Compositions and methods for treating macrophage activation syndrome

Abstract

The present disclosure provides compositions and methods for treating and preventing macrophage activation syndrome (MAS) or hemophagocytic lymphohistiocytosis (HLH). The methods involve administering to a patient in need thereof an antibody or fragment thereof having specificity for the human GM-CSF protein. The methods involve administering to a patient in need thereof an antibody or fragment thereof having specificity for the human GM-CSF protein, e.g., IMH001. Treatment using anti-GM-CSF antibodies according to the present disclosure has shown effective effects in slowing disease progression.

Description

[Background technology]

[0001] Hemophagocytic lymphohistiocytosis (HLH) is a fatal autosomal recessive disorder that develops in childhood. It is characterized by fever, hepatosplenomegaly, cytopenias, and widespread infiltration of vital organs by activated lymphocytes and macrophages. Macrophage activation syndrome (MAS), also known as secondary HLH, is a rare, severe, and life-threatening disorder. MAS is considered a hyperinflammatory "cytokine storm" state associated with rheumatic diseases or other triggers. The exact mechanism of MAS remains unclear. However, regardless of the initiating events in these diseases, the final pathology is defined by an overactive immune system that leads to chronic inflammation and hemophagocytosis. MAS is characterized by fever, hyperferritinemia, hemophagocytosis, cytopenias (including pancytopenia), and hepatosplenomegaly.

[0002] Conventional treatments for HLH / MAS rely on multiple immunosuppressive schemes using dexamethasone or other steroids, cyclosporine, and etoposide. These therapies themselves are toxic and increase the mortality rate of patients with the disease. Therefore, patients with HLH / MAS are in dire need of newer, safer therapies. Summary of the Invention

[0003] The present disclosure provides compositions and methods for treating and preventing hemophagocytic lymphohistiocytosis (HLH) or macrophage activation syndrome (MAS). The methods involve administering to a patient in need thereof an antibody or fragment thereof having specificity for the human GM-CSF protein, e.g., IMH001. Treatment with anti-GM-CSF antibodies according to the present disclosure has shown effective effects in slowing disease progression.

[0004] One embodiment of the present disclosure provides a method for treating or preventing macrophage activation syndrome (MAS) or hemophagocytic lymphohistiocytosis (HLH) in a patient in need thereof, the method comprising administering to the patient an antibody or fragment thereof having specificity for human GM-CSF protein.

[0005] One embodiment of the present disclosure provides an antibody or fragment thereof having specificity for human GM-CSF protein for use in treating or preventing macrophage activation syndrome (MAS) or hemophagocytic lymphohistiocytosis (HLH) in a patient in need thereof.

[0006] One embodiment of the present disclosure provides the use of an antibody or fragment thereof having specificity for human GM-CSF protein in the manufacture of a medicament for treating or preventing macrophage activation syndrome (MAS) or hemophagocytic lymphohistiocytosis (HLH) in a patient in need thereof.

[0007] In some embodiments, the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 1 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the antibody comprises a human IgG1 Fc.

[0008] In some embodiments, the patient has a disease or condition selected from the group consisting of systemic onset juvenile idiopathic arthritis (SoJIA), systemic lupus erythematosus (SLE), Kawasaki disease, and adult-onset Still's disease.

[0009] In some embodiments, the patient has a blood ferritin level of at least 300 ng / ml, or at least 350 ng / ml, 400 ng / ml, 450 ng / ml, 500 ng / ml, 550 ng / ml, 600 ng / ml, 650 ng / ml, 684 ng / ml, 700 ng / ml, 720 ng / ml, 740 ng / ml, 750 ng / ml, 760 ng / ml, 784 ng / ml, 800 ng / ml, 820 ng / ml, 840 ng / ml, 850 ng / ml, 860 ng / ml, 884 ng / ml, 900 ng / ml, 950 ng / ml, or 1000 ng / ml.

[0010] In some embodiments, the patient has a hemoglobin level less than 11 g / dL, or less than 10.5 g / dL, 10 g / dL, 9.5 g / dL, 9 g / dL, 8.5 g / dL, 8 g / dL, 7.5 g / dL, or 7 g / dL.

[0011] In some embodiments, the patient has a platelet count of 140×10 9 / L or lower than 130 x 10 9 / L, 120 x 10 9 / L, 110 x 10 9 / L, 100 x 10 9 / L, 90 x 10 9 / L, 80 x 10 9 / L, 70 x 10 9 / L or 60 x 10 9 / L lower.

[0012] In some embodiments, the patient has a neutrophil count of 1.4 x 10 9 / L or less than 1.3 × 10 9 / L, 1.2 × 10 9 / L, 1.1 × 10 9 / L, 1.0 × 10 9 / L, 0.9 × 10 9 / L, 0.8 × 10 9 / L, 0.7 × 10 9 / L or 0.6 x 10 9 / L lower.

[0013] In some embodiments, the patient has a fasting triglyceride level greater than 2.49 mmol / L, or greater than 2.55 mmol / L, 2.6 mmol / L, 2.66 mmol / L, 2.72 mmol / L, 2.77 mmol / L, 2.83 mmol / L, 2.89 mmol / L, 2.94 mmol / L, 3 mmol / L, 3.06 mmol / L, 3.11 mmol / L, 3.17 mmol / L, 3.23 mmol / L, 3.28 mmol / L, 3.34 mmol / L, or 3.4 mmol / L.

[0014] In some embodiments, the patient has a blood fibrinogen level less than 1.5 g / L, or less than 1.4 g / L, 1.3 g / L, 1.2 g / L, 1.1 g / L, 1.0 g / L, 0.9 g / L, 0.8 g / L, 0.7 g / L, 0.6 g / L, or 0.5 g / L.

[0015] In some embodiments, the patient has elevated levels of soluble CD163 compared to healthy individuals. In some embodiments, the patient has elevated levels of soluble IL-2 receptor compared to healthy individuals.

[0016] In some embodiments, the antibody or antigen-binding fragment thereof is administered at 0.3 mg / kg to 10 mg / kg. In some embodiments, the antibody or antigen-binding fragment thereof is administered once a week, once every two weeks, or once a month. In some embodiments, the antibody or antigen-binding fragment thereof is administered intravenously or subcutaneously.

[0017] In some embodiments, the antibody is provided in a formulation comprising 20 mg / ml to 200 mg / ml of antibody, 10 mM to 30 mM of buffer, 130 mM to 250 mM of tonicity adjusting agent, and 0.01% (w / v) to 0.03% (w / v) of surfactant, wherein the formulation has a pH of 4.5 to 7.5.

[0018] In some embodiments, the formulation comprises 50-150 mg / ml antibody, 10-20 mM histidine, 200-220 mM sucrose, and 0.01-0.03% (w / v) polysorbate 80, and has a pH of about 5.5-6.1.

[0019] In some examples, the formulation comprises about 100 mg / ml antibody, about 20 mM histidine, about 220 mM sucrose, and about 0.02% (w / v) polysorbate 80, and has a pH of about 5.8.

[0020] In some embodiments, the method further comprises administering to the subject a glucocorticoid, cyclosporine, or anakinra. In some embodiments, the glucocorticoid is selected from the group consisting of cortisol, cortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, deflazacort, fludrocortisone acetate, deoxycorticosterone acetate, aldosterone, and beclometasone. [Brief explanation of the drawings]

[0021] [Figure 1] The weight change in the treatment groups is shown. [Figure 2] Changes in RBC (Panel A), HGB (Panel B), NEUT% (Panel C), and MONO% (Panel D) in the treatment groups are shown. [Figure 3] Total bone marrow viable cell changes in treatment groups are shown. [Figure 4]Fibrinogen (Panel A), triglyceride (Panel B), and ferritin (Panel C) levels for treatment groups are shown. [Figure 5] Histopathological evaluation of hemophagocytosis (Panel A), inflammatory infiltrate (Panel B), and lymphocyte exhaustion (Panel C) in treatment groups is shown. DETAILED DESCRIPTION OF THE INVENTION

[0022] Before describing the present disclosure in detail, it should be understood that the present disclosure is not limited to the particular compositions or biological systems described, as these can, of course, vary. It should be further understood that the terminology used herein is for the purpose of describing particular examples only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a / an" and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to a "molecule" optionally includes combinations of two or more such molecules, and the like.

[0023] As used herein, the term "about" refers to a normal error range for the relevant numerical value, which is readily known to one of ordinary skill in the art. As used herein, reference to "about" a value or parameter includes (and describes) examples of the value or parameter itself. In cases of doubt, or in the absence of a commonly accepted understanding in the art of the error range for a value or parameter, "about" means ±1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the value or parameter.

[0024] It should be understood that aspects and embodiments of the present disclosure described herein include "comprising aspects and embodiments," "consisting of aspects and embodiments," and "consisting essentially of aspects and embodiments."

[0025] As used herein, the term "antibody" generally refers to immunoglobulin molecules and multimers thereof (e.g., IgM) comprising four polypeptide chains (two heavy (H) chains and two light (L) chains linked together by disulfide bonds); however, immunoglobulin molecules consisting only of heavy chains (i.e., lacking light chains) are also included within the definition of the term "antibody." Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions may be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0026] Unless otherwise specified, as used herein, the term "antibody" should be understood to cover intact antibody molecules and antigen-binding fragments thereof. As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or abbreviated as "antibody portion" or "antibody fragment") refers to one or more fragments of an antibody, e.g., F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc., that retain the ability to specifically bind to human GM-CSF or an epitope thereof.

[0027] The term "specifically binds" and the like means that an antibody or an antigen-binding fragment thereof and an antigen form a relatively stable complex under physiological conditions. Specific binding is at least about 1 × 10 -8The specific binding may be characterized by a dissociation constant M or greater. Methods for determining whether two molecules specifically bind are known in the art, and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. However, an isolated antibody that specifically binds human GM-CSF may have cross-reactivity with other antigens, such as GM-CSF (orthologs) from other species. In the context of the present disclosure, a multispecific (e.g., bispecific) antibody that binds human GM-CSF and one or more additional antigens is considered to "specifically bind" human GM-CSF. And, an isolated antibody may be substantially free of other cellular material or chemicals.

[0028] The term "drug formulation" refers to a formulation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain other ingredients that are unacceptably toxic to the subject to which the formulation is administered. Such formulations are sterile.

[0029] As used herein, the term "treat" or "treatment" refers to both therapeutic treatment and preventative or prophylactic measures, where the objective is to prevent or alleviate (reduce) the progression of a physiological change or disorder, such as cancer. Beneficial or desired clinical results include alleviation of symptoms, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), delaying or slowing of disease progression, improvement or palliation of the disease state, and remission (whether partial or complete remission), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to the condition or disorder, or those in whom the condition or disorder is to be prevented.

[0030] As used herein, the terms "prevention" or "prophylaxis" refer to prophylactic or protective treatment against a disease or disease state. Prevention of a disease or disease state can include, for example, a decrease (e.g., alleviation) of one or more symptoms of the disease or disease state compared to a reference level (e.g., a symptom in a similar subject not receiving the treatment). Prevention can further include a delay in the onset of one or more symptoms of the disease or disease state compared to a reference level (e.g., the onset of one or more symptoms in a similar subject not receiving the treatment). In an example, the disease is a disease described herein.

[0031] "Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject in need of diagnosis, prognosis, or therapy, particularly mammalian subjects. Mammalian subjects include humans, domestic, farm, and zoo animals, sport or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, cows, etc. Treatment of macrophage activation syndrome (MAS) or hemophagocytic lymphohistiocytosis (HLH)

[0032] The present inventors tested the anti-GM-CSF antibody IMH001 in animal models of macrophage activation syndrome (MAS) or hemophagocytic lymphohistiocytosis (HLH). Animals with MAS are immunocompromised and express human GM-CSF and IL-3 cytokines. The animals showed progressive disease with many of the hallmarks of MAS, rapidly developing progressive anemia and weight loss.

[0033] When treated with IMH001, animals showed reduced weight loss (and therefore weight gain), improved anemia (improved red blood cell count, hemoglobin, and hematocrit), and inhibited ferritin increase compared to controls. Thus, such in vivo data demonstrated the efficacy of IMH001 in treating MAS or HLH.

[0034] Therefore, according to one embodiment of the present technology, a method for treating or preventing macrophage activation syndrome (MAS) or hemophagocytic lymphohistiocytosis (HLH) is provided. In some embodiments, the method involves administering an anti-GM-CSF antibody or antigen-binding fragment to a patient in need thereof. As used herein, the term "GM-CSF" refers to human granulocyte-macrophage colony-stimulating factor. GM-CSF, also known as colony-stimulating factor 2 (CSF2), is a monomeric glycoprotein secreted by macrophages, T cells, mast cells, NK cells, endothelial cells, and fibroblasts, where it functions as a cytokine. Naturally occurring drug analogs of GM-CSF are also called sargramostim and molgramostim. Antibodies against human GM-CSF are described, for example, in WO2006122797, WO2015028657, and WO2018050111.

[0035] In some examples, the anti-GM-CSF antibody is IMH001, which is a humanized IgG1 antibody that comprises a heavy chain variable region (VH) of SEQ ID NO:1 and a light chain variable region (VL) of SEQ ID NO:2.

[0036] In some embodiments, the patient has MAS or HLH. In some embodiments, the patient is at risk for MAS or HLH. In some embodiments, the HLH / MAS patient or at risk for HLH / MAS has a disease or condition, such as systemic onset juvenile idiopathic arthritis (SoJIA), systemic lupus erythematosus (SLE), Kawasaki disease, or adult-onset Still's disease.

[0037] "Systemic-onset juvenile idiopathic arthritis (SoJIA)" (or juvenile-onset form of Still's disease) is a type of juvenile idiopathic arthritis (JIA) that, in addition to arthritis, also has extra-articular symptoms such as fever and rash. It was originally called systemic juvenile rheumatoid arthritis or Still's disease. The predominant symptoms are extra-articular, such as high fever, rheumatic rash, hepatosplenomegaly, lymphadenopathy, and anemia. Other symptoms include inflammation of the pleura, pericardium, and cardiac muscle tissue, and peritoneal inflammation is also seen.

[0038] Systemic lupus erythematosus (SLE) is an autoimmune disease in which the body's immune system mistakenly attacks healthy tissue in many parts of the body. Symptoms vary from patient to patient and range from mild to severe. Common symptoms include joint pain and swelling, fever, chest pain, hair loss, mouth sores, swollen lymph nodes, fatigue, and a red rash, most commonly on the face. There are generally periods of disease called flares, and periods of remission when symptoms are very minimal.

[0039] Kawasaki disease is a syndrome of unknown cause that causes fever and affects children under the age of five. It is a form of vasculitis, in which blood vessels throughout the body become inflamed. The fever usually lasts for more than five days, and conventional medications do not work. Other common symptoms include enlarged lymph nodes in the neck, a rash in the genital area, lips, palms, or soles, and redness of the eyes. Within three weeks of onset, the skin on the hands and feet peels, usually followed by recovery. In some children, coronary artery aneurysms form in the heart.

[0040] Adult-onset Still's disease (AOSD) is a rare systemic autoinflammatory disorder characterized by the classic triad of fever, joint pain, and a pronounced salmon-pink papular rash. It is considered a diagnosis of exclusion. Levels of the iron-binding protein ferritin can be dramatically elevated in such disorders.

[0041] In some embodiments, the patient has abnormally high blood ferritin levels, ie, at least 300 ng / ml, or at least 350 ng / ml, 400 ng / ml, 450 ng / ml, 500 ng / ml, 550 ng / ml, 600 ng / ml, 650 ng / ml, 684 ng / ml, 700 ng / ml, 720 ng / ml, 740 ng / ml, 750 ng / ml, 760 ng / ml, 784 ng / ml, 800 ng / ml, 820 ng / ml, 840 ng / ml, 850 ng / ml, 860 ng / ml, 884 ng / ml, 900 ng / ml, 950 ng / ml, or 1000 ng / ml.

[0042] In some embodiments, administration of an anti-GM-CSF antibody provided herein reduces blood ferritin levels by at least 10%, at least 20%, at least 30%, at least 50%, or at least 70% compared to untreated controls. In some embodiments, administration of an anti-GM-CSF antibody provided herein reverses abnormally high blood ferritin levels in a patient.

[0043] In some embodiments, the patient has an abnormally low hemoglobin level, ie, less than 11 g / dL, or less than 10.5 g / dL, 10 g / dL, 9.5 g / dL, 9 g / dL, 8.5 g / dL, 8 g / dL, 7.5 g / dL, or 7 g / dL.

[0044] In some embodiments, administration of an anti-GM-CSF antibody provided herein increases blood hemoglobin levels by at least 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, or 8 times compared to an untreated control. In some embodiments, administration of an anti-GM-CSF antibody provided herein reverses abnormally low hemoglobin levels in a patient.

[0045] In some embodiments, the patient has an abnormally low platelet count. In some embodiments, the platelet count is 140×10 9 / L or lower than 130 x 10 9 / L, 120 x 10 9 / L, 110 x 10 9 / L, 100 x 10 9 / L, 90 x 10 9 / L, 80 x 10 9 / L, 70 x 10 9 / L or 60 x 10 9 / L lower.

[0046] In some embodiments, administering an anti-GM-CSF antibody provided herein increases platelet counts by at least 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, or 8 times compared to an untreated control. In some embodiments, administering an anti-GM-CSF antibody provided herein reverses abnormally low platelet counts in a patient.

[0047] In some embodiments, the patient has an abnormally low neutrophil count. In some embodiments, the neutrophil count is 1.4 x 10 9 / L or less than 1.3 × 10 9 / L, 1.2 × 10 9 / L, 1.1 × 10 9 / L, 1.0 × 10 9 / L, 0.9 × 10 9 / L, 0.8 × 10 9 / L, 0.7 × 10 9 / L or 0.6 x 10 9 / L lower.

[0048] In some embodiments, administration of an anti-GM-CSF antibody provided herein increases neutrophil counts by at least 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, or 8 times compared to an untreated control. In some embodiments, administration of an anti-GM-CSF antibody provided herein reverses abnormally low neutrophil counts in a patient.

[0049] In some embodiments, the patient has abnormally high fasting triglyceride levels, ie, greater than 2.49 mmol / L, or greater than 2.55 mmol / L, 2.6 mmol / L, 2.66 mmol / L, 2.72 mmol / L, 2.77 mmol / L, 2.83 mmol / L, 2.89 mmol / L, 2.94 mmol / L, 3 mmol / L, 3.06 mmol / L, 3.11 mmol / L, 3.17 mmol / L, 3.23 mmol / L, 3.28 mmol / L, 3.34 mmol / L, or 3.4 mmol / L. In some embodiments, fasting triglyceride levels are greater than 220 mg / dl, or greater than 225 mg / dl, 230 mg / dl, 235 mg / dl, 240 mg / dl, 245 mg / dl, 250 mg / dl, 255 mg / dl, 260 mg / dl, 265 mg / dl, 270 mg / dl, 275 mg / dl, 280 mg / dl, 285 mg / dl, 290 mg / dl, 295 mg / dl, or 300 mg / dl.

[0050] In some embodiments, administration of an anti-GM-CSF antibody provided herein reduces fasting triglyceride levels by at least 10%, at least 20%, at least 30%, at least 50%, or at least 70% compared to untreated controls, hi some embodiments, administration of an anti-GM-CSF antibody provided herein reverses abnormally high fasting triglyceride levels in a patient.

[0051] In some embodiments, the patient has abnormally low blood fibrinogen levels, ie, blood fibrinogen levels below 1.5 g / L, or below 1.4 g / L, 1.3 g / L, 1.2 g / L, 1.1 g / L, 1.0 g / L, 0.9 g / L, 0.8 g / L, 0.7 g / L, 0.6 g / L, or 0.5 g / L.

[0052] In some embodiments, administering an anti-GM-CSF antibody provided herein increases blood fibrinogen levels by at least 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, or 8 fold. In some embodiments, administering an anti-GM-CSF antibody provided herein reverses abnormally low blood fibrinogen levels in a patient.

[0053] In some embodiments, administration of an anti-GM-CSF antibody according to the present disclosure reduces or reverses elevated levels of hemophagocytosis in a patient.

[0054] In some embodiments, administration of an anti-GM-CSF antibody according to the present disclosure reduces or reverses high levels of inflammatory infiltrate in a patient.

[0055] In some embodiments, administration of an anti-GM-CSF antibody according to the present disclosure reduces or reverses elevated lymphocyte exhaustion in a patient.

[0056] In some embodiments, administration of an anti-GM-CSF antibody provided herein increases live bone marrow cells.

[0057] In some embodiments, the patient has macrophage activation. In some embodiments, the patient has elevated soluble CD163 levels compared to a healthy individual. In some embodiments, the patient has lymphocyte activation. In some embodiments, the patient has elevated soluble IL-2 receptor levels compared to a healthy individual.

[0058] In some embodiments, the patient meets two or more of the above criteria, hi some embodiments, the patient meets three, four, five or more of the above criteria.

[0059] The amount of an antibody of the present disclosure effective in treatment. The precise dose to be employed in the formulation will further depend on the route of administration and the severity of the disease, disorder, or condition, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0060] As a general guide, the dose of an antigen-binding polypeptide of the present disclosure administered to a patient is generally between 0.1 mg / kg and 100 mg / kg of patient body weight, between 0.1 mg / kg and 20 mg / kg of patient body weight, or between 1 mg / kg and 10 mg / kg of patient body weight. Due to the immune response to foreign polypeptides, human antibodies generally have a longer half-life in the human body than antibodies from other species. Therefore, the dose of human antibodies may generally be reduced and the frequency of administration may be reduced. Furthermore, the dosage and frequency of administration of antibodies of the present disclosure can be reduced by enhancing antibody uptake and tissue penetration (e.g., brain entry) through modifications (e.g., lipidation).

[0061] In some embodiments, the dose of IMH001 is 0.1 to 25 mg / kg, 0.3 to 20 mg / kg, 0.3 to 15 mg / kg, 0.3 to 10 mg / kg, 0.5 to 20 mg / kg, 1 to 20 mg / kg, 5 to 20 mg / kg, 6 to 20 mg / kg, 6 to 10 mg / kg, or 5 to 10 mg / kg per administration. In some embodiments, the dose is at least 0.3 mg / kg, or at least 0.6 mg / kg, 1 mg / kg, 1.3 mg / kg, 1.6 mg / kg, 2 mg / kg, 2.3 mg / kg, 2.6 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg. In some embodiments, the dosage is 15 mg / kg, 14 mg / kg, 13 mg / kg, 12 mg / kg, 11 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, or 2 mg / kg or less. In some embodiments, the dosage administered is 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 12 mg / kg, 15 mg / kg, 20 mg / kg, or 25 mg / kg.

[0062] In some embodiments, administration occurs once a day, once every two days, once every three days, once every four days, once a week, twice a week, once every two weeks, once every three weeks, once a month, or once every two months.

[0063] Methods of administering antibodies include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The antibodies or compositions can be administered by any convenient route, for example, by infusion or bolus injection, for absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other bioactive agents. Thus, pharmaceutical compositions containing antibodies of the present disclosure can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (e.g., by powder, ointment, drops, or transdermal patch), bucally, or as an oral or nasal spray.

[0064] As used herein, the term "parenteral" refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0065] Administration may be systemic or local. Antibodies of the present disclosure are desirably introduced into the central nervous system by any suitable route, including intraventricular and intrathecal injection, which may be facilitated, for example, by an intraventricular catheter attached to a reservoir (e.g., an Ommaya reservoir). Pulmonary administration may also be employed, for example, by use of an inhaler or nebulizer, and formulation with an aerosol.

[0066] Desirably, the antibodies or compositions of the present disclosure are administered locally to the area in need of treatment, for example, but not limited to, by local infusion during surgery, topical application (e.g., in combination with a wound dressing after surgery), injection, catheter, suppository, or implant, which may be made of a porous, non-porous, or gelatinous material, including a membrane such as a silicone sialastic membrane or fiber. Preferably, when administering a protein (including an antibody) of the present disclosure, care must be taken to use a material to which the protein does not absorb.

[0067] In another embodiment, the antibody or composition may be delivered in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533; Treat et al., Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; ibid. throughout).

[0068] In yet another embodiment, the antibody or composition may be delivered in a controlled release system. In one embodiment, a pump may be used (see Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, a polymeric material may be used (Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, FL (1974); Controlled Drug Bioavailability, Drug Product Design and See, Controlled Drug Bioavailability, Pharmaceutical Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61; also see, Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105. In yet another embodiment, a controlled-release system may be placed in the vicinity of the therapeutic target (i.e., the brain), thereby requiring only a fraction of the systemic dose. (See, e.g., Goodson, Medical Applications of Controlled Release, Ibid., Vol. 2, pp. 115-138 (1984).) Other controlled-release systems were discussed in the review by Langer (1990, Science 249:1527-1533). Compositions and Formulations

[0069] In some embodiments, the patient may further be administered a second agent, non-limiting examples of which include glucocorticoids, cyclosporine, and anakinra.

[0070] Examples of glucocorticoids include cortisol (hydrocortisone), cortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, deflazacort, fludrocortisone acetate, deoxycorticosterone acetate, aldosterone, and beclomethasone.

[0071] The formulated antibody is preferably substantially pure and, desirably, substantially homogeneous (e.g., free from contaminating proteins, etc.). A "substantially pure" antibody refers to a composition that comprises at least about 90% antibody by weight, based on the total weight of protein in the composition, and preferably at least about 95% antibody by weight, and a "substantially homogeneous" antibody refers to a composition that comprises at least about 99% antibody by weight, based on the total weight of protein in the composition.

[0072] The term "pharmaceutical formulation" refers to a formulation containing an anti-GM-CSF antibody, which formulation is in a form that allows the biological activity of the active ingredient to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0073] The formulations may be liquid or aqueous. Liquid formulations are aqueous solutions or suspensions made in a suitable aqueous solvent, such as water or aqueous / organic mixtures, such as water-alcohol mixtures.

[0074] In some embodiments, the liquid pharmaceutical formulation has a concentration of 20 mg / ml to 200 mg / ml, e.g., 30 mg / ml to 200 mg / ml, 40 mg / ml to 200 mg / ml, 50 mg / ml to 200 mg / ml, 60 mg / ml to 200 mg / ml, 70 mg / ml to 200 mg / ml, 80 mg / ml to 200 mg / ml, 90 mg / ml to 200 mg / ml, 100 mg / ml to 200 mg / ml, 110 mg / ml to 200 mg / ml, l~200mg / ml, 120mg / ml~200mg / ml, 130mg / ml~200mg / ml, 140mg / ml~200mg / ml, 150mg / ml~200mg / ml, 160mg / ml~20 0mg / ml, 170mg / ml~200mg / ml, 180mg / ml~200mg / ml, 190mg / ml~200mg / ml, 20mg / ml~190mg / ml, 20mg / ml~180mg / ml , 20mg / ml~170mg / ml, 20mg / ml~160mg / ml, 20mg / ml~150mg / ml, 20mg / ml~140mg / ml, 20mg / ml~130mg / ml, 20mg / ml~ 120mg / ml, 20mg / ml~110mg / ml, 20mg / ml~100mg / ml, 20mg / ml~90mg / ml, 20mg / ml~80mg / ml, 20mg / ml~70mg / ml, 20m g / ml~60mg / ml, 20mg / ml~50mg / ml, 20mg / ml~40mg / ml, 30mg / ml~190mg / ml, 40mg / ml~180mg / ml, 50mg / ml~170mg / m l, containing antibodies of 60mg / ml~160mg / ml, 70mg / ml~150mg / ml, 80mg / ml~140mg / ml, 90mg / ml~130mg / ml, 100mg / ml~120mg / ml. In some embodiments, the liquid pharmaceutical formulation comprises 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, 100 mg / ml, 110 mg / ml, 120 mg / ml, 130 mg / ml, 140 mg / ml, 150 mg / ml, 160 mg / ml, 170 mg / ml, 180 mg / ml, 190 mg / ml or 200 mg / ml of the antibody.

[0075] In another example, the pharmaceutical formulation further comprises another excipient. As used herein, the term "excipient" refers to an inert substance generally used as a drug diluent, vehicle, preservative, adhesive, or stabilizer, which imparts beneficial physical properties to the formulation, such as increasing protein stability, increasing protein solubility, or reducing viscosity.

[0076] "Excipients" include, for example, stabilizers such as human serum albumin (HSA), bovine serum albumin (BSA), α-casein, globulin, α-lactalbumin, LDH, lysozyme, myoglobin, ovalbumin, and RNAse A; buffers such as citric acid, HEPES, PBS, histidine, potassium acetate, potassium citrate, potassium phosphate (KH2PO4), sodium acetate, sodium bicarbonate, sodium citrate, sodium phosphate (NaH2PO4), Tris base, and Tris-HCl; and buffers such as glycine, alanine (α-alanine, β-alanine), arginine, betaine, leucine, lysine, glutamic acid, aspartic acid, histidine, proline, 4-hydroxyproline, sarcosine, γ-aminobutyric acid (GABA), and opioids. amino acids / metabolites such as amines (alanopine, octopine, strombine) and trimethylamine N-oxide (TMAO); surfactants such as polysorbate 20 and polysorbate 80 and poloxamer 407; lipid molecules such as phosphatidylcholine, ethanolamine and acetyltryptophan esters; polymers such as polyethylene glycol (PEG) and polyvinylpyrrolidone (PVP) 10, 24, 40;Low molecular weight excipients such as 4-pentanediol, octulose, propylene glycol, raffinose, ribose, sucrose, trehalose, xylitol and xylose, and other excipients such as cellulose, β-cyclodextrin, dextran (10 kd), dextran (40 kd), dextran (70 kd), ficoll, gelatin, hydroxypropylmethylcellulose, hydroxyethyl starch, maltodextrin, methylcellulose, PEG (6 kd), polydextrose, polyvinylpyrrolidone (PVP) kl5 (10 kd), PVP (40 kd), PVP k30 (40 kd), PVP High molecular weight excipients such as k90 (1000 kd), Sephadex G-200 and starch, antioxidants such as ascorbic acid, cysteine ​​hydrochloride, thioglycerol, thioglycolic acid, thiosorbitol and glutathione, reducing agents such as cysteine ​​hydrochloride, dithiothreitol and other thiols or thiophenes, chelating agents such as EDTA, EGTA, glutamic acid and aspartic acid, e.g. Ca, 2 +, Ni 2 +, Mg 2 +, Mn 2 +, inorganic salts / metals such as Na2SO4, (NH4)2SO4, Na2HPO4 / NaH2PO4, K2HPO4 / KH2PO4, MgSO4, and NaF; organic salts such as sodium acetate, sodium polyethylene, sodium caprylate / octanoate, proprionate, lactate, succinate, and citrate; and organic solvents such as acetonitrile, dimethyl sulfoxide (DMSO), and ethanol. For additional information regarding excipients, see Remington's Pharmaceutical Sciences (Joseph P. Remington, 18th ed., Mack Publishing Co., Easton, Pa.), which is incorporated herein in its entirety.

[0077] "Isotonic" means that the formulation has an osmotic pressure approximately equal to that of human blood. Isotonic formulations generally have an osmotic pressure of about 250 to about 350 mOsm (e.g., about 250 to about 340, about 250 to about 330, about 250 to about 320, about 250 to about 310, about 250 to about 300, about 260 to about 350, about 270 to about 350, about 280 to about 350, about 290 to about 350, about 300 to about 350, about 260 to about 340, about 270 to about 330, about 280 to about 320, about 290 to about 310 mOsm). Isotonicity may be measured, for example, by vapor pressure or freeze osmometry. In some embodiments, the isotonicity is about 300 mOsm. The tonicity adjusting agent may be one or more selected from the group consisting of sucrose, trehalose, mannitol, arginine, and sodium chloride. In some embodiments, the tonicity adjusting agent ranges from about 130 to 250 mM (e.g., about 130 mM to about 240 mM, about 150 mM to about 240 mM, about 180 mM to about 240 mM, about 200 mM to about 240 mM, about 130 mM to about 220 mM, about 150 mM to about 220 mM, about 180 mM to about 220 mM, about 130 mM to about 210 mM, about 150 mM to about 210 mM, about 180 mM to about 210 mM, about 200 mM to about 220 mM, or about 200 to about 210 mM). In some embodiments, the tonicity adjusting agent is about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, or about 250 mM. In some embodiments, the tonicity adjusting agent comprises sucrose or trehalose.

[0078] As used herein, "pH buffer" refers to a buffered solution that resists pH changes by the action of its acid-base conjugate components. Preferably, the buffer of the present disclosure has a pH within the range of about 4.5 to about 7.5 (preferably about 5.0 to about 7.0, e.g., about 5.0 to about 6.9, about 5.2 to about 6.8, about 5.3 to about 6.7, about 5.4 to about 6.6, about 5.5 to about 6.5, about 5.6 to about 6.4, about 5.7 to about 6.3, about 5.8 to about 6.2, about 5.9 to about 6.1, about 5.5 to about 6.4, about 5.5 to about 6.3, about 5.5 to about 6.2, about 5.5 to about 6.1, or about 5.5 to about 6.0). In some embodiments, the buffer has a pH of about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or 7.0. In one embodiment, the buffer has a pH of 5.8. The required pH level can be achieved by several methods, including, but not limited to, the addition of an appropriate buffer.

[0079] In some embodiments, the pH buffer comprises histidine, acetate, citrate, and succinate, hi some embodiments, the pH buffer comprises about 10 to about 30 mM histidine and / or about 10 to about 30 mM acetate, e.g., about 10 to about 25 mM, about 10 to about 20 mM, or about 15 mM to about 20 mM histidine and / or about 10 to about 25 mM, about 10 mM to about 20 mM, or about 15 mM to about 20 mM acetate. In some embodiments, the pH buffer comprises about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM histidine or acetate.

[0080] In some embodiments, the pH buffer comprises L-histidine and histidine hydrochloride monohydrate. In some embodiments, the pH buffer comprises 20 mM histidine, the histidine being selected from the group consisting of about 4.4 mM L-histidine and about 15.6 mM histidine hydrochloride monohydrate, about 5.2 mM L-histidine and about 14.8 mM histidine hydrochloride monohydrate, about 6.2 mM L-histidine and about 13.8 mM histidine hydrochloride monohydrate, and about It contains 6.8 mM L-histidine and about 13.2 mM histidine hydrochloride monohydrate, about 7.8 mM L-histidine and about 12.2 mM histidine hydrochloride monohydrate, about 8.4 mM L-histidine hydrochloride monohydrate and about 11.6 mM histidine hydrochloride monohydrate, or about 9 mM L-histidine and about 11 mM histidine hydrochloride monohydrate.

[0081] As used herein, "surfactant" refers to a surface-active agent, preferably a nonionic surfactant. Examples of surfactants herein include polysorbates (e.g., polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, and polysorbate 85), poloxamers (e.g., poloxamer 188 and poloxamer 407), Triton®, sodium dodecyl sulfate (SDS), sodium lauryl sulfate, sodium octyl glucoside (sodium octyli), and the like. glycoside), lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine or stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine or stearyl-sarcosine, linoleyl-betaine, myristyl-betaine or cetyl-betaine, lauramidopropyl-betaine, cocamidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmidopropyl-betaine or isostearamidopropyl-betaine (e.g., lauramidopropyl), myristamidopropyl-dimethylamine, palmidopropyl-dimethylamine or isostearamidopropyl-dimethylamine, sodium cocoyl methyl taurate or disodium oleoyl methyl taurate, and MONAQUAT TM series (Mona Industries, Inc., Paterson, NJ), polyethylene glycol, polypropylene glycol, and copolymers of ethylene glycol and propylene glycol (e.g., Piuronics, PF68, etc.), and the like.

[0082] The concentration of the surfactant is generally about 0.0001% (w / v) to about 1.0% (w / v), about 0.01% (w / v) to about 0.5% (w / v), e.g., about 0.015% (w / v) to about 0.03% (w / v), about 0.02% (w / v) to about 0.03% (w / v), about 0.025% (w / v) to about 0.03% (w / v), about 0.01% (w / v) to about 0.025% (w / v), about 0.01% (w / v) to about 0.02% (w / v), or about 0.01% (w / v) to about 0.015% (w / v). In one embodiment, the surfactant herein comprises polysorbate 80 or polysorbate 20. In some embodiments, the surfactant comprises about 0.01% (w / v), 0.015% (w / v), 0.02% (w / v), 0.025% (w / v), or 0.03% (w / v) polysorbate 80 or polysorbate 20. In some embodiments, the surfactant comprises about 0.02% (w / v) polysorbate 80.

[0083] In some embodiments, the liquid pharmaceutical formulation further comprises an antioxidant, a preservative, or a mixture thereof.

[0084] The term "antioxidant" refers to a reagent that inhibits oxidation of other molecules. Examples of antioxidants include ascorbic acid, citrate, lipoic acid, uric acid, cysteine ​​hydrochloride, monothioglycerol, thioglycerol, thioglycolic acid, thiosorbitol, tocopherol, carotenes, lycopene, and glutathione; reducing agents such as cysteine ​​hydrochloride, dithiothreitol; phosphonate compounds such as etidronic acid, deferoxamine, and malate; and other thiols or thiophenes and methionine. In another embodiment, the antioxidant is a metal chelator. Metal chelators include, but are not limited to, ethylenediaminetetraacetate ("EDTA"), ethylene glycol tetraacetic acid ("EGTA"), thiamine tetrahydrofurfuryl disulfide ("TTFD"), and 2,3-dimercaptosuccinic acid ("DMSA"). In some embodiments, the formulation comprises about 1 mM to about 50 mM of antioxidant, hi one embodiment, the formulation comprises about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, or about 45 mM of antioxidant.

[0085] The term "preservative" refers to a pharmaceutically acceptable excipient that prevents the growth of microorganisms within the composition. More particularly, the present disclosure provides multi-dose liquid compositions containing a preservative, which protects the composition from microbial contamination.

[0086] In one embodiment, the preservative is present in the composition in an amount of 0.001% (w / v) to 2% (w / v). In one embodiment, the preservative is present in the composition in an amount of 0.002% (w / v) to 1% (w / v). In one embodiment, the one or more preservatives are selected from phenol, m-cresol, benzyl alcohol, chlorobutanol, ethanol, phenoxyethanol, p-chloro-m-cresol, methylparaben, propylparaben, benzalkonium chloride, thimerosal, or any combination thereof. In one embodiment, the one or more preservatives are selected from phenol, m-cresol, benzyl alcohol, and chlorobutanol.

[0087] The viscosity of anti-GM-CSF antibody formulations may be controlled for subcutaneous, intravenous, or intramuscular administration. Viscosity may be affected by protein concentration and pH. For example, viscosity may increase as protein concentration increases. Increasing pH can reduce the viscosity of anti-GM-CSF antibody formulations. In some protein formulations, sodium chloride is added to reduce the viscosity of the formulation. Other components that can affect the viscosity of anti-GM-CSF antibody formulations are amino acids, such as histidine and arginine.

[0088] The liquid pharmaceutical formulations described herein may have a variety of viscosities. Methods for measuring the viscosity of liquid pharmaceutical formulations are known to those of skill in the art and may include, for example, a rheometer (e.g., an Anton Paar MCR301 Rheometer with a 50 mM, 40 mM, or 20 mM cone accessory). In some embodiments of the present disclosure, the viscosity is reported at the high shear limit of a shear rate of 1000 / sec. In some embodiments, the liquid pharmaceutical formulation has a viscosity between 1.0 cP ± 10% and 20 cP ± 10%. In some embodiments, the liquid pharmaceutical formulation has a viscosity of less than 20 cP, less than 18 cP, less than 15 cP, less than 13 cP, or less than 11 cP. As will be appreciated by those of skill in the art, viscosity is temperature dependent; therefore, unless otherwise specified, viscosities herein are measured at 25°C. In some embodiments, the viscosity of the liquid pharmaceutical formulation at 25° C. is 1.0 cP±10%, 2.0 cP±10%, 3.0 cP±10%, 3.1 cP±10%, 3.2 cP±10%, 3.5 cP±10%, 3.6 cP±10%, 3.8 cP±10%, 4.0 cP±10%, 5.0 cP±10%, 5.3 cP±10%, 6.0 cP±10%, 6.3 cP±10%, 7.0 cP±10%, 7.0 cP±10%, 8.0 cP±10%, 8.0 cP±10%, 9.0 cP±10%, 9.0 cP±10%, 10 ... P±10%, 6.4cP±10%, 6.8cP±10%, 7.0cP±10%, 7.1cP±10%, 7.4cP±10%, 8.0cP±10%, 9.0cP±10%, 10.0cP±10%, 11.0cP±10%, 12.0cP±10%, 13.0cP±10%, 14.0cP±10%, 15.0cP±10% or 16cP±10%.

[0089] In some embodiments, the liquid pharmaceutical formulation further comprises a viscosity modifier. In one embodiment, the viscosity modifier is an amino acid. In one embodiment, the viscosity modifier is L-proline. In some embodiments, the viscosity modifier has a concentration of 1%±0.2% to 5%±1% w / v. In one embodiment, the viscosity modifier is proline at a concentration of 1.5%±0.3% or about 1.5%. In one embodiment, the viscosity modifier is proline at a concentration of 3%±0.6% or about 3%.

[0090] In a first aspect, the present disclosure provides a novel liquid pharmaceutical formulation, comprising: a) an anti-GM-CSF antibody having a concentration of 20 mg / ml to 200 mg / ml as an antibody; b) acetate or histidine at a concentration of 10 mM to 30 mM as a buffer; c) sucrose or trehalose having a concentration of 130 mM to 250 mM as an isotonicity adjuster; d) containing polysorbate 80 or polysorbate 20 as a surfactant at a concentration of 0.01% (w / v) to 0.03% (w / v), Here, the pH of the formulation is from about 4.5 to about 7.5, preferably from about 5.5 to about 6.1.

[0091] In a further embodiment, the formulation does not include other excipients.

[0092] In a preferred embodiment, the present disclosure provides a novel liquid pharmaceutical formulation, comprising: a) an anti-GM-CSF antibody having a concentration of 50 mg / ml to 150 mg / ml; b) histidine at a concentration of 10 mM to 20 mM; c) sucrose having a concentration of 200 mM to 220 mM; d) polysorbate 80 at a concentration of 0.01% (w / v) to 0.03% (w / v), Here, the formulation has a pH of 5.5 to 6.1.

[0093] In a further embodiment, the formulation does not include other excipients.

[0094] In a more preferred embodiment, the present disclosure provides a novel liquid pharmaceutical formulation, comprising: a) an anti-GM-CSF antibody at a concentration of 100 mg / ml, comprising a heavy chain variable region CDR1 (HCDR1) of SEQ ID NO:1, an HCDR2 of SEQ ID NO:2, an HCDR3 of SEQ ID NO:3, a light chain variable region CDR1 (LCDR1) of SEQ ID NO:4, an LCDR2 of SEQ ID NO:5, and an LCDR3 of SEQ ID NO:6; b) histidine at a concentration of 20 mM; c) sucrose at a concentration of 220 mM; d) Polysorbate 80 at a concentration of 0.02% (w / v), Here, the formulation has a pH of 5.8.

[0095] In a further embodiment, the formulation does not include other excipients.

[0096] The pharmaceutical formulations may be administered to a patient by parenteral routes, such as injection (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, etc.), or by transdermal, mucosal, nasal, pulmonary, or oral administration. Many reusable pen or automated syringe delivery devices can be used to subcutaneously deliver the pharmaceutical formulations of the present disclosure. Examples include the AUTOPEN TM (Owen Mumford, Inc., Woodstock, UK), DISETRONIC TM Pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25 TM Pen, HUMALOG TM Pen, HUMALIN 70 / 30 TM Penn (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN TM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR TM (Novo Nordisk, Copenhagen, Denmark), BD TMPen (BD (Becton Dickinson), Franklin Lakes, New Jersey), OPTIPEN TM , OPTIPEN PRO TM , OPTIPEN STARLET TM and OPTICLIK TM (Sanofi-Aventis, Frankfurt, Germany). An example of a disposable pen or automatic syringe delivery device used for subcutaneous delivery of the drug compositions of the present disclosure is the SOLOSTAR TM Pen (Sanofi-Aventis), FLEXPEN TM (Novo Nordisk) and KWIKPEN TM (Lilly), SURECLICK TM Auto syringe (Amgen, Thousand Oaks, Calif.), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP) and HUMIRA TM Penn (Abbott Labs, Abbott Park, Ill.), but is not limited to it.

[0097] The use of microinfusers to deliver pharmaceutical formulations of the present disclosure is also contemplated herein. As used herein, the term "microinfuser" refers to a subcutaneous delivery device designed to slowly administer large volumes (e.g., up to about 2.5 mL or more) of therapeutic formulations over an extended period of time (e.g., about 10, 15, 20, 25, 30 minutes or more). See, e.g., U.S. Pat. No. 6,629,949, U.S. Pat. No. 6,659,982, and Meehan et al., J. Controlled Release 46:107-116 (1996). Microinfusers are particularly applicable to the delivery of large doses of therapeutic proteins contained in high concentrations (e.g., about 100, 125, 150, 175, 200 mg / mL or more) or viscous solutions.

[0098] In some embodiments, the present disclosure provides a pre-filled syringe containing any one of the liquid formulations described herein. In some embodiments, the syringe is a 1 mL or 2.25 mL long glass syringe equipped with a 27-gauge thin-walled needle, a rubber plunger coated with a fluorocarbon compound, and a rubber needle protector cap.

[0099] In one aspect, the liquid pharmaceutical formulations of the present disclosure may be stored at room temperature, refrigerated (e.g., 2-8°C), or frozen (e.g., at -20°C or -70°C).

[0100] In some embodiments, the formulation of any one of the above embodiments has an attribute selected from the group consisting of: (i) the formulation is stable when stored for an extended period of time at 50° C., 40° C., 25° C., 5° C., −20° C., −30° C., and −80° C.; (ii) the formulation has a low viscosity (viscosity less than 10 cP); (iv) the formulation is isotonic under physiological conditions; (v) the formulation is stable and compatible with intravenous or subcutaneous delivery devices and procedures; and (vi) the formulation is stable when stored for an extended period of time in a glass vial or a pre-filled syringe.

[0101] In one embodiment, the formulation substantially retains its physical and chemical stability and its biological activity upon storage. The storage period is generally selected based on the expected shelf life of the formulation. Several analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, pp. 247-301, edited by Vincent Lee, published by Marcel Dekker, Inc., New York, NY (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured at a selected temperature over a selected period of time. For example, a liquid formulation remains stable at about 40°C for at least about 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks. In another embodiment, the liquid formulation remains stable for at least about 1 month, at least about 3 months, at least about 6 months, at least about 9 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, or at least about 36 months at about 5° C. and / or 25° C., and / or remains stable for at least about 1 month, at least about 3 months, at least about 6 months, at least about 9 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months, at least about 42 months, or at least about 48 months at about −20° C. and / or −70° C. Furthermore, in some embodiments, the liquid formulation may remain stable after being frozen (e.g., to −80° C.) and thawed (e.g., after one, two, or three freeze-thaw cycles).

[0102] The stability of liquid formulations may be qualitatively and / or quantitatively assessed in several different ways, including assessment of dimer, multimer and / or aggregate formation (e.g., size exclusion chromatography (SEC), matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS), analytical ultracentrifugation, light scattering (photon correlation spectroscopy, dynamic light scattering (DLS), static light scattering, multi-angle laser light scattering (MALLS)), flow-based microscopic imaging, and the like. imaging), electrical impedance (Coulter) counting, light obscuration or other liquid particle counting systems, by measuring turbidity and / or visual inspection), cation exchange chromatography (CEX), isoelectric focusing (IEF) (e.g., capillary technique (cIEF)) or capillary zone electrophoresis to assess charge heterogeneity, amino- or carboxyl-terminal sequence analysis, mass spectrometry, SDS-PAGE or SEC analysis to compare fragmented, intact and multimeric (i.e., dimers, trimers, etc.) antibodies, peptide map (e.g., trypsin or LYS-C) analysis, assessment of antibody biological activity or antigen-binding function, and similar methods. The stability of solid-state formulations may be assessed qualitatively and / or quantitatively in several different ways, including direct tests such as X-ray powder diffraction (XRPD) to identify crystalline structure, using Fourier transform infrared spectroscopy (FTIR) to assess antibody structure in the solid state, and measuring thermal transitions (e.g., melting, glass transition) in lyophilized solids using differential scanning calorimetry (DSC) and indirect tests (e.g., measuring water content by the Karl Fisher test) to estimate potential chemical instability, e.g., due to hydrolysis.The instability may be related to any one or more of aggregation (e.g., non-covalent soluble aggregation, covalent soluble aggregation (e.g., disulfide bond rearrangement / scrambling), insoluble aggregation), deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), shearing / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, N-terminal extension, C-terminal processing, differential glycosylation, and the like. example Example 1. Inhibition of MAS progression and improvement of symptoms by IMH001

[0103] This study was conducted to evaluate the in vivo efficacy of the GM-CSF neutralizing antibody IMH001 (see sequence in Table 1) in delaying disease progression in the NOG-EXL mouse macrophage activation syndrome (MAS) model (induced by CBMC transplantation). By transplanting human umbilical cord blood (UCB) into NOG-EXL mice, such a lymphocyte-independent, effector-driven MAS mouse model can be established. NOG-EXL (NOD.Cg-Prkdc) scid IL2rg tm1Sug Tg(SV40 / HTLV-IL3, CSF2) is NOG(NOD.Cg-Prkdc scid IL2rg tm1Sug The NOG-EXL mouse is a genetically modified strain based on the NOG-EXL mouse, which is immunodeficient and persistently overexpresses human IL-3 and GM-CSF, which can enrich myeloid cell populations, including DCs, monocytes, and macrophages, after human stem cell transplantation. A total of 46 NOG-EXL mice and 8 NOG mice were used in this study.

[0104] [Table 1]

[0105] The model exhibited progressive disease with many MAS characteristics and rapidly evolved into progressive anemia without the need for exogenous immune stimulation, and therefore can be used to explore the contribution of the monocyte / macrophage arm and identify new therapeutic strategies for MAS / HLH. 1.1 Method

[0106] Five- to six-week-old NOG-EXL mice were irradiated with 1 Gy of radiation before reconstitution with OKT3-pretreated umbilical cord blood mononuclear cells (CBMCs) administered intravenously through the tail vein. The day of reconstitution was defined as day 0. Starting one week after transplantation, OKT3-pretreated CBMC-transplanted NOG-EXL mice were treated with ip doses of PBS or 10 mg / kg IMH001 once every two weeks. Animal weights, CBC counts, and serum ferritin were measured at the indicated time points.

[0107] One week after reconstitution (day 7), four NOG mice (G1) and 14 NOG-EXL mice (6 G2 mice and 8 G3 mice, respectively) were treated twice weekly for 15 weeks. The grouping and treatment regimen are shown in Table 2 below.

[0108] [Table 2]

[0109] Body weight: After CBMC transplantation, the body weight of the mice was measured and recorded once a week.

[0110] CBC counts: Whole blood was collected from mice at weeks 4, 8, and 16 and counted using a CBC machine. Total bone marrow viable cells were also counted at the end of the study at week 16. Mice were anesthetized with 3% to 4% isoflurane prior to blood collection.

[0111] Cytokines measured by the MSD method: Serum was collected at weeks 8 and 10 and used to measure IL-1β, IL-6, TNF-α, GM-CSF, IFN-γ, and IL-12. Before blood collection, mice were anesthetized with 3% to 4% isoflurane.

[0112] Serum samples were collected from mice at weeks 8 and 16 for ferritin, fibrinogen, and triglycerides. These were then measured using ELISA kits. Before blood collection, mice were anesthetized with 3% to 4% isoflurane.

[0113] Experimental endpoint collection: The endpoint was day 112 / week 16. Spleens were collected from all mice in each group and weighed. Cell smears from bone marrow, liver, and spleen sections were collected and subjected to Wright Giemsa staining to detect the presence of hemophagocytosis. Activated lymphoid tissues, spleen / lymph nodes, liver, and meninges were collected and subjected to H&E sections to detect inflammatory cell infiltration and reduction of lymphoid follicles in the spleen / lymph nodes. statistics

[0114] All statistical analysis methods for data in this study were consistent with the experimental design, and results are expressed as mean ± SEM. First, homogeneity of variance was analyzed using the F test (IDBS E-WorkBook 10.5.0 (64-bit) (c) IDBS). If homogeneity of variance was confirmed (p > 0.05), one-way ANOVA was performed. The T test was used to compare two groups, and Dunnett's multiple comparison test was used for multiple group comparisons. If variances were heterogeneous (p ≤ 0.05), the Kruskal-Wallis test was used for nonparametric comparisons. The U test was used for comparisons of two groups (Kruskal-Wallis parametric tests were significant, p ≤ 0.05), and Dunn's multiple comparison test was used for comparisons of multiple groups (Kruskal-Wallis parametric tests were not significant, p > 0.05). A p < 0.05 was considered significant. 1.2 Results

[0115] Shortly after OKT3-pretreated CBMCs were transplanted into NOG-EXL mice, the mice developed MAS, characterized by weight loss, a gradual decline in red blood cell (RBC) count, hemoglobin (HGB) and hematocrit (HCT), and elevated ferritin. 1.2.1 Weight change

[0116] However, IMH001 treatment completely reversed disease progression. After CBMC transplantation, animals were weighed weekly.

[0117] No obvious abnormalities were observed in any group during the experiment. Figure 1 shows the weight changes in each group at each time point. As the disease progressed, sustained weight loss was observed in CBMC-transplanted NOG or NOG-EXL mice. Compared with the vehicle control group (G2), the body weight of the IMH001-treated group (G3: 10 mg / kg) significantly increased from day 63 to day 112 (p<0.05). These results indicated that IMH001 treatment effectively ameliorated MAS-induced weight loss in animals. 1.2.2 Peripheral blood and bone marrow cell counts

[0118] Whole blood was collected from mice for complete blood count (CBC) analysis at weeks 4, 8, 12, and 16. Total bone marrow viable cells were also counted at the end of the study (week 16). Figure 2 shows the RBC, HGB, NEUT, and MONO counts for each group at each time point. With disease progression, a sustained decrease in peripheral RBC counts, HGB levels, and NEUT percentages was observed in NOG-EXL:CBMC-transplanted mice, while there were no significant changes in PLT counts and MONO percentages. After IMH001 treatment, a recovery in RBC and NEUT reductions was observed, with significantly higher RBC cell counts and HGB levels compared with untreated controls, particularly on day 56 (6.45 vs. 8.17, p<0.001). The monocyte percentage in the IMH001-treated group also decreased, which was considered to be a pharmacological effect of IMH001.

[0119] Figure 3 shows the total bone marrow viable cells. Compared to NOG-EXL-untreated mice, there was a tendency for the total bone marrow cells to increase on day 112 (mean 9.55 vs. 11.6, p=0.182). The number of peripheral RBCs in the MAS control group continued to decrease. IMH001 treatment effectively improved the number of RBCs in peripheral blood, accompanied by an increase in HGB. 1.2.3 Ferritin, fibrinogen and triglycerides

[0120] Serum was collected from mice at weeks 8 and 16. Figure 4 shows the ferritin, fibrinogen, and triglyceride levels in each group at each time point. In NOG-EXL CBMC-implanted mice, a clear increase in ferritin, one of the indicators of macrophage activation, was observed as the disease progressed. However, this phenomenon was not observed in NOG CBMC-implanted mice. Furthermore, IMH001 treatment effectively controlled ferritin levels compared with untreated mice, and this phenomenon was observed at weeks 8 and 16. No significant changes in fibrinogen and triglycerides were observed during disease progression or after IMH001 treatment.

[0121] The results showed a significant increase in ferritin levels in the peripheral blood of the MAS control group, indicating macrophage activation. IMH001 treatment can significantly reduce ferritin levels in the peripheral blood. 1.2.4 Histopathological evaluation

[0122] At the end of the study (week 16), cytological smears from bone marrow, liver, and spleen sections were collected and stained with Wright-Giemsa to detect the presence of hemophagocytosis. Activated lymphoid tissue / lymph nodes and spleen were collected and stained with H&E to assess lymphocyte exhaustion, and H&E staining was performed on the liver and brain / meninges to detect inflammatory cell infiltration. The results are shown in Figure 5.

[0123] Hemophagocytosis: Hemosiderin-containing macrophages were observed in splenic red pulp sections and bone marrow smears from the MAS control group, indicating hemophagocytosis. IMH001 treatment can effectively prevent hemophagocytosis associated with MAS.

[0124] Lymphocyte exhaustion: The number of lymphoid follicles and germinal centers in the spleen and the number of lymphocytes in the lymph nodes of NOG-EXL mice were reduced, indicating lymphocyte exhaustion. IMH001 treatment can effectively reduce lymphocyte exhaustion. Furthermore, a decrease in inflammatory infiltration was observed in the liver and meninges after IMH001 treatment.

[0125] Results showed that 16 weeks after CBMC transplantation, NOG-EXL mice showed increased hemophagocytosis, lymphocyte exhaustion, and increased inflammation compared with NOG mice. The above histopathological parameters were significantly improved in IMH001-treated mice.

[0126] Hemophagocytosis: In the splenic red pulp sections and bone marrow smears of the MAS control group, hemosiderin-containing macrophages were observed, indicating hemophagocytosis.

[0127] Lymphocyte exhaustion: The number of lymphoid follicles and germinal centers in the spleen and the number of lymphocytes in the lymph nodes of NOG-EXL mice were reduced, indicating lymphocyte exhaustion.

[0128] The study showed that IMH001 treatment was effective in delaying disease progression in all of the above parameters compared to tocilizumab. Example 2. Development of IMH001 formulation

[0129] This example developed a suitable formulation of IMH001 for clinical use in the treatment of MAS.

[0130] IMH001 was first evaluated in three different buffer systems: sodium acetate, histidine hydrochloride, and sodium dihydrogen phosphate, at several different pH levels. All three buffer systems were able to stabilize the antibody, and a pH of 5.5 to 6.0 appeared to be more beneficial than 6.5 or higher. Histidine hydrochloride and pH 5.8 were selected for further testing.

[0131] Next, several excipients were evaluated, including sucrose, trehalose, mannitol, proline, arginine hydrochloride, glycine, sodium chloride, PS20, and PS80. Based on the changes in stability (CEX-HPLC) results after 1 week at 50°C, a comparison was made between formulations with different excipients. Results showed that sucrose and proline performed similarly and were slightly superior to the other formulations. Sucrose was selected for further development.

[0132] However, it was unclear whether the selected buffer and excipients were suitable for subcutaneous injection of IMH001 protein at high concentrations. Therefore, the solubility and viscosity of the protein at concentrations of 100 mg / ml and 150 mg / ml in a histidine hydrochloride buffer system (pH 5.8) were evaluated.

[0133] In a 20 mmol / L histidine hydrochloride buffer system (pH 5.8), when the protein concentrations were 100 mg / ml and 150 mg / ml, and the samples were left at 4°C and 25°C for 48 hours, no significant changes were observed in the appearance color, clarity, OD350, protein concentration, or viscosity, where the viscosity was less than 10 cP and the clarity was less than 6 NTU.

[0134] The ability of PS80 (Polysorbate 80) to improve the durability and stability of candidate formulations at a protein concentration of 100 mg / ml under different concentrations of sucrose was then tested. The viscosity of all samples was acceptable, and the viscosity decreased with decreasing sucrose concentration.

[0135] After further adjusting the concentration of each component, a formulation of 100 mg / ml IMH001, 20 mM histidine, 220 mM sucrose, and 0.02% (w / v) polysorbate 80 was determined to be superior to other candidate formulations, with a pH of 5.8, and was used as the stock solution for the following in vivo studies. * * *

[0136] The scope of the present disclosure is not limited by the specific examples described; these specific examples are intended as single illustrations of each aspect of the present disclosure, and any functionally equivalent compositions or methods are within the scope of the present disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of the present disclosure without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure is intended to cover modifications and variations of the present disclosure provided they come within the scope of the appended claims and their equivalents.

[0137] All publications, patents, and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A method for treating or preventing macrophage activation syndrome (MAS) or hemophagocytic lymphohistiocytosis (HLH) in a patient in need thereof, comprising administering to the patient an antibody or a fragment thereof having specificity for human GM-CSF protein.

2. 2. The method of claim 1, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 1 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

2.

3. The method of claim 2 , wherein the antibody comprises a human IgG1 Fc.

4. 10. The method of any one of the preceding claims, wherein the patient has a disease or condition selected from the group consisting of systemic onset juvenile idiopathic arthritis (SoJIA), systemic lupus erythematosus (SLE), Kawasaki disease, and adult-onset Still's disease.

5. 10. The method of any one of the preceding claims, wherein the patient has a blood ferritin level of at least 300ng / ml, or at least 350ng / ml, 400ng / ml, 450ng / ml, 500ng / ml, 550ng / ml, 600ng / ml, 650ng / ml, 684ng / ml, 700ng / ml, 720ng / ml, 740ng / ml, 750ng / ml, 760ng / ml, 784ng / ml, 800ng / ml, 820ng / ml, 840ng / ml, 850ng / ml, 860ng / ml, 884ng / ml, 900ng / ml, 950ng / ml or 1000ng / ml.

6. 10. The method of any one of the preceding claims, wherein the patient has a hemoglobin level lower than 11 g / dL, or lower than 10.5 g / dL, 10 g / dL, 9.5 g / dL, 9 g / dL, 8.5 g / dL, 8 g / dL, 7.5 g / dL or 7 g / dL.

7. The patient had a platelet count of 140 x 10 ９ / L or less than 130 × 10 ９ / L, 120 x 10 ９ / L, 110 x 10 ９ / L, 100 x 10 ９ / L, 90 x 10 ９ / L, 80 x 10 ９ / L, 70 x 10 ９ / L or 60 x 10 ９ 10. The method of claim 1, wherein the saturation is less than 1000 saturations per 1000 saturates.

8. The patient had a neutrophil count of 1.4 x 10 ９ / L or lower than 1.3 × 10 ９ / L, 1.2 x 10 ９ / L, 1.1 x 10 ９ / L, 1.0 x 10 ９ / L, 0.9 x 10 ９ / L, 0.8 x 10 ９ / L, 0.7 x 10 ９ / L or 0.6 x 10 ９ 10. The method of claim 1, wherein the .alpha.-hydroxybenzoate concentration is less than 1000 ppm.

9. 10. The method of any one of the preceding claims, wherein the patient has a fasting triglyceride level greater than 2.49mmol / L, or greater than 2.55mmol / L, 2.6mmol / L, 2.66mmol / L, 2.72mmol / L, 2.77mmol / L, 2.83mmol / L, 2.89mmol / L, 2.94mmol / L, 3mmol / L, 3.06mmol / L, 3.11mmol / L, 3.17mmol / L, 3.23mmol / L, 3.28mmol / L, 3.34mmol / L or 3.4mmol / L.

10. 10. The method of any one of the preceding claims, wherein the patient has a blood fibrinogen level lower than 1.5 g / L, or lower than 1.4 g / L, 1.3 g / L, 1.2 g / L, 1.1 g / L, 1.0 g / L, 0.9 g / L, 0.8 g / L, 0.7 g / L, 0.6 g / L or 0.5 g / L.

11. 10. The method of any one of the preceding claims, wherein the patient has elevated levels of soluble CD163 compared to healthy individuals.

12. 10. The method of any one of the preceding claims, wherein the patient has elevated levels of soluble IL-2 receptor compared to healthy individuals.

13. The method of any one of claims 1 to 12, wherein the antibody or antigen-binding fragment thereof is administered at 0.3 mg / kg to 25 mg / kg.

14. The method of any one of claims 1 to 12, wherein the antibody or antigen-binding fragment thereof is administered at 5 mg / kg to 20 mg / kg.

15. The method of any one of claims 1 to 12, wherein the antibody or antigen-binding fragment thereof is administered at 5 mg / kg to 10 mg / kg.

16. The method of any one of claims 1 to 12, wherein the antibody or antigen-binding fragment thereof is administered at 6 mg / kg to 10 mg / kg.

17. 10. The method of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is administered once a week, twice a week, once every two weeks, or once a month.

18. 10. The method of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is administered intravenously or subcutaneously.

19. The antibody, (a) 20 mg / ml to 200 mg / ml of the antibody; (b) 10 mM to 30 mM of a buffering agent; (c) 130 mM to 250 mM of a tonicity adjuster; (d) 0.01% (w / v) to 0.03% (w / v) of a surfactant; 19. The method of any one of claims 3 to 18, wherein the formulation has a pH of 4.5 to 7.

5.

20. 20. The method of claim 19, wherein the formulation comprises 50-150 mg / ml of the antibody, 10-20 mM histidine, 200-220 mM sucrose, and 0.01-0.03% (w / v) polysorbate 80, and has a pH of about 5.5-6.

1.

21. 21. The method of claim 20, wherein the formulation comprises about 100 mg / ml of the antibody, about 20 mM histidine, about 220 mM sucrose, and about 0.02% (w / v) polysorbate 80, and has a pH of about 5.

8.

22. 10. The method of any one of the preceding claims, wherein the method further comprises administering to the patient a glucocorticoid, cyclosporine or anakinra.

23. 23. The method of claim 22, wherein the glucocorticoid is selected from the group consisting of cortisol, cortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, deflazacort, fludrocortisone acetate, deoxycorticosterone acetate, aldosterone, and beclomethasone.

Images