Novotny et al., Proc Natl Acad Sci USA 88:8646-8650, 1991 (Novotny et al., Proc Natl Acad Sci USA 88:8646-8650, 1991) describes how to fuse two V-domains (called α and β) of T cell receptor and express them as single-chain polypeptides, and how to change surface residues to reduce the hydrophobicity directly similar to antibody scFv. Other publications describing the production of single-chain T cell receptors or multimeric T cell receptors including two V-α and V-β domains include WO1999 / 045110 or WO2011 / 107595.

[0271] Other non-antibody proteins including antigen binding domains include proteins with V-like domains, which are typically monomeric. Examples of proteins including such V-like domains include CTLA-4, CD28, and ICOS. Further disclosures of proteins including such V-like domains are included in WO1999 / 045110.

[0272] Adnectin

[0273] In one example, the compound of the present disclosure is adinectin. Adinectin is based on the tenth fibronectin type III of human fibronectin ( 10 Fn3) domains, wherein the loop regions are altered to confer antigen binding. For example, one can engineer 10 The three loops at one end of the β-sandwich of the Fn3 domain enable Adicatenin to specifically recognize antigens. For more details, see US20080139791 or WO2005 / 056764.

[0274] Anticalin

[0275] In another example, the compound of the present disclosure is anticalin. Anticalin is derived from lipocalin, which is a family of extracellular proteins that transport small hydrophobic molecules such as steroids, bilirubin, retinoids and lipids. Lipocalin has a rigid β-sheet secondary structure with multiple loops at the open end of the cone structure, which can be engineered to bind antigens. Such engineered lipocalin is called anticalin. For further description of anticalin, see US7250297B1 or US20070224633.

[0276] Affibody

[0277] In another example, the compound of the present disclosure is an affibody. An affibody is a scaffold of the Z domain (antigen binding domain) of protein A derived from Staphylococcus aureus, which can be engineered to bind antigens. The Z domain consists of a three-helix bundle of approximately 58 amino acids. Libraries have been generated by randomization of surface residues. See EP1641818 for more details.

[0278] Avimer

[0279] In another example, the compounds of the present disclosure are Avimers. Avimers are multi-domain proteins derived from the A-domain backbone family. The natural domain of about 35 amino acids adopts a defined disulfide bond structure. Diversity is generated by reorganizing the natural variation displayed by the A-domain family. For more details, see WO2002088171.

[0280] Ankyrin repeat proteins (DARPins)

[0281] In another example, the compounds disclosed herein are designed ankyrin repeat proteins (DARPins). Ankyrin repeat proteins are derived from ankyrin, a family of proteins that mediate the attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two α-helices and a β-turn. They can be engineered to bind different target antigens by randomizing the residues in the first α-helix and β-turn of each repeat. Their binding interface can be increased by increasing the number of components (affinity maturation method). See US20040132028 for more details.

[0282] Soluble G-CSFR

[0283] The present disclosure also contemplates soluble forms of G-CSFR that compete with naturally occurring membrane-associated G-CSFR for G-CSF interaction. Soluble forms of the receptor can be readily prepared by one skilled in the art, see, e.g., U.S. Patent Application No. 5,589,456 and Honjo et al., Acta Crystallograph Sect F Struct Biol Cryst Commun. 61(Pt8):788-790, 2005.

[0284] Deimmunized Antibodies and Proteins

[0285] The disclosure also encompasses deimmunized antibodies or proteins. Deimmunized antibodies and proteins have one or more epitopes, for example, B cell epitopes or T cell epitopes that are removed (i.e., mutated), thereby reducing the likelihood that mammals will produce an immune response to the antibody or protein. Methods for producing deimmunized antibodies and proteins are known in the art and are described in, for example, WO2000 / 34317, WO2004 / 108158, and WO2004 / 064724.

[0286] Methods for introducing appropriate mutations and expressing and assaying the resulting proteins will be apparent to those of skill in the art based on the description herein.

[0287] Protein mutation

[0288] The disclosure also contemplates mutant forms of the proteins of the disclosure. In this regard, the data provided herein indicate sites that can be altered in addition to the exemplary changes that can be made within the CDRs of the proteins of the disclosure. It will be appreciated by those skilled in the art that, in the context of the disclosure, changes can be made additionally or alternatively within the framework regions containing the variable regions of the proteins without inhibiting or significantly reducing their function.

[0289] For example, compared to the sequences described herein, such mutant proteins include one or more conservative amino acid substitutions. In some examples, the protein includes 30 or less or 20 or less or 10 or less (e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2) conservative amino acid substitutions. "Conservative amino acid substitutions" are amino acid residues that are replaced by amino acid residues with similar side chains and / or hydrophilicity and / or hydrophilicity.

[0290] In one example, the mutant protein has only one or two or three or four or five or six conservative amino acid changes, or no more than one or two or three or four or five or six conservative amino acid changes, when compared to the naturally occurring protein. Details of conservative amino acid changes are provided below. As those skilled in the art will know, for example, based on the disclosure herein, it is reasonable to predict that such minor changes will not alter the activity of the protein.

[0291] Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0292] The present disclosure also encompasses non-conservative amino acid changes (e.g., substitutions) in the proteins (e.g., CDRs, such as CDR3) of the present disclosure. For example, the inventors have identified several non-conservative amino acid substitutions that can be made while maintaining the activity of the proteins of the present disclosure. In one example, the protein includes less than 6, or 5, or 4, or 3, or 2, or 1 non-conservative amino acid substitutions, such as in CDR3 (e.g., CDR3).

[0293] The present disclosure also encompasses one or more insertions or deletions compared to the sequences described herein. In some examples, the protein includes 10 or fewer, such as 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 insertions and / or deletions.

[0294] Constant region

[0295] The present disclosure encompasses proteins and / or antibodies described herein that include antibody constant regions. This includes antigen-binding fragments of antibodies fused to Fc.

[0296] The constant region sequences that can be used to generate the proteins of the present disclosure can be obtained from many different sources. In some examples, the constant region of the protein or a portion thereof is derived from a human antibody. The constant region or a portion thereof can be derived from any antibody class, including IgM, IgG, IgD, IgA and IgE, and any antibody isotype, including IgG1, IgG2, IgG3 and IgG4. In one example, the constant region is a human isotype IgG4 or a stable IgG4 constant region.

[0297] In one example, the Fc region of the constant region has the ability of inducing effector functions reduced compared to natural or wild-type human IgG1 or IgG3 Fc regions. In one example, the effector functions are antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC). Methods for assessing the effector function levels of proteins containing Fc regions are known in the art and / or described herein.

[0298] In one example, the Fc region is an IgG4 Fc region (i.e., a constant region from IgG4), e.g., a human IgG4 Fc region. The sequence of a suitable IgG4 Fc region is apparent to those skilled in the art and / or can be obtained in publicly available databases (e.g., literature obtained from the National Center for Biotechnology Information).

[0299] In one example, constant region is a stable IgG4 constant region.The term "stable IgG4 constant region" is understood to refer to an IgG4 constant region that has been modified to reduce the exchange of Fab arms or to carry out Fab arm exchange or to form a tendency of half antibodies or to form a tendency of half antibodies."Fab arm exchange" refers to a protein modification of human IgG4, in which the IgG4 heavy chain and the attached light chain (half molecule) are exchanged for the heavy chain-light chain pair from another IgG4 molecule. Therefore, IgG4 molecules can obtain two different Fab arms (causing bispecific molecules) that recognize two different antigens. Fab arm exchange occurs naturally in vivo, and can be induced in vitro by purified blood cells or reducing agents (such as reduced glutathione). When IgG4 antibody dissociates to form two molecules each containing a single heavy chain and a single light chain, a "half antibody" is formed.

[0300] In one example, a stabilized IgG4 constant region includes a proline at position 241 of the hinge region according to the Kabat system (Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 1987 and / or 1991). This position corresponds to position 228 of the hinge region according to the EU numbering system (Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 2001 and Edelman et al., Proc. Natl. Acad. USA, 63, 78-85, 1969). In human IgG4, this residue is typically serine. After serine replaces proline, the IgG4 hinge region includes the sequence CPPC. In this regard, those skilled in the art will know that the "hinge region" is the proline-rich portion of the constant region of the heavy chain of an antibody, which connects the Fc and Fab regions that confer mobility to the two Fab arms of the antibody. The hinge region includes the cysteine ​​residues that participate in the inter-heavy chain disulfide bonds. According to the numbering system of Kabat, it is generally defined as an extension from Glu226 to Pro243 of human IgG1. By placing the first and last cysteine ​​residues that form the inter-heavy chain disulfide (SS) bond in the same position, the hinge region of other IgG isotypes can be compared with the IgG1 sequence (see, e.g., WO2010 / 080538).

[0301] Other examples of stable IgG4 antibodies are antibodies (such as described in WO2006 / 033386) in which the arginine at position 409 is replaced by lysine, threonine, methionine or leucine in the heavy chain constant region of human IgG4 (according to the EU numbering system). The Fc region of the constant region can additionally or alternatively include a residue selected from the group consisting of the following at a position corresponding to 405: alanine, valine, glycine, isoleucine and leucine (according to the EU numbering system). Optionally, the hinge region includes proline (i.e., CPPC sequence) (as described above) at position 241.

[0302] In another example, the Fc region is a region modified to have reduced effector function, i.e., a "non-immunostimulatory Fc region." For example, the Fc region is an IgG1 Fc region comprising substitutions at one or more positions selected from 268, 309, 330, and 331. In another example, the Fc region is an IgG1 Fc region comprising one or more of the following alterations: E233P, L234V, L235A, and G236 deletions and / or one or more of the following alterations: A327G, A330S, and P331S (Armour et al., Eur J Immunol. 29:2613-2624, 1999; Shields et al., J Biol Chem. 276(9):6591-604, 2001). Other examples of non-immunostimulatory Fc regions are described in, e.g., Dall'Acqua et al., J Immunol. 177: 1129-1138 2006; and / or Hezareh J Virol; 75: 12161-12168, 2001.

[0303] In another example, the Fc region is a chimeric Fc region, e.g., comprising at least one C from an IgG4 antibody. H 2 domains and at least one C from an IgG1 antibody H3 domains, wherein the Fc region comprises a substitution at one or more amino acid positions selected from 240, 262, 264, 266, 297, 299, 307, 309, 323, 399, 409 and 427 (EU numbering) (e.g., as described in WO2010 / 085682). Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299A, 299K, 307P, 309K, 309M, 309P, 323F, 399S and 427F.

[0304] Other changes

[0305] The present disclosure also encompasses additional modifications to the antibodies or proteins of the present disclosure.

[0306] For example, the antibody includes one or more amino acid substitutions that increase the half-life of the protein. For example, the antibody includes an Fc region, and the Fc region includes one or more amino acid substitutions that increase the affinity of the Fc region to the nascent Fc region. For example, the Fc region has an increased affinity for FcRn at a relatively low pH (e.g., about pH 6.0) to promote Fc / FcRn binding in the endosome. In one example, compared with the affinity of the Fc region at about pH 7.4, the Fc region has an increased affinity for FcRn at about pH 6, which promotes Fc to be released into the blood after cell recycling. These amino acid substitutions can be used to extend the half-life of the protein by reducing clearance from the blood.

[0307] Exemplary amino acid substitutions include T250Q and / or M428L or T252A, T254S and T266F or M252Y, S254T and T256E or H433K and N434F according to the EU numbering system. Additional or alternative amino acid substitutions are described in, for example, US20070135620 or US7083784.

[0308] In one example, the protein of the present disclosure further includes albumin, a functional fragment or variant thereof. In one example, albumin, a functional fragment or variant thereof is serum albumin, such as human serum albumin. In one example, albumin, a functional fragment or variant thereof includes one or more amino acid substitutions, deletions or insertions, for example, no more than 5 or 4 or 3 or 2 or 1 substitutions. Amino acid substitutions suitable for use in the present disclosure will be apparent to those skilled in the art and include naturally occurring substitutions and engineered substitutions, such as those described in WO2011051489, WO2014072481, WO2011103076, WO2012112188, WO2013075066, WO2015063611 and WO2014179657.

[0309] Protein production

[0310] In one example, a protein described herein according to any example is produced by culturing a hybridoma under conditions sufficient to produce the protein, eg, as described herein and / or known in the art.

[0311] Recombinant expression

[0312] In another example, the protein described herein according to any example is recombinant.

[0313] In the case of a recombinant protein, the nucleic acid encoding the protein can be cloned into an expression construct or vector, which is then transfected into a host cell such as an E. coli cell, a yeast cell, an insect cell, or a mammalian cell, such as a simian COS cell, a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a myeloma cell that does not otherwise produce protein. Exemplary cells for expressing proteins are CHO cells, myeloma cells, or HEK cells. Molecular cloning techniques for achieving these purposes are known in the art and are described in, for example, Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date) by Ausubel et al., Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date) or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). A variety of cloning and in vitro amplification methods are suitable for constructing recombinant nucleic acids. Methods for producing recombinant antibodies are also known in the art, see, for example, US4816567 or US5530101.

[0314] After isolation, the nucleic acid operably linked to the promoter is inserted into an expression construct or expression vector for further cloning (amplification of the DNA) or for expression in a cell-free system or cells.

[0315] As used herein, the term "promoter" is considered in its broadest context and includes transcriptional regulatory sequences of genomic genes, including TATA boxes or initiation elements required for precise transcription initiation, with or without additional regulatory elements (e.g., upstream activation sequences, transcription factor binding sites, enhancers and silencers) that alter nucleic acid expression (e.g., in response to development and / or external stimuli, or in a tissue-specific manner). In this article, the term "promoter" is also used to describe recombinant, synthetic or fused nucleic acids or derivatives that confer, activate or enhance expression of nucleic acids operably connected thereto. Exemplary promoters may contain additional copies of one or more specific regulatory elements to further enhance expression of the nucleic acid and / or alter spatial expression and / or temporal expression of the nucleic acid.

[0316] As used herein, the term "operably linked" means that a promoter is positioned relative to a nucleic acid such that expression of the nucleic acid is controlled by the promoter.

[0317] Many vectors for expression in cells are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, a sequence encoding a protein (e.g., from the information provided herein), an enhancer element, a promoter, and a transcription termination sequence. Suitable sequences for expressing proteins are known to those skilled in the art. Exemplary signal sequences include prokaryotic secretion signals (e.g., pelB, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II, etc.), yeast secretion signals (e.g., invertase leader, alpha factor leader, or acid phosphatase leader), or mammalian secretion signals (e.g., herpes simplex gD signal).

[0318] Exemplary promoters active in mammalian cells include cytomegalovirus immediate early promoter (CMV-IE), human elongation factor 1-alpha promoter (EF1), small nuclear RNA promoters (U1a and U1b), alpha-myosin heavy chain promoter, simian virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), adenovirus major late promoter, beta-actin promoter; hybrid regulatory elements including CMV enhancer / beta-actin promoter or immunoglobulin promoter or active fragments thereof. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); and human embryonic kidney cell lines (293 or 293 cells grown in suspension culture as subclones; baby hamster kidney cells (BHK, ATCC CCL 10); or Chinese hamster ovary cells (CHO).

[0319] Typical promoters suitable for expression in yeast cells, such as yeast cells selected from Pichia pastoris, Saccharomyces cerevisiae and S. pombe, include but are not limited to ADH1 promoter, GAL1 promoter, GAL4 promoter, CUP1 promoter, PHO5 promoter, nmt promoter, RPR1 promoter or TEF1 promoter.

[0320] The means for introducing the nucleic acid of separation or the expression construct comprising the nucleic acid into the cell for expression are well known to those skilled in the art. The technology for a given cell depends on known successful technology. The method for introducing recombinant DNA into the cell comprises microinjection, DEAE-dextran-mediated transfection, liposome-mediated transfection, for example, using lipofectamine (Gibco, MD, the U.S.) and / or cellfectin (Gibco, MD, the U.S.), PEG-mediated DNA uptake, electroporation and microparticle bombardment, for example, by using tungsten or gold particles (Agracetus Inc., WI, the U.S.) coated with DNA to carry out.

[0321] Host cells used to produce proteins can be cultured in a variety of culture media, depending on the cell type used. Commercially available culture media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma)), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing mammalian cells. Culture media for culturing other cell types discussed herein are known in the art.

[0322] Protein separation

[0323] Methods for isolating proteins are known in the art and / or described herein.

[0324] When the protein is secreted into the culture medium, the supernatant from such expression system can first be concentrated using a commercially available protein concentration filter (e.g., Amicon or Millipore Pellicon ultrafiltration device). Protease inhibitors such as PMSF can be included in any of the preceding steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of exogenous contaminants. Alternatively, or additionally, the supernatant can be filtered and / or separated from the cells expressing the protein, for example, using continuous centrifugation.

[0325] Proteins prepared from cells can be purified using, for example, ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., protein A affinity chromatography or protein G chromatography), or any combination of the foregoing. These methods are known in the art and are described, for example, in WO 99 / 57134 or Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988).

[0326] Those skilled in the art will also appreciate that the protein can be modified to include a tag that facilitates purification or detection, such as a polyhistidine tag, e.g., a hexahistidine tag, or an influenza virus hemagglutinin (HA) tag, or a simian virus 5 (V5) tag, or a FLAG tag, or a glutathione S-transferase (GST) tag. The resulting protein is then purified using methods known in the art (e.g., affinity purification). For example, a protein containing a hexa-his tag is purified by contacting a sample of the protein containing the hexa-his tag with nickel-nitrilotriacetic acid (Ni-NTA) that specifically binds to the hexa-his tag immobilized on a solid or semisolid support, washing the sample to remove unbound protein, and then eluting the bound protein. Alternatively, or in addition, a ligand or antibody that binds to the tag is used in an affinity purification method.

[0327] Nucleic acid-based G-CSF signaling inhibitors

[0328] In one example of the present disclosure, the therapeutic and / or preventive methods as described herein according to any example of the present disclosure involve reducing the expression of G-CSF and / or G-CSFR. For example, such methods involve administering a compound that reduces transcription and / or translation of a nucleic acid encoding G-CSF or G-CSFR. In one example, the compound that inhibits G-CSF signaling is a nucleic acid, e.g., an antisense polynucleotide, a ribozyme, a PNA, an interfering RNA, a siRNA, a microRNA.

[0329] In another example, the compound that inhibits G-CSF signaling is a nucleic acid encoding a protein compound (eg, an antibody or antigen-binding fragment thereof) that inhibits G-CSF signaling.

[0330] Antisense

[0331] The term "antisense nucleic acid" is understood to refer to a DNA or RNA or its derivative (e.g., LNA or PNA) or a combination thereof that is complementary to at least a portion of a specific mRNA molecule encoding a polypeptide as described in any example of the present disclosure and that can interfere with post-transcriptional events (e.g., mRNA translation). The use of antisense methods is known in the art (see, e.g., Hartmann and Endres (editor), Manual of Antisense Methodology, Kluwer (1999)).

[0332] The antisense nucleic acids disclosed herein will hybridize to the target nucleic acid under physiological conditions. Antisense nucleic acids include sequences corresponding to structural genes or coding regions or sequences that affect control of gene expression or splicing. For example, the antisense nucleic acid may correspond to the targeted coding region of a nucleic acid encoding G-CSF or G-CSFR, or the 5'-untranslated region (UTR) or the 3'-UTR or a combination thereof. It may be partially complementary to an intron sequence, which may be spliced ​​out during or after transcription, such as spliced ​​out only with an exon sequence of the target gene. The length of the antisense sequence should be at least 19 consecutive nucleotides of the nucleic acid encoding G-CSF or G-CSFR, such as at least 50 nucleotides, such as at least 100, 200, 500 or 1000 nucleotides. Full-length sequences complementary to the entire gene transcript may be used. The length may be 100-2000 nucleotides. The degree of identity of the antisense sequence to the target transcript should be at least 90%, such as 95-100%.

[0333] Exemplary antisense nucleic acids to G-CSF or G-CSFR are described, for example, in WO2011032204.

[0334] Catalytic nucleic acid

[0335] The term "catalytic nucleic acid" refers to a DNA molecule or a DNA-containing molecule (also referred to as "deoxyribozyme" or "DNAzyme") or an RNA or RNA-containing molecule (also referred to as "ribozyme" or "RNAzyme") that specifically recognizes different substrates and catalyzes the chemical modification of the substrate. The nucleic acid base in the catalytic nucleic acid can be the base A, C, G, T (U for RNA).

[0336] Typically, the catalytic nucleic acid comprises an antisense sequence for specific recognition of a target nucleic acid and a nucleic acid cleaving enzyme activity (also referred to herein as a "catalytic domain"). The types of ribozymes useful in the present disclosure are hammerhead ribozymes and hairpin ribozymes.

[0337] RNA interference

[0338] RNA interference (RNAi) can be used to specifically inhibit the production of a particular protein. Without being limited by theory, the technique relies on the presence of a dsRNA molecule comprising a sequence substantially identical to the mRNA of the gene of interest or a portion thereof, in this case the mRNA encoding G-CSF or G-CSFR. The dsRNA can be conveniently produced from a single promoter in a recombinant vector host cell, wherein the sense and antisense sequences are flanked by unrelated sequences that allow the sense and antisense sequences to hybridize to form the dsRNA molecule, the unrelated sequences forming a loop structure. The design and generation of suitable dsRNA molecules for use in the present disclosure are well within the capabilities of those skilled in the art, with particular regard to WO99 / 32619, WO99 / 53050, WO99 / 49029 and WO01 / 34815. Such dsRNA molecules for RNAi include, but are not limited to, short hairpin RNA (shRNA) and bifunctional shRNA.

[0339] The length of the sense and antisense sequences that hybridize should each be at least 19 consecutive nucleotides, such as at least 30 or 50 nucleotides, such as at least 100, 200, 500 or 1000 nucleotides. The full-length sequence corresponding to the entire gene transcript can be used. The length can be 100-2000 nucleotides. The degree of identity of the sense and antisense sequences to the target transcript should be at least 85%, such as at least 90%, such as 95-100%.

[0340] Exemplary small interfering RNA ("siRNA") molecules include a nucleotide sequence identical to about 19-21 consecutive nucleotides of a target mRNA. For example, the siRNA sequence begins with the dinucleotide AA, includes a GC content of about 30-70% (e.g., 30-60%, such as 40-60%, such as about 45%-55%), and does not have a high percentage identity with any nucleotide sequence other than the target in the genome of the mammal to be introduced, such as determined by a standard BLAST search.

[0341] Aptamer

[0342] In another example, the compound is a nucleic acid aptamer (adaptive oligomer). An aptamer is a single-stranded oligonucleotide or oligonucleotide analog that is capable of forming a secondary and / or tertiary structure that provides the ability to bind to a specific target molecule (e.g., a protein or small molecule, such as G-CSF or G-CSFR). Thus, an aptamer is an oligonucleotide similar to an antibody. Typically, an aptamer comprises about 15 to about 100 nucleotides, such as about 15 to about 40 nucleotides, such as about 20 to about 40 nucleotides, because oligonucleotides with lengths falling within these ranges can be prepared by conventional techniques.

[0343] Aptamers can be isolated or identified from aptamer libraries. Aptamer libraries are generated, for example, by cloning random oligonucleotides into vectors (or expression vectors in the case of RNA aptamers), wherein the random sequences are flanked by known sequences that provide PCR primer binding sites. Aptamers that provide the desired biological activity (e.g., specific binding to G-CSF or G-CSFR) are selected. For example, aptamers with increased activity are selected using SELEX (systematic evolution of ligands by exponential enrichment). Suitable methods for generating and / or screening aptamer libraries are described, for example, in Elloington and Szostak, Nature 346:818-22, 1990; US 5270163; and / or US 5475096.

[0344] Determine the activity of compounds

[0345] Binds to G-CSFR and its mutants

[0346] From the disclosure herein, it will be apparent to those skilled in the art that some compounds of the present disclosure bind to the ligand binding domain of hG-CSFR and specific mutant forms of the ligand binding domain of hG-CSFR (e.g., SEQ ID NO: 1 with or without certain point mutations) and / or bind to human and cynomolgus monkey G-CSFR. Methods for assessing binding to proteins are known in the art, for example as described in Scopes (In: Protein purification: principles and practice, Third Edition, Springer Verlag, 1994). Such methods generally involve labeling the protein and contacting it with an immobilized compound. After washing to remove non-specifically bound proteins, the amount of label is detected, thereby detecting the amount of bound protein. Of course, the protein can be immobilized and the compound that inhibits G-CSF signaling can be labeled. Panning-type assays can also be used. Alternatively or in addition, surface plasmon resonance assays can be used.

[0347] The above assays can also be used to detect the level of binding of a compound to hG-CSFR or its ligand binding domain (eg, SEQ ID NO: 1) or a mutant form thereof.

[0348] In one example, a protein of the present disclosure binds to a polypeptide of SEQ ID NO: 1, wherein alanine is substituted for lysine at position 167 of SEQ ID NO: 1 and / or wherein alanine is substituted for histidine at position 168 of SEQ ID NO: 1, at a level substantially the same as its binding to SEQ ID NO: 1 (e.g., within 10% or 5% or 1%).

[0349] In one example, a protein of the present disclosure binds to a polypeptide of SEQ ID NO: 1, wherein the level of alanine substitution for arginine at position 287 of SEQ ID NO: 1 is at least about 100-fold, 150-fold, 160-fold, or 200-fold lower than its binding to the polypeptide of SEQ ID NO: 1. In one example, a protein of the present disclosure binds to a polypeptide of SEQ ID NO: 1, wherein the level of alanine substitution for arginine at position 287 of SEQ ID NO: 1 is at least about 160-fold lower than its binding to the polypeptide of SEQ ID NO: 1.

[0350] In one example, a protein of the present disclosure binds to a polypeptide of SEQ ID NO: 1, wherein alanine is substituted for histidine at position 237 of SEQ ID NO: 1, at a level that is at least about 20-fold, 40-fold, 50-fold, or 60-fold lower than its binding to the polypeptide of SEQ ID NO: 1. In one example, a protein of the present disclosure binds to a polypeptide of SEQ ID NO: 1, wherein alanine is substituted for histidine at position 237 of SEQ ID NO: 1, at a level that is at least about 50-fold lower than its binding to the polypeptide of SEQ ID NO: 1.

[0351] In one example, a protein of the present disclosure binds to a polypeptide of SEQ ID NO: 1, wherein alanine is substituted for methionine at position 198 of SEQ ID NO: 1, at a level that is at least about 20-fold, 40-fold, 60-fold, or 70-fold lower than its binding to the polypeptide of SEQ ID NO: 1. In one example, a protein of the present disclosure binds to a polypeptide of SEQ ID NO: 1, wherein alanine is substituted for methionine at position 198 of SEQ ID NO: 1, at a level that is at least about 40-fold lower than its binding to the polypeptide of SEQ ID NO: 1.

[0352] In one example, a protein of the present disclosure binds to a polypeptide of SEQ ID NO: 1, wherein alanine is substituted for tyrosine at position 172 of SEQ ID NO: 1, at a level that is at least about 20-fold, 30-fold, or 40-fold lower than its binding to a polypeptide of SEQ ID NO: 1. In one example, a protein of the present disclosure binds to a polypeptide of SEQ ID NO: 1, wherein alanine is substituted for tyrosine at position 172 of SEQ ID NO: 1, at a level that is at least about 40-fold lower than its binding to a polypeptide of SEQ ID NO: 1.

[0353] In one example, a protein of the present disclosure binds to a polypeptide of SEQ ID NO: 1, wherein the level of alanine substitution for leucine at position 171 of SEQ ID NO: 1 is at least about 100-fold, 120-fold, 130-fold, or 140-fold lower than its binding to the polypeptide of SEQ ID NO: 1. In one example, a protein of the present disclosure binds to a polypeptide of SEQ ID NO: 1, wherein alanine substitution for leucine at position 171 of SEQ ID NO: 1 is at least about 140-fold lower than its binding to the polypeptide of SEQ ID NO: 1.

[0354] In one example, a protein of the present disclosure binds to a polypeptide of SEQ ID NO: 1, wherein alanine is substituted for leucine at position 111 of SEQ ID NO: 1, at a level that is at least about 20-fold, 40-fold, 60-fold, or 70-fold lower than its binding to a polypeptide of SEQ ID NO: 1. In one example, a protein of the present disclosure binds to a polypeptide of SEQ ID NO: 1, wherein alanine is substituted for leucine at position 111 of SEQ ID NO: 1, at a level that is at least about 60-fold lower than its binding to a polypeptide of SEQ ID NO: 1.

[0355] In one example, a protein of the present disclosure binds to a polypeptide of SEQ ID NO: 1 in which alanine replaces the histidine at position 168 of SEQ ID NO: 1 at a level no less than 5-fold, 4-fold, 3-fold, 2-fold, or 1-fold lower than its binding to the polypeptide of SEQ ID NO: 1.

[0356] In one example, a protein of the present disclosure binds to a polypeptide of SEQ ID NO: 1 in which alanine replaces lysine at position 167 of SEQ ID NO: 1 at a level no more than 5-fold, or 4-fold, or 3-fold, or 2-fold, or 1-fold lower than its binding to the polypeptide of SEQ ID NO: 1.

[0357] The level of binding is conveniently determined using a biosensor.

[0358] The present disclosure encompasses any combination of the foregoing features. In one example, the protein described herein has all of the binding features set forth in the first seven paragraphs.

[0359] Epitope mapping

[0360] In another example, the epitope bound by the protein described herein is mapped. Epitope mapping methods are obvious to those skilled in the art. For example, a series of overlapping peptides, such as peptides of 10-15 amino acids, are prepared across the hG-CSFR sequence or a region thereof that includes the epitope of interest. The protein is then contacted with each peptide and the peptides that bind to it are determined. This allows the determination of peptides that include an epitope that binds to the protein. If multiple non-contiguous peptides are bound by the protein, the protein may bind to a conformational epitope.

[0361] Alternatively, or additionally, amino acid residues within hG-CSFR are mutated, for example by alanine scanning mutagenesis, and mutations that reduce or prevent protein binding are identified. Any mutation that reduces or prevents protein binding may be within an epitope to which the protein binds.

[0362] Another method is exemplified herein, which involves binding hG-CSFR or a region thereof to an immobilized protein of the present disclosure and digesting the resulting complex with a protease. The peptides remaining bound to the immobilized protein are then isolated and analyzed, for example, using mass spectrometry, to determine their sequence.

[0363] Another method involves converting hydrogen in hG-CSFR or a region thereof to deuterons and binding the resulting protein to an immobilized protein of the present disclosure. The deuterons are then converted back to hydrogen, the hG-CSFR or a region thereof is isolated, digested with an enzyme and analyzed, for example, using mass spectrometry, to identify those regions containing deuterons that have been protected from conversion to hydrogen by binding to a protein described herein.

[0364] Optionally, the dissociation constant (Kd) of the protein for hG-CSFR or its epitope is determined. The "Kd" or "Kd value" of the hG-CSFR binding protein is measured in one example by a radiolabeled or fluorescently labeled hG-CSFR binding assay. The assay equilibrates the protein with a minimal concentration of labeled G-CSFR in the presence of a titration series of unlabeled hG-CSFR. After washing to remove unbound hG-CSFR, the amount of labeling is determined, which indicates the Kd of the protein.

[0365] According to another example, the Kd or Kd value is measured by using a surface plasmon resonance assay, for example, using a BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) with immobilized hG-CSFR or a region thereof.

[0366] In some examples, proteins with a similar Kd or higher than C1.2 or C1.2G are selected because they are likely to compete for binding to hG-CSFR.

[0367] Determine competitive binding

[0368] The determination of proteins that competitively inhibit the binding of monoclonal antibody C1.2 or C1.2G will be apparent to those skilled in the art. For example, C1.2 or C1.2G is conjugated to a detectable label, such as a fluorescent label or a radioactive label. The labeled antibody and test protein are then mixed and contacted with hG-CSFR or a region thereof (e.g., a polypeptide comprising SEQ ID NO: 1) or a cell expressing it. The level of labeled C1.2 or C1.2G is then determined and compared with the level determined when the labeled antibody is contacted with hG-CSFR, region or cell in the absence of the protein. If the level of labeled C1.2 or C1.2G in the presence of the test protein is reduced compared to the absence of the protein, the protein is considered to competitively inhibit the binding of C1.2 or C1.2G to hG-CSFR.

[0369] Optionally, the test protein is conjugated to a different tag of C1.2 or C1.2G. Such alternative tags allow detection of the level of binding of the test protein to hG-CSFR or a region thereof or to cells.

[0370] In another example, before contacting hG-CSFR or a region thereof or a cell with C1.2 or C1.2G, a protein is allowed to bind to hG-CSFR or a region thereof (e.g., a polypeptide comprising SEQ ID NO: 1) or a cell expressing it. A reduction in the amount of C1.2 or C1.2G bound in the presence of the protein compared to the absence of the protein indicates that the protein competitively inhibits the binding of C1.2 or C1.2G to hG-CSFR. A reciprocal assay can also be performed using a labeled protein and first allowing C1.2 or C1.2G to bind to G-CSFR. In this case, a reduction in the amount of labeled protein bound to hG-CSFR in the presence of C1.2 or C1.2G compared to the absence of C1.2 or C1.2G indicates that the protein competitively inhibits the binding of C1.2 or C1.2G to hG-CSFR.

[0371] Any of the foregoing assays can be performed with the hG-CSFR and / or mutant forms of SEQ ID NO: 1 described herein and / or the ligand binding region of hG-CSFR bound to C1.2 or C1.2G.

[0372] Determine Neutralization

[0373] In some examples of the present disclosure, the compound is capable of neutralizing hG-CSFR signaling.

[0374] Various assays are known in the art for evaluating the ability of a compound to neutralize signaling of a ligand through a receptor.

[0375] In one example, a compound that inhibits G-CSF signaling reduces or prevents G-CSF binding to hG-CSFR. These assays can be performed as competitive binding assays as described herein using labeled G-CSF and / or labeled proteins.

[0376] In another example, when CD34 + Compounds that inhibit G-CSF signaling reduce the formation of CFU-G when bone marrow cells are present. + Bone marrow cells are cultured in a semisolid cell culture medium in the presence or absence of a test compound in the presence of G-CSF (e.g., about 10 ng / ml cell culture medium) and optionally stem cell factor (e.g., about 10 ng / ml cell culture medium). After sufficient time for granulocyte colonies (CFU-G) to form, the number of clones or colonies is determined. A decrease in the number of colonies in the presence of a compound that inhibits G-CSF signaling compared to in the absence of a compound that inhibits G-CSF signaling indicates that the compound that inhibits G-CSF signaling neutralizes G-CSF signaling. By testing multiple concentrations of a compound that inhibits G-CSF signaling, the IC 50 , that is, IC 50 is the concentration at which 50% of maximal inhibition of CFU-G formation occurs. In one example, IC 50 0.2 nM or less, such as 0.1 nM or less, such as 0.09 nM or less, or 0.08 nM or less, or 0.07 nM or less, or 0.06 nM or less, or 0.05 nM or less. In one example, IC 50 is 0.04 nM or less. In another example, IC 50 0.02nM or less. 50 In relation to any of the CFU-G assays described herein.

[0377] In another example, a compound that inhibits G-CSF signaling reduces the proliferation of hG-CSFR expressing cells (e.g., BaF3 cells) cultured in the presence of G-CSF. Cells are cultured in the presence of G-CSF (e.g., 0.5 ng / ml) and in the presence or absence of a test compound. Methods for evaluating cell proliferation are known in the art and include, for example, MTT reduction and thymidine nucleotide incorporation. Compounds that reduce proliferation levels compared to levels observed in the absence of the compound are believed to neutralize G-CSF signaling. By testing multiple concentrations of the compound, the IC 50 , which is the concentration that produces 50% maximal inhibition of cell proliferation. In one example, IC 50 is 6 nM or less, such as 5.9 nM or less. In another example, IC 50 2 nM or less, or 1 nM or less, or 0.7 nM or less, or 0.6 nM or less, or 0.5 nM or less. 50 In connection with any of the cell proliferation assays described herein.

[0378] In another example, the compound that inhibits G-CSF signaling reduces the mobilization of hematopoietic stem cells and / or endothelial progenitor cells in vivo after administration of G-CSF and / or, for example, reduces the number of neutrophils in vivo after administration of G-CSF (however, this is not required). For example, the compound that inhibits G-CSF signaling is optionally administered before, during, or after administration of G-CSF or a modified form thereof (e.g., pegylated G-CSF or filgrastim). The number of hematopoietic stem cells (e.g., expressing CD34 and / or Thy1) and / or endothelial progenitor cells (e.g., expressing CD34 and VEGFR2) and / or neutrophils (identified morphologically and / or expressing, for example, CD10, CD14, CD31, and / or CD88) is assessed. It is believed that the compound that reduces the cell level compared to the level observed in the absence of the compound neutralizes G-CSF signaling. In one example, the compound that inhibits G-CSF signaling reduces the number of neutrophils without inducing neutropenia.

[0379] Other methods for assessing neutralization of G-CSF signaling are contemplated by the present disclosure.

[0380] Determining effector function

[0381] As discussed herein, some proteins of the disclosure have reduced effector function.Methods for assessing ADCC activity are known in the art.

[0382] In one example, using 51 Cr release assay, Europium release assay or 35The level of ADCC activity is assessed by S release assays. In each of these assays, cells expressing G-CSFR are incubated with one or more compounds that inhibit G-CSF signaling under conditions and for a period of time sufficient to allow the compounds to be taken up by the cells. 35 In the case of S release assays, cells expressing hG-CSFR can be 35 The cells are then cultured with S-labeled methionine and / or cysteine ​​for a time sufficient to allow the labeled amino acid to be incorporated into newly synthesized proteins. The cells are then cultured in the presence or absence of the protein and in the presence of immune effector cells, such as peripheral blood mononuclear cells (PBMCs) and / or NK cells. 51 Cr, Europium and / or 35 S, and little or no change in the presence of the protein compared to the absence of the protein (or a reduction in the level of the compound compared to the level observed in the presence of an anti-hG-CSFR antibody including human IgG1 Fc) indicates that the protein has reduced effector function. Exemplary publications disclosing assays for assessing the level of ADCC induced by a protein include Hellstrom et al. Proc. Natl Acad. Sci. USA 83:7059-7063, 1986 and Bruggemann et al. J. Exp. Med. 166:1351-1361, 1987.

[0383] Other assays used to assess the level of ADCC induced by a protein include ACTI for flow cytometry. TM Non-radioactive cytotoxicity assay (CellTechnology, Inc., California, USA) or CytoTox Non-radioactive cytotoxicity assay (Promega, WI, USA).

[0384] A CIq binding assay can also be performed to confirm that the protein is able to bind CIq and can induce CDC. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al, J. Immunol. Methods 202: 163, 1996).

[0385] Determine half-life

[0386] Some proteins encompassed by the present disclosure have improved half-lives, e.g., modified to extend their half-lives compared to unmodified proteins. Methods for determining proteins with improved half-lives will be apparent to those skilled in the art. For example, the ability of a protein to bind to a neonatal Fc receptor (FcRn) is assessed. In this regard, increased binding affinity to FcRn increases the serum half-life of the molecule (see, e.g., Kim et al., Eur J Immunol., 24:2429, 1994).

[0387] The half-life of the proteins of the present disclosure can also be measured by pharmacokinetic studies, for example, according to the method described by Kim et al., Eur J of Immunol 24:542, 1994. According to this method, a radiolabeled protein is injected intravenously into mice, and its plasma concentration is measured regularly as a function of time, for example, from 3 minutes to 72 hours after injection. The clearance curve thus obtained should be biphasic, i.e., an α phase and a β phase. In order to determine the in vivo half-life of a protein, the β phase clearance is calculated and compared with the wild-type or unmodified protein.

[0388] Therapeutic effects

[0389] In vivo assays can be used to assess the efficacy of compounds for treating ischemia-reperfusion injury. For example, by using mouse models such as Bohl et al., Am J Physiol Heart Circulatory Physiology (2009), Hartsock et al., Journal of Visualized Experiments (2016), Greenberg et al., Methods Enzymol (2008), or Zhang et al., Journal of the Federation of American Societies for Experimental Biology (2016), Zhang et al., The FASEB Journal (2016).

[0390] Composition

[0391] In some instances, the compounds described herein can be administered orally, parenterally, by inhalation spray, adsorption, absorption, topically, rectally, nasally, buccally, vaginally, intraventricularly, via an implanted reservoir in a dosage form containing a conventional non-toxic pharmaceutically acceptable carrier, or in any other convenient dosage form. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion techniques.

[0392] Methods for preparing compounds into a form suitable for administration (e.g., pharmaceutical compositions) are known in the art and include, for example, those described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa., 1990) and US Pharmacopeia: National Formulary (Mack Publishing Company, Easton, Pa., 1984).

[0393] The pharmaceutical compositions disclosed herein are particularly useful for parenteral administration, such as intravenous administration or subcutaneous administration or administration to a body cavity or lumen of an organ or joint. The composition for administration will generally include a solution of a compound that inhibits G-CSF signaling dissolved in a pharmaceutically acceptable carrier (e.g., an aqueous carrier). Various aqueous carriers can be used, for example, buffered saline, etc. The composition can contain pharmaceutically acceptable auxiliary substances as needed to approach physiological conditions, such as pH regulators and buffers, toxicity regulators, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the compounds disclosed herein in these preparations can vary widely, and will be selected mainly based on the volume of fluid, viscosity, body weight, etc. according to the specific mode of administration selected and the needs of the patient. Exemplary carriers include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as mixed oils and ethyl oleate can also be used. Liposomes can also be used as carriers. The vehicle can contain a small amount of additives that enhance isotonicity and chemical stability, such as buffers and preservatives.

[0394] When formulated, the compounds of the present disclosure will be administered in a manner compatible with the dosage formulation and in an amount effective for treatment / prevention. The preparations are easily administered in various dosage forms, such as injectable solutions of the above types, but other pharmaceutically acceptable forms are also contemplated, such as tablets, pills, capsules or other solids for oral administration, suppositories, vaginal suppositories, nasal solutions or sprays, aerosols, inhalants, liposome forms, etc. Drug "slow-release" capsules or compositions may also be used. Sustained-release formulations are typically designed to provide constant drug levels over an extended period of time and can be used to deliver compounds of the present disclosure.

[0395] WO2002 / 080967 describes compositions and methods for administering aerosolized compositions including antibodies for treating, for example, asthma, which are also suitable for administering the proteins of the present disclosure.

[0396] Combination therapy

[0397] In one example, a compound of the present disclosure is administered in combination with another compound useful for treating a disease or condition described herein, either as a combined or additional treatment step or as an additional component of a therapeutic formulation.

[0398] For example, the other compound is an anti-inflammatory compound. Alternatively or additionally, the other compound is an immunosuppressant. Alternatively or additionally, the other compound is a corticosteroid, such as prednisone and / or prednisolone. Alternatively or additionally, the other compound is methotrexate. Alternatively or additionally, the other compound is cyclophosphamide. Alternatively or additionally, the other compound is mycophenolate mofetil. Alternatively or additionally, another compound is tissue plasminogen activator (t-PA).

[0399] In some examples, the additional compound inhibits or reduces the expression or activity of one or more of:

[0400] (i) Kidney injury molecule 1 (KIM-1);

[0401] (ii) neutrophil gelatinase-associated lipocalin (NGAL);

[0402] (iii) interleukin 1β (IL-1β);

[0403] (iv) interleukin-6 (IL-6);

[0404] (v) tumor necrosis factor α (TNFα);

[0405] (vi) complement component 5a receptor 1 (C5AR1);

[0406] (vii) macrophage inflammatory protein 2-α (MIP2-α);

[0407] (viii) intercellular adhesion molecule 1 (ICAM-1);

[0408] (ix) E-selectin;

[0409] (x) CXC motif chemokine ligand 1 (CXCL1);

[0410] (xi) interleukin 8 receptor beta (IL-8Rβ); and

[0411] (xii) Monocyte chemoattractant protein 1 (MCP-1).

[0412] In some examples, the other compound is hydrogen sulfide. In some examples, the other compound is cyclosporine. In some examples, the other compound is TRO40303, as described in Le Lamer et al., J Transl Med (2014). In some examples, the other compound is superoxide dismutase. In some examples, the other compound is a cannabinoid or a synthetic analog thereof.

[0413] In some instances, the other compound is a compound commonly used in transplantation procedures.

[0414] In one example, the compound that inhibits G-CSF signaling is administered simultaneously with another compound. In one example, the compound that inhibits G-CSF signaling is administered before the other compound. In one example, the compound that inhibits G-CSF signaling is administered after the other compound.

[0415] In some examples, a compound that inhibits G-CSF signaling is administered in combination with a cell. In some examples, the cell is a stem cell, such as a mesenchymal stem cell.

[0416] In some examples, compounds that inhibit G-CSF signaling are administered in combination with gene therapy.

[0417] Dosage and time of administration

[0418] The appropriate dose of the compounds of the present disclosure will vary depending on the specific compound, the condition to be treated, and / or the subject to be treated. It is within the ability of a skilled physician to determine an appropriate dose, for example by starting with a suboptimal dose and gradually changing the dose to determine an optimal or useful dose. Alternatively, to determine an appropriate dose for treatment / prevention, data from cell culture assays or animal studies are used, where an appropriate dose is within the range of an ED value of an active compound that includes little or no toxicity. 50The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The therapeutically / preventive effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve an IC value including that determined in cell culture. 50 The circulating plasma concentration range of the compound (i.e., the concentration of the compound that achieves a half-maximal inhibition of symptoms) can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0419] In some examples, the compound that inhibits G-CSF signaling is administered systemically. In some examples, the compound that inhibits G-CSF signaling is administered locally.

[0420] Those skilled in the art will appreciate that in order to prevent or treat ischemia-reperfusion injury in a subject, the compound that inhibits G-CSF signaling does not have to be directly administered to the subject. For example, the compound can be administered to an organ donor or organ (e.g., a harvested organ) prior to transplantation to the subject, thereby reducing the effects of ischemia-reperfusion injury generated in the subject after transplantation. Therefore, ischemia-reperfusion injury in a subject can be treated without directly administering a compound that inhibits G-CSF signaling to the subject.

[0421] In some examples, a compound that inhibits G-CSF signaling is administered to a subject. In some examples, the subject is an organ transplant recipient.

[0422] In some instances, the methods of the present disclosure include administering a prophylactically or therapeutically effective amount of a compound described herein.

[0423] The term "therapeutically effective amount" is an amount that, when administered, improves the prognosis and / or status of a subject and / or reduces or inhibits one or more symptoms of a clinical condition described herein to a level below that observed and accepted as a clinical diagnosis or clinical feature of the condition. The amount to be administered will depend on the specific characteristics of the condition to be treated, the type and stage of the condition to be treated, the mode of administration, and the characteristics of the subject, such as general health, other diseases, age, sex, genotype, and body weight. One skilled in the art will be able to determine an appropriate dose based on these and other factors. Therefore, the term should not be interpreted as limiting the present disclosure to a specific amount, such as the weight or amount of a compound, but rather, the present disclosure encompasses any amount of a compound that inhibits G-CSF signaling that is sufficient to achieve the described result in a subject. In one example, a therapeutically effective amount of a compound that inhibits G-CSF signaling does not induce neutropenia.

[0424] As used herein, the term "preventive effective amount" shall refer to an amount of a compound sufficient to prevent or inhibit or delay the onset of one or more detectable symptoms of a clinical condition. One skilled in the art will appreciate that such an amount will vary depending on, for example, the specific compound administered and / or the specific subject and / or the type or severity or level of the condition and / or the cause (genetic or other) of the condition. Thus, the term should not be interpreted as limiting the disclosure to a specific amount, such as the weight or amount of a compound, but rather, the disclosure encompasses any amount of a compound that inhibits G-CSF signaling that is sufficient to achieve the described result in a subject. In one example, a preventive effective amount of a compound that inhibits G-CSF signaling does not induce neutropenia.

[0425] For in vivo administration of the compounds described herein, normal dosages may vary from about 10 ng / kg up to about 100 mg / kg or more of the individual's body weight per day. Exemplary dosages and ranges thereof are described herein. For repeated administration over several days or longer, depending on the severity of the disease or condition to be treated, treatment may be continued until the desired symptom suppression is achieved.

[0426] In some examples, the compound that inhibits G-CSF signaling is administered at an initial (or loading) dose of about 0.1 mg / kg to about 30 mg / kg, for example, about 1 mg / kg to about 10 mg / kg, or about 2 mg / kg to about 8 mg / kg, or about 4 mg / kg to about 6 mg / kg. Then, the compound that inhibits G-CSF signaling can be administered at a lower maintenance dose of about 0.01 mg / kg to about 5 mg / kg, for example, about 0.05 mg / kg to about 1 mg / kg, for example, about 0.1 mg / kg to about 1 mg / kg, for example, about 0.1 mg / kg or 0.5 mg / kg or 1 mg / kg or 2 mg / kg or 3 mg / kg or 4 mg / kg or 5 mg / kg. The maintenance dose can be administered every 7-30 days, for example, every 10-15 days, for example, every 10 or 11 or 12 or 13 or 14 or 15 days. In this regard, a maintenance dose can be used to maintain the therapeutically effective level of the compound in the subject's blood for a period of time, for example after an organ transplant operation.

[0427] In some examples, the compound that inhibits G-CSF signaling is administered at a dose of about 0.1 mg / kg to about 1 mg / kg. In some examples, the compound that inhibits G-CSF signaling is administered at a dose of about 0.4 mg / kg to about 0.5 mg / kg. In one example, the compound that inhibits G-CSF signaling is administered at a dose of about 0.1 mg / kg. In one example, the compound that inhibits G-CSF signaling is administered at a dose of about 0.2 mg / kg. In one example, the compound that inhibits G-CSF signaling is administered at a dose of about 0.3 mg / kg. In one example, the compound that inhibits G-CSF signaling is administered at a dose of about 0.4 mg / kg. In one example, the compound that inhibits G-CSF signaling is administered at a dose of about 0.5 mg / kg. In one example, the compound that inhibits G-CSF signaling is administered at a dose of about 0.6 mg / kg. In one example, the compound that inhibits G-CSF signaling is administered at a dose of about 0.7 mg / kg. In one example, the compound that inhibits G-CSF signaling is administered at a dose of about 0.8 mg / kg. In one example, the compound that inhibits G-CSF signaling is administered at a dose of about 1 mg / kg.

[0428] In some examples, the compound that inhibits G-CSF signaling is administered at a dose of about 0.01 mg / kg to about 50 mg / kg, such as about 0.1 mg / kg to about 40 mg / kg, such as about 2 mg / kg to about 30 mg / kg, such as about 5 mg / kg to about 25 mg / kg.

[0429] In some examples, the compound that inhibits G-CSF signaling is administered at a dose of about 2 mg / kg to about 8 mg / kg. In one example, the compound that inhibits G-CSF signaling is administered at a dose of about 4 mg / kg to about 6 mg / kg.

[0430] In some examples, the compound that inhibits G-CSF signaling is administered at a dose of about 10 mg / kg to about 40 mg / kg. In one example, the compound that inhibits G-CSF signaling is administered at a dose of about 20 mg / kg to about 30 mg / kg.

[0431] In some examples, the compound that inhibits G-CSF signaling is administered at a dose of about 5 mg / kg. In some examples, the compound that inhibits G-CSF signaling is administered at a dose of about 10 mg / kg. In some examples, the compound that inhibits G-CSF signaling is administered at a dose of about 25 mg / kg.

[0432] In some examples, the compound that inhibits G-CSF signaling is administered without a higher loading dose or a lower maintenance dose.

[0433] In some examples, the compound that inhibits G-CSF signaling is administered in a single dose.

[0434] In some examples, multiple doses are administered, for example, every 7-30 days, such as every 10-22 days, for example every 10-15 days, for example every 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 days. For example, a compound that inhibits G-CSF signaling is administered every 7 days or every 14 days or every 21 days.

[0435] In some examples, at the initiation of treatment, a compound that inhibits G-CSF signaling is administered to the mammal for 7 consecutive days, or 6 consecutive days, or 5 consecutive days, or 4 consecutive days.

[0436] In the case of a mammal that does not respond adequately to treatment, multiple doses may be administered over a week. Alternatively, or in addition, increasing doses may be administered.

[0437] In another example, for a mammal experiencing an adverse effect, the initial (or loading) dose may be divided over several days within a week or over consecutive days.

[0438] Administration of the compounds according to the methods of the present disclosure may be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of administration is therapeutic or preventive, and other factors known to those skilled in the art. Administration of the compounds may be substantially continuous over a preselected period of time, or may be a series of spaced doses during or after the progression of the condition.

[0439] In some examples, the compound that inhibits G-CSF signaling is administered before ischemia. Therefore, in some examples, the compound that inhibits G-CSF signaling is administered between 0 days before ischemia (e.g., immediately before ischemia) and 7 days before ischemia. For example, the compound that inhibits G-CSF signaling is administered between 0 days and 6 days, 5 days, or 4 days before ischemia. For example, the compound that inhibits G-CSF signaling is administered 0-48 hours before ischemia. In some examples, the compound that inhibits G-CSF signaling is administered between 12 and 36 hours before ischemia. In some examples, the compound that inhibits G-CSF signaling is administered about 24 hours before ischemia. In other examples, the compound that inhibits G-CSF signaling is administered after ischemia. In some examples, the compound that inhibits G-CSF signaling is administered before and after ischemia. In some examples, the compound that inhibits G-CSF signaling is administered during ischemia, such as during tissue or organ transplant surgery.

[0440] In some examples, the compound that inhibits G-CSF signaling is administered at least 1 hour before ischemia (e.g., up to 7 days or more before ischemia). In some examples, the compound that inhibits G-CSF signaling is administered at least 2 hours, or at least 4 hours, or at least 6 hours, or at least 8 hours, or at least 10 hours, or at least 12 hours, or at least 14 hours, or at least 16 hours, or at least 18 hours, or at least 20 hours, or at least 22 hours, or at least 24 hours before ischemia.

[0441] In some examples, the compound that inhibits G-CSF signaling is administered before reperfusion. Therefore, in some examples, the compound that inhibits G-CSF signaling is administered between 0 days (e.g., immediate reperfusion) and 7 days before reperfusion. For example, the compound that inhibits G-CSF signaling is administered between 0 days and 6 days, 5 days, or 4 days before reperfusion. For example, the compound that inhibits G-CSF signaling is administered between 0 and 48 hours before reperfusion. In some examples, the compound that inhibits G-CSF signaling is administered between 12 and 36 hours before reperfusion. In some examples, the compound that inhibits G-CSF signaling is administered about 24 hours before reperfusion. In other examples, the compound that inhibits G-CSF signaling is administered after reperfusion. In some examples, the compound that inhibits G-CSF signaling is administered before and after reperfusion.

[0442] In some examples, the compound that inhibits G-CSF signaling is administered at least 1 hour before reperfusion. In some examples, the compound that inhibits G-CSF signaling is administered at least 2 hours, or at least 4 hours, or at least 6 hours, or at least 8 hours, or at least 10 hours, or at least 12 hours, or at least 14 hours, or at least 16 hours, or at least 18 hours, or at least 20 hours, or at least 22 hours, or at least 24 hours (e.g., up to 7 days or longer) before reperfusion.

[0443] Tissue or organ transplantation

[0444] When the ischemia-reperfusion injury is due to or associated with a tissue or organ transplant, the compound that inhibits G-CSF signaling can be administered before, during, or after the transplant. Thus, in some examples, the compound that inhibits G-CSF signaling is administered before the transplant. In some examples, the compound that inhibits G-CSF signaling is administered during the transplant, such as before the clamp is released, when blood flow is restored to the transplanted organ. In some examples, the compound that inhibits G-CSF signaling is administered after the transplant.

[0445] In some examples, a compound that inhibits G-CSF signaling is administered to a subject, wherein the subject is a tissue or organ transplant recipient. In some examples, a compound that inhibits G-CSF signaling is administered to a tissue or organ transplant recipient prior to reperfusion. In one example, a compound that inhibits G-CSF signaling is administered to a tissue or organ transplant recipient after reperfusion.

[0446] In some examples, a compound that inhibits G-CSF signaling is administered to a tissue or organ transplant donor prior to tissue or organ harvest.

[0447] In some examples, the tissue or organ transplant donor is a living donor. In some examples, the donor is a deceased donor. According to the quality of the organ and / or the order and mechanism of cessation of circulatory and respiratory functions, deceased donors can be divided into several groups. The classification of deceased donors is described in Panduranga & Akinlolu (Clinical Journal of the American Society of Nephrology, 4: 1827-1831, 200 (Clin J Am Soc Nephrol 4: 1827-1831, 200)).

[0448] In some examples, the donor is a brain-dead donation (DBD) donor, also known as a 'brain-dead' donor. A DBD donor is a donor who is primary brain dead, but whose circulatory system remains functional, either naturally or through medical measures (e.g., mechanical ventilation, medications, intra-aortic balloon pumps, or extracorporeal machine oxygenation devices). In some examples, a compound that inhibits G-CSF signaling is administered to a DBD donor prior to organ harvest and is distributed through the blood circulation to potential transplant organs.

[0449] In some examples, the donor is a donation from circulatory death (DCD) donor, also known as a 'donation from cardiac death' donor or a 'no heartbeat donor'. A DCD donor is a donor whose circulatory function has been lost (e.g., cardiac death) before organ harvesting. In some examples, artificial circulation may be required, for example, by a heart-lung bypass apparatus to deliver compounds administered to a DCD donor to an organ intended for harvesting and transplantation. DCD donors can be further divided into other subgroups. In some examples, a DCD donor is a controlled DCD (cDCD) donor. A cDCD donor is a donor whose life support will be withdrawn and whose family has given written consent for organ donation in a controlled environment in an operating room. In some examples, a DCD donor is an uncontrolled DCD (uDCD) donor. A uDCD donor is, for example, a donor who has had a catheter placed in the femoral blood vessels and peritoneum to cool the organ before obtaining consent for organ donation, until the donor expires in the emergency room or elsewhere in the hospital before obtaining consent. A uDCD donor is also a donor who has consented to organ donation but suffers a cardiac arrest requiring CPR during organ procurement.

[0450] In some examples, the donor is an extended criteria donor (ECD). An ECD is a donor who was 60 years of age or older or 50 to 59 years of age at the time of death and had any two of the following three criteria: (1) cause of death was cerebrovascular accident; (2) history of hypertension; (3) terminal serum creatinine greater than 1.5 mg / dL.

[0451] In some examples, the donor is a standard donor (SCD). An SCD is a donor younger than 50 years old. Generally, all deceased donors who donate after brain death and do not meet any of the ECD criteria (see above) are considered SCD.

[0452] In some examples, the compound that inhibits G-CSF signaling is administered shortly after brain death is determined, or as soon as possible after brain death is feasible taking into account legal, ethical and patient considerations. The attending physician or transplant surgeon will recognize the various factors that affect the time to harvest an organ from a suitable donor and transplant the harvested organ into a suitable recipient. Therefore, in some examples, the compound that inhibits G-CSF signaling is administered to the donor at any time between the occurrence of brain death and the removal of the organ for transplantation.

[0453] In some examples, a compound that inhibits G-CSF signaling is administered in vitro to a harvested tissue or organ prior to tissue or organ transplantation. For example, a harvested tissue or organ can be perfused or infused with a solution containing a compound that inhibits G-CSF signaling prior to transplantation.

[0454] Reagent test kit

[0455] Another example of the present disclosure provides a kit containing a compound for treating ischemia-reperfusion injury as described above.

[0456] In one example, the kit includes (a) a container comprising a compound that inhibits G-CSF signaling as described herein, optionally in a pharmaceutically acceptable carrier or diluent; and (b) a package insert having instructions for reducing the effects of ischemia-reperfusion injury in a subject.

[0457] According to this example of the present disclosure, the package insert is on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. These containers can be formed of a variety of materials such as glass or plastic. The container holds or contains a composition that effectively treats ischemia-reperfusion injury and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is a compound that inhibits G-CSF signaling that inhibits G-CSF signaling. The label or package insert indicates that the composition is used to treat a subject suitable for treatment, such as an organ transplant recipient, with specific instructions for the dosage and interval of the compound provided and any other drug. The kit may further include an additional container containing a pharmaceutically acceptable dilution buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and / or dextrose solution. The kit may further include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles and syringes.

[0458] The present disclosure includes the following non-limiting examples.

[0459] Example

[0460] Example 1: Methods and Materials

[0461] Mouse

[0462] Adult male C57BL / 6 wild-type mice aged 10–12 weeks were obtained from the Animal Resource Center, Perth, Australia. Mice were housed in an approved animal facility within St. Vincent's Hospital Melbourne, and all experiments involving animals were approved by the Animal Ethics Committee of St. Vincent's Hospital Melbourne.

[0463] Warm renal ischemia-reperfusion injury model

[0464] Mice were anesthetized by ip administration of 85mg / kg ketamine and 15mg / kg xylazine. After the mice were anesthetized, they were placed on a heating pad to maintain their core body temperature at 37°C during surgery. A midline abdominal incision was made and the renal pedicle was bluntly dissected. After right nephrectomy, a microvascular clamp (Roboz, Rockville, MD) was placed on the left renal pedicle for 22min, while the animals were kept in an incubator at 37°C and fully hydrated. The clamp was removed after ischemia and the kidney was observed to confirm complete reperfusion. The surgical wound was then sutured into two layers with 5-0 silk. During surgery, warm saline (100ml / kg) was instilled into the peritoneal cavity. The mice were allowed to recover for 2h under a heating lamp and then kept on a heating pad. The mice were euthanized 24h after reperfusion, and blood and kidney samples were obtained. Sham-operated mice underwent only right nephrectomy without IR.

[0465] Antibody

[0466] Anti-mouse G-CSFR (Ch5E2-VR81-mIgG1κ), isotype control (BM4, mouse isotype IgG1κ) or anti-C5 antibody (muBB5.1-mIgG1κ) (Rother R, et al. Nat Biotechnol. 2007) were injected intraperitoneally at a dose of 100 μg / mouse 24 h before the start of ischemia. In a dose titration study, anti-mouse G-CSFR (Ch5E2-VR81-mIgG1κ) was injected intraperitoneally 24 h before local ischemia at 200 μg / mouse or 500 μg / mouse. The isotype control was injected at the highest dose, therefore 500 μg / mouse. VR81 is a mouse monoclonal IgG1κ antibody raised against the extracellular domain of mouse G-CSFR, which blocks G-CSF binding to G-CSFR as described (Campbell et al. Journal of Immunology, 197(11)(2016)4392-4402). In this regard, VR81 is a mouse surrogate antibody for C1.2 and C1.2G described herein and in WO2012171057.

[0467] Assessment of renal function

[0468] Renal function was assessed by measuring serum creatinine and serum or plasma urea 24 h after reperfusion. Creatinine was measured using a kinetic colorimetric assay based on the Jaffé method, analyzed on a Roche COBAS Integra 400Plus analyzer. Cell Biolabs' urea assay kit is based on the Berthelot reaction. Urea is first degraded to ammonia and carbon dioxide, which further react with an alkaline developer to produce a blue-green product that can be measured with a standard spectrophotometric plate reader at an optical density between 580-630 nm.

[0469] Renal histology and scoring

[0470] Renal tissue sections (4 mm) were fixed with 10% formalin, embedded in paraffin, and then stained with H&E. Histological scores were assessed using a semiquantitative method (Lu B et al. Nephrology 2008). Briefly, three high-power fields of each of the cortex, cortical-medullary junction, and medulla in a minimum of two sections were evaluated in a blinded manner. The extent of tubular necrosis was graded and a modified scoring system was used: 0 = normal kidney; 1 = minimal (#10% invasion); 2 = mild (10-25% invasion); 3 = moderate (26-50% invasion); 4 = severe (51-75% invasion); and 5 = very severe (75% invasion).

[0471] Complement activation and deposition

[0472] Activation of mouse plasma complement was measured using the Mouse C5a DuoSet ELISA Kit (DY2150; R&D Systems, Minneapolis, MN). C5b-9 deposition in kidney sections was determined by immunofluorescence (see below).

[0473] Immunofluorescence

[0474] The quick-frozen biopsy samples were cut into 5 mm thick slices, dried, and immediately processed or stored at 280 ° C until further analysis. After acetone fixation and hydration, the slices were stained using rabbit anti-mouse C9 Alexa488 (Bioss antibodies, Woburn, MA), rat anti-mouse Ly-6G FITC (Hycult Bio-tech), rat anti-mouse F4 / 80 FITC (Bio-Rad, Raleigh, NC) or FITC-conjugated isotype matching control Ab: rat IgG1 (BD Biosciences, San Diego, CA). Fluorescence / confocal microscope (Nikon A1R) was used to analyze slides. Unprocessed TIFF images were subjected to quantitative analysis of fluorescence intensity (original integrated density) using ImageJ software version 10.2 (National Institutes of Health).

[0475] RNA extraction, complementary DNA synthesis, quantitative real-time polymerase chain reaction

[0476] According to the manufacturer's instructions, total RNA was isolated from frozen mouse kidney using PureLink RNA mini kit (Life Technologies, Carlsbad, CA). Total RNA quality and quantity were determined using NanoDrop spectrophotometer (NanoDrop Technologies, Oxfordshire, UK). First-strand complementary DNA synthesis was performed by incubating 1.0 μg of total RNA at 70 ° C for 10 minutes in a 50-μL reaction mixture containing 0.5 μg Oligo (dT) primer and 1 μg random primer (all from Invitrogen, Carlsbad, CA). Then a 50-μL reaction mixture containing 10 mM dNTP, 300 U SuperScript III recombinant ribonuclease inhibitor, 60 U RNaseOUT recombinant ribonuclease inhibitor, 0.1 M DTT and first-strand buffer (all from Invitrogen) was added. Reverse transcription was performed at 42 ° C for 1 hour and at 70 ° C for 10 minutes. Complementary DNA was stored at -20 ° C. Real-time polymerase chain reaction was performed using the TaqMan Universal PCR Master Mix System according to the manufacturer's instructions (Applied Biosystem, Bedford, MA).

[0477] Kidney digestion and peripheral blood preparation

[0478] With scalpel, 1 / 8 piece of kidney is cut into smaller piece, and it is digested 30 minutes in 500ul collagenase type I (10ug / ml in PBS2mM EDTA) under shaking condition at 37 ℃.The tissue digestion thing is separated cell by 70um filter.Use RCRB buffer (156mM NH4Cl, 11.9mM NaHCO3, 0.097mM EDTA, pH7.3) lysis red blood cell.Finally, cell is resuspended in PBS2mM EDTA.

[0479] Peripheral blood was collected into EDTA-coated tubes, erythrocytes were lysed using RCRB buffer (as described above), and cells were resuspended in PBS 2 mM EDTA.

[0480] Flow cytometry

[0481] Single cell suspensions of kidney and peripheral blood were resuspended in PBS containing 2 mM EDTA. Cells were stained with the following Ab's: PE-conjugated anti-mouse Gr-1 (R86-8C5; BD Biosciences), allophycocyanin Cy7-conjugated anti-mouse Ly6G (1A8; BD Biosciences), FITC-conjugated anti-mouse CD11b (M1 / 70; BD Biosciences), PECy7-conjugated anti-mouse CD69 (H1.2F3; BD Biosciences), eFluor450-conjugated anti-mouse CD62L (MEL-14; eBiosecience), allophycocyanin-conjugated anti-mouse CD45.2 (104; eBioseciences). FcR blocking anti-mouse CD16 / 32 (93; Biolegend) was added to reduce nonspecific binding, and dead cells were excluded using fixable live / dead yellow (ThermoFisher Scientific). Samples were fixed overnight using BD Cytofix fixation buffer (BD Biosciences) and analyzed on a BD Fortessa using FlowJo software.

[0482] Statistical analysis

[0483] Statistical tests were performed using GraphPad Prism software. Multiple groups were compared using one-way ANOVA with post-test Bonferroni correction or Kruskal-Wallis test with Dunn's multiple comparison test. Two groups were compared using unpaired Student t test (two-tailed) or Mann-Whitney U test. Results are expressed as mean ± SEM. p values ​​< 0.05 were considered statistically significant.

[0484] Example 2: Inhibition of G-CSF signaling reduces serum creatinine and plasma urea concentrations in a warm renal IRI model

[0485] To evaluate whether inhibition of G-CSF signaling reduces renal injury in mice subjected to warm renal ischemia-reperfusion injury (IRI), analysis of serum creatinine and plasma / serum urea concentrations was performed.

[0486] A significant decrease in serum creatinine concentration was observed in mice treated with 100 μg of anti-G-CSFR antibody (VR81) (n=8; mean±SEM; 66.13±25.18) compared to serum creatinine concentration in mice treated with 100 μg of isotype control antibody (n=8; mean±SEM; 134.38±20.57). Figure 1 , Table 1).

[0487] A significant decrease in plasma urea concentration was observed in mice treated with anti-G-CSFR antibody (VR81) (n=8; mean±SEM; 159.02±44.42) compared to isotype control antibody (n=8; mean±SEM; 292.49±21.35). Figure 2 , Table 1). The concentrations of serum creatinine and plasma urea in mice treated with anti-G-CSFR antibody (VR81) before IRI were comparable to those observed in mice that were not treated or operated on (sham) ( Figure 1 and 2 ; Table 1). In addition, the concentrations of serum creatinine and plasma urea in mice treated with VR81 were not statistically different from those observed in mice treated with anti-C5 antibody (BB5.1) ( Fig. 9 and 10 ).

[0488] Table 1. Serum creatinine and plasma urea levels in mice treated with 100 μg of anti-G-CSFR antibody (VR81) or isotype control antibody via the intraperitoneal (IP)-thermal renal IRI model.

[0489]

[0490] Example 3: Inhibition of G-CSF signaling reduces inflammatory infiltration in renal tissue in a hot renal IRI model

[0491] To evaluate the effect of administration of anti-G-CSFR antibody (VR81) to mice subjected to warm renal ischemia-reperfusion injury (IRI), the infiltration of neutrophils and macrophages into renal tissues was measured by immunofluorescence microscopy according to Example 1.

[0492] Mice treated with 100 μg of anti-G-CSFR antibody (VR81) had significantly reduced infiltration of neutrophils and macrophages within the tubular interstitium at the corticomedullary junction of the kidney compared to mice treated with 100 μg of isotype control antibody. Little / no infiltration was observed in sham-operated mice ( Figure 3 and Figure 4 ). One-way ANOVA (with Bonferroni correction) was used to test significance.

[0493] In separate experiments, the infiltration of neutrophils and macrophages was also significantly reduced in mice treated with 200 μg and 500 μg of VR81 compared with mice treated with 500 μg of isotype control antibody (Tables 2 and Figure 5 ).

[0494] Table 2 Reduction of neutrophil and macrophage infiltration in tubular interstitium at the corticomedullary junction of the kidney D'Agostino & Pearson Composite Normal Test. One-way ANOVA Kruskal-Wallis test for VR81 vs. Ab control (*p<0.05, **p<0.01), Dunn's multiple comparison test.

[0495]

[0496] Example 4: Inhibition of G-CSF signaling reduces necrosis in the renal cortex in a thermal renal IRI model

[0497] Renal injury in the renal cortex was assessed in a warm renal IRI model of mice treated with 100 μg of anti-G-CSFR antibody (VR81). The extent of tubular necrosis was graded and expressed as tubular injury (TI) score according to Table 3. TI was determined by two independent operators blinded to the treatment group, assessing the percentage of tubular injury invasion (necrosis, cast formation, cell swelling and dilation).

[0498] A trend toward reduced tubular damage was observed in the kidneys of mice treated with anti-G-CSFR antibody (VR81) ( Figure 6 ; Shown are the means of two independent assessments per mouse). As expected, sham-operated mice showed no tubular damage.

[0499] Table 3: Reference table defining the percentage of necrotic cells in the renal cortex and the corresponding tubular injury (TI) score.

[0500] Tubular injury (TI) score % Tubular necrosis (cortex only) 0 normal 1 <10 2 10-25 3 26-50 4 51-75 5 >75

[0501] Example 5: KIM-1, NGAL, IL-6, IL-8Rβ / CXCR2, MCP-1 / CCL2, CXCL1, MIP-2 / CXCL2, ICAM-1 and C5aR mRNA expression in the renal tissue thermal renal IRI model

[0502] In the initial experiments, mice were injected with 100 μg / mouse of anti-G-CSFR antibody VR81 24 h before induction of ischemia / reperfusion injury in the warm renal IRI model. After reperfusion, RNA was isolated from the kidneys and qRT-PCR was performed as described in Example 1. The mRNA transcript levels of KIM-1, NGAL, IL-6, IL-8Rβ / CXCR2, MCP-1 / CCL2, ICAM-1, C5aR, IL-1β, TNFα, CXCL1, MIP-2 / CXCL2, and E-selectin were determined. The transcript levels of these genes were quantified and normalized to GAPDH.

[0503] Compared with treatment with isotype control antibody, treatment with anti-G-CSFR antibody (VR81) at a dose of 100 μg / mouse for 24h reduced gene expression of NGAL, IL-6, IL-8Rβ / CXCR2, ICAM-1, C5aR, MIP-2 / CXCL2, MCP-1 / CCL2, E-selectin, and CXCL1. These data indicate that inhibition of G-CSF signaling is effective in reducing the inflammatory response associated with IRI. A similar pattern of reduced expression of the above genes was observed in the kidneys of mice treated with anti-C5 antibody (BB5.1).

[0504] Initial results were confirmed using doses of 200 μg / mouse or 500 μg / mouse VR81. Table 4 and Figure 7 show that treatment with VR81 at a dose of 500 μg / mouse significantly reduced the expression of KIM-1, NGAL, IL-6, IL-8Rβ / CXCR2, ICAM-1, C5aR, MIP-2 / CXCL2, MCP-1 / CCL2, and CXCL1 compared to treatment with an isotype control antibody.

[0505]

[0506] Example 6: Inhibition of G-CSF signaling reduces neutrophil infiltration into the kidney in a hot renal IRI model According to the method described in Example 1, renal neutrophils (CD11b + Gr1 + ) and leukocytes (CD11b - Gr1 - CD45.2 + ). The percentage of live cells was determined based on PI staining of the total cell population.

[0507] Compared with mice treated with isotype control antibody, reduced neutrophil numbers were observed in the kidneys of mice treated with 100 μg / mouse of anti-GCSFR antibody (VR81) or anti-C5 antibody (BB5.1) (and sham-treated mice). Figure 8 A). The number of peripheral blood neutrophils was similar in all experimental groups ( Figure 8 B).

[0508] Similarly, a significant decrease in the number of neutrophils observed in the kidneys of mice treated with 200 μg / mouse or 500 μg / mouse VR81 was observed compared to mice treated with an isotype control antibody ( Fig.13 A). However, no significant reduction in neutrophils was observed in peripheral blood relative to the isotype control antibody ( Fig.13 B).

[0509] These data indicate that treatment with anti-G-CSFR antibody (VR81) reduces neutrophil infiltration into the kidney and does not significantly affect the number of neutrophils in peripheral blood.

[0510] No significant differences were observed in the expression of neutrophil CD69 or CD62L in the kidney or peripheral blood of mice in all experimental groups.

[0511] No increase in leukocyte percentage (CD11b - Gr1 - CD45.2 + ) significantly different.

[0512] Example 7: VR81 Dose Titration in the Warm Renal IRI Model

[0513] In the dose titration study in the warm renal IRI model described in Example 1, anti-mouse G-CSFR antibody (VR81) was injected at a dose of 200 μg / mouse or 500 μg / mouse 24 h before ischemia-reperfusion injury. Mice treated with 500 μg / mouse of isotype control antibody were used as controls.

[0514] As shown in Table 5 and Fig.11 and 12 As shown, serum creatinine and serum urea levels were significantly reduced in mice treated with 500 μg VR81, but not in mice treated with 200 μg VR81 or 500 μg isotype control antibody.

[0515] Table 5 Serum creatinine and serum urea levels in mice given 200 μg and 500 μg VR81 / mouse. D'Agostino & Pearson Omnibus Normal Test. One-way ANOVA VR81 vs. Ab Control (*p<0.05, **p<0.01), Dunn's Multiple Comparison Test.

[0516] Creatinine (μM) Urea (mg / dL) Control (500ug) 127.3±19.5 507.7±75.2 VR81(200ug) 116.0±38.5 344±94.2 VR81(500ug) 22.4±1.0* 84.42±3.27**

[0517] Example 8: Inhibition of G-CSF signaling reduces complement C5 activation and C9 deposition in a hot renal IRI model

[0518] To evaluate the effect of administering anti-G-CSFR antibody (VR81) to mice subjected to warm renal ischemia-reperfusion injury (IRI), complement C5 activation was measured by analyzing C5a in plasma using a mouse C5a ELISA kit, and C5b-9 deposition was measured by immunofluorescence staining of kidney sections according to Example 1.

[0519] As shown in Table 6 and Fig.14 As shown in A, plasma C5a levels were significantly reduced in mice treated with 500 μg VR81 compared with mice treated with 500 μg isotype control antibody.

[0520] As shown in Table 6 and Fig.14 As shown in B, mice treated with 500 μg VR81 also had a significant reduction in C5B-9 deposition in the kidneys compared to mice treated with 500 μg isotype control antibody.

[0521] Table 6 Complement C5 activation and C5b-9 deposition in mice treated with 200 μg or 500 μg VR81 (*p<0.05, **p<0.02, ***p<0.001 vs. Ab control)

[0522]

[0523] Example 9: Administration of a G-CSF signaling inhibitor 1 h before ischemia reduces the frequency of neutrophils in the kidney and blood in the warm renal IRI model

[0524] To evaluate the effect of anti-G-CSFR antibody (VR81) administered to mice 1 hour before ischemia in the warm renal IRI model, the frequency of neutrophils (CD11b+Grl+Ly6G+) in the kidney and blood was measured by tissue digestion and flow cytometry according to the method described in Example 1. 500 μg / mouse anti-C5 antibody, BB5.1, was used for comparison.

[0525] Compared with treatment with isotype control antibody, treatment with 500 μg / mouse anti-G-CSFR antibody VR81 1 h before ischemia significantly reduced the frequency of neutrophils in blood and kidneys ( Fig.15 Treatment with 500 μg / mouse of control antibody BB5.1 did not significantly reduce the frequency of neutrophils in the kidney compared with treatment with isotype control antibody. These results demonstrate that treatment with G-CSF signaling inhibitors 1 h before ischemia is less effective than treatment 24 h before ischemia ( Fig.13 ), but was sufficient to significantly reduce neutrophil counts in the blood and kidneys.

Claims

1. A method for preventing or treating ischemia-reperfusion injury in a subject, the method comprising administering a compound that inhibits granulocyte colony stimulating factor (G-CSF) signaling.

2. The method of claim 1, wherein the ischemia-reperfusion injury is due to or associated with tissue or organ transplantation.

3. The method of claim 2, wherein the organ transplant is a kidney transplant.

4. The method of claim 2 or 3, wherein the compound that inhibits G-CSF signaling is administered to the subject, wherein the subject is a tissue or organ transplant recipient.

5. The method of any one of claims 2 to 4, wherein the compound that inhibits G-CSF signaling is administered to a tissue or organ transplant donor prior to organ harvesting and / or to a tissue or organ prior to transplantation.

6. A method for tissue or organ transplantation or for improving the outcome of tissue or organ transplantation or improving the function of a transplanted tissue or organ or for preventing delayed transplant function, the method comprising administering a compound that inhibits G-CSF signaling to a tissue or organ transplant recipient before or at the time of transplanting the tissue or organ, and then transplanting the tissue or organ into the tissue or organ transplant recipient.

7. A method of tissue or organ transplantation or for improving the outcome of tissue or organ transplantation or improving the function of a transplanted tissue or organ or for preventing delayed transplant function, the method comprising administering a compound that inhibits G-CSF signaling to a tissue or organ transplant donor prior to collecting the tissue or organ; collecting the tissue or organ and transplanting the tissue or organ into a tissue or organ transplant recipient.

8. A method of tissue or organ transplantation or for improving the outcome of tissue or organ transplantation or improving the function of transplanted tissue or organ or for preventing delayed transplant function, the method comprising administering a compound that inhibits G-CSF signaling to harvested tissue or organ in vitro, and transplanting the harvested tissue or organ into a tissue or organ transplant recipient.

9. The method of any one of claims 1 to 8, wherein the compound that inhibits G-CSF signaling is administered prior to ischemia or reperfusion.

10. The method of claim 9, wherein the compound that inhibits G-CSF signaling is administered 0-48 hours prior to ischemia or reperfusion.

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