Methods, compositions and dosing regimens for treating or preventing interferon-gamma related indications

Abstract

To provide methods, compositions and dosing regimens for treating or preventing interferon-gamma related indications.SOLUTION: The disclosure relates generally to methods, compositions and dosing regimens for treating, preventing and / or delaying the onset or progression of, or alleviating, a symptom associated with elevated IFN-γ levels. The disclosure relates generally to methods and compositions for treating, preventing and / or delaying the onset or progression of, or alleviating, a symptom associated with elevated levels of interferon gamma (IFNγ, IFN-gamma), such as hemophagocytic lymphohistiocytosis (HLH), hemorrhagic fever, CAR-T cell therapy, transplant failure, transplant rejection, Graft-versus-host disease (GvHD), and / or an inflammatory disorder associated with transplant rejection.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 62 / 411,783, filed October 24, 2016, the contents of which are incorporated by reference herein in their entirety.

[0002] The present disclosure generally relates to methods and compositions for treating, preventing, and / or delaying the onset or progression of, or alleviating, conditions associated with elevated levels of interferon gamma (IFNγ, IFN-gamma), such as, for example, hemophagocytic lymphohistiocytosis (HLH), hemorrhagic fever, CAR-T cell therapy, transplant failure, graft rejection, graft-versus-host disease (GvHD), and / or inflammatory disorders associated with graft rejection. The present disclosure also relates to methods and compositions for extending the survival of transplanted biological materials. [Background technology]

[0003] Human interferon gamma (IFNγ, IFN-gamma) is a lymphokine produced by activated T-lymphocytes and natural killer cells. Human interferon gamma (IFNγ, IFN-gamma) exhibits antiproliferative and immunomodulatory activity, binding to IFNγ-R, a heterodimeric receptor on the majority of primary cells of the immune system, and triggering a cascade of events that lead to inflammation. The immunomodulatory activity of IFNγ is known to have beneficial effects in several clinical conditions. However, there are also many clinical situations in which IFNγ activity is known to have adverse effects. For example, autoimmune diseases are associated with high IFNγ levels in blood and affected tissues from autoimmune patients. IFNγ activity has also been linked to disease states such as cachexia and septic shock.

[0004] IFNγ has been implicated in a number of disorders, and anti-IFNγ agents are under development as therapeutics. Summary of the Invention [Means for solving the problem]

[0005] In various embodiments, the present invention provides a number of variable dose treatment regimens for treating a disease, disorder or condition associated with elevated IFN-g levels.

[0006] In one embodiment, the present invention provides a method for treating primary hemophagocytic lymphohistiocytosis (HLH) in humans by intravenously administering a first and a second dose of an antibody that binds to interferon gamma (IFNγ) to a subject. The subject is an adult subject or a pediatric subject. The first dose is 1.0 or 3.0 mg / kg of the subject's body weight, and the second dose is 3.0, 6.0 or 10.0 mg / kg of the subject's body weight. Optionally, a third dose of 1.0 mg / kg of the subject's body weight is administered.

[0007] In another embodiment, the present invention provides a method for treating secondary hemophagocytic lymphohistiocytosis (HLH) in a human pediatric subject by intravenously administering to the subject a first and a second dose of an antibody that binds interferon gamma (IFNγ). The first dose is 6.0 mg / kg of the subject's body weight and the second dose is 3.0 mg / kg of the subject's body weight. Optionally, a third dose of 6.0 mg / kg of the subject's body weight is administered.

[0008] In a further aspect, the present invention provides a method for treating secondary hemophagocytic lymphohistiocytosis (HLH) in a human adult subject by intravenously administering to the subject a first dose and a second dose of an antibody that binds to interferon gamma (IFNγ). The first dose is 3.0 mg / kg of the subject's body weight or 6.0 mg / kg of the subject's body weight, and the second dose is 10 mg / kg or less of the subject's body weight. For example, the second dose is 1.0, 3.0, 6.0 or 10.0 mg / kg. Optionally, a third dose is administered that is less than 10.0 mg / kg of the subject's body weight. For example, the third dose is 1.0, 3.0 or 6.0 mg / kg of the subject's body weight.

[0009] In yet another embodiment, the present invention provides a method for treating a condition in a human subject by intravenously administering to the subject a first and a second dose of an antibody that binds interferon gamma (IFNγ). The subject is an adult or pediatric subject. The condition is graft rejection, such as solid organ transplantation disorder or bone marrow acute graft rejection. The condition is graft-versus-host disease, paraneoplastic cerebellar degeneration, hemorrhagic fever, sarcoidosis, or adult-onset Still's disease. Alternatively, the method is administered to the subject after receiving CART cell therapy. The first dose is 1.0-10 mg / kg of the subject's body weight, and the second dose is 1.0-10 mg / kg of the subject's body weight. For example, the second dose is 1.0, 3.0, 6.0 or 10.0 mg / kg. The second dose is preferably higher or lower than the first dose. Optionally, a third dose of 1.0 to 10 mg / kg of the subject's body weight is administered, e.g., the first, second, or third dose is 1.0, 3.0, 6.0, or 10.0 mg / kg of the subject's body weight.

[0010] The antibody that binds to interferon gamma (IFNγ) has a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO: 1); a variable heavy chain complementarity determining region 2 (VH CDR3) comprising the amino acid sequence of AISGSGGSTYYADSVKG (SEQ ID NO: 2); and variable heavy chain complementarity determining region 3 (VH CDR3) comprising the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO:3); variable light chain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO:4); variable light chain complementarity determining region 2 (VL CDR2) region comprising the amino acid sequence of EDNQRPS (SEQ ID NO:5); and variable light chain complementarity determining region 3 (VL CDR3) region comprising the amino acid sequence of QSYDGSNRWM (SEQ ID NO:6). For example, the antibody comprises a heavy chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO:47, and a light chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO:48.

[0011] The antibody doses are administered within 1 hour, 6 hours or 12 hours.

[0012] The second dose is administered every 3 days for the first treatment period after the first dose.Furthermore, the second dose is administered for the second treatment period after the completion of the first treatment period.The second treatment period is, for example, twice a week.

[0013] The antibody dose is administered as a single injection.

[0014] The antibodies are administered as monotherapy or in combination therapy.

[0015] Optionally, the method further comprises administering dexamethasone immediately prior to dosing with the antibody. The dexamethasone is at least 10 mg / m 2 or at least 5 mg / m 2 is administered at a dose of

[0016] Subjects had not previously received treatment for HLH.

[0017] In various embodiments, the method further comprises administering to the subject at least a second agent, the second agent being a therapeutic agent, an anti-inflammatory agent, and / or an immunosuppressant agent.

[0018] Also included in the present invention is an injectable pharmaceutical formulation containing per mL: 5 mg or 25 mg fully human anti-interferon gamma (IFNγ) monoclonal antibody; 1.55 mg L-histidine, 3.14 mg L-histidine monohydrochloride monohydrate, 7.31 mg sodium chloride (NaCl), and 0.05 mg polysorbate 80, and having a pH between 5.8 and 6.2.

[0019] In a further aspect, the present invention provides a unit dose vial containing 20 ml of a fully human anti-interferon gamma (IFNγ) monoclonal antibody solution suitable for injection, the antibody having a concentration of 5 mg / ml or 25 mg / ml and the pH of the solution being between 5.8 and 6.2. The antibody is solubilized in the solution, and therefore the solution is clear, colorless and without precipitate.

[0020] In another aspect, the invention provides a unit dose vial containing 10 ml or 20 ml of a fully human anti-interferon gamma (IFNγ) monoclonal antibody solution suitable for injection, the antibody having a concentration of 25 mg / ml and the pH of the solution being between 5.8 and 6.2. The antibody is solubilized in the solution, and therefore the solution is clear, colorless, and free of precipitate.

[0021] In yet another aspect, the present invention provides a unit dose vial containing 2 ml or 10 ml of a fully human anti-interferon gamma (IFNγ) monoclonal antibody solution suitable for injection, the antibody having a concentration of 5 mg / ml and the pH of the solution being between 5.8 and 6.2. The antibody is solubilized in the solution, and therefore the solution is clear, colorless, and free of precipitate.

[0022] An antibody that binds to interferon gamma (IFNγ) comprises: a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO:1); a variable heavy chain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence of AISGSGGGSTYYADSVKG (SEQ ID NO:2); and a variable heavy chain complementarity determining region 3 (VH CDR3) comprising the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO:3); a variable light chain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO:4); a variable light chain complementarity determining region 2 (VL CDR2) region comprising the amino acid sequence of EDNQRPS (SEQ ID NO:5); and a variable light chain complementarity determining region 3 (VL CDR3) region comprising the amino acid sequence of QSYDGSNRWM (SEQ ID NO:6). For example, the antibody comprises a heavy chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO:47, and a light chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO:48. Any of the above aspects or embodiments may be combined with any other aspect or embodiment. Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains.Methods and materials similar or equivalent to those described herein can be used to implement the present invention, and suitable methods and materials are described below.All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will control.In addition, the materials, methods, and examples described herein are merely illustrative and are not intended to be limiting.

[0023] Other features and advantages of the invention will become apparent from, and are encompassed by, the following detailed description and claims. [Brief description of the drawings]

[0024] [Figure 1]FIG. 1 is a graph showing the correlation between pre-dose serum CXCL9 levels and total IFNγ levels 24 hours after infusion of NI-0501 antibody in an ongoing Phase 2 pilot study in patients with primary HLH.

[0025] [Diagram 2] FIG. 2 is a graph showing the correlation between serum CXCL9 levels 24 hours after infusion of NI-0501 antibody and total IFNγ pre-dose levels in an ongoing Phase 2 pilot study in patients with primary HLH.

[0026] [Diagram 3] 3A and 3B are a series of graphs showing the correlation between serum CXCL9 and IFNγ levels in patients with macrophage activation syndrome (MAS) secondary to systemic juvenile idiopathic arthritis (sJIA) and in patients with active sJIA.

[0027] [Figure 4-1] Figures 4A-1, 4A-2, 4B-1, 4B-2, 4C-1, 4C-2, 4D-1, and 4D-2 are a series of graphs showing correlations of IFNγ and serum CXCL9 levels with clinical parameters in patients with active sJIA and MAS secondary to sJIA. [Figure 4-2] Figures 4A-1, 4A-2, 4B-1, 4B-2, 4C-1, 4C-2, 4D-1, and 4D-2 are a series of graphs showing correlations of IFNγ and serum CXCL9 levels with clinical parameters in patients with active sJIA and MAS secondary to sJIA.

[0028] [Diagram 5] FIG. 5 is a graph showing that IFNγ was fully neutralized as indicated by undetectable levels of IFNγ-induced chemokines.

[0029] [Figure 6]FIG. 6 is a graph showing improvement in HLH disease activity during treatment with NI-0501 (at 2 weeks and end of treatment): percent of patients with platelet count >100×109 / L, neutrophil count >1×109 / L, fibrinogen >1.5 g / L, and at least a 25% decrease in ferritin.

[0030] [Figure 7] Figures 7A and 7B are a series of graphs showing the correlation between pre-dose CXCL9 levels and total IFNγ levels 24 hours after NI-0501 infusion. The insert shown in Figure 7B shows examples of individual IFNγ and CXCL9 profiles during treatment with NI-0501.

[0031] [Figure 8] Figures 8A, 8B, 8C, and 8D are a series of graphs showing serum levels of IFNγ and CXCL9, CXCL10, and CXCL11 in individual patients for whom paired samples were available during active MAS and during active sJIA without MAS (Act sJIA) at the time of sampling. Significance levels (p) were obtained using the Wilcoxon rank test for paired samples.

[0032] [Figure 9] Figures 9A and 9B are a series of graphs showing changes in white blood cell (WBC) and platelet (PLT) counts and ferritin levels (Figure 9A) and serum levels of IFNγ, CXCL9, CXCL10, and CXCL11 (Figure 9B) in one patient who had three episodes of MAS during the course of sJIA.

[0033] [Figure 10-1]Figures 10A, 10B, 10C, 10D, 10E, 10F, 10G, 10H, 10I, and 10J are a series of graphs showing the correlation of IFNγ and CXCL9 levels with ferritin levels, neutrophil and platelet counts, and with LDH and ALT levels in patients with active MAS at the time of sampling (red circles) and in patients with active sJIA without MAS at the time of sampling (black triangles). The Spearman correlation coefficient (Rs) and significance level (p) for each correlation are shown in Table 3. [Figure 10-2] Figures 10A, 10B, 10C, 10D, 10E, 10F, 10G, 10H, 10I, and 10J are a series of graphs showing the correlation of IFNγ and CXCL9 levels with ferritin levels, neutrophil and platelet counts, and with LDH and ALT levels in patients with active MAS at the time of sampling (red circles) and in patients with active sJIA without MAS at the time of sampling (black triangles). The Spearman correlation coefficient (Rs) and significance level (p) for each correlation are shown in Table 3.

[0034] [Figure 11-1] 11A, 11B, 11C, 11D, 11E, and 11F are a series of graphs showing the relationship between IFNγ and CXCL9 and CXCL10 production in MAS. Panel A: Correlation between levels of IFNγ and levels of CXCL9 and CXCL10 in patients with MAS at the time of sampling. The Spearman correlation coefficient (Rs) and significance level (p) for each correlation are shown in Table 3. [Figure 11-2] 11A, 11B, 11C, 11D, 11E, and 11F are a series of graphs showing the relationship between IFNγ and CXCL9 and CXCL10 production in MAS. Panel A: Correlation between levels of IFNγ and levels of CXCL9 and CXCL10 in patients with MAS at the time of sampling. The Spearman correlation coefficient (Rs) and significance level (p) for each correlation are shown in Table 3.

[0035] [Figure 12]FIG. 12 is a schematic diagram of the screening, treatment, and follow-up portions of the study presented in Example 7.

[0036] [Figure 13] 13A and 13B are graphs showing the effect of NI-0501 administration on body temperature in two patients with a body temperature >37.5° C. at the start of treatment with NI-0501.

[0037] [Figure 14] FIG. 14 is a series of graphs and tables showing the effect of NI-0501 administration on neutrophil counts in patients.

[0038] [Figure 15] FIG. 15 is a series of graphs and tables showing the effect of NI-0501 administration on platelet counts in patients.

[0039] [Figure 16] FIG. 16 is a series of graphs and tables showing the effect of NI-0501 administration on serum levels of ferritin in patients.

[0040] [Figure 17] FIG. 17 is a series of graphs and tables showing the effect of NI-0501 administration on glucocorticoid tapering in patients.

[0041] [Figure 18] Figure 18 is a graph showing that administration of NI-0510 maintained IFNγ neutralization until the time of HSCT. The HLH response to treatment with NI-0501 also persisted until transplant.

[0042] [Figure 19] FIG. 19 is a schematic diagram of the screening, treatment, and follow-up portions of the study presented in Example 8. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] The compositions and methods presented herein use a fully human IgG1 anti-interferon gamma (IFNγ) monoclonal antibody (mAb), referred to herein as NI-0501, which binds and neutralizes IFNγ. NI-0501 binds to soluble and receptor (IFNγR1)-bound forms of IFNγ. Because NI-0501 is a human IgG1, it retains the properties of this immunoglobulin isotype, including the ability to associate with Fcγ receptors and bind complement. IFNγ is one of the most potent and versatile cytokines of the immune system. IFNγ is crucial for innate and adaptive immunity against viral and intracellular bacterial infections. After binding to its receptor, IFNγ acts to generate a variety of physiological and cellular responses. Over the last two decades, numerous studies have implicated IFNγ in the pathogenesis and maintenance of inflammatory diseases (e.g., Billiau A., "Interferon-gamma: biology and role in pathogenesis." Adv. Immunol. 1996;62:61-130; Schoenborn JR, Wilson CB., "Regulation of interferon-gamma during innate and adaptive immune responses." Adv. Immunol. 2007;96:41-101; and Zhang SY, Boisson-Dupuis S, Chapgier A, et al., "Inborn errors of interferon (IFN)-mediated immunity in (See humans: insights into the respective roles of IFN-alpha / beta, IFN-gamma, and IFN-lambda in host defense., Immunol. Rev., 2008;226:29-40.) IFNγ is produced primarily by natural killer (NK) and natural killer T (NKT) cells as part of the innate immune response, following the development of antigen-specific immunity by CD4 Th1 and CD8 cytotoxic T lymphocyte (CTL) effector T cells.

[0044] The compositions and methods presented herein are useful in treating diseases, disorders and conditions associated with elevated IFNγ.Diseases, disorders and conditions suitable for treatment or prevention using the compositions and methods of the present invention include, for example, hemophagocytic lymphohistiocytosis (HLH), graft-versus-host disease, paraneoplastic cerebellar degeneration, hemorrhagic fever, sarcoidosis, adult-onset Still's disease, transplant failure, graft rejection, and / or inflammatory disorders associated with graft rejection.Graft rejection includes solid organ transplantation disorder, bone marrow acute graft rejection.In addition, the compositions and methods are also useful in treating or mitigating the side effects of CAR-T cell therapy.

[0045] HLH is a syndrome characterized by severe dysfunction or absence of cytotoxic function by NK and CD8+ T cells, accompanied by marked activation of the immune system. HLH includes primary (genetic / familial) HLH and secondary HLH, both of which are clinically explained by dysregulation of the immune system leading to severe hypercytokinemia with deleterious consequences for various tissues and organs (Henter JI, Elinder G, Soder O, et al., "Hypercytokinemia in familial hemophagocytic lymphohistiocytosis." Blood, 1991;78:2918-2922). HLH classification is shown in Table 9 below. HLH occurs in both adult and pediatric patients. [Table 9]

[0046] Primary HLH is a heterogeneous autosomal recessive genetic disorder. It is mostly seen in infancy and early childhood, with an estimated prevalence of 1 in 50,000 live births in Europe (Henter JI, Elinder G, Soder O, Ost A. Incidence in Sweden and clinical features of familial hemophagocytic lymphohistiocytosis., Acta Paediatr. Scand., 1991;80:428-435). The disease is always fatal, and if untreated, the median survival is less than 2 months after the onset of symptoms (Janka GE. Familial hemophagocytic lymphohistiocytosis., Eur. J. Pediatr., 1983;140:221-230; and Arico M, Janka G, Fischer A, Henter JI, Blanche S, Elinder G, Martinetti M, Rusca MP Hemophagocytic lymphohistiocytosis Report of 122 children from the International Registry. FHL Study Group of the Histiocyte Society., Leukemia., February 1996;10(2):197-203).

[0047] The cytotoxic dysfunction present in HLH leads to hypercytokinemia and hemophagocytosis, which in turn cause all the classic symptoms of HLH (Dhote R, Simon J, Papo T, et al. Reactive hemophagocytic syndrome in adult systemic disease: report of twenty-six cases and literature review. Arthritis Rheum. 2003;49:633-639; Risdall RJ, McKenna RW, Nesbit ME, et al. Virus-associated hemophagocytic syndrome: a benign histiocytic proliferation distinct from malignant histiocytosis. Cancer 1979;44:993-1002; and Risdall RJ, Brunning RD, Hernandez JI, Gordon DH. Bacteria-associated hemophagocytic syndrome. Cancer, 1984;54:2968-2972). Typical symptoms of HLH include, for example, prolonged fever, splenomegaly, hepatomegaly, cytopenia, hyperferritinemia, hypertriglyceridemia, hypofibrinogenemia, hemophagocytosis, hypercytokinemia, and / or lymphohistiocytic infiltration, bone marrow hypoplasia, and meningeal infiltration.

[0048] Cytokines that are elevated in HLH patients include IFNγ, interleukin 6 (IL-6), IL-10, tumor necrosis factor (TNF)α, IL-8, macrophage colony-stimulating factor (MCSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0049] HLH can also occur during the course of infection, rheumatic or neoplastic disease, in which case it is referred to as secondary HLH. Secondary HLH presents with the same signs and symptoms as the primary form and can be equally severe. Current treatment of secondary HLH aims to address the underlying cause of the disease. This is certainly the case in the case of HLH caused by infections such as leishmaniasis. Of note, the presence of certain infections, especially viral infections such as those caused by CMV or EBV, is very often a trigger for the manifestation of the primary form of HLH. This finding is supported by evidence in animal models of primary HLH that infection with lymphocytic choriomeningitis virus (LCMV) is required for disease development (Jordan MB, Hildeman D, Kappler J, Marrack P. An animal model of hemophagocytic lymphohistiocytosis (HLH): CD8+ T cells and interferon gamma are essential for the disorder. Blood, 2004;104:735-743; Pachlopnik SJ, Ho CH, Chretien F, et al. Neutralization of IFNgamma defeats haemophagocytosis in LCMV-infected perforin- and Rab27a-deficient mice. EMBO Mol. Med., 2009;1:112-124; Kogl T, Muller J, Jessen B, et al. Hemophagocytic lymphohistiocytosis in Syntaxin-11-deficient mice: T-cell exhaustion limits fatal disease., Blood., 2013;121:604-613; and Sepulveda FE, Debeume F, Menasche G, et al., Distinct severity of HLH in both human and murine mutants with complete loss of cytotoxic effector PRF1, RAB27A, and STX11, Blood., 2013; vol. 121: 595-603).

[0050] When HLH manifests during neoplastic diseases, particularly hematological malignancies, often the severity of the patient's condition necessitates immediate treatment of the HLH before the underlying disease can be specifically addressed.

[0051] The presence of signs and symptoms of HLH in patients suffering from rheumatic diseases such as systemic juvenile idiopathic arthritis (sJIA) and systemic lupus erythematosus (SLE), often referred to by rheumatologists as macrophage activation syndrome (MAS), may precede the appearance of the rheumatic disease itself. The majority of patients with MAS have impaired NK and perforin function tests, and a significant number of patients show polymorphic or heterozygous mutations in PRF1 and UNC13D. MAS is an extremely severe and life-threatening condition, but it usually resolves once appropriate treatment is initiated, consisting in most cases of corticosteroids and cyclosporine. However, in approximately 15% of patients developing MAS, the disease may be difficult to control, and the use of etoposide may be considered (Minoia F, Davi S, Horne AC, et al. Clinical Features, Treatment, and Outcome of Macrophage Activation Syndrome Complicating Systemic Juvenile Idiopathic Arthritis: A Multinational, Multicenter Study of 362 Patients. Arthritis & Rheumatism, 2014;66:3160-3169).

[0052] Although primary HLH is primarily understood as a childhood disease, HLH is a condition that can be seen in adults, and increasing awareness indicates that this may occur more frequently than previously understood. In the majority of adult patients, the disease arises during malignancy (mainly non-Hodgkin's lymphoma), infection, autoinflammatory or autoimmune disease, and iatrogenic immunodeficiency.

[0053] Currently, there are no drugs approved for the treatment of HLH. However, experts in the field have established guidelines for managing patients with HLH (Henter JI, Horne AC, Arico' M, Egeler RM, Filipovich AH, Imashuku S, Ladisch S, McClain K, Webb D, Winiarski J, and Janka, Diagnostic and Therapeutic Guidelines for Hemophagocytic Lymphohistiocytosis Blood Cancer, 2007;48:124-131; Henter JI, Samuelsson-Horne A, Arico M, et al., Treatment of hemophagocytic lymphohistiocytosis with HLH-94 immunochemotherapy and bone marrow transplantation., Blood, 2002;100:2367-2373; and Jordan MB, Allen CE, Weitzman S, Filipovich AH, McClain KL. How I treat hemophagocytic lymphohistiocytosis., Blood, 2011;118:4041-4052).

[0054] Management of primary HLH patients currently involves the following steps (Henter et al., Blood Cancer, 2007): (i) induction treatment for 8 weeks with a combination of corticosteroids and immunosuppressants (e.g., etoposide, CsA, alemtuzumab, antithymocyte globulin); (ii) maintenance treatment until transplant; and (iii) transplant in all patients with an identified genetic defect and, ultimately, in cases of very severe HLH without a disease-associated mutation.

[0055] The primary goal of induction therapy is to suppress the life-threatening inflammatory process that characterizes HLH, thereby enabling transplantation in patients who require it (Horne A, Janka G, Maarten ER, et al. Haematopoietic stem cell transplantation in haemophagocytic lymphohistiocytosis. Br. J. Haematol. 2005;129:622-630). Transplantation is the only curative treatment for HLH associated with highly penetrant genetic mutations (Henter et al., Blood, 2002).

[0056] Despite the adoption of such guidelines, the overall mortality rate for primary HLH remains approximately 40-50% (Henter et al., Blood, 2002; Trottestam H, Horne A, Arico M, et al., Chemoimmunotherapy for hemophagocytic lymphohistiocytosis: long-term results of the HLH-94 treatment protocol., Blood, 2011; 118:4577-4584).

[0057] The need to use drugs with serious short-term and long-term safety issues during the induction period further contributes to the already high mortality rate. The compositions and methods presented herein have been developed as targeted treatments that ensure efficacy with less toxicity.

[0058] Over the last few years, there has been increasing evidence of a central role for IFNγ in the development of HLH (Henter JI, Elinder G, Soder O, et al. Hypercytokinemia in familial hemophagocytic lymphohistiocytosis. Blood, 1991;78:2918-2922; Jordan MB, Hildeman D, Kappler J, Marrack P. An animal model of hemophagocytic lymphohistiocytosis (HLH): CD8+ T cells and interferon gamma are essential for the disorder. Blood, 2004;104:735-743; Pachlopnik SJ, Ho CH, Chretien F, et al. Neutralization of IFNgamma defeats haemophagocytosis in LCMV-infected perforin- and Rab27a-deficient mice., EMBO Mol. Med., 2009;1:112-124;Behrens EM, Canna SW, Slade K, et al., Repeated TLR9 stimulation results in macrophage activation syndrome-like disease in mice., J. Clin. Invest, 2011;121:2264-2277;Xu XJ, Tang YM, Song H, MD, Yang SL, Xu WQ, Zhao N, Shi SW, Shen HP, Mao JQ, Zhang LY, and Pan BH, Diagnostic Accuracy of a Specific Cytokine Pattern in Hemophagocytic Lymphohistiocytosis, Children J Pediatr, 2011;and Risma K, Jordan MB. Hemophagocytic lymphohistiocytosis: updates and evolving concepts., Curr. Opin. Pediatr., 2012; vol. 24: pp. 9-15).

[0059] The genetic mutations that characterize the primary form of HLH all affect proteins involved in the same process, ultimately impairing cytotoxic activity. Perforin mutations were the first to be identified in HLH patients.

[0060] Perforin knockout (KO) mice are considered a relevant model for human disease. Indeed, when infected with LCMV, these mice develop all of the diagnostic and many of the clinical and laboratory hallmarks of the human disease and die if untreated. For these reasons, perforin KO mice have been used to study the pathophysiology of HLH. The HLH-like pathology that occurs in perforin KO mice is dependent on CD8+ T cells and IFNγ produced in response to antigenic stimulation.

[0061] It has been demonstrated that neutralization of high circulating levels of IFNγ by administration of anti-IFNγ antibodies not only reverses clinical and laboratory abnormalities but also dramatically improves survival, whereas removal of any other cytokine had no effect on survival (Jordan et al., Blood, 2004; Pachlopnik et al., EMBO Mol. Med., 2009).

[0062] Two models of secondary HLH have been investigated in the context of the NI-0501 development program. In one model, repeated administration of CpG (leading to TLR9 stimulation) is used to mimic chronic severe overstimulation in healthy mice (i.e. with normal genes for cytotoxic pathways) as a model of HLH secondary to infection. These mice do not necessarily die, but develop typical clinical and laboratory features of HLH. Neutralization of IFNγ by administration of anti-IFNγ antibodies reverses the clinical and laboratory features of the disease. Interestingly, this model demonstrates that administration of anti-IFNγ antibodies also leads to sufficient neutralization of the effects of IFNγ in relevant target tissues such as the liver and spleen (manuscript in preparation).

[0063] To test the physiopathology of secondary HLH occurring in the context of rheumatic diseases, we generated an animal model using IL-6 transgenic mice that express high levels of IL-6 similar to those occurring in patients with sJIA, the rheumatic disease most frequently associated with secondary forms of HLH. When challenged with Toll-like receptor (TLR) ligands, these mice die with many of the hallmarks of the human disease (Strippolli R, Carvallo F, Scianaro R, et al. Amplification of the response to Toll-like receptor ligands by prolonged exposure to interleukin-6 in mice: Implication for the pathogenesis of macrophage activation syndrome. Arthritis & Rheumatism, 2012;64:1680-1688). Neutralizing IFNγ by administration of anti-IFNγ antibodies markedly improved survival and reverted laboratory parameters in these mice (Prencipe G, et al., Manuscript in preparation).

[0064] The importance of IFNγ in HLH is further strengthened by the high concentration of circulating IFNγ levels in primary HLH patients (Henter et al., Blood, 1991; Xu et al., J Pedatr, 2011). In a series of 71 patients monitored from HLH diagnosis to treatment and follow-up, all patients had IFNγ levels above the upper limit of normal (17.3 pg / mL), notably, 53.5% had levels above 1000 pg / mL. It was also reported that IFNγ levels rise quickly initially and can fall from >5000 pg / mL to normal within 48 hours of effective treatment of HLH.

[0065] More recently, an observational study in patients with secondary forms of HLH demonstrated high levels of IFNγ in both patients with HLH secondary to infection and in patients with HLH occurring in the context of sJIA. The levels of CXCL9, CXCL10 and CXCL11, three chemokines known to be induced by IFNγ, were also significantly elevated. Notably, the levels of IFNγ and the levels of the three IFNγ chemokines were found to significantly correlate with laboratory parameters of disease severity, such as ferritin, platelet count and transaminases (Bracaglia et al., submitted).

[0066] Since hypercytokinemia and tissue infiltration by activated lymphocytes and histiocytes are involved in all HLH conditions and depend on CD8+ T cell hyperactivity and high IFNγ levels, neutralization of IFNγ constitutes a rational therapeutic approach. Indeed, there are currently no drugs available that specifically target CD8+ T cells, and targeting individual cytokines downstream of IFNγ is not always feasible.

[0067] Therefore, based on data from animal models of primary and secondary HLH, in which the pivotal role played by IFNγ in the pathogenesis of this disease is confirmed, as well as from observations made in both patients with primary HLH and those with secondary HLH, neutralization of IFNγ provides a robust rationale for developing targeted treatments against HLH that should be effective with no or limited toxicity.

[0068] The present disclosure also provides compositions and methods useful in identifying or alternatively accurately discriminating between patient populations suffering from disorders in which the patients have elevated levels of CXCL9 alone or in combination with one or more additional interferon gamma (IFNγ)-related biomarkers. In particular, the present disclosure provides compositions and methods for detecting CXCL9 levels as a biomarker for IFNγ production in hemophagocytic lymphohistiocytosis (HLH), secondary HLH, and / or macrophage activation syndrome (MAS).

[0069] A growing body of evidence in animal models indicates a central pathogenic role for IFNγ in primary hemophagocytic lymphohistiocytosis (HLH). High levels of IFNγ are also found in humans with HLH. It has previously been reported that high levels of IFNγ and three IFNγ-related chemokines, CXCL9, CXCL10 and CXCL11, are observed in patients with active MAS, a form of secondary HLH that occurs in the setting of systemic juvenile idiopathic arthritis (sJIA) (e.g., Bracaglia C., Caiello J. et al., 2011). See I, De Graaf K. et al., Pediatric Rheumatology, 2014, Vol. 12(Suppl 1):O3. Indirect evidence in mice suggests that IFNγ is produced mostly in peripheral tissues and may be at relatively low levels in the blood.

[0070] The term macrophage activation syndrome (MAS) refers to a severe and potentially fatal complication of chronic inflammatory rheumatic diseases. MAS commonly occurs in the setting of systemic juvenile idiopathic arthritis (sJIA), with 10-20% of patients developing the syndrome during the course of the disease. Although more rare, MAS can also occur in systemic lupus erythematosus, Kawasaki disease, and other autoimmune and autoinflammatory disorders. In sJIA, MAS commonly occurs during the active phase of disease, including at disease onset. An infectious trigger can be identified in a high percentage of patients. Typical features of MAS include fever, splenomegaly, bleeding, and signs of liver, central nervous system, and kidney involvement that can lead to multiple organ failure. Laboratory abnormalities include decreased white blood cells, platelets, and hemoglobin, hypertransaminasemia, marked increase in ferritin, and evidence of intravascular activation of the coagulation system (Ravelli, A. et al., Macrophage activation syndrome as part of systemic juvenile idiopathic arthritis: diagnosis, genetics, pathophysiology and treatment., Genes Immun., 13(4):289-98). MAS causes significant morbidity and mortality, accounting for a relevant portion of deaths attributable to sJIA (Minoia, F. et al., Clinical features, treatment, and outcome of macrophage activation syndrome complicating systemic juvenile idiopathic arthritis: a multinational, multicenter study of 362 patients. Arthritis Rheumatol 2014;66(11):3160-9;Hashkes, PJ et al., Mortality outcomes in pediatric rheumatology in the US. Arthritis Rheum 2010;62(2):599-608).A better understanding of disease pathogenesis, together with the resulting identification of new therapeutic targets and possible development of targeted therapies, may lead to significant improvements in the management and outcome of MAS.

[0071] MAS shares most clinical features and laboratory abnormalities with hemophagocytic lymphohistiocytosis (HLH) and is in fact currently classified among secondary or reactive HLH (sec-HLH) (Jordan, MB, et al., How I treat hemophagocytic lymphohistiocytosis., Blood, 2011, 118(15):4041-52). The primary form of HLH (p-HLH) is caused by mutations in genes encoding proteins involved in granule exocytosis, including PRF1, UNC13D, STXBP2, STX11, RAB27A and XIAP, which generally lead to defective cytotoxic activity of CD8+ lymphocytes and NK cells. According to the current classification, HLH is defined as secondary or reactive in the absence of an identifiable genetic cause and / or familial inheritance. Sec-HLH may occur in the absence of a demonstrable trigger or in the context of infection, malignancy, or rheumatic disease, the latter commonly referred to as MAS. The genetic basis for the development of MAS is gradually being elucidated, and several studies have demonstrated an association between MAS, and sec-HLH in general, and heterozygosity for low-penetrance variants or mutations in the same causative genes as p-HLH (Kaufman, KM, et al., Whole-exome sequencing reveals overlap between macrophage activation syndrome in systemic juvenile idiopathic arthritis and familial hemophagocytic lymphohistiocytosis., Arthritis Rheumatol, 2014, 66(12):3486-95; Vastert, SJ, et al., Mutations in the perforin gene can be linked to macrophage activation syndrome in patients with systemic onset juvenile idiopathic arthritis., Rheumatology(Oxford) 2010,49(3):441-9.; Zhang, K. et al., Macrophage activation syndrome in patients with systemic juvenile idiopathic arthritis is associated with MUNC 13-4 polymorphisms., Arthritis Rheum 2008,58(9):2892-6; and Zhang, M. et al., Genetic defects in cytolysis in macrophage activation syndrome. Curr Rheumatol Rep 2014,16(9):439; and Bracaglia C, Sieni E, Da Ros M, et al., Mutations of familial hemophagocytic lymphohistiocytosis (FHL) related genes and abnormalities of cytotoxicity function tests in patients with macrophage activation syndrome (MAS) occurring in systemic juvenile idiopathic arthritis (sJIA). Pediatric Rheumatology, 2014, vol. 12(Suppl 1):53. These similarities in genetic background between p-HLH and MAS further support a shared pathogenic mechanism.

[0072] Studies in patients with p-HLH as well as in mouse models of p-HLH support the hypothesis that defective cytotoxic activity and abnormalities in antigen-presenting cell (APC)-CD8+ T cell crosstalk lead to defective silencing of immune responses and abnormal T cell activation. This leads to uncontrolled immune activation and production of proinflammatory cytokines by T lymphocytes and macrophages, leading to organ damage. Studies in animal models of p-HLH performed in perforin-deficient and Rab27-deficient mice indicate a crucial role for interferon-gamma (IFNγ) produced by activated CD8+ T cells. In perforin-deficient mice, neutralization of IFNγ leads to survival of an otherwise fatal syndrome along with reversal of biochemical and hematological abnormalities (Jordan, MB, et al., An animal model of hemophagocytic lymphohistiocytosis (HLH): CD8+T cells and interferon gamma are essential for the disorder., Blood, 2004, 104(3):735-43; Pachlopnik Schmid, J., et al., Neutralization of IFNgamma defeats haemophagocytosis in LCMV-infected perforin- and Rab27a-deficient mice., EMBO Mol Med, 2009, 1(2):112-24). In Rab27-deficient mice, where the disease is not lethal, neutralization of IFNγ leads to a marked improvement in peripheral organ involvement, including the central nervous system (Pachlopnik, 2009).High circulating IFNγ levels have also been found in patients with HLH diagnosed according to the HLH 2004 diagnostic guidelines (My, LT et al., Comprehensive analyses and characterization of haemophagocytic lymphohistiocytosis in Vietnamese children. Br J Haematol, 2010,148(2):301-10; Takada, H. et al., Increased serum levels of. interferon-gamma-inducible protein 10 and monokine induced by gamma interferon in patients with haemophagocytic lymphohistiocytosis., Clin Exp Immunol, 2003, 133(3):448-53; Tang, Y. et al., Early diagnosis and prognostic significance of a specific Th1 / Th2 cytokine pattern in children with haemophagocytic syndrome. Br J Haematol 2008, 143(1):84-91;Xu, XJ et al., Diagnostic accuracy of a specific cytokine pattern in hemophagocytic lymphohistiocytosis in children., J Pediatr 2012, 160(6):984-90e1), and therefore not necessarily based on the presence of a genetic mutation. It should be noted that these studies included a variable but significant proportion of patients without a demonstrable genetic cause (Ibid).

[0073] To search for biomarkers of in vivo production of IFNγ, the study presented herein was designed to evaluate the correlation of serum levels of IFNγ and three IFNγ-related chemokines with themselves and with laboratory parameters of disease activity in patients with active MAS. Specifically, circulating levels of IFNγ, CXCL9, CXCL10, CXCL11 and IL-6 were measured in patients with sJIA, of which about 37% (20 out of 54) had MAS at the time of sampling. The correlation of IFNγ levels with CXCL9, CXCL10 and CXCL11 levels as well as the relationship between circulating levels and disease activity parameters were evaluated. In some embodiments, the biomarker is total IFNγ levels, which is useful as a pharmacodynamic biomarker.

[0074] As demonstrated herein, the levels of IFNγ and three IFNγ-related chemokines, CXCL9, CXCL10, and CXCL11, were significantly elevated in active MAS compared to active sJIA without MAS at the time of sampling. In active MAS, laboratory parameters of disease severity, such as ferritin, neutrophils, platelets, alanine aminotransferase, and lactate dehydrogenase, were significantly correlated with IFNγ and CXCL9, to a lesser extent with CXCL10 and CXCL11, and were not correlated with IL-6 levels. In patients with active sJIA without MAS, there was no significant correlation between laboratory parameters and cytokine levels. In active MAS I, IFNγ levels were significantly correlated with CXCL9 levels, to a lesser extent with CXCL10 levels, and were not correlated with CXCL11 levels.

[0075] High levels of IFNγ and CXCL9 present in patients with active MAS are significantly correlated with laboratory parameters of disease severity. In patients with active MAS, IFNγ and CXCL9 are closely correlated. Since CXCL9 has been shown to be induced only by IFNγ and not by other interferons (see, for example, Groom JR and Luster AD, Immunol Cell Biol, 2011, February;89(2):207-15), the findings disclosed herein demonstrate that CXCL9 is a biomarker of IFNγ production in MAS.

[0076] The studies presented herein also demonstrate that levels of IFNγ and three chemokines known to be induced by IFNγ, chemokine (CXC motif) ligand 9 (CXCL9), CXL10 and CXCL11, are elevated in patients with MAS complicating sJIA but not in patients with active sJIA without MAS. Furthermore, in these patients, levels of IFNγ, CXCL9, CXCL10 and CXCL11 correlated with laboratory parameters of disease severity.

[0077] The present disclosure provides compositions and methods using neutralizing anti-IFNγ antibodies or antigen-binding fragments thereof to treat, prevent, and / or delay the onset or progression of, or alleviate symptoms associated with transplant failure, transplant rejection, and / or inflammatory disorders associated with transplant rejection, such as solid organ transplant disorder, bone marrow acute graft rejection, etc. The present disclosure provides compositions and methods using neutralizing anti-IFNγ antibodies or antigen-binding fragments thereof to treat, inhibit, delay the progression of, or alternatively reverse, symptoms of graft-versus-host disease (GvHD) in subjects who have undergone or are undergoing a transplant involving biological material or a series of transplants involving biological material. The present disclosure provides compositions and methods using neutralizing anti-IFNγ antibodies or antigen-binding fragments thereof to prolong the survival of transplanted biological material. The present disclosure provides compositions and methods using neutralizing anti-IFNγ antibodies or antigen-binding fragments thereof to treat, prevent, and / or delay the onset or progression of, or alleviate symptoms associated with paraneoplastic cerebellar degeneration, hemorrhagic fever, sarcoidosis, adult-onset Still's disease, or CART-T cell therapy.

[0078] The compositions and methods presented herein are useful in the transplantation of any biological material, including, for example, cells, tissue(s), bone marrow, and / or organ(s), including, as non-limiting examples, the heart, kidney, pancreas, liver, and / or intestine. In some embodiments, the transplanted biological material is an allogeneic biological material. In some embodiments, the transplanted biological material is bone marrow. In some embodiments, the transplanted biological material is a population of hematopoietic stem cells. In some embodiments, the transplanted biological material is or is derived from one or more hepatic cells.

[0079] In the compositions and methods provided herein, NI-0501 is administered to a subject in need thereof to treat, prevent, and / or delay the onset or progression of or alleviate symptoms associated with transplant failure, transplant rejection, and / or inflammatory disorders associated with transplant rejection.In some embodiments, transplant rejection, also referred to herein as transplant failure, is acute.In some embodiments, transplant rejection is hyperacute.

[0080] Neutralizing anti-IFNγ antibodies of the invention include, for example, the heavy chain complementarity determining regions (CDRs) shown in Table 1A below, the light chain CDRs shown in Table 1B below, and combinations thereof. The amino acids encompassing the complementarity determining regions (CDRs) as defined by Chothia et al., 1989, EA Kabat et al., 1991 are highlighted below in underlined and italicized text (see Chothia, C et al., Nature, 342:877-883 (1989); Kabat, EA et al., Sequences of Protein of immunological interest, 5th ed., US Department of Health and Human Services, US Government Printing Office (1991)). Table 1A. VH CDR sequences from antibody clones that bind and neutralize IFNγ [Table 1A-1] [Table 1A-2] Table 1B. VL CDR sequences from antibody clones that bind and neutralize IFNγ [Table 1B-1] [Table 1B-2]

[0081] Exemplary antibodies of the invention include, for example, the anti-IFNγ antibodies described in PCT Publication No. WO2006 / 109191, the contents of which are incorporated herein by reference in their entirety.

[0082] Exemplary antibodies of the invention include, for example, an antibody designated herein as NI-0501, which binds to human IFNγ. The heavy, light, variable heavy (VH) and variable light (VL) chain sequences of the NI-0501 antibody are shown below, with the CDR sequences of the VH and VL amino acid sequences underlined: [ka] [ka] [ka]

[0083] Suitable anti-IFNγ antibodies include those described in U.S. Patent No. 7,700,098, the entirety of which is incorporated herein by reference. Some exemplary antibodies include ARC1.2R3P2_A6 ("A6"), ARC1.2R3P2_B4 ("B4"), ARC1.2R3P2_B9 ("B9"), ARC1.2R3P2_C9 ("C9"), ARC1.2R3P2_C10 ("C10"), ARC1.2R3P2_D3 ("D3"), ARC1.2R3P2_D6 ("D6"), , antibodies designated ARC1.2R3P2_D8 ("D8"), ARC1.2R3P2_E1 ("E1"), ARC1.2R3P2_F8 ("F8"), ARC1.2R3P2_F9 ("F9"), ARC1.2R3P2_G7 ("G7"), ARC1.2R3P2_G9 ("G9"), and ARC1.2R3P2_G10 ("G10"). The sequences of these antibodies are shown below. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0084] In some embodiments, the IFNγ antibody is formatted as an IgG isotype. In some embodiments, the IFNγ antibody is formatted as an IgG1 isotype.

[0085] In some embodiments, the IFNγ antibody of the invention specifically binds to human and / or cynomolgus IFNγ, and the antibody binds to the same epitope as the NI-0501 antibody, the A6 antibody, the B4 antibody, the B9 antibody, the C9 antibody, the C10 antibody, the D3 antibody, the D6 antibody, the D8 antibody, the E1 antibody, the F8 antibody, the F9 antibody, the G7 antibody, the G9 antibody, and / or the G10 antibody. Treatment Method

[0086] The compositions and methods are useful in treating any of a variety of disorders associated with interferon-gamma (IFNγ) expression and / or activity, including abnormal IFNγ expression and / or activity. The compositions and methods of the present disclosure are useful in treating hemophagocytic lymphohistiocytosis (HLH). HLH is a rare, severe and life-threatening disease of pathological immune activation characterized by clinical signs and symptoms of extreme inflammation (fever, splenomegaly, cytopenia, coagulopathy) that lead to the development of abnormal immune-mediated pathology that can ultimately cause multiple organ failure and death through tissue damage (Henter JI, Elinder G, Soder O, Hansson M, et al.: Hypercytokinemia in familial hemophagocytic lymphohistiocytosis., Blood, 1991, 78:2918-2922). HLH includes primary (genetic / familial) HLH and secondary HLH.

[0087] Primary HLH is a heterogeneous autosomal recessive genetic disorder that is mostly seen in infancy and early childhood, with an estimated prevalence in Europe of 1 per 50,000 live births (Janka GE:Familial hemophagocytic lymphohistiocytosis., Eur. J. Pediatr., 1983, vol. 140:221-230). The disease is invariably fatal, and if untreated, the median survival is less than 2 months after the onset of symptoms (Filipovich AH:Hemophagocytic lymphohistiocytosis (HLH) and related disorders., Hematology Am Soc Hematol Educ Program, 2009:127-131).

[0088] The genetic defects in primary HHL all affect genes involved in the cytotoxic pathways of NK cells and / or cytotoxic lymphocytes required for the elimination of activated macrophages, encoding proteins for perforin synthesis, cytolytic granule maturation, granule exocytosis and release granule exocytosis or function (Filipovich, A., K. McClain, and A. Grom., 2010, Histiocytic disorders: recent insights into pathophysiology and practical guidelines., Biol., Blood Marrow Transplant., vol. 16(1 sup.):S82-S89). In approximately 20-40% of patients with primary HLH, the cytotoxic dysfunction that characterizes the HLH syndrome is due to mutations in the gene encoding perforin (PRF1), a cytolytic protein of cytotoxic granules that is a key regulator of T-cell and natural killer cell-mediated cytolysis. In about 10% of patients, the disease is caused by mutations in the UNC13D gene, which encodes a protein involved in the release of perforin into target cells. In addition, some immunodeficiency syndromes, such as Griscelli syndrome type 2 (GS-2) and Chediak-Higashi syndrome (CHS), frequently coexist with HLH (Janka GE, Lehmberg K: Hemophagocytic lymphohistiocytosis: pathogenesis and treatment., Hematology Am Soc Hematol Educ. Program, 2013, Vol. 2013: 605-611).

[0089] Secondary forms of HLH can arise during the course of infection, autoimmune / rheumatic disease, or in association with malignancy. Secondary forms present with the same signs and symptoms as primary HLH and can be equally severe.

[0090] The compositions and methods of the present disclosure are useful in the treatment of secondary HLH.The compositions and methods of the present disclosure are useful in the treatment of macrophage activation syndrome (MAS).

[0091] MAS is a severe, potentially life-threatening, complication of rheumatic diseases caused by excessive activation and expansion of T lymphocytes and macrophages. Uncontrolled expansion of these immune cells leads to a marked hypercytokinemia and a hyperinflammatory state with fever, cytopenia, hepatosplenomegaly, liver dysfunction, coagulopathy and hyperferritinemia that can progress to multiple organ failure and death (Schulert GS, Grom AA: Pathogenesis of macrophage activation syndrome and potential for cytokine-directed therapies. Annu. Rev. Med. 2015, 66:145-159). MAS is classified among the secondary or acquired forms of HLH because of its strong clinical and pathological similarities to HLH. Indeed, it has recently been demonstrated that the majority of patients with MAS have impaired NK and perforin function tests, and that a significant number of MAS patients display polymorphisms or heterozygous mutations in PRF1 and UNC13D (Zhang M, Behrens EM, Atkinson TP, Shakoory B et al.: Genetic defects in cytolysis in macrophage activation syndrome., Curr Rheumatol Rep, 2014, 16:439).

[0092] MAS occurs most frequently in patients with sJIA and more rarely in those with systemic lupus erythematosus (SLE), but has also been described, albeit more rarely, in patients with vasculitis, particularly Kawasaki disease. Approximately 7-17% of patients with SJIA develop overt MAS (Sawhney S, Woo P, Murray KJ: Macrophage activation syndrome: a potentially fatal complication of rheumatic disorders. Arch. Dis. Child. 2001, 85:421-426; Moradinejad MH, Ziaee V: The incidence of of macrophage activation syndrome in children with rheumatic disorders., Minerva Pediatr. 2011;63:459-466), and some evidence suggests that as many as one-third of patients with active systemic disease may have asymptomatic MAS (Behrens EM, Beukelman T, Paessler M, Cron RQ: Occult macrophage activation syndrome in patients with systemic juvenile idiopathic arthritis. J. Rheumatol. 2007;34:1133-1138).

[0093] MAS is potentially fatal, so timely diagnosis and immediate therapeutic intervention are essential for the proper management of this disease. The reported mortality rate of MAS reaches 20-30%, and it remains the leading source of mortality in pediatric rheumatology (Grom et al., 2013). AA, Horne A, De Benedetti F: Macrophage activation syndrome in the era of biology. therapy., Nat Rev Rheumatol., March 24, 2016, doi:10.1038 / nrrheum.2015.179).

[0094] Different sets of criteria for the diagnosis of MAS in patients with sJIA have been proposed. The HLH-2004 diagnostic guidelines (Henter J, Horne A, Arico M, Egeler RM, et al.: HLH-2004: Diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer, 2007, 48: 124-131), developed mainly for primary (hereditary) forms of HLH, are sometimes recommended. However, these show some limitations and do not apply to patients with sJIA. For example, these patients often have elevated white blood cell and platelet counts and elevated serum levels of fibrinogen as part of the sJIA inflammatory response, such that criteria such as cytopenias and hypofibrinogenemia below the threshold required by HLH-2004 only become evident in the later stages of MAS (Schulert GS, Grom AA: Pathogenesis of macrophage activation syndrome and potential for cytokine-directed therapies. Annu. Rev. Med. 2015, 66:145-159). In a significant proportion of patients with MAS, hemophagocytosis may not be present at presentation (Minoia F, Davi S, Horne A, Demirkaya E, et al.: Clinical features, treatment, and outcome of macrophage activation syndrome complicating systemic juvenile idiopathic arthritis: a multinational, multicenter study of 362 patients. Arthritis & rheumatology (Hoboken, NJ) 2014;66:3160-3169). Furthermore, hemophagocytosis, NK cell activity, and sCD25 are not routinely assessed in MAS.

[0095] An alternative approach is based on the application of Preliminary Diagnostic Guidelines (PDGs) for MAS complicating sJIA, which were generated by analysis of a cohort of patients with MAS compared with a group of patients with sJIA1 flares.

[0096] Recently, the HLH-2004 diagnostic guidelines and preliminary diagnostic guidelines for sJIA-associated MAS were compared in a large patient cohort for their ability to distinguish sJIA / MAS from sJIA (in the absence of MAS) and systemic infections (Davi S, Minoia F, Pistorio A, Horne A, et al.: Performance of current guidelines for diagnosis of macrophage activation syndrome complicating systemic juvenile idiopathic arthritis. Arthritis & rheumatology (Hoboken, NJ) 2014;66:2871-2880). Despite some limitations due to its retrospective nature, this study seems to indicate that the preliminary MAS guidelines achieve the best balance of sensitivity and specificity, and the best agreement with the diagnosis made by the treating physician. The sensitivity of the HLH-2004 criteria set was <30%. Nevertheless, it has also been reported that the proportion of patients fulfilling each single criterion for PDG is highly variable and that some clinical features (e.g., CNS dysfunction and bleeding) may be manifested at later stages of MAS, making them less sensitive in early MAS (Lehmberg K, Pink I, Eulenburg C, Beutel K, et al.: Differentiating macrophage activation syndrome in systemic juvenile idiopathic arthritis from other forms of hemophagocytic lymphohistiocytosis., The Journal of pediatrics, 2013, vol. 162: 1245-1251).

[0097] More recently, a diagnostic score (HScore) was developed and validated in a retrospective cohort of 312 patients, of whom 162 were determined to have reactive hemophagocytic syndrome (Fardet L, Galicier L, Lambotte O, Marzac C, Aumont C, Chahwan D, Coppo P, Hejblum G: Development and validation of the HScore, a score for the diagnosis of reactive hemophagocytic syndrome. Arthritis & rheumatology (Hoboken, NJ) 2014;66:2613-2620).

[0098] Nine variables (three clinical variables [i.e., known underlying immunosuppression, hypertension, and organomegaly], five biological variables [i.e., triglycerides, ferritin, serum glutamic oxaloacetic transaminase, fibrinogen levels, and cytopenias], and one cytological variable [i.e., hemophagocytic features on bone marrow aspirate]) were retained with HScore, and the probability of having hemophagocytic syndrome ranged from <1% for an HScore ≤ 90 to > 99% for an HScore ≥ 250.

[0099] Until final consensus on validated diagnostic criteria for MAS can be achieved, the challenge of distinguishing MAS from conditions with overlapping features, such as SJIA flares or sepsis-like syndromes, remains critical in clinical diagnosis by expert physicians.

[0100] Currently, there are no drugs approved for the treatment of MAS. High-dose glucocorticoids are usually the first-line treatment for MAS. In patients who fail to respond to glucocorticoids, cyclosporine A (CsA) has been proposed as an additional treatment (Stephan JL, Kone-Paut I, Galambrun C, Mouy R, Bader-Meunier B, Prieur AM: Reactive haemophagocytic syndrome in children with inflammatory disorders. A retrospective study of 24 patients., Rheumatology (Oxford, England), 2001, vol. 40: pp. 1285-1292).

[0101] As part of the HLH-94 treatment protocol developed to treat pHLH, administration of etoposide is also considered in patients in whom high-dose glucocorticoids have failed. However, the potential toxicity of the drug remains a major concern. Other current first-line HLH treatments include dexamethasone. However, treatments such as etoposide and / or dexamethasone are myelosuppressive and / or extensively immunosuppressive. Currently, there is no standard of care for second-line HLH treatment, and treatments such as alemtuzumab / ATG are severely immunosuppressive, and survival is thought to be very poor with these treatments.

[0102] The utility of biologics that inhibit the IL-1, IL-6R, or TNFα pathways in the treatment of MAS remains unclear. Biologics that inhibit these pathways have been reported to be effective in isolated cases, but few patients develop MAS in the setting of these treatments (Stern A, Riley R, Buckley L: Worsening of macrophage activation syndrome in a patient with adult onset Still's disease after initiation of etanercept therapy. J Clin Rheumatol 2001, 7:252-256; Ramanan AV, Schneider R: Macrophage activation syndrome following initiation of etanercept in a child with systemic juvenile onset rheumatoid arthritis., J. Rheumatol., 2003, 30:401-403; De Benedetti F, Brunner HI, Ruperto N, Kenwright A, et al.: Randomized trial of tocilizumab in systemic juvenile idiopathic arthritis., N. Engl. J. Med., 2012, 367:2385-2395; Ruperto N, Brunner HI, Quartier P, Constantin T, et al.: Two randomized trials of canakinumab in systemic juvenile idiopathic arthritis. N. Engl. J. Med. 2012, 367:2396-2406), as well as patients who do not respond to these treatments, have been reported, indicating that inhibition of IL-1, IL-6R or TNFα does not provide complete protection from MAS development nor effective treatment for the end-stage syndrome.

[0103] A large retrospective multicenter study investigated the clinical, laboratory, and histopathological characteristics of MAS / sJIA as well as current treatment and outcome in a total of 362 patients (Minoia F, Davi S, Horne A, Demirkaya E, et al.: Clinical features, treatment, and outcome of macrophage activation syndrome complicating systemic juvenile idiopathic arthritis: a multinational, multicenter study of 362 patients. Arthritis & rheumatology (Hoboken, NJ) 2014;66:3160-3169). At disease onset, MAS occurred in the setting of active sJIA in approximately half of patients or during an sJIA flare in 30%. An infectious trigger was identified in one-third of patients. Among the 24 patients for whom the type of infection was reported, EBV was the most common causative agent (25%). In 11 patients (3.8%), MAS was considered to be related to treatment side effects, 8 of which involved biologic agents targeting the IL-6 (N=4), IL-1 (N=3) or TNFα (N=1) pathways. Nearly all patients received glucocorticoids. Cyclosporine, biologic agents and etoposide were given in 61%, 15% and 12% of patients, respectively.

[0104] Thus, the identification of effective therapeutic regimens for MAS is an area of ​​high unmet medical need. More than 50% of patients with sJIA and MAS may not respond to systemic glucocorticoids alone or require prolonged treatment at high doses, with significant associated morbidity. If patients fail to respond to glucocorticoids, good evidence-based data on the efficacy of additional treatments such as CsA or etoposide are not available. The course of MAS can rapidly become irreversible and lead to a fatal outcome. Current data suggest that the mortality rate of sJIA-associated MAS is 8%, with approximately one-third of patients requiring ICU admission. Recent discoveries regarding the central role of IFNγ in disease pathogenesis suggest that IFNγ blockade potentially represents a novel therapeutic target.

[0105] The compositions and methods, including the NI-0501 compositions, of the present disclosure offer advantages over current treatments for primary and secondary HLH.

[0106] MAS and HLH are characterized by persistent immune cell activation and a cytokine storm of proinflammatory cytokines with a concomitant overproduction of IFNγ, TNFα, IL-1 and IL-6 (Henter JI, Elinder G, Soder O, Hansson M et al: Hypercytokinemia in familial hemophagocytic lymphohistiocytosis. Blood, 1991, Vol. 78: 2918-2922 pages; Imashuku S, Hibi S, Fujiwara F, Todo S: Hyper-interleukin(IL)-6-naemia in haemophagocytic lymphohistiocytosis. Br. J. Haematol., 1996, Vol. 93: 803-807 pages; Xu X, Tang Y, Song H, Yang S et al: Diagnostic accuracy of a specific cytokine pattern in hemophagocytic lymphohistiocytosis in children. J. Pediatr., 2012, Vol. 160: 984-90 pages e1; Put K, Avau A, Brisse E, Mitera T et al: Cytokines in systemic juvenile idiopathic arthritis and haemophagocytic lymphohistiocytosis: tipping the balance between interleukin-18 and interferon-γ. Rheumatology(Oxford), 2015). In recent years, HLH (Jordan MB, Hildeman D, Kappler J, Marrack P: An animal model of hemophagocytic lymphohistiocytosis (HLH): CD8+ T cells and interferon gamma are essential for the disorder. Blood, 2004, Vol. 104: 735-743 pages; Pachlopnik Schmid J, Ho C, Chretien F, Lefebvre JM, et al.: Neutralization of IFNgamma defeats haemophagocytosis in LCMV-infected perforin- and Rab27a-deficient mice. EMBO Mol Med, 2009, 1:112-124; Zoller EE, Lykens JE, Terrell CE, Aliberti J, et al.: Hemophagocytosis causes a consumptive anemia of inflammation., J. Exp. Med., 2011, 208:1203-1214) and MAS (Behrens EM, Canna SW, Slade K, Rao S, et al.: Repeated TLR9 stimulation results in macrophage activation syndrome-like disease in mice., J. Accumulating evidence supports a central role for IFNγ in the development of both inflammatory bowel disease and inflammatory bowel disease (Clin. Invest. 2011;121:2264-2277).

[0107] Regarding primary HLH, perforin knockout mice are considered a relevant model since they develop all of the diagnostic and many of the clinical and laboratory features of the human disease upon infection with LCMV. The HLH-like disease that develops in mice is dependent on CD8+ T cells and IFNγ produced in response to antigenic stimulation (Imashuku S, Hibi S, Fujiwara F, Todo S: Hyper-interleukin (IL)-6-naemia in haemophagocytic lymphohistiocytosis. Br. J. Haematol. 1996, 93:803-807). It has been demonstrated that when high circulating levels of IFNγ are neutralized by administration of anti-IFNγ antibodies, not only are clinical and laboratory abnormalities reversed, but survival rates are also dramatically improved. In contrast, removal of many other cytokines did not affect survival (Imashuku S, Hibi S, Fujiwara F, Todo S: Hyper-interleukin (IL)-6-naemia in haemophagocytic lymphohistiocytosis. Br. J. Haematol. 1996, 93:803-807). S:Hyper-interleukin (IL)-6-naemia in haemophagocytic lymphohistiocytosis., Br. J. Haematol., 1996, 93:803-807; Xu X, Tang Y, Song H, Yang S, et al.: Diagnostic accuracy of The importance of IFNγ in HLH is further strengthened by the high circulating IFNγ levels found in these patients (Henter JI, Elinder G, Soder O, Hansson M, et al.: Hypercytokinemia in familial hemophagocytic lymphohistiocytosis., Blood, 1991, 78:2918-2922; Xu X, Tang Y, Song H, Yang S, et al.: Diagnostic accuracy of a specific cytokine pattern in hemophagocytic lymphohistiocytosis in children., J. Pediatr., 2012, 160:984-90e1). In a series of 71 patients monitored from HLH diagnosis through treatment and follow-up, all patients had IFNγ levels above the upper limit of normal (17.3 pg / mL), notably, 53.5% had levels above 1000 pg / mL. It was also reported that IFNγ levels rose quickly initially and could decline from >5000 pg / mL to normal within 48 hours of effective treatment of HLH.

[0108] To elucidate the potential pathogenic role of IFNγ, two animal models of secondary HLH were investigated in the context of the NI-0501 development program. First, in a mouse model mimicking infection-driven HLH, repeated administration of CpG induced hypercytokinemia that leads to clinical (e.g., weight loss, splenomegaly) and laboratory features (e.g., cytopenia, hyperferritinemia) of HLH33 via activation of TLR9. Neutralizing IFNγ by administration of anti-IFNγ antibodies reverted the clinical and laboratory features of the disease. Neutralization of IFNγ was also shown to be complete in relevant target tissues such as the liver and spleen. Interestingly, administration of anti-IFNγ antibodies revealed that the amount of IFNγ was 500-2,000 times higher than the amount of IFNγ measured in the blood, likely better reflecting IFNγ production in tissues. Two IFNγ-inducible chemokines (CXCL9 and CXCL10) were upregulated in both blood and liver after TLR9 stimulation, and a significant correlation was observed between serum levels of IFNγ and serum concentrations of CXCL9 and CXCL10. Neutralization of IFNγ induced a significant decrease in serum CXCL9 and CXCL10, as well as their mRNA levels in the liver (Buatois V, Chatel L, Cons L, Lory S, et al.: IFNγ drives disease in the TLR9-mediated secondary HLH in mice: rationale for a new therapeutic target in secondary HLH, in preparation).

[0109] Secondly, an animal model of IL-6 transgenic mice expressing high levels of IL-6 was tested as this mimics the condition of patients with sJIA, the rheumatic disease most frequently associated with secondary forms of HLH. When challenged with Toll-like receptor (TLR) ligands, increased lethality, increased inflammatory cytokine production and hyperactivation of inflammatory signalling pathways were observed. Furthermore, these mice showed a drop in platelet and neutrophil counts and increased sCD25, ferritin and LDH levels, similar to many of the features commonly present in patients with MAS (Strippoli R, Carvello F, Scianaro R, De Pasquale L, et al.: Amplification of the response to Toll-like receptor (Ligands by prolonged exposure to interleukin-6 in mice: implication for the pathogenesis of macrophage activation syndrome., Arthritis Rheum., 2012, 64:1680-1688). In these mice, neutralization of IFNγ by administration of anti-IFNγ antibodies significantly improved survival and restored laboratory parameters (Prencipe G et al., manuscript in preparation).

[0110] Similar evidence has been gathered recently in observational studies performed in patients with secondary forms of HLH, either secondary to infection or of unknown etiology (pHLH was excluded by normal cytotoxic activity, the absence of known genetic mutations causing pHLH, and no family history) or in patients with MAS occurring in the setting of sJIA.

[0111] In 14 patients with secondary HLH (7 of whom had an identifiable underlying infection), serum samples were analyzed during active end-stage disease and during disease remission. Levels of IFNγ, CXCL9 and CXCL10 were significantly higher during active compared with disease remission (IFNγ: 34.7 vs. <3.5 pg / ml; CXCL9: 33598 vs. 745 pg / ml; CXCL10: 4420 vs. 132 pg / ml; median). IFNγ levels correlated significantly with levels of CXCL9 (p=0.0018) and to a lesser extent with levels of CXCL10 (p=0.014). IFNγ and chemokines (especially CXCL9) levels significantly correlated with parameters of disease severity such as neutrophil and platelet counts, ferritin and ALT, further supporting the pathogenic role of IFNγ in secondary HLH and the potential use of chemokines as relevant biomarkers of disease (Buatois V, Chatel L, Cons L, Lory S, et al.: IFNγ drives disease in the TLR9-mediated secondary HLH in mice: rationale for a new therapeutic target in secondary HLH).

[0112] Similar findings were noted in patients with MAS arising in patients with sJIA. Serum concentrations of IFNγ, IFNγ-inducible chemokines (CXCL9, CXCL10, CXCL11) and IL-6 were measured in 54 patients with sJIA, 20 of whom had MAS. Levels of IL-6 were similar in patients with end-stage MAS and in patients with active sJIA but without MAS at the time of sampling. In contrast, circulating IFNγ and chemokine levels were significantly higher in MAS, especially for CXCL9, with median levels approximately 15-fold higher in patients with MAS compared to those with active sJIA without MAS (13392 vs. 837 pg / mL; p=0.005). Of note, only in patients with MAS, significant correlations were demonstrated between CXCL9 levels and commonly abnormal parameters such as ferritin (p=0.041), neutrophil (p=0.010) and platelet (p=0.022) counts, ALT (p=0.044) and LDH (p=0.013). IFNγ levels also correlated with laboratory parameters of disease severity, with the exception of LDH, where statistical significance was not achieved (Bracaglia et al., manuscript in preparation).

[0113] Taken together, these data provide a robust rationale for neutralization of IFNγ as a targeted therapy for secondary HLH and MAS and for its investigation in the clinical setting.

[0114] The compositions and methods, including the NI-0501 composition of the present disclosure, are advantageous over current treatments for sJIA. For example, the compositions and methods, including the NI-0501 composition of the present disclosure, are useful in treating MAS / sHLH in sJIA patients, with the primary goal of achieving MAS remission.

[0115] The rationale for identifying this patient population as benefiting from treatment with NI-0501 and for evaluating its efficacy in MAS / sHLH is based on several factors. First, preclinical data obtained in animal models relevant to MAS in sJIA showed that IFNγ neutralization significantly improved survival and reversed changes in laboratory parameters. Second, observational data in patients with MAS / sHLH demonstrate the presence of high levels of IFNγ and, more importantly, highly elevated levels of the IFNγ-inducible chemokines, CXCL9, CXCL10 and CXCL11. Third, in patients with MAS / sHLH, concentrations of IFNγ and CXCL9 significantly correlate with disease parameters such as ferritin, platelet counts and transaminases. Secondly, in previous studies, where all administered infusates were well tolerated, a favorable tolerability profile and no relevant safety concerns were observed in pHLH patients, confirming the observations made in healthy volunteers, no infections caused by pathogens known to be beneficial for IFNγ neutralization were reported, and any infections that occurred in some of the pHLH patients were not related to treatment with NI-0501, but rather to their immune status, duration of disease and previous or concurrent treatments. Fifthly, preliminary data from previous clinical trials showed a favorable impact on disease parameters, visible within the first day of treatment: typical clinical signs and symptoms of HLH began to improve rapidly after the first dose of NI-0501 (fever within hours, splenomegaly / hepatomegaly within days), and treatment with NI-0501 allowed 10 patients to proceed to HSCT out of 18 patients evaluable at the cut-off. Second, evidence from PK modeling and simulation approaches demonstrates that a predictable pharmacokinetic profile of NI-0501 and neutralization of IFNγ are achieved and maintained. Finally, in the unlikely event that disease is not adequately controlled by NI-0501, conventional therapy (e.g., CsA) can be immediately initiated without the need for a drug holiday.

[0116] In conclusion, based on preclinical and clinical evidence, there is a strong rationale for neutralizing IFNγ in MAS / sHLH secondary to rheumatic diseases, and preliminary data in patients with pHLH indicate a favorable benefit-risk profile of NI-0501 with significant improvements in normalizing HLH features.

[0117] NI-0501 therefore represents an innovative and effective therapeutic approach in the management of this severe and life-threatening complication of rheumatic diseases, potentially limiting the side effects of long-term high-dose glucocorticoid treatment. Administration of anti-IFNγ antibody

[0118] It will be understood that administration of the therapeutic entities according to the present invention will be with suitable carriers, excipients, and other agents that are incorporated into the formulation to provide improved transport, delivery, tolerability, etc. Many suitable formulations can be found in formularies known to all pharmacists, such as Remington's Pharmaceutical Sciences (15th Edition, Mack Publishing Co., 1999). Publishing Company, Easton, PA (1975), particularly Chapter 87 therein by Blaug, Seymour. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as Lipofectin™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be appropriate in treatments and therapies according to the invention, provided that the active ingredient therein is not inactivated by the formulation and that the formulation is physiologically compatible and tolerated for the route of administration. For additional information relating to formulations, excipients and carriers well known to pharmacists, see Baldrick P., "Pharmaceutical excipient development: the need for preclinical guidance.” Regul. Toxicol Pharmacol., vol. 32(2): pp. 210-8 (2000). Wang W., “Lyophilization and development of solid protein pharmaceuticals.”, Int. See also J. Pharm. 203(1-2):1-60 (2000); Charman WN, "Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts." J Pharm Sci. 89(8):967-78 (2000); Powell et al., "Compendium of excipients for parenteral formulations," PDA J Pharm Sci Technol. 52:238-311 (1998) and citations therein.

[0119] Efficacy of treatment is determined in association with any known method for diagnosing or treating the particular immune-related disorder. Alleviation of one or more symptoms of the immune-related disorder indicates that the antibody confers clinical benefit.

[0120] The antibodies of the present invention, including polyclonal, monoclonal, humanized and fully human antibodies, can be used as therapeutic agents. Such agents are generally used to treat or prevent diseases or pathologies associated with ectopic expression or activation of a given target in a subject. An antibody preparation, preferably one with high specificity and high affinity for its target antigen, is administered to a subject and generally has an effect due to binding with the target. The administration of the antibody can suppress, inhibit or interfere with the signaling function of the target. The administration of the antibody can suppress, inhibit or interfere with the binding of the target to the endogenous ligand to which it naturally binds.

[0121] The therapeutically effective amount of the antibody of the present invention generally relates to the amount necessary to achieve the therapeutic purpose. As mentioned above, this may be the binding interaction of the antibody with its target antigen, which in certain cases interferes with the function of the target. The amount that needs to be administered further depends on the binding affinity of the antibody to its specific antigen, and also on the rate at which the administered antibody is depleted from the free volume of the other subject to which it is administered. The general range for therapeutically effective dosing of the antibody or antibody fragment of the present invention may be, as a non-limiting example, from about 0.1 mg per kg of body weight to about 50 mg per kg of body weight. Preferred doses include 1, 3, 6, 10 mg per kg of body weight. The general dosing frequency may range, for example, from once a day to twice a week. Treatment may last for 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks or longer.

[0122] The antibody or fragment thereof of the present invention can be administered in the form of a pharmaceutical composition to treat various diseases and disorders. Principles and considerations involved in the preparation of such compositions, as well as guidance in the selection of components, can be found, for example, in Remington: The Science And Practice Of Pharmacy, 19th Edition (eds. Alfonso R. Gennaro et al.) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drugs. Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0123] The formulation may contain more than one active compound, for example, anti-IFNγ antagonists, as required for the particular indication being treated, preferably with complementary activities that do not adversely affect each other. Alternatively or in addition, the composition may contain agents that enhance its function, such as cytotoxic agents, cytokines, chemotherapeutic agents, or growth inhibitory agents. Such molecules are suitably present in combination in amounts effective for the intended purpose.

[0124] In one embodiment, the active compound, for example, anti-IFNγ antagonist, is administered in combination therapy, i.e., in combination with one or more additional drugs useful for treating pathological conditions or disorders.In this context, the term "in combination" means that the drugs are given substantially simultaneously, simultaneously or sequentially.When given sequentially, it is preferred that the first compound of the two compounds is still detectable at effective concentration at the treatment site when the administration of the second compound begins.

[0125] For example, a combination therapy may include one or more neutralizing anti-IFNγ antibodies of the invention co-formulated and / or co-administered with one or more additional therapeutic agents, such as one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and / or cytotoxic or cytostatic agents, as described in more detail below. Such combination therapy may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding toxicities or complications that may be associated with various monotherapies.

[0126] In some embodiments, the additional agent is an immunosuppressant. In some embodiments, the immunosuppressant is cyclosporine A (CsA). In some embodiments, the subject receives CsA prior to administration of NI-0501. In some embodiments, the additional agent includes at least etoposide. In some embodiments, the subject receives etoposide prior to administration of NI-0501.

[0127] In some embodiments, the additional agent is intrathecal methotrexate and / or a glucocorticoid. In some embodiments, the subject has received intrathecal methotrexate and / or a glucocorticoid prior to administration of NI-0501.

[0128] In some embodiments, the additional agent is IV immunoglobulin (IVIG). In some embodiments, in subjects with documented immunoglobulin deficiency, IVIG is administered as a replacement treatment. In some embodiments, in subjects with documented immunoglobulin deficiency, IVIG is administered at a dose of 0.5 g / kg every 4 weeks or more frequently to maintain adequate IgG levels.

[0129] In some embodiments, the one or more additional medications are analgesic treatments, transfusions of blood products, electrolyte and glucose infusions, antibiotics, antifungal and antiviral treatments, and / or general supportive care.

[0130] When using antibody fragments, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein and / or interferes with or alternatively antagonizes IFNγ signaling is preferred. For example, peptide molecules that retain the ability to bind to the target protein sequence can be designed based on the variable region sequence of the antibody. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology (see, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)). The formulation may also contain more than one active compound, preferably those with complementary activities that do not adversely affect each other, depending on the needs of the particular indication being treated. Alternatively or in addition, the composition may contain agents that enhance its function, such as cytotoxic agents, cytokines, chemotherapeutic agents, or growth inhibitory agents. Such molecules are suitably present in combination in amounts effective for the intended purpose. Medication Regimen

[0131] The present invention further provides a dosing regimen for treating, preventing, and / or delaying the onset or progression of, or alleviating, symptoms associated with elevated IFN-γ. The dosing regimen is a multiple variable dose regimen. An antibody that binds interferon gamma (IFNγ) at a dose of 1.0-10 mg per kg of subject body weight. The doses are administered as at least a first and a second dose. The second dose is lower or higher than the first dose.

[0132] A number of variable dose regimens for treating primary hemophagocytic lymphohistiocytosis (HLH) in a human subject include an induction dose or first dose and a treatment dose or second dose of an anti-IFN-g monoclonal antibody. The subject is an adult subject or a pediatric subject.

[0133] The first dose is 1.0 or 3.0 mg per kg of body weight. The second dose is 3.0, 6.0 or 10.0 mg per kg of body weight. The first dose is 1.0 mg of antibody per kg of body weight and the second dose is 3.0, 6.0 or 10.0 mg per kg of body weight. Alternatively, the first dose is 3.0 mg of antibody per kg of body weight and the second dose is 6.0 or 10.0 mg per kg of body weight.

[0134] The first dose is administered once as a single dose, or alternatively, the first dose is administered more than once over an induction treatment period.

[0135] The second dose is administered for one or more treatment periods. The second dose is administered for a first treatment period. During the first treatment period, the second dose is administered every 3 days after the first dose. The first treatment period lasts for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks or longer. The first treatment period is preferably 2 weeks. Optionally, the second dose is administered for a second treatment period after completion of the first treatment period. During the second treatment period, the second dose is administered twice weekly. The second treatment period lasts for about 1-20 weeks, 2-20 weeks, 3-20 weeks, 4-20 weeks, 5-20 weeks, 6-20 weeks, 1-10 weeks, 2-10 weeks, 3-10 weeks, 4-10 weeks, 5-10 weeks, 6-10 weeks. The second treatment period is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks or longer.

[0136] In some embodiments, the second dose is increased or decreased during the course of the first or second treatment period. The increased or decreased dose is referred to as the third dose. The third dose can be 1.0, 3.0 or 6.0 mg per kg of body weight. The third dose is administered for the remainder of the first and second treatment periods. Alternatively, the third dose is administered for the third treatment period. In some embodiments, the third dose is referred to as a maintenance dose, and the third treatment period is referred to as a maintenance period.

[0137] A multiple-dose regimen for treating secondary hemophagocytic lymphohistiocytosis (HLH) in human children includes an induction dose or first dose and a treatment dose or second dose of an anti-IFN-g monoclonal antibody.

[0138] The first dose is 6.0 mg / kg body weight. The second dose is 3.0 mg / kg body weight. 10.0 mg / kg body weight.

[0139] The first dose is administered once as a single dose, or alternatively, the first dose is administered more than once over an induction treatment period.

[0140] The second dose is administered for one or more treatment periods. The second dose is administered for a first treatment period. During the first treatment period, the second dose is administered every 3 days after the first dose. The first treatment period lasts for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks or longer. The first treatment period is preferably 2 weeks. Optionally, the second dose is administered for a second treatment period after completion of the first treatment period. During the second treatment period, the second dose is administered twice weekly. The second treatment period lasts for about 1-20 weeks, 2-20 weeks, 3-20 weeks, 4-20 weeks, 5-20 weeks, 6-20 weeks, 1-10 weeks, 2-10 weeks, 3-10 weeks, 4-10 weeks, 5-10 weeks, 6-10 weeks. The second treatment period is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks or longer.

[0141] In some embodiments, the second dose is increased or decreased during the course of the first or second treatment period. The increased or decreased dose is referred to as the third dose. Preferably, the third dose is 6.0 mg / kg body weight. The third dose is administered for the remaining first and second treatment periods. Alternatively, the third dose is administered for the third treatment period. In some embodiments, the third dose is referred to as a maintenance dose, and the third treatment period is referred to as a maintenance period.

[0142] A multiple-dose regimen for treating secondary hemophagocytic lymphohistiocytosis (HLH) in human adults includes an induction dose or first dose and a treatment dose or second dose of an anti-IFN-g monoclonal antibody.

[0143] The first dose is 3.0 or 6.0 mg per kg of body weight. The first dose is preferably 6.0 mg per kg of body weight. The second dose is 6.0 or 10.0 mg per kg of body weight. The second dose is preferably 10.0 mg per kg of body weight. The first dose is 6.0 mg of antibody per kg of body weight, and the second dose is 10.0 mg per kg of body weight. The first dose is administered once as a single dose. Alternatively, the first dose is administered more than once over the induction treatment period.

[0144] The second dose is administered for one or more treatment periods. The second dose is administered for a first treatment period. During the first treatment period, the second dose is administered every 3 days after the first dose. The first treatment period lasts for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks or longer. The first treatment period is preferably 2 weeks. Optionally, the second dose is administered for a second treatment period after completion of the first treatment period. During the second treatment period, the second dose is administered twice weekly. The second treatment period lasts for about 1-20 weeks, 2-20 weeks, 3-20 weeks, 4-20 weeks, 5-20 weeks, 6-20 weeks, 1-10 weeks, 2-10 weeks, 3-10 weeks, 4-10 weeks, 5-10 weeks, 6-10 weeks. The second treatment period is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks or longer.

[0145] In some embodiments, the second dose is increased or decreased during the course of the first or second treatment period. The increased or decreased dose is referred to as the third dose. The third dose can be 1.0, 3.0 or 6.0 mg per kg of body weight. The third dose is administered for the remainder of the first and second treatment periods. Alternatively, the third dose is administered for the third treatment period. In some embodiments, the third dose is referred to as a maintenance dose, and the third treatment period is referred to as a maintenance period.

[0146] The multiple variable dose regimen for treating a condition in a human subject includes an induction dose or a first dose and a treatment dose or a second dose of an anti-IFN-g monoclonal antibody. The subject is an adult subject or a pediatric subject. The condition is associated with elevated IFNg levels. The condition is solid organ transplantation disorder or bone marrow acute graft rejection, graft rejection such as graft-versus-host disease, paraneoplastic cerebellar degeneration, hemorrhagic fever, sarcoidosis, adult-onset Still's disease. In other embodiments, the dosing regimen is administered to the subject after receiving CAR-T cell therapy.

[0147] The first dose is 1.0-10 mg per kg of body weight. For example, the first dose is 1.0, 3.0, 6.0, or 10 mg of antibody per kg of body weight. The second dose is higher or lower than the first dose. The second dose is 1.0-10 mg per kg of body weight. For example, the second dose is 1.0, 3.0, 6.0, or 10 mg of antibody per kg of body weight. The first dose is 1.0 mg per kg of body weight and the second dose is 3.0, 6.0, or 10.0 mg per kg of body weight. Alternatively, the first dose is 3.0 mg of antibody per kg of body weight and the second dose is 6.0 or 10.0 mg per kg of body weight. The first dose is 6.0 mg of antibody per kg of body weight and the second dose is 10.0 mg per kg of body weight.

[0148] The first dose is administered once as a single dose, or alternatively, the first dose is administered more than once over an induction treatment period.

[0149] The second dose is administered for one or more treatment periods. The second dose is administered for a first treatment period. During the first treatment period, the second dose is administered every 3 days after the first dose. The first treatment period lasts for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks or longer. The first treatment period is preferably 2 weeks. Optionally, the second dose is administered for a second treatment period after completion of the first treatment period. During the second treatment period, the second dose is administered twice weekly. The second treatment period lasts for about 1-20 weeks, 2-20 weeks, 3-20 weeks, 4-20 weeks, 5-20 weeks, 6-20 weeks, 1-10 weeks, 2-10 weeks, 3-10 weeks, 4-10 weeks, 5-10 weeks, 6-10 weeks. The second treatment period is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks or longer.

[0150] In some embodiments, the second dose is increased or decreased during the course of the first or second treatment period. The increased or decreased dose is referred to as the third dose. The third dose can be 1.0, 3.0, 6.0 or 10.0 mg per kg of body weight. The third dose is administered for the remainder of the first and second treatment periods. Alternatively, the third dose is administered for the third treatment period. In some embodiments, the third dose is referred to as a maintenance dose, and the third treatment period is referred to as a maintenance period.

[0151] In the multiple variable dosing regimens according to the present invention, the first and second period for the second dose can be adjusted from time to time, for example, based on the health condition of the subject.For example, the adjustment of the time between doses can be adjusted to ±1, 2, 3 or 4 days.±2 days is preferred.

[0152] In many variable dosing regimens according to the present invention, the dose is administered in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours. It is preferred to administer the dose in 1 hour. The duration of the dose infusion is based on several factors known in the art, such as age, weight, physical condition or adverse reactions to the therapeutic agent. The duration of the dose infusion is generally a rapid infusion rate that is tolerated by the subject (i.e., does not cause adverse reactions).

[0153] The dose is administered as a single injection. The antibody is administered as a monotherapy or combination therapy for the disease or condition to be treated. For example, the subject is administered a second drug. The second drug is, for example, an anti-inflammatory drug and / or an immunosuppressant.

[0154] Optionally, the subject has received dexamethasone immediately prior to dosing with the antibody. The dexamethasone is at least 10 mg / m 2 Alternatively, dexamethasone is administered at a dose of at least 5 mg / m 2 is administered at a dose of

[0155] In some embodiments, the subject has not previously undergone treatment for HLH. Pharmaceutical Compositions

[0156] The antibodies or soluble chimeric polypeptides of the present invention (also referred to herein as "active compounds"), as well as derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions generally comprise the antibody or soluble chimeric polypeptide and a pharma- ceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers can be found in Remington's Pharmaceutical Sciences, a standard reference text in the field. The latest edition of Pharmaceutical Sciences, which is incorporated herein by reference.Preferred examples of such carrier or diluent include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin.Non-aqueous vehicles such as liposomes and fixed oils can also be used.The use of such media and agents for pharmacoactive substances is well known in the art.Except insofar as any conventional media or agent is incompatible with the active compound, its use in the composition is intended.Supplementary active compounds can also be incorporated into the composition.

[0157] The pharmaceutical composition of the present invention is formulated to suit its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. The solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents such as water for injection, saline solution, non-volatile oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate, or phosphate, and agents for adjusting tonicity, e.g., sodium chloride or dextrose. The pH can be adjusted using acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes, or multiple dose vials.

[0158] The preferred route of administration is by injection, such as intravenous injection. Intravenous injection can be a rapid infusion or a slow infusion. For example, the infusion is for a period of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours. The duration of the infusion is based on a number of factors known in the art, such as age, weight, physical condition, or adverse reactions to the therapeutic agent. The duration of the infusion is generally a rapid infusion rate that is tolerated by the subject (i.e., does not cause adverse reactions).

[0159] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble), or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. The agent that delays absorption, such as aluminum monostearate and gelatin, can be included in the composition to bring about sustained absorption of the injectable composition.

[0160] Sterile injection can be prepared by incorporating the required amount of active compound in a suitable solvent with one or a combination of ingredients listed above as necessary, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains a basic dispersion medium and other ingredients required from those listed above.In the case of sterile powder for preparing sterile injection, the preparation method is vacuum drying and freeze-drying, which obtains the powder of active ingredient and any additional desired ingredients from the solution that has been previously sterilized and filtered.

[0161] A preferred injectable pharmaceutical composition contains excipients such as g L-histidine, 3 L-histidine monohydrochloride monohydrate, sodium chloride (NaCl), polysorbate 80, and the like.

[0162] The pH of the injectable pharmaceutical composition is between about 5.8 and 6.2. The pH of the injectable pharmaceutical composition is preferably pH 6.0.

[0163] In some embodiments, an anti-IFN-γ antibody such as NI-0501 is formulated as follows (per ml): NI-051 5 mg, L-histidine 1.55 mg, L-histidine monohydrochloride monohydrate 3.14 mg, sodium chloride (NaCl) 7.31 mg, and polysorbate 80 0.05 mg, pH 6.0±0.2.

[0164] In some embodiments, the pharmaceutical composition is packaged in a unit dose. The unit dose is packaged in a container. The container is a glass or plastic container. The container is a syringe, a vial, an injection bottle, an ampoule, or a carpoule. The container can hold a volume of 1 to 25 ml. For example, the container can hold a volume of 2, 5, 10, or 20 ml.

[0165] The unit dose of the fully human anti-interferon gamma (IFNγ) monoclonal antibody (e.g., NI-501) is 5-25 mg / ml. The unit dose is preferably 5 mg / ml or 25 mg / ml. The antibody is in a solution (e.g., water for injection) having per ml: 1.55 mg L-histidine, 3.14 mg L-histidine monohydrochloride monohydrate, 7.31 mg sodium chloride (NaCl), and 0.05 mg polysorbate 80.

[0166] In some embodiments, the invention provides a unit dose container with 20 ml of fully human anti-interferon gamma (IFNγ) monoclonal antibody solution, with a concentration of 5 mg / ml or 25 mg / ml of antibody and a pH of the solution between 5.8 and 6.2. The antibody is solubilized in the solution, so the solution is clear, colorless, and free of precipitate. The antibody is preferably in a solution (e.g., water for injection) with per ml: 1.55 mg L-histidine, 3.14 mg L-histidine monohydrochloride monohydrate, 7.31 mg sodium chloride (NaCl), and 0.05 mg polysorbate 80.

[0167] In another embodiment, the invention provides a unit dose container having 10 ml or 20 ml of fully human anti-interferon gamma (IFNγ) monoclonal antibody solution, with a concentration of 25 mg / ml of antibody and a pH of the solution between 5.8 and 6.2. The antibody is solubilized in the solution, so the solution is clear, colorless, and free of precipitate. The antibody is preferably in a solution (e.g., water for injection) having per ml: 1.55 mg L-histidine, 3.14 mg L-histidine monohydrochloride monohydrate, 7.31 mg sodium chloride (NaCl), and 0.05 mg polysorbate 80.

[0168] In another embodiment, the invention provides a unit dose container having 2 ml or 10 ml of fully human anti-interferon gamma (IFNγ) monoclonal antibody solution, with a concentration of 5 mg / ml antibody and a pH of the solution between 5.8 and 6.2. The antibody is solubilized in the solution, so the solution is clear, colorless, and free of precipitate. The antibody is preferably in a solution (e.g., water for injection) having per ml: 1.55 mg L-histidine, 3.14 mg L-histidine monohydrochloride monohydrate, 7.31 mg sodium chloride (NaCl), and 0.05 mg polysorbate 80.

[0169] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound can be incorporated with an excipient and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, where the compound in the fluid carrier is orally applied, swirled in the mouth, expectorated, or swallowed. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like may contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0170] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0171] Systemic administration may be by transmucosal or transdermal means.For transmucosal or transdermal administration, a penetrant that is appropriate for the barrier to be permeated is used in the formulation.Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.Transmucosal administration can be achieved by using nasal sprays or suppositories.For transdermal administration, active compound is formulated into ointments, salves, gels, or creams as generally known in the art.

[0172] The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0173] In one embodiment, the active compound is prepared with a carrier that protects the compound from rapid elimination from the body, for example, sustained release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be clear to those skilled in the art. Materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0174] Oral or parenteral compositions are particularly advantageously formulated into unit dosage form for ease of administration and uniformity of dosage.Unit dosage form, as used herein, refers to a physically separate unit suitable as a unit dosage for the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce desired therapeutic effect in association with required pharmaceutical carrier.The specification of the unit dosage form of the present invention is determined by and directly depends on the unique characteristics of active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations of the technical field of compounding such active compound to treat individuals.

[0175] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. Detection of CXCL9 and other biomarkers

[0176] The levels of CXCL9 and other biomarkers are detected using any of a variety of standard detection techniques. A detection agent can be used to detect the presence of a given target (or a protein fragment thereof) in a sample. In some embodiments, the detection agent contains a detectable label. In some embodiments, the detection agent is an antibody (or a fragment thereof) or a probe. In some embodiments, the agent or probe is labeled. The term "labeling", in reference to a probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin.

[0177] The term "biological sample" includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Thus, the use of the term "biological sample" includes blood and blood fractions or components, including serum, plasma, or lymph. A biological fluid may be a fluid isolated from anywhere within the subject's body, preferably from a peripheral location, including, but not limited to, blood, plasma, serum, synovial fluid, urine, sputum, spinal fluid, cerebrospinal fluid, pleural fluid, respiratory fluid, intestinal fluid, and urogenital fluid, saliva, organ system fluid, ascites, tumor cyst fluid, amniotic fluid, and combinations thereof. Biological samples also include experimentally isolated fractions of all the aforementioned fluids. Biological samples also include solutions or mixtures containing homogenized solid materials, such as feces, tissues, and biopsy samples. The detection method of the present invention can be used to detect analyte mRNA, protein, or genomic DNA in biological samples in vitro as well as in vivo. For example, in vitro techniques for detecting analyte mRNA include Northern hybridization and in situ hybridization. In vitro techniques for detecting analyte protein include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence. In vitro techniques for detecting analyte genomic DNA include Southern hybridization. Procedures for carrying out immunoassays are described, for example, in "ELISA: Theory and Practice: Methods in Molecular Biology", Vol. 42, J.R. Crowther (ed.) Human Press, Totowa, NJ, 1995; "Immunoassay", E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and "Practice and Theory of Enzyme Immunoassays", P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985.Additionally, in vivo techniques for detecting analyte proteins include introducing into a subject a labeled anti-analyte protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in the subject can be detected by standard imaging techniques. Definition:

[0178] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. In general, the nomenclature utilized in connection with, and techniques related to, cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly realized in the art or as described herein. The techniques and procedures described above are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). The nomenclature utilized in connection with, and the laboratory procedures and techniques related to, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0179] The term "dose," as used herein, refers to the amount of an anti-IFN-gamma antibody administered to a subject.

[0180] The term "multiple variable doses" encompasses different doses of anti-IFN-gamma antibody administered to a subject for therapeutic treatment. "Multiple variable dose regimen" or "multiple variable dose therapy" describes a treatment schedule based on administering different amounts of anti-IFN-gamma antibody at different times throughout the course of treatment. In one embodiment, the invention describes a multiple variable dose treatment method comprising an induction phase and a treatment phase, where the anti-IFN-gamma antibody is administered at a higher or lower dose during the induction phase than during the treatment phase. In another embodiment, the invention describes a multiple variable dose treatment method comprising an induction phase, a first treatment phase, and a second treatment phase, where the anti-IFN-gamma antibody is administered at a higher or lower dose during the induction phase than during the treatment phase. The anti-IFN-gamma antibody may be administered at a higher or lower dose during the first treatment phase than during the second treatment phase. The dose is preferably the same in the first and second treatment phases.

[0181] The term "induction phase" or "loading phase" as used herein refers to a period of treatment that involves administering an IFN-gamma antibody to a subject to achieve a threshold level. During the induction phase, at least one induction dose of IFN-gamma is administered to a subject suffering from a disorder in which IFN-gamma is detrimental.

[0182] The term "threshold level" as used herein refers to a therapeutically effective level of IFN-gamma antibody in a subject. The threshold level is achieved by administering at least one induction dose during the induction phase of treatment. Any number of induction doses can be administered to achieve a threshold level of IFN-gamma. Once the threshold level is achieved, the treatment phase begins.

[0183] The terms "induction dose" or "loading dose" are used interchangeably herein and refer to a first dose of anti-IFN-gamma antibody, which is larger or smaller than a maintenance or treatment dose. An induction dose may be a single dose or a set of doses. An induction dose is often used to achieve a steady state amount of drug in the body and can also be used to quickly achieve a maintenance drug level. The induction dose is followed by administration of a smaller or larger dose of anti-IFN-gamma antibody, i.e., a treatment dose. An induction dose is administered during the induction phase of treatment.

[0184] The term "treatment phase" or "maintenance phase" as used herein refers to a period of treatment that includes administering an anti-IFN-gamma antibody to a subject to maintain a desired therapeutic effect. The treatment phase follows an induction phase and thus begins once a threshold level is achieved. There may be more than one treatment phase, which is a treatment phase that is shortened or extended to maintain a certain threshold or achieve a desired clinical response. Preferably, in the treatment phase, after an induction dose (e.g., an initial dose), the substance is administered every three days. Alternatively, the substance is administered once or twice a week.

[0185] The term "treatment dose" or "maintenance dose" is the amount of anti-IFN-gamma antibody taken by a subject to maintain or persist the desired therapeutic effect. A treatment dose is administered after an induction dose. A treatment dose may be a single dose or may be a set of doses. A treatment dose is administered during the treatment phase of therapy. A treatment dose may be smaller or larger than an induction dose and may be equal to each other if administered consecutively. If there is more than one treatment phase, there may also be more than one treatment dose.

[0186] A "dosage regimen" or "dosing regimen" includes a treatment regimen based on a set of determined doses.

[0187] The term "dosing," as used herein, refers to the administration of a substance (e.g., an anti-IFN-gamma antibody) to achieve a therapeutic goal (e.g., treatment of an IFN-gamma associated disorder).

[0188] The terms "biweekly dosing regimen," "biweekly dosing," and "biweekly administration," as used herein, refer to the time course over which a substance (e.g., an anti-IFN-gamma antibody) is administered to a subject to achieve a therapeutic objective (e.g., treatment of an IFN-gamma associated disorder). A biweekly dosing regimen does not include a weekly dosing regimen.

[0189] The term "combination," as in the phrase "a combination of a first agent and a second agent," includes simultaneous administration of the first agent and the second agent, which may be, for example, dissolved or mixed in the same pharma- ceutically acceptable carrier, or the administration of the first agent followed by the administration of the second agent, or the administration of the second agent followed by the administration of the first agent. Thus, the present invention includes methods of combination therapeutic treatment and combination pharmaceutical compositions.

[0190] The term "concurrent" as in the phrase "concurrent therapeutic treatment" includes administering an agent in the presence of a second agent. Concurrent therapeutic treatment methods include methods of concurrently administering a first, second, third, or additional agent. Concurrent therapeutic treatment methods also include methods of administering a first or additional agent in the presence of a second or additional agent, where the second or additional agent may, for example, have been previously administered. Concurrent therapeutic treatment methods may be performed stepwise by different practitioners. For example, one practitioner may administer a first agent to a subject, and a second practitioner may administer a second agent to a subject, and the administering steps may be performed at the same or near the same time, or at separate times as long as the first agent (and additional agent) is administered in the presence of the second agent (and additional agent). The practitioner and the subject may be the same entity (e.g., human).

[0191] The term "combination therapy," as used herein, refers to the administration of two or more therapeutic agents, such as an anti-IFN-gamma antibody and another drug, such as a DMARD or NSAID. The other drug(s) can be administered simultaneously with, prior to, or following administration of the anti-IFN-gamma antibody. Embodiment

[0192] In the compositions and methods presented herein, NI-0501 is formulated as a sterile concentrate for injection (per mL). In some embodiments, NI-0501 is formulated as follows: NI-051 5mg, L-histidine 1.55mg, L-histidine monohydrochloride monohydrate 3.14mg, sodium chloride (NaCl) 7.31mg, and polysorbate 80 0.05mg, pH 5.8 to 6.2. In some embodiments, NI-0501 is formulated as follows: NI-051 5mg, L-histidine 1.55mg, L-histidine monohydrochloride monohydrate 3.14mg, sodium chloride (NaCl) 7.31mg, and polysorbate 80 0.05mg, pH 6.0.

[0193] In the compositions and methods presented herein, NI-0501 is administered to a subject in need thereof to treat, prevent, and / or delay the onset or progression of, or alleviate, symptoms associated with HLH.In some embodiments, NI-0501 is administered to a subject in need thereof at an initial dose of 1 mg / kg over 1 hour by IV infusion.In certain patient populations, such as low-weight and / or very young patient populations, IV infusion can last longer than 1 hour, for example, at least 90 minutes, at least 2 hours, or at least 3 hours or longer.

[0194] In some embodiments, NI-0501 is administered to a subject in need thereof at an initial dose of 1 mg / kg over 1 hour with at least one additional IV infusion after the initial IV infusion. In some embodiments, the at least one additional IV infusion is at a higher dose than the initial dose of 1 mg / kg. In some embodiments, the dosage of the at least one additional IV infusion is 3 mg / kg. In some embodiments, the at least one additional IV infusion is administered at least 3 days after the initial IV infusion. In some embodiments, the at least one additional IV infusion is administered at a time selected from the group consisting of 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial infusion, and 15 days after the initial infusion. In some embodiments, the at least one additional IV infusion is administered 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial infusion, and 15 days after the initial infusion.

[0195] In some embodiments, NI-0501 is administered to a subject in need thereof at an initial dose of 1 mg / kg over 1 hour with at least one additional series of IV infusions after the initial IV infusion, wherein the series of additional IV infusions includes at least one series of twice weekly IV infusions. In some embodiments, at least one series of twice weekly IV infusions is administered at a dose higher than the initial dose of 1 mg / kg. In some embodiments, at least one series of twice weekly IV infusions is administered at a dose of 3 mg / kg. In some embodiments, at least one additional IV infusion is administered at least 3 weeks after the initial IV infusion. In some embodiments, at least one additional IV infusion is administered at a time selected from the group consisting of 3 weeks after the initial IV infusion, 4 weeks after the initial IV infusion, 5 weeks after the initial IV infusion, 6 weeks after the initial infusion, 7 weeks after the initial infusion, and 8 weeks after the initial infusion. In some embodiments, at least one additional IV infusion is administered 3 weeks after the first IV infusion, 4 weeks after the first IV infusion, 5 weeks after the first IV infusion, 6 weeks after the first infusion, 7 weeks after the first infusion, and 8 weeks after the first infusion.

[0196] In some embodiments, NI-0501 is administered to a subject in need thereof by at least two additional IV infusions after the first IV infusion. In some embodiments, the at least two additional IV infusions are at a dose higher than the initial dose of 1 mg / kg. In some embodiments, the first additional IV infusion and the second additional IV infusion are administered at the same dosage. In some embodiments, the first additional IV infusion and the second additional IV infusion are administered at the same dosage higher than the initial dose. In some embodiments, at least one of the first and second additional IV infusions is administered at a dosage of 3 mg / kg. In some embodiments, the first additional IV infusion is administered at least 3 days after the first IV infusion. In some embodiments, the first additional IV infusion is administered at a time selected from the group consisting of 3 days after the first IV infusion, 6 days after the first IV infusion, 9 days after the first IV infusion, 12 days after the first infusion, and 15 days after the first infusion. In some embodiments, the first additional IV infusion is administered 3 days after the first IV infusion, 6 days after the first IV infusion, 9 days after the first IV infusion, 12 days after the first infusion, and 15 days after the first infusion. In some embodiments, the second additional IV infusion is administered at a time selected from the group consisting of 3 weeks after the first IV infusion, 4 weeks after the first IV infusion, 5 weeks after the first IV infusion, 6 weeks after the first infusion, 7 weeks after the first infusion, and 8 weeks after the first infusion. In some embodiments, the second additional IV infusion is administered 3 weeks after the first IV infusion, 4 weeks after the first IV infusion, 5 weeks after the first IV infusion, 6 weeks after the first infusion, 7 weeks after the first infusion, and 8 weeks after the first infusion. In some embodiments, the first additional IV infusion is administered 3 days after the first IV infusion, 6 days after the first IV infusion, 9 days after the first IV infusion, 12 days after the first infusion, and 15 days after the first infusion, and the second additional IV infusion is administered 3 weeks after the first IV infusion, 4 weeks after the first IV infusion, 5 weeks after the first IV infusion, 6 weeks after the first infusion, 7 weeks after the first infusion, and 8 weeks after the first infusion.

[0197] In some embodiments, the first additional IV infusion and the second additional IV infusion are administered at different dosages. In some embodiments, the first additional IV infusion and the second additional IV infusion are administered at different dosages, where the dosage of the second additional IV infusion is higher than the first additional IV infusion. In some embodiments, the first additional IV infusion and the second additional IV infusion are administered at different dosages, where the dosage of the second additional IV infusion is higher than the first additional IV infusion, and where both the dosages of the first and second additional IV infusions are higher than the first dosage. In some embodiments, at least one of the first and second additional IV infusions is administered at a dosage of 3 mg / kg. In some embodiments, the first additional IV infusion is administered at a dosage of 3 mg / kg, and the second additional IV infusion is administered at a dosage of 6 mg / kg. In some embodiments, the first additional IV infusion is administered at least 3 days after the first IV infusion. In some embodiments, the first additional IV infusion is administered at a time selected from the group consisting of 3 days after the first IV infusion, 6 days after the first IV infusion, 9 days after the first IV infusion, 12 days after the first infusion, and 15 days after the first infusion. In some embodiments, the first additional IV infusion is administered at a time selected from the group consisting of 3 weeks after the first IV infusion, 4 weeks after the first IV infusion, 5 weeks after the first IV infusion, 6 weeks after the first IV infusion, 7 weeks after the first infusion, and 8 weeks after the first infusion. In some embodiments, the second additional IV infusion is administered at a time selected from the group consisting of 3 weeks after the first IV infusion, 4 weeks after the first IV infusion, 5 weeks after the first IV infusion, 6 weeks after the first infusion, 7 weeks after the first infusion, and 8 weeks after the first infusion. In some embodiments, the second additional IV infusion is administered at a time selected from the group consisting of 3 weeks after the first IV infusion, 4 weeks after the first IV infusion, 5 weeks after the first IV infusion, 6 weeks after the first infusion, 7 weeks after the first infusion, and 8 weeks after the first infusion.In some embodiments, the first additional IV infusion is administered 3 days after the first IV infusion, 6 days after the first IV infusion, 9 days after the first IV infusion, 12 days after the first infusion, and 15 days after the first infusion, and the second additional IV infusion is administered 3 weeks after the first IV infusion, 4 weeks after the first IV infusion, 5 weeks after the first IV infusion, 6 weeks after the first infusion, 7 weeks after the first infusion, and 8 weeks after the first infusion.

[0198] In some embodiments, the first additional IV infusion comprises at least a first series of twice-weekly IV infusions, and the second additional IV infusion comprises at least a second series of twice-weekly IV infusions. In some embodiments, the first series of twice-weekly IV infusions and the second series of twice-weekly IV infusions are administered at a dose higher than the initial dose of 1 mg / kg. In some embodiments, the first series of twice-weekly IV infusions is administered at a dose of 3 mg / kg, and the second series of twice-weekly IV infusions is administered at a dose of 6 mg / kg. In some embodiments, the first series of additional IV infusions is administered at least 3 days after the first IV infusion. In some embodiments, the first series of additional IV infusions is administered at a time selected from the group consisting of 3 days after the first IV infusion, 6 days after the first IV infusion, 9 days after the first IV infusion, 12 days after the first infusion, and 15 days after the first infusion. In some embodiments, the first series of additional IV infusions is administered 3 days after the first IV infusion, 6 days after the first IV infusion, 9 days after the first IV infusion, 12 days after the first infusion, and 15 days after the first infusion. In some embodiments, the second series of additional IV infusions is administered at a time selected from the group consisting of 3 weeks after the first IV infusion, 4 weeks after the first IV infusion, 5 weeks after the first IV infusion, 6 weeks after the first infusion, 7 weeks after the first infusion, and 8 weeks after the first infusion. In some embodiments, the second series of additional IV infusions is administered 3 weeks after the first IV infusion, 4 weeks after the first IV infusion, 5 weeks after the first IV infusion, 6 weeks after the first infusion, 7 weeks after the first infusion, and 8 weeks after the first infusion. In some embodiments, a first series of additional IV infusions is administered 3 days after the first IV infusion, 6 days after the first IV infusion, 9 days after the first IV infusion, 12 days after the first infusion, and 15 days after the first infusion, and a second series of additional IV infusions is administered 3 weeks after the first IV infusion, 4 weeks after the first IV infusion, 5 weeks after the first IV infusion, 6 weeks after the first infusion, 7 weeks after the first infusion, and 8 weeks after the first infusion.

[0199] In some embodiments, after the first dose, infusions are administered every 3 days until 15 days after the first dose. In some embodiments, after the first dose, infusions are administered every 3 days until 15 days after the first dose, and then twice weekly infusions are administered starting at least 15 days after the first dose. In some embodiments, the dosage of the infusion is increased to 3 mg / kg at any time after the first dose. In some embodiments, after a minimum of two infusions at 3 mg / kg, the dose of NI-0501 is increased to 6 mg / kg for up to four infusions.

[0200] In the compositions and methods provided herein, NI-0501 is administered to a subject in need thereof to treat, prevent, and / or delay the onset or progression of, or alleviate, symptoms associated with HLH. In some embodiments, NI-0501 is administered to a subject in need thereof at an initial dose of 1 mg / kg over 1 hour by IV infusion. In some embodiments, after the initial dose, infusions are administered every 3 days until 15 days after the initial dose. In some embodiments, after the initial dose, infusions are administered every 3 days until 15 days after the initial dose, and then twice weekly infusions are administered starting at least 15 days after the initial dose. In some embodiments, the dosage of the infusion is increased to 3 mg / kg at any time after the initial dose. In some embodiments, after a minimum of two infusions at 3 mg / kg, the dose of NI-0501 is increased to 6 mg / kg over up to four infusions.

[0201] In the compositions and methods provided herein, NI-0501 is administered to a subject in need thereof to treat, prevent, and / or delay the onset or progression of, or alleviate, symptoms associated with HLH. In some embodiments, NI-0501 is administered to a subject in need thereof by IV infusion at a dose greater than 6 mg / kg. In some embodiments, NI-0501 is administered to a subject in need thereof by IV infusion at a second dose greater than 6 mg / kg after the first dose. In some embodiments, the second dose is at least 10 mg / kg. In some embodiments, the second dose is 10 mg / kg. In some embodiments, the second dose is 10 mg / kg and is repeated daily. In some embodiments, the second dose is 10 mg / kg and is repeated daily for one week. In some embodiments, the second dose is 10 mg / kg and is repeated daily for two weeks. In some embodiments, the second dose is 10 mg / kg and is repeated daily for more than two weeks.

[0202] In some embodiments, NI-0501 is administered to a subject in need thereof to treat, prevent, and / or delay the onset or progression of, or alleviate symptoms associated with secondary HLH. In some embodiments, NI-0501 is administered to a subject in need thereof to treat, prevent, and / or delay the onset or progression of, or alleviate symptoms associated with secondary HLH in the context of sJIA. In some embodiments, NI-0501 is administered to a subject in need thereof as an initial dose of 6 mg / kg. In some embodiments, treatment with NI-0501 is continued with subsequent doses of NI-0501. In some embodiments, treatment with NI-0501 is continued with subsequent doses of NI-0501 of 3 mg / kg every 3 days for at least 4 weeks (i.e., until SD27).

[0203] In some embodiments, once the desired clinical outcome is achieved, treatment with NI-0501 is reduced, stopped, or alternatively shortened. In some embodiments, treatment with NI-0501 is shortened once a complete clinical response, i.e., MAS remission, is demonstrated.

[0204] In some embodiments, after 4 weeks, treatment with NI-0501 is continued as maintenance as needed for up to another 4 weeks (i.e., until SD56) until MAS remission is achieved. In some embodiments, after 4 weeks, treatment with NI-0501 is continued as maintenance as needed for up to another 4 weeks (i.e., until SD56) until MAS remission is achieved, with the possibility of reducing the dose to 1 mg / kg and extending the interval between injections to weekly dosing.

[0205] In the compositions and methods provided herein, NI-0501 is administered to a subject in need thereof to treat, prevent, and / or delay the onset or progression of, or alleviate, symptoms associated with HLH, where the subject has been receiving background dexamethasone. In some embodiments, the subject is a treatment naive patient (i.e., has not previously been treated for HLH) and is receiving at least 10 mg / m dexamethasone. 2 In some embodiments, the subject is receiving NI-0501 as a second line HLH treatment and is receiving dexamethasone at a dose of 10 mg / m 2 ~5mg / m 2 In some embodiments, the subject is receiving NI-0501 as a second line HLH treatment and is receiving dexamethasone at least 5 mg / m 2 In some embodiments, the subject is receiving NI-0501 as a second line HLH treatment and is receiving dexamethasone at a dose of 5 mg / m 2 Administer in doses less than 100 mg / kg.

[0206] In some embodiments, NI-0501 is administered before and / or during and / or after treatment in combination with one or more additional agents, such as, by way of non-limiting example, therapeutic agents, anti-inflammatory agents, and / or immunosuppressants. In some embodiments, the second agent is an agent known to be used in the treatment of HLH. In some embodiments, the additional agent includes at least etoposide. In some embodiments, NI-0501 and the additional agent are formulated into a single therapeutic composition, and NI-0501 and the additional agent are administered simultaneously. Alternatively, NI-0501 and the additional agent are separate from each other, e.g., each is formulated into a separate therapeutic composition, and NI-0501 and the additional agent are administered simultaneously, or NI-0501 and the additional agent are administered at different times during the treatment regimen. For example, NI-0501 is administered before administration of the additional agent, NI-0501 is administered after administration of the additional agent, or NI-0501 and the additional agent are administered alternately. As described herein, NI-0501 and the additional agent are administered in a single dose or in multiple doses.

[0207] In some embodiments, NI-0501 and additional agent(s) are administered simultaneously. For example, NI-0501 and additional agent(s) can be formulated in a single composition, or can be administered as two or more separate compositions. In some embodiments, NI-0501 and additional agent(s) are administered sequentially, or NI-0501 and additional agent(s) are administered at different times during a treatment regimen.

[0208] In some embodiments, the additional agent is an immunosuppressant. In some embodiments, the immunosuppressant is cyclosporine A (CsA). In some embodiments, the subject receives CsA prior to administration of NI-0501. In some embodiments, the additional agent includes at least etoposide. In some embodiments, the subject receives etoposide prior to administration of NI-0501.

[0209] In some embodiments, the additional agent is intrathecal methotrexate and / or a glucocorticoid. In some embodiments, the subject has received intrathecal methotrexate and / or a glucocorticoid prior to administration of NI-0501.

[0210] In some embodiments, the additional agent is IV immunoglobulin (IVIG). In some embodiments, in subjects with documented immunoglobulin deficiency, IVIG is administered as a replacement treatment. In some embodiments, in subjects with documented immunoglobulin deficiency, IVIG is administered at a dose of 0.5 g / kg every 4 weeks or more frequently to maintain adequate IgG levels.

[0211] In some embodiments, the one or more additional medications are analgesic treatments, transfusions of blood products, electrolyte and glucose infusions, antibiotics, antifungal and antiviral treatments, and / or general supportive care.

[0212] The compositions and methods presented herein are useful in treating, preventing, and / or delaying the onset or progression of, or alleviating, symptoms associated with transplant failure, transplant rejection, and / or inflammatory disorders associated with transplant rejection.The compositions and methods presented herein are useful in treating, inhibiting, delaying the progression of, or alternatively reversing, symptoms of graft-versus-host disease (GvHD) in subjects who have received or are receiving a transplant containing biological material or a series of transplants containing biological material.The compositions and methods presented herein are useful in extending the survival of transplanted biological material.

[0213] The compositions and methods presented herein are useful in transplantation of any biological material, including, for example, cells, tissue(s), bone marrow, and / or organ(s) or at least a portion of an organ, including, as non-limiting examples, the heart, kidney, pancreas, liver, and / or intestine. In some embodiments, the transplanted biological material is an allogeneic biological material. In some embodiments, the transplanted biological material is bone marrow. In some embodiments, the transplanted biological material is a population of hematopoietic stem cells. In some embodiments, the transplanted biological material is or is derived from one or more hepatic cells.

[0214] In the compositions and methods provided herein, NI-0501 is administered to a subject in need thereof to treat, prevent, and / or delay the onset or progression of or alleviate symptoms associated with transplant failure, transplant rejection, and / or inflammatory disorders associated with transplant rejection.In some embodiments, transplant rejection, also referred to herein as transplant failure, is acute.In some embodiments, transplant rejection is hyperacute.

[0215] In the compositions and methods provided herein, NI-0501 is formulated as a sterile concentrate for injection (per mL). In some embodiments, NI-0501 is formulated as follows: NI-051 5mg, L-histidine 1.55mg, L-histidine monohydrochloride monohydrate 3.14mg, sodium chloride (NaCl) 7.31mg, and polysorbate 80 0.05mg, pH 5.8 to 6.2. In some embodiments, NI-0501 is formulated as follows: NI-051 5mg, L-histidine 1.55mg, L-histidine monohydrochloride monohydrate 3.14mg, sodium chloride (NaCl) 7.31mg, and polysorbate 80 0.05mg, pH 6.0.

[0216] In some embodiments, graft rejection is chronic.In some embodiments, NI-0501 is administered to the subject in need thereof with an initial dose of 1mg / kg over 1 hour by IV infusion.In certain patient populations, such as low body weight and / or very young patient populations, IV infusion can last longer than 1 hour, for example, at least 90 minutes, at least 2 hours, or at least 3 hours or longer.

[0217] In some embodiments, NI-0501 is administered to a subject in need thereof at an initial dose of 1 mg / kg over 1 hour with at least one additional IV infusion after the initial IV infusion. In some embodiments, the at least one additional IV infusion is at a higher dose than the initial dose of 1 mg / kg. In some embodiments, the dosage of the at least one additional IV infusion is 3 mg / kg. In some embodiments, the at least one additional IV infusion is administered at least 3 days after the initial IV infusion. In some embodiments, the at least one additional IV infusion is administered at a time selected from the group consisting of 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial infusion, and 15 days after the initial infusion. In some embodiments, the at least one additional IV infusion is administered 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial infusion, and 15 days after the initial infusion.

[0218] In some embodiments, NI-0501 is administered to a subject in need thereof at an initial dose of 1 mg / kg over 1 hour with at least one additional series of IV infusions after the initial IV infusion, wherein the series of additional IV infusions includes at least one series of twice weekly IV infusions. In some embodiments, at least one series of twice weekly IV infusions is administered at a dose higher than the initial dose of 1 mg / kg. In some embodiments, at least one series of twice weekly IV infusions is administered at a dose of 3 mg / kg. In some embodiments, at least one additional IV infusion is administered at least 3 weeks after the initial IV infusion. In some embodiments, at least one additional IV infusion is administered at a time selected from the group consisting of 3 weeks after the initial IV infusion, 4 weeks after the initial IV infusion, 5 weeks after the initial IV infusion, 6 weeks after the initial infusion, 7 weeks after the initial infusion, and 8 weeks after the initial infusion. In some embodiments, at least one additional IV infusion is administered 3 weeks after the first IV infusion, 4 weeks after the first IV infusion, 5 weeks after the first IV infusion, 6 weeks after the first infusion, 7 weeks after the first infusion, and 8 weeks after the first infusion.

[0219] In some embodiments, NI-0501 is administered to a subject in need thereof by at least two additional IV infusions after the first IV infusion. In some embodiments, the at least two additional IV infusions are at a dose higher than the initial dose of 1 mg / kg. In some embodiments, the first additional IV infusion and the second additional IV infusion are administered at the same dosage. In some embodiments, the first additional IV infusion and the second additional IV infusion are administered at the same dosage higher than the initial dose. In some embodiments, at least one of the first and second additional IV infusions is administered at a dosage of 3 mg / kg. In some embodiments, the first additional IV infusion is administered at least 3 days after the first IV infusion. In some embodiments, the first additional IV infusion is administered at a time selected from the group consisting of 3 days after the first IV infusion, 6 days after the first IV infusion, 9 days after the first IV infusion, 12 days after the first infusion, and 15 days after the first infusion. In some embodiments, the first additional IV infusion is administered 3 days after the first IV infusion, 6 days after the first IV infusion, 9 days after the first IV infusion, 12 days after the first infusion, and 15 days after the first infusion. In some embodiments, the second additional IV infusion is administered at a time selected from the group consisting of 3 weeks after the first IV infusion, 4 weeks after the first IV infusion, 5 weeks after the first IV infusion, 6 weeks after the first infusion, 7 weeks after the first infusion, and 8 weeks after the first infusion. In some embodiments, the second additional IV infusion is administered 3 weeks after the first IV infusion, 4 weeks after the first IV infusion, 5 weeks after the first IV infusion, 6 weeks after the first infusion, 7 weeks after the first infusion, and 8 weeks after the first infusion. In some embodiments, the first additional IV infusion is administered 3 days after the first IV infusion, 6 days after the first IV infusion, 9 days after the first IV infusion, 12 days after the first infusion, and 15 days after the first infusion, and the second additional IV infusion is administered 3 weeks after the first IV infusion, 4 weeks after the first IV infusion, 5 weeks after the first IV infusion, 6 weeks after the first infusion, 7 weeks after the first infusion, and 8 weeks after the first infusion.

[0220] In some embodiments, the first additional IV infusion and the second additional IV infusion are administered at different dosages. In some embodiments, the first additional IV infusion and the second additional IV infusion are administered at different dosages, where the dosage of the second additional IV infusion is higher than the first additional IV infusion. In some embodiments, the first additional IV infusion and the second additional IV infusion are administered at different dosages, where the dosage of the second additional IV infusion is higher than the first additional IV infusion, and where both the dosages of the first and second additional IV infusions are higher than the first dosage. In some embodiments, at least one of the first and second additional IV infusions is administered at a dosage of 3 mg / kg. In some embodiments, the first additional IV infusion is administered at a dosage of 3 mg / kg, and the second additional IV infusion is administered at a dosage of 6 mg / kg. In some embodiments, the first additional IV infusion is administered at least 3 days after the first IV infusion. In some embodiments, the first additional IV infusion is administered at a time selected from the group consisting of 3 days after the first IV infusion, 6 days after the first IV infusion, 9 days after the first IV infusion, 12 days after the first infusion, and 15 days after the first infusion. In some embodiments, the first additional IV infusion is administered at a time selected from the group consisting of 3 weeks after the first IV infusion, 4 weeks after the first IV infusion, 5 weeks after the first IV infusion, 6 weeks after the first IV infusion, 7 weeks after the first infusion, and 8 weeks after the first infusion. In some embodiments, the second additional IV infusion is administered at a time selected from the group consisting of 3 weeks after the first IV infusion, 4 weeks after the first IV infusion, 5 weeks after the first IV infusion, 6 weeks after the first infusion, 7 weeks after the first infusion, and 8 weeks after the first infusion. In some embodiments, the second additional IV infusion is administered at a time selected from the group consisting of 3 weeks after the first IV infusion, 4 weeks after the first IV infusion, 5 weeks after the first IV infusion, 6 weeks after the first infusion, 7 weeks after the first infusion, and 8 weeks after the first infusion.In some embodiments, the first additional IV infusion is administered 3 days after the first IV infusion, 6 days after the first IV infusion, 9 days after the first IV infusion, 12 days after the first infusion, and 15 days after the first infusion, and the second additional IV infusion is administered 3 weeks after the first IV infusion, 4 weeks after the first IV infusion, 5 weeks after the first IV infusion, 6 weeks after the first infusion, 7 weeks after the first infusion, and 8 weeks after the first infusion.

[0221] In some embodiments, the first additional IV infusion comprises at least a first series of twice-weekly IV infusions, and the second additional IV infusion comprises at least a second series of twice-weekly IV infusions. In some embodiments, the first series of twice-weekly IV infusions and the second series of twice-weekly IV infusions are administered at a dose higher than the initial dose of 1 mg / kg. In some embodiments, the first series of twice-weekly IV infusions is administered at a dose of 3 mg / kg, and the second series of twice-weekly IV infusions is administered at a dose of 6 mg / kg. In some embodiments, the first series of additional IV infusions is administered at least 3 days after the first IV infusion. In some embodiments, the first series of additional IV infusions is administered at a time selected from the group consisting of 3 days after the first IV infusion, 6 days after the first IV infusion, 9 days after the first IV infusion, 12 days after the first infusion, and 15 days after the first infusion. In some embodiments, the first series of additional IV infusions is administered 3 days after the first IV infusion, 6 days after the first IV infusion, 9 days after the first IV infusion, 12 days after the first infusion, and 15 days after the first infusion. In some embodiments, the second series of additional IV infusions is administered at a time selected from the group consisting of 3 weeks after the first IV infusion, 4 weeks after the first IV infusion, 5 weeks after the first IV infusion, 6 weeks after the first infusion, 7 weeks after the first infusion, and 8 weeks after the first infusion. In some embodiments, the second series of additional IV infusions is administered 3 weeks after the first IV infusion, 4 weeks after the first IV infusion, 5 weeks after the first IV infusion, 6 weeks after the first infusion, 7 weeks after the first infusion, and 8 weeks after the first infusion. In some embodiments, a first series of additional IV infusions is administered 3 days after the first IV infusion, 6 days after the first IV infusion, 9 days after the first IV infusion, 12 days after the first infusion, and 15 days after the first infusion, and a second series of additional IV infusions is administered 3 weeks after the first IV infusion, 4 weeks after the first IV infusion, 5 weeks after the first IV infusion, 6 weeks after the first infusion, 7 weeks after the first infusion, and 8 weeks after the first infusion.

[0222] In some embodiments, after the first dose, infusions are administered every 3 days until 15 days after the first dose. In some embodiments, after the first dose, infusions are administered every 3 days until 15 days after the first dose, and then twice weekly infusions are administered starting at least 15 days after the first dose. In some embodiments, the dosage of the infusion is increased to 3 mg / kg at any time after the first dose. In some embodiments, after a minimum of two infusions at 3 mg / kg, the dose of NI-0501 is increased to 6 mg / kg for up to four infusions.

[0223] The present disclosure also provides compositions and methods useful in identifying or alternatively accurately distinguishing patient populations suffering from disorders, where the patients have elevated levels of CXCL9 alone or in combination with one or more additional interferon gamma (IFNγ)-related biomarkers. In particular, the present disclosure provides compositions and methods for detecting CXCL9 levels as a biomarker for IFNγ production in patients suffering from or suspected of suffering from hemophagocytic lymphohistiocytosis (HLH). In particular, the present disclosure provides compositions and methods for detecting CXCL9 levels as a biomarker for IFNγ production in patients suffering from or suspected of suffering from secondary hemophagocytic lymphohistiocytosis (HLH). In some embodiments, the compositions and methods are used to detect CXCL9 levels as a biomarker for IFNγ production in patients suffering from or suspected of suffering from macrophage activation syndrome (MAS). In some embodiments, the compositions and methods are used to detect CXCL9 levels as a biomarker for IFNγ production in patients suffering from or suspected of suffering from MAS in the context of an autoimmune disease or inflammatory disorder. In some embodiments, the compositions and methods are used to detect CXCL9 levels as a biomarker for IFNγ production in patients suffering from or suspected of suffering from MAS in the context of a systemic autoimmune disease or inflammatory disorder. In some embodiments, the compositions and methods are used to detect CXCL9 levels as a biomarker for IFNγ production in patients suffering from or suspected of suffering from MAS in the context of systemic juvenile idiopathic arthritis (sJIA). In some embodiments, the compositions and methods are used to detect CXCL9 levels as a biomarker for IFNγ production in patients suffering from or suspected of suffering from MAS in the context of systemic lupus erythematosus (SLE).

[0224] Patients identified as having elevated levels of CXCL9 are identified as suitable candidates for treatment with agents (e.g., antibodies or other polypeptide-based therapeutics, peptide-based therapeutics, small molecule inhibitors, nucleic acid-based therapeutics and derivatives thereof) that interfere with or alternatively antagonize one or more biological activities of IFNγ, such as IFNγ signaling, and neutralize at least one biological activity of IFNγ.

[0225] In some patients suffering from or suspected of suffering from the disorder, fluids and other biological samples contain elevated levels of CXCL9 alone or in combination with other IFNγ-related biomarkers, such as CXCL10 and / or CXCL11.

[0226] CXCL9 and these other biomarkers are indicators of IFNγ production in vivo. Thus, IFNγ activity is blocked or inhibited by the use of anti-IFNγ antagonists, such as neutralizing anti-IFNγ antibodies or other polypeptide-based therapeutics, peptide-based therapeutics, small molecule inhibitors, nucleic acid-based therapeutics and derivatives thereof, which interfere with, inhibit, reduce or alternatively antagonize IFNγ signaling. Thus, the compositions and methods are useful in treating, delaying the progression of, or alternatively reversing the symptoms of disorders that depend on, are driven by, are associated with, or are alternatively affected by abnormalities, such as elevations, abnormalities in pro-inflammatory cytokine production, and / or combinations thereof, by administering anti-IFNγ antagonists, such as neutralizing anti-IFNγ antibodies or other polypeptide-based therapeutics, peptide-based therapeutics, small molecule inhibitors, nucleic acid-based therapeutics and derivatives thereof, to patients who show elevated levels of expression of CXCL9 and / or other biomarkers. Patients who are likely to be suitable candidates for treatment with anti-IFNγ antagonists, such as neutralizing anti-IFNγ antibodies as described herein, are identified by detecting the level of CXCL9 alone or in combination with one or more IFNγ-related ligands or other biomarkers. In some embodiments, patients who do not have elevated levels of CXCL9 alone or in combination with other IFNγ-related biomarkers can still be treated with anti-IFNγ antagonists, including neutralizing anti-IFNγ antibodies or other polypeptide-based therapeutics, peptide-based therapeutics, small molecule inhibitors, nucleic acid-based therapeutics and derivatives thereof, as described herein.

[0227] Patients with elevated levels of CXCL9 alone or in combination with one or more additional IFNγ-related biomarkers are identified as suitable candidates for treatment with one or more anti-IFNγ antagonists, such as the neutralizing anti-IFNγ antibodies described herein.As used herein, the phrase "elevated level of expression" refers to a level of expression that is higher than the baseline level of expression of CXCL9 alone or in combination with one or more additional biomarkers in a sample from a patient who is not affected or suspected of being affected by primary or secondary HLH or HLH-related disorder, or in another control sample.In some embodiments, the elevated level of expression of CXCL9 and / or other biomarkers is a significant level of elevation.

[0228] The detected level of CXCL9 alone or in combination with one or more other IFNγ-related biomarkers is useful for accurately identifying or alternatively stratifying patient populations. In some embodiments, the detected level is used to determine the dosage of anti-IFNγ antagonist to be administered to a given patient. In some embodiments, the detected level is used to categorize or alternatively stratify patient populations. For example, patients can be classified as having "severe" or high-grade MAS, or conversely, having non-severe or low-grade MAS, based on the detected level of CXCL9.

[0229] The sample is, for example, blood or blood components, for example, serum, plasma. In some embodiments, the sample is another body fluid, such as, for example, but not limited to, urine, synovial fluid, bronchoalveolar fluid, cerebrospinal fluid, bronchoalveolar lavage fluid (BAL), and / or saliva. In some embodiments, the biological sample is CSF. In some embodiments, the biological sample is CSF from a HLH patient.

[0230] In addition to detecting the level of IFNγ and / or other IFNγ-related biomarkers, patients suitable for treatment with anti-IFNγ antagonists can also be identified by evaluating any of several additional biological and clinical parameters that improve the sensitivity and specificity of biomarkers to identify or alternatively accurately discriminate patient populations. Alternatively, these additional biological and clinical parameters can be used alone as a means to identify patients who are suitable candidates for treatment with anti-IFNγ antagonists or other appropriate therapies. These biological and clinical parameters include, by way of non-limiting example, any of the following: ferritin level, neutrophil count, platelet count, alanine aminotransferase level, and / or lactate dehydrogenase level.

[0231] Disorders for which the compositions and methods of the invention are useful include any disorder in which there is an abnormality, eg, elevation, of IFNγ expression and / or activity, particularly HLH, including secondary HLH, MAS, and / or sJIA.

[0232] By way of non-limiting example, the methods and compositions provided herein are suitable for diagnosing and / or treating disorders such as primary and / or secondary HLH disorders. Suitable autoimmune and / or inflammatory disorders include, by way of non-limiting example, primary and / or secondary HLH disorders associated with aberrant IFNγ activity and / or expression.

[0233] Once a patient is identified as having an elevated level of CXCL9 alone or in combination with one or more IFNγ-related biomarkers, the patient is then treated with an anti-IFNγ antagonist.For example, the anti-IFNγ antagonist is a neutralizing anti-IFNγ antibody or its immunologically active (e.g., antigen-binding) fragment.Suitable neutralizing anti-IFNγ antibodies include any of the anti-IFNγ antibodies described herein.

[0234] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises a variable heavy chain complementarity determining region 1 (VH CDR1) comprising an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SYAMS (SEQ ID NO:1); a variable heavy chain complementarity determining region 2 (VH CDR2) comprising an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AISGSGGGSTYYADSVKG (SEQ ID NO:2); and a variable heavy chain complementarity determining region 3 (VH CDR4) comprising an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO:3). CDR3); variable light chain complementarity determining region 1 (VL CDR1) comprising an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO:4); variable light chain complementarity determining region 2 (VL CDR2) comprising an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of EDNQRPS (SEQ ID NO:5); and variable light chain complementarity determining region 3 (VL CDR3) comprising an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of QSYDGSNRWM (SEQ ID NO:6).

[0235] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises a VH CDR1 comprising the amino acid sequence of SYAMS (SEQ ID NO:1); a VH CDR2 region comprising the amino acid sequence of AISGSGGGSTYYADSVKG (SEQ ID NO:2); and a VH CDR3 region comprising the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO:3); a variable light chain complementarity determining region 1 (VL CDR1) region comprising the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO:4); a VL CDR2 region comprising the amino acid sequence of EDNQRPS (SEQ ID NO:5); and a VL CDR3 region comprising the amino acid sequence of QSYDGSNRWM (SEQ ID NO:6).

[0236] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises a heavy chain comprising a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein the combination is one of the three heavy chain CDR sequences shown in one row of Table 1A (VH CDR1, VH CDR2, VH CDR3, VH CDR4, VH CDR5, VH CDR6, VH CDR7, VH CDR8, VH CDR9, VH CDR10, VH CDR11, VH CDR12, VH CDR13, VH CDR14, VH CDR15, VH CDR16, VH CDR17, VH CDR18, VH CDR19, VH CDR110, VH CDR111, VH CDR12, VH CDR132, VH CDR14, VH CDR15, VH CDR16, VH CDR1 The heavy chain comprises a combination of VH CDR1, VH CDR2, VH CDR3).

[0237] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises a light chain that comprises a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination is one of the three light chain CDR sequences shown in one row of Table 1B (VL CDR1, VL CDR2, VL CDR3, VL CDR4, VL CDR5, VL CDR6, VL CDR7, VL CDR8, VL CDR9, VL CDR10, VL CDR11, VL CDR12, VL CDR13, VL CDR14, VL CDR15, VL CDR16, VL CDR17, VL CDR18, VL CDR19, VL CDR110, VL CDR111, VL CDR121, VL CDR132, VL CDR14, VL CDR153, VL CDR164, V The light chain comprises a combination of VL CDR1, VL CDR2, VL CDR3).

[0238] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises a heavy chain comprising a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein the combination is one of the three heavy chain CDR sequences shown in one row of Table 1A (VH CDR1, VH CDR2, VH CDR3, VH CDR4, VH CDR5, VH CDR6, VH CDR7, VH CDR8, VH CDR9, VH CDR10, VH CDR11, VH CDR12, VH CDR13, VH CDR14, VH CDR15, VH CDR16, VH CDR17, VH CDR18, VH CDR19, VH CDR110, VH CDR111, VH CDR12, VH CDR132, VH CDR14, VH CDR15, VH CDR16, VH CDR1 and a light chain comprising a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination is a combination of the three light chain CDR sequences (VL CDR1, VL CDR2, VL CDR3) shown in one row of Table 1B.

[0239] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to the heavy chain variable amino acid sequence of SEQ ID NO:47.

[0240] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to the light chain variable amino acid sequence of SEQ ID NO:48.

[0241] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to a heavy chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO:47, and an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to a light chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO:48.

[0242] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises a heavy chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO:47.

[0243] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises a light chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO:48.

[0244] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises a heavy chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO:47, and a light chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO:48.

[0245] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to the heavy chain amino acid sequence of SEQ ID NO:44.

[0246] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to the light chain amino acid sequence of SEQ ID NO:46.

[0247] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to a heavy chain amino acid sequence that is the amino acid sequence of SEQ ID NO:44, and an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to a light chain amino acid sequence that is the amino acid sequence of SEQ ID NO:46.

[0248] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises a heavy chain amino acid sequence that is the amino acid sequence of SEQ ID NO:44.

[0249] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises a light chain amino acid sequence that is the amino acid sequence of SEQ ID NO:46.

[0250] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises a heavy chain amino acid sequence that is the amino acid sequence of SEQ ID NO:44 and a light chain amino acid sequence that is the amino acid sequence of SEQ ID NO:46.

[0251] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to a heavy chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, and 102.

[0252] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to a light chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, and 104.

[0253] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to a heavy chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, and 102, and an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to a light chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, and 104. In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises a heavy chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, and 102.

[0254] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises a light chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, and 104.

[0255] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises a heavy chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, and 102, and a light chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, and 104.

[0256] In some embodiments, the anti-IFNγ antibody or its immunologically active fragment is administered in a therapeutically effective amount. The therapeutically effective amount of the antibody of the present invention generally relates to the amount necessary to achieve a therapeutic purpose. This therapeutic purpose may, in certain cases, be the binding interaction of the antibody with its target antigen that interferes with the function of the target. The general range for therapeutically effective dosing of the antibody or antibody fragment of the present invention may be, as a non-limiting example, from about 0.1 mg per kg body weight to about 50 mg per kg body weight. The general dosing frequency may range, for example, from twice a day to once a week.

[0257] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof is administered at an initial dose or loading dose ranging from about 0.5 mg / kg to about 2 mg / kg, e.g., from about 0.5 mg / kg to about 1.5 mg / kg, and / or from about 0.5 mg / kg to about 1.0 mg / kg. In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof is administered at an initial dose of about 1.0 mg / kg.

[0258] In some embodiments, the anti-IFNγ antibody or its immunologically active fragment is administered as an initial loading dose, followed by one or more maintenance doses. In some embodiments, the one or more maintenance doses are substantially the same dosage as the initial loading dose. In some embodiments, the one or more maintenance doses are lower dosage than the initial loading dose. In some embodiments, the one or more maintenance doses are higher dosage than the initial loading dose.

[0259] In some embodiments, one or more maintenance doses include at least two or more doses, and each maintenance dose is the same dose.In some embodiments, two or more maintenance doses are substantially similar to the initial loading dose.In some embodiments, two or more maintenance doses are greater than the initial loading dose.In some embodiments, two or more maintenance doses are less than the initial loading dose.

[0260] In some embodiments, the one or more maintenance doses include at least two or more doses, and each maintenance dose is not the same dose. In some embodiments, the two or more maintenance doses are administered in ascending doses. In some embodiments, the two or more maintenance doses are administered in decreasing doses.

[0261] In some embodiments, the one or more maintenance doses include at least two or more doses, and each maintenance dose is administered at a periodic time interval. In some embodiments, the two or more doses are administered at increasing time intervals. In some embodiments, the two or more doses are administered at decreasing time intervals.

[0262] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof is administered at an initial loading dose ranging from about 0.5 mg / kg to about 2 mg / kg, e.g., from about 0.5 mg / kg to about 1.5 mg / kg, and / or from about 0.5 mg / kg to about 1.0 mg / kg, followed by at least one, e.g., two or more, three or more, four or more, or five or more maintenance doses. In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof is administered at an initial loading dose of about 1.0 mg / kg, followed by at least one, e.g., two or more, three or more, four or more, or five or more maintenance doses.

[0263] Pharmaceutical compositions according to the invention can include an anti-IFNγ antibody of the invention and a carrier. These pharmaceutical compositions can be included in a kit, such as a diagnostic kit.

[0264] The present invention also provides a kit for carrying out any of the methods presented herein.For example, in some embodiments, the kit comprises a detection reagent specific for CXCL9 alone or in combination with one or more IFNγ-related biomarkers and a means for detecting the detection reagent.

[0265] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES

[0266] Example 1 CXCL9 levels as a biomarker for IFN-.GAMMA. production in macrophage activation syndrome (MAS) To search for potential biomarkers of in vivo production of IFNγ, the study presented here was designed to evaluate the correlation of serum levels of IFNγ and three IFNγ-related chemokines with themselves and with laboratory parameters of disease activity in patients with active MAS.

[0267] Circulating levels of IFNγ, CXCL9, CXCL10, CXCL11 and IL-6 were measured using Luminex multiplex assays in patients with sJIA, 20 of whom had MAS at the time of sampling. Associations between these circulating levels and disease activity parameters were assessed, along with correlations between levels of IFNγ and levels of CXCL9, CXCL10 and CXCL11.

[0268] In active MAS, levels of IFNγ and three IFNγ-related chemokines (CXCL9, CXCL10 and CXCL11) were significantly elevated at the time of sampling compared to active sJIA without MAS (all p-values ​​<0.005). In active MAS, laboratory parameters of disease severity (ferritin, neutrophils, platelets, alanine aminotransferase and lactate dehydrogenase) correlated significantly with IFNγ and CXCL9, to a lesser extent with CXCL10 and CXCL11, and no correlation was found with IL-6 levels. As shown in Table 7 below, in patients with active sJIA without MAS, there was no significant correlation between laboratory parameters and cytokine levels. In active MAS, IFNγ levels correlated significantly with CXCL9 levels (r=0.69; r 2 = 0.47; p = 0.001), and correlated to a lesser extent with levels of CXCL10 (r = 0.53; r 2 = 0.28; p = 0.015), but did not correlate with CXCL11 levels (r = -0.04; p = 0.886). Table 7. Correlation of laboratory parameters of disease activity with IFNγ, CXCL9, CXCL10, CXCL11, and IL-6 in patients with MAS and in patients with active sJIA. [Table 7]

[0269] High levels of IFNγ and CXCL9 present in patients with active MAS correlate significantly with laboratory parameters of disease severity. In patients with active MAS, IFNγ and CXCL9 are closely correlated. Because CXCL9 has been shown to be induced only by IFNγ and not by other interferons (see, e.g., Groom JR and Luster AD Immunol Cell Biol, February 2011;89(2):207-15), these findings demonstrate that CXCL9 is a biomarker of IFNγ production in MAS. Example 2 Correlation between CXCL9 and IFN-gamma levels in patients with primary hemophagocytic lymphohistiocytosis (HLH)

[0270] The studies presented herein are from an ongoing Phase 2 pilot study in patients with primary HLH who received the NI-0501 antibody, as well as from patients who received the NI-0501 antibody under compassionate use.

[0271] Serum levels of CXCL9 and IFNγ in samples from six primary HLH patients and three compassionate use patients were measured by Luminex and Meso Scale Discovery (MSD) technology, respectively, as shown in Figure 1. Correlation between CXCL9 concentration and total IFNγ concentration was performed. Statistics were performed and p-values ​​were obtained using the Spearman test.

[0272] Pre-dose serum levels of CXCL9 and IFNγ were measured by Luminex and MSD technology, respectively, in samples from six primary HLH patients and three compassionate use patients, as shown in Figure 2. Correlation between CXCL9 and total IFNγ concentrations was performed. Statistics were performed and p-values ​​were obtained using the Spearman test. Example 3 Correlation between CXCL9 and IFN-gamma levels in patients with secondary hemophagocytic lymphohistiocytosis (HLH)

[0273] The studies presented herein are derived from observational studies in patients with secondary HLH who received NI-0501 antibody, as well as from patients who received NI-0501 antibody on a compassionate use basis.

[0274] Specifically, these are patients with systemic juvenile idiopathic arthritis (sJIA) in which macrophage activation syndrome (MAS, a form of secondary HLH) has occurred. For these patients, there is also a correlation between CXCL9 or IFNγ and disease parameters such as ferritin, platelet count (PLT), neutrophil count (Neu), and alanine aminotransferase (ALT).

[0275] As shown in Figure 3A and Figure 3B, CXCL9 and IFNγ serum levels were measured by multiplex assay using Luminex technology from samples from 19 patients with MAS secondary to sJIA and 24 patients with active sJIA at the time of sampling. Correlation between CXCL9 and IFNγ concentrations was performed. Statistics were performed and p-values ​​were obtained using the Spearman test.

[0276] As shown in Figures 4A-1, 4A-2, 4B-1, 4B-2, 4C-1, 4C-2, 4D-1, and 4D-2, serum levels of CXCL9 and IFNγ were measured by multiplex assay using Luminex technology from patients with MAS secondary to sJIA and from patients with active sJIA at the time of sampling. Correlation of IFNγ or CXCL9 levels with ferritin, platelet count, neutrophil count, or ALT (alanine aminotransferase) was performed. Statistics were performed and p-values ​​were obtained using the Spearman test. Example 4 Correlation between CXCL9 and IFN-gamma levels in patients with severe hemophagocytic lymphohistiocytosis (HLH)

[0277] The study presented here is from a patient who received the NI-0501 antibody under compassionate use. The patient presented with NLRC4-associated disease and symptoms of severe hemophagocytic lymphohistiocytosis (HLH). Mutations in the NLRC4 gene have recently been reported to cause recurrent macrophage activation syndrome and increased production of IL-18, which is known to induce IFNγ.

[0278] The patient in this study had the following characteristics: Onset at 20 days of age with fever, skin rash, marked hepatosplenomegaly, pancytopenia, hypofibrinogenemia, hypertriglyceridemia, markedly elevated ferritin and sCD25. Multiple organ failure followed, necessitating admission to ICU. HLH diagnosis was based on six of eight HLH-2004 criteria. Primary HLH causing genes (PRF1, UNC13D, STXBP2, STX11, RAB27A, XIAP) and functional tests (perforin expression, degranulation and cytotoxicity) were negative. High-dose iv glucocorticoids and iv cyclosporine A with progressive improvement in performance status and laboratory abnormalities. HLH reactivation induced by infection (Candida Albicans and Klebsiella Pneumoniae sepsis), rapid deterioration in performance status, and new ICU admission. Treatment with etoposide and / or ATG was not considered due to the presence of active infection in already immunocompromised subjects.

[0279] Not only were measurable serum levels of IFNγ and high serum levels of the IFNγ-inducible chemokines, CXCL9 and CXCL10, demonstrated, but also significantly elevated serum levels of IL-18 (Table 8). [Table 8]

[0280] Dexamethasone (13.6 mg / m 2 ) and iv cyclosporine A. Compassionate use treatment with NI-0501 was initiated. NI-0501 was administered pharmacokinetically every 3 days, then every 7 days. No infusion reactions were observed. NI0501 was well tolerated. Clinical features and laboratory abnormalities of HLH progressively improved. The active ongoing infection was rapidly cleared. After 5 months of treatment, the patient remained in excellent condition. The patient was treated with oral cyclosporine A (6 mg / kg) and prednisone (0.3 mg / kg, 0.9 mg / m 2The patient continued to receive 100 mg of bronchodilator therapy (equivalent to 100 mg of dexamethasone). All HLH parameters normalized.

[0281] The subject still exhibited episodes of inflammation of unknown etiology. Analysis of NLRC4 revealed a novel missense mutation (T337N). Elevated serum IL-18 was demonstrated, confirming the relevance of the NLRC4 mutation. High production of IFNγ was demonstrated by high levels of IFNγ complexed with NI-0501. IFNγ was fully neutralized as shown by undetectable levels of IFNγ-inducible chemokines (Figure 5 and Table 8). Circulating levels of IL-18 were persistently elevated.

[0282] Thus, this study demonstrates that blockade of IFNγ with NI-0501 in patients with severe refractory HLH (due to NLRC4 mutations) is well tolerated, has no safety concerns, allows control of all HLH features, allows rapid glucocorticoid tapering, and is associated with resolution of ongoing active infection. Example 5 A Targeted Approach to Treat Hemophagocytic Lymphohistiocytosis (HLH) with NI-0501

[0283] The study presented here is from a pilot phase 2 study in children with primary HLH. Primary HLH (pHLH) is a rare immunoregulatory disorder that is invariably fatal if untreated. pHLH is driven by pathological immune activation, leading to the development of fever, splenomegaly, cytopenias and coagulopathy, which may cause multiple organ failure and death. Based on data from mouse models of primary and secondary HLH (sHLH) treated with anti-IFNγ antibodies, as well as observational studies in patients with HLH, high production of IFNγ appears to be a pivotal factor driving disease development. Immunochemotherapy, primarily etoposide-based regimens, is currently the only pharmacological approach to control HLH and guide patients to curative allogeneic hematopoietic stem cell transplantation (allo-HSCT). Despite recent attempts to further intensify treatment regimens, mortality and morbidity remain high, due in part to drug-related toxicity.

[0284] As described above, NI-0501 is a fully human, high affinity, anti-IFNγ mAb that binds to and neutralizes human IFNγ, providing a novel targeted approach to control HLH.

[0285] METHODS: An open-label Phase 2 study was conducted in the United States and Europe to evaluate the safety and efficacy of NI-0501 in children with confirmed or suspected pHLH. NI-0501 was administered at an initial dose of 1 mg / kg every 3 days, with initial background dexamethasone 5-10 mg / m 2 Methods: PK data and / or clinical response to chemotherapy guided possible dose escalation. Duration of treatment ranged from 4 to 8 weeks. Ability to proceed to allo-HSCT, relevant HLH disease parameters, and 8-week survival were assessed.

[0286] Study Population: A total of 13 patients were enrolled: 8F / 5M, median age 1.0 years (range 2.5 months to 13 years). Twelve patients who had reactivated, had an inadequate response, or were intolerant to treatment after receiving conventional therapy received NI-0501 as second-line treatment. One patient was treated with NI-0501 as first-line. Nine patients had known HLH gene defects (three had FHL2, two had FHL3, two had GS-2, one had XLP1, and one had XLP2). The majority of patients were in the severe end-stage of the HLH spectrum, had compromised performance status, and had significant toxicity from previous HLH treatment. Twelve of the 13 patients had elevated ferritin, 8 had elevated sCD25, 10 had cytopenias, 8 had splenomegaly, and 9 had hypofibrinogenemia and hypertriglyceridemia. Liver dysfunction and CNS complications were present in 7 and 3 patients, respectively.

[0287] Results: Overall, treatment with NI-0501 significantly improved HLH disease activity parameters (Figure 6), with adequate responses achieved in 9 of 13 patients. Six patients proceeded to HSCT. Two patients with good HLH control plan to proceed to HSCT once a suitable donor is identified. In one patient (disease control achieved with first-line NI-0501), HSCT is not yet planned given the absence of a causative HLH gene mutation. Eleven of 13 patients were alive at 8 weeks. CNS signs and symptoms resolved in two evaluable patients. It was possible to reduce the dexamethasone dose by more than 50% in 50% of patients during the first 4 weeks of treatment with NI-0501.

[0288] Assessment of biomarkers, in particular CXCL9, a chemokine known to be exquisitely induced by IFNγ, not only allowed the demonstration of complete IFNγ neutralization but also correlated with IFNγ production and appears to be a new parameter for diagnosing HLH (Figures 7A and 7B).

[0289] NI-0501 was well tolerated and no safety concerns were identified. No infections known to benefit from IFNγ neutralization were reported, and no infections occurred in patients who had not received prior chemotherapy. Seven patients reported at least one SAE, all of which were assessed by the DMC as not related to NI-0501 administration. No unexpected events due to "off-target" effects of NI-0501 (e.g., bone marrow toxicity, hemodynamic effects) were observed.

[0290] Conclusions: Targeted neutralization of IFNγ with NI-0501 offers an innovative and potentially less toxic approach to managing HLH. The results of this study demonstrate that NI-0501 is a safe and effective treatment option in patients with primary HLH who have responded inadequately to or are intolerant to conventional therapy. Furthermore, treatment with NI-0501 was not associated with any of the typical short- or long-term toxicities associated with etoposide-based regimens. Evaluation of NI-0501 as a first-line treatment in patients with pHLH is ongoing and it is anticipated that similar significant clinical benefits may be realized. Example 6 Elevated circulating levels of interferon-gamma and interferon-inducible chemokines characterize patients with macrophage activation syndrome complicating systemic JIA

[0291] Interferon gamma (IFNγ) is a central mediator in a mouse model of primary hemophagocytic lymphohistiocytosis (HLH). Given the similarities between primary HLH and secondary HLH (sec-HLH), including macrophage activation syndrome (MAS), we analyzed IFNγ levels and its biological activity in patients with systemic juvenile idiopathic arthritis (sJIA) and MAS.

[0292] In the study presented herein, Luminex multiplex assays were used to evaluate serum levels of IL-1β, IL-6, IFNγ, and the IFN-inducible and / or IFN-related chemokines, CXCL9, CXCL10, and CXCL11, in patients with sec-HLH (n=11) and sJIA (n=54), 20 of whom had MAS at the time of sampling. Expression of IFNγ-inducible chemokines (CXCL9 and CXCL10 mRNA levels in liver and spleen) and their correlation with serum ferritin levels were evaluated in an IL-6 transgenic mouse model in which features of MAS are induced by TLR4 stimulation with LPS.

[0293] As shown in more detail below, during MAS, also referred to herein as active MAS, and sec-HLH, circulating levels of IFNγ and IFN-inducible chemokines were significantly elevated. IFNγ and IFN-inducible chemokines levels were significantly higher in patients with MAS compared to patients with active sJIA without MAS. In this latter group, IFNγ and IFNγ-inducible chemokines were comparable to patients with clinically inactive sJIA. During MAS, laboratory abnormalities characterizing this syndrome, including ferritin and alanine transferase levels, as well as neutrophil and platelet counts, significantly correlated with IFNγ and CXCL9 levels. In a mouse model of MAS, serum levels of ferritin significantly correlated with CXCL9 mRNA levels in the liver and spleen.

[0294] Thus, the studies presented below demonstrate that high levels of IFNγ and IFN-inducible chemokines and their correlation, particularly with CXCL9, with the severity of laboratory abnormalities in MAS suggest that IFNγ plays a central role in MAS.Elevated circulating levels of interferon-γ and interferon-inducible chemokines characterize patients with macrophage activation syndrome complicating systemic JIA.

[0295] Materials and Methods: Patients and Samples. Peripheral blood samples were collected from patients with sJIA with or without MAS in three Paediatric Rheumatology Centers: the Ospedale Pediatrico Bambino Gesu in Rome, the Istituto Giannina Gaslini in Genoa and the Cincinnati Children's Hospital Medical Center. Fifty-four patients with sJIA (age at onset 7.9 years, interquartile range 4.6-13.6 years; 48% female) who fulfilled the ILAR classification criteria for systemic arthritis were studied (Petty, RE et al., International League of Associations for Rheumatology classification of juvenile idiopathic arthritis: revised 2nd edition, Edmonton, 2001; J Rheumatol, 2004, 31(2):390-2). Twenty of the SJIA patients had samples taken during episodes of active end-stage MAS diagnosed by the treating physicians at each of the three centers. A posteriori analysis showed that 17 of these 20 episodes (85%) fulfilled the newly proposed classification criteria for MAS (Minoia F, Davi S, Bovis F, et al. Development of new classification criteria for macrophage activation syndrome complicating systemic juvenile idiopathic arthritis. Pediatric Rheumatology 2014 Vol 12(Suppl 1):O1). Samples were available from 28 patients with active SJIA without evidence of MAS. Thirty-five samples were available from 35 sJIA patients (with and without history of MAS) during clinically inactive disease as defined according to the Wallace criteria (Wallace, CA, et al. Preliminary criteria for clinical remission for select categories of juvenile idiopathic arthritis. J Rheumatol, 2004, Volume 31 (Issue 11): 2290-4).

[0296] As IFNγ has been shown to be elevated in patients with sec-HLH (excluding rheumatic diseases), samples were also taken from 11 patients with sec-HLH seen at Ospedale Pediatrico Bambino Gesu (age at onset 8.6 years, interquartile range 4.1-12.9 years; 36% female) and used as positive controls. All sec-HLH patients fulfilled the 2004-HLH diagnostic guidelines (Henter, JI et al., HLH-2004: Diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer, 2007, 48(2):124-31), with six patients fulfilling five criteria and five patients fulfilling four criteria. It should be noted that the levels of sCD25 in U / ml were not available as this test is not routinely performed in the center where these patients were recruited. The diagnosis of primary HLH was excluded based on the absence of family history, the absence of pathogenic mutations in genes known to cause HLH, and the presence of normal functional tests (NK activity, perforin expression, and CD107 degranulation).All 11 patients with sec-HLH each contributed one sample obtained during active disease.

[0297] Clinical and laboratory characteristics of all patients with regard to diagnosis and at the time of sampling were collected in a centralized web database by the responsible investigator at each center. Of the 20 MAS patients sampled during active disease, 6 had not received any treatment at the time of sampling, and the remaining 14 patients had already received one of the specific treatments for MAS, including pulsed glucocorticoids, cyclosporine A, anakinra, or cyclophosphamide. Of the 11 patients with sec-HLH with active disease, 6 had not yet received any specific treatment at the time of sampling, and the remaining 5 patients had already received at least one of the above treatments. The Ethical Committee of the Ospedale Pediatrico Bambino Gesu approved the study. Written informed consent was collected for all participants.

[0298] Cytokine quantification. Levels of IL-6, IL-1β, IFNγ, CXCL9, CXCL10 and CXCL11 were analyzed by Luminex® multiplex bead technology. Reagents were purchased from Millipore and all reagents were provided with the Milliplex® MAP kit. Reagents were prepared according to the manufacturer's protocol. 25 μl / well of standards, blanks and quality check samples were added in duplicate to a Milliplex MAP 96-well plate, followed by 25 μl of serum matrix. 25 μl of assay buffer was added to each sample well, followed by 25 μl of sample. Samples are added in duplicate or triplicate depending on the sample volume available. Plates were measured on a Luminex 200® system (Luminex Corp.). Raw data were acquired using x PONENT software version 3.1 (Luminex Corp.) and data were analyzed using Milliplex Analyst software version 3.5.5.0 (Millipore). Raw data obtained with Milliplex Analyst software were then further analyzed with Luminex analysis-specific macros (NI-Sc-ESM-MAC-012-v01 and Sc-ESM-MAC-013-v01).

[0299] Animal experiments. The generation and phenotype of IL-6 transgenic mice, as well as the characteristics of the MAS-like syndrome induced by administration of TLR ligands, have been described previously (Stripoli, R. et al., Amplification of the response to Toll-like receptor ligands by prolonged exposure to interleukin-6 in mice: implication for the pathogenesis of macrophage activation syndrome., Arthritis Rheum, 2012, 64(5):1680-8). Mice were maintained under specific pathogen-free conditions and handled in accordance with the National Police. The study protocol was approved by the local ethical committee. All experiments were performed on mice between 10 and 14 weeks of age. Mice were intraperitoneally administered a single dose of 5 μg per gram of body weight of lipopolysaccharide (LPS, E. coli serotype 055:B5; Sigma-Aldrich). Mice were sacrificed 30 hours later. Total RNA was extracted from spleen and liver tissues using Trizol (Life technologies). cDNA was obtained using the Superscript Vilo kit (Invitrogen). Real-time PCR assays were performed using TaqMan Universal PCR Master Mix (Applied Biosystems) with mouse Cxcl9 and Cxcl10 gene expression assays (Applied Biosystems). Gene expression data were normalized using mouse Hprt (Applied Biosystems). Data were analyzed using 2 -Δct The serum ferritin concentrations were determined using a commercially available ELISA kit (ALPCO Diagnostics) according to the manufacturer's instructions.

[0300] Statistical analysis. Statistical analysis was performed using GraphPad Prism 5 software. Continuous variables (quantitative demographic, clinical and laboratory data) were expressed as median and interquartile range (IQR) and compared using the Mann-Whitney U test. The Wilcoxon signed rank test was used to compare two paired groups without assuming that the distribution of pre-post differences followed a Gaussian distribution. Spearman rank correlation was used to evaluate the relationship with laboratory parameters. A p-value <0.05 was considered statistically significant.

[0301] Results: Elevated levels of IFNγ and IFNγ-inducible chemokines in patients with MAS. When comparing patients with active sJIA without MAS at the time of sampling with patients sampled during clinically inactive disease, we found that IL-6 levels were significantly higher (p<0.01) in patients with active sJIA compared with those with clinically inactive disease, as expected (de Benedetti, F. et al., Correlation of serum interleukin-6 levels with joint involvement and thrombocytosis in systemic juvenile rheumatoid arthritis., Arthritis Rheum, 1991, 34(9):1158-63). As reported in several previous studies of active SJIA, serum IL-1β levels were below the limit of detection in the majority of patients, regardless of disease activity status. Of note, levels of IFNγ and the three IFNγ-inducible chemokines did not differ between patients with clinically active sJIA and those with clinically inactive disease.

[0302] When comparing patients with MAS at sampling with patients with active sJIA without MAS at sampling, the levels of IL-1β and IL-6 were comparable, suggesting that the levels of these two cytokines, known to play a central role in active sJIA, are not elevated during end-stage MAS. It should be noted that circulating IL-1β levels were below the limit of quantification (i.e., 3.5 pg / ml) in the majority of patients with sJIA with or without MAS. In contrast, circulating IFNγ levels were significantly higher in patients with active MAS compared with patients with active sJIA without MAS at sampling. The levels of three IFNγ-related chemokines, CXCL9, CXCL10, and CXCL11, were also significantly higher in patients with active MAS compared with patients with active sJIA without MAS at sampling. This difference was particularly clear for CXCL9, whose median levels were approximately 15-fold higher in patients with MAS compared with patients with active sJIA without MAS.

[0303] In patients with sec-HLH, the levels of IFNγ and the three IFNγ-related chemokines were significantly elevated. The levels of IFNγ and the IFNγ-related chemokines were nearly indistinguishable from those in patients with MAS, and the differences were not statistically significant. Incidentally, the levels of IFNγ and the three IFNγ-inducible chemokines in patients with active MAS and in patients with active sec-HLH were comparable to those in untreated and previously treated patients.

[0304] The levels of IFNγ and CXCL9, CXCL10 and CXCL11 are associated with the presence of MAS in individual patients. Figures 8A-8D show the levels of IFNγ and CXCL9, CXCL10 and CXCL11 in individual patients for whom paired samples were available during active MAS and during active sJIA without MAS. Consistent with the results obtained in the cross-sectional analysis, the levels of IFNγ and the three IFNγ-inducible chemokines were significantly higher in samples obtained by paired sample analysis during MAS. Furthermore, in some patients, samples were available both before and after MAS episodes, demonstrating that IFNγ and IFNγ-inducible chemokine levels return to normal once MAS clinical symptoms resolve. For example, one patient in this study experienced three episodes of MAS and serum samples were obtained during these episodes as well as during disease phases without MAS at the time of sampling. In this patient, elevated levels of IFNγ and the three IFNγ-related chemokines were found only during MAS episodes, further confirming the association of increased production of IFNγ and the three IFNγ-inducible chemokines with active MAS (Figures 9A-9B).

[0305] Levels of IFNγ and IFNγ-related chemokines correlate with laboratory abnormalities in MAS. We then investigated the correlation of levels of IFNγ and the three IFNγ-inducible chemokines with laboratory parameters of MAS at the time of sampling. In patients with active sJIA without MAS, levels of IFNγ and the three IFNγ-inducible chemokines did not correlate with laboratory parameters of MAS, with one exception: levels of CXCL9, CXCL10, and CXCL11 correlated with ALT levels with r values ​​ranging from 0.17 to 0.25. 2(Table 2). The significance of this association is unclear, but it should be noted that ALT levels were within the normal range in all patients with active sJIA without MAS. No significant correlations of IL-1 and IL-6 with laboratory features of MAS were found in patients with MAS at sampling. In contrast, in patients with MAS at sampling, levels of IFNγ and IFNγ-inducible chemokines were associated with levels of ferritin, neutrophil and platelet counts, as well as elevated LDH and ALT, all of which are typically abnormal in patients with MAS (Table 2). Correlations with laboratory abnormalities were particularly evident for IFNγ and CXCL9, with the only exception being the correlation between IFNγ and LDH, which did not reach statistical significance (Table 2 and Figures 10A-10J). Again, as noted above, these correlations were absent in patients with active s-JIA without MAS at sampling. One patient in this group had remarkably high levels of IFNγ (336.2 pg / ml), CXCL9 (549,400 pg / ml) and CXCL10 (35,066 pg / ml). This patient had particularly severe MAS and was admitted to the intensive care unit with severe central nervous system complications. This finding provides further support for the hypothesis that there is a strong association between IFNγ and CXCL9 levels and disease severity. Taken together, these results indicate that increased production of IFNγ and IFNγ-related chemokines is a hallmark of active MAS that correlates strongly with the severity of MAS laboratory abnormalities. [Table 2-1] [Table 2-2] [Table 3]

[0306] Correlation between IFNγ and the levels of IFNγ-inducible chemokines in patients with MAS. To further characterize the relationship between IFNγ and the three IFNγ-inducible chemokines in patients with MAS, the correlation between IFNγ levels and the levels of each individual chemokine was evaluated. Notably, CXCL9 appears to be primarily and specifically induced by IFNγ, whereas CXCL10 and CXCL11 are also induced by type I interferons. Consistent with this, in patients with active MAS, circulating levels of IFNγ were significantly correlated with CXCL9 (r = 0.693; r 2 = 0.48; p = 0.001), whereas the correlation with CXCL10 levels was weaker (r = 0.535; r 2 = 0.29; p = 0.015) (Figures 11A-11F). The correlation with CXCL11 levels was also weak and did not reach statistical significance (r = 0.447; r 2 = 0.20; p = 0.08) (not shown).

[0307] IFNγ-induced chemokines correlate with disease activity in a mouse model of MAS. To further investigate the association of IFNγ-induced chemokine production with MAS, the expression of these chemokines in target tissues (liver and spleen) was investigated in a mouse model of MAS. In this model, clinical and laboratory features of MAS are induced by mimicking acute infection with the TLR4 agonist lipopolysaccharide (LPS) in the background of high levels of IL-6 in IL-6 transgenic mice (Strippoli et al., Arthritis Rheum, 2012). This approach recapitulates what occurs in patients with sJIA: infection can induce MAS / HLH in the presence of active disease, which is indeed characterized by high levels of IL-6. After induction with LPS, high mRNA levels of CXCL9 and CXCL10 were present in the liver and spleen of IL-6 transgenic mice. In particular, serum levels of ferritin are significantly correlated with the levels of expression of CXCL9 in the spleen and liver and the levels of expression of CXCL10 in the liver, indicating a relationship between upstream events related to IFNγ in target tissues (i.e., CXCL9 and CXCL10 production in the liver and spleen) and typical downstream laboratory abnormalities such as high ferritin levels. Taken together, data from patients with MAS and MAS mouse models indicate a clear relationship between increased production of IFNγ and increased expression of CXCL9, and to a lesser extent CXCL10, as well as laboratory abnormalities of MAS.

[0308] Studies in both patients and animal models of p-HLH have demonstrated a central role for IFNγ in disease pathogenesis. However, the role of IFNγ in sec-HLH, including MAS in the context of sJIA, remains unclear. This study conclusively demonstrates that high levels of IFNγ and IFNγ-inducible chemokines were present in patients with MAS occurring in sJIA. Furthermore, levels of IFNγ, CXCL9 and CXCL10 strongly correlated with laboratory parameters of MAS severity. This study found that serum levels of IFNγ and three IFNγ-related chemokines were comparable in patients with active sJIA and those with clinically inactive disease. This result argues against a pathogenic role for IFNγ in sJIA and is, in fact, in agreement with some findings by other authors. Three gene expression studies have failed to find a significant IFNγ-inducible signature in peripheral blood mononuclear cells (PBMCs) from patients with active sJIA without MAS at the time of sampling (Fall, N. et al., Gene expression profiling of peripheral blood from patients with untreated new-onset systemic juvenile idiopathic arthritis reveals molecular heterogeneity that may predict macrophage activation syndrome., Arthritis Rheum, 2007, 56(11): 3793-804; Ogilvie, EM et al., Specific gene expression profiles in systemic juvenile idiopathic arthritis., Arthritis Rheum, 2007, 56(6): 1954-65; Pascual, V. et al., Role of interleukin-1 (IL-1) in the pathogenesis of systemic onset juvenile idiopathic arthritis and clinical response to IL-1 blockade. J Exp Med 2005, 201(9):1479-86. After ex vivo stimulation of PBMCs, the number of IFNγ-producing cells in patients with active sJIA was similar to that in controls (Lasiglie, D. et al., Role of IL-1 beta in the development of human T (H) 17 cells: lesson from NLPR3 mutated patients., PLoS One, 2011, 6(5):e20014. Consistently, neither patients with active nor inactive SJIA show elevated serum or synovial fluid IFNγ levels (de Jager, W. et al., Blood and synovial fluid cytokine signatures in patients with juvenile idiopathic arthritis: a cross-sectional study., Ann Rheum Dis, 2007, 66(5):589-98). CXCL9 and CXCL10 are barely detectable in synovial tissue from patients with sJIA, whereas high levels of these chemokines can be found in synovial tissue from patients with oligoarticular or polyarticular JIA, supporting the lack of a role for IFNγ in sJIA joint inflammation (Sikora, KA et al., The limited role of interferon-gamma in systemic juvenile idiopathic arthritis: a cross-sectional study., Ann Rheum Dis, 2007, 66(5):589-98). arthritis cannot be explained by cellular hyporesponsiveness., Arthritis Rheum, 2012, 64(11):3799-808. Recent data in mice show that immunostimulation of IFNγ knockout mice with complete Freund's adjuvant results in a systemic inflammatory syndrome that includes features of sJIA, supporting the limited role of IFNγ in sJIA (Avau, A. et al., Systemic juvenile idiopathic arthritis-like syndrome in mice following stimulation of the immune system with friend's complete adjuvant: regulation by interferon-gamma., Arthritis Rheumatol, 2014, Vol. 66(5): 1340-51).

[0309] In sharp contrast, this study showed significantly higher levels of IFNγ and IFNγ-related chemokines in patients with active MAS at the time of sampling compared to patients with active sJIA without MAS at the time of sampling. This was also confirmed in individual patients with staged samples obtained both during active MAS and during active sJIA without MAS. Coincidentally, this study did not find significantly elevated levels of IL-6 or IL-1β in patients sampled during MAS, nor any association with laboratory parameters of MAS, thereby concluding that these cytokines are crucially involved in the pathogenesis of sJIA (De Benedetti, F. et al., Randomized trial of tocilizumab in systemic juvenile idiopathic arthritis., N Engl J Med, 2012, 367(25):2385-95; Ruperto, N. et al., Two randomized trials of canakinumab in systemic juvenile idiopathic arthritis., N Engl J Med, 2012, 367(25):2396-406, suggesting that cytokines may not be critical in maintaining MAS. This finding of elevated levels of IFNγ and IFNγ-related chemokines is consistent with some previous findings. Shimizu et al. reported that levels of neopterin, a catabolic product of guanosine triphosphate synthesized by human macrophages upon stimulation with IFNγ, were higher in patients with MAS during sJIA compared with patients with active sJIA without MAS (Shimizu, M. et al., Distinct cytokine profiles of systemic-onset juvenile idiopathic arthritis-associated macrophage activation syndrome with particular emphasis on the role of interleukin-18 in its pathogenesis., Rheumatology (Oxford), 2010, 49(9):1645-53). More recently, Put et al. reported elevated levels of IFNγ and CXCL10 in five patients with primary and secondary HLH, three of whom had MAS during the course of sJIA (Put, K. et al., Cytokines in systemic juvenile idiopathic arthritis and haemophagocytic lymphohistiocytosis: tipping the balance between interleukin-18 and interferon-gamma., Rheumatology (Oxford), 2015). Consistent with these results, five patients with active sJIA without MAS at the time of sampling had significantly lower levels of IFNγ and CXCL10 (Put et al., Rheumatology, 2015).

[0310] Interestingly, this study found that not only were the levels of IFNγ and IFNγ-related chemokines significantly elevated, but that their levels, especially that of CXCL9, were closely correlated with the laboratory features of MAS, indicating a link with disease severity. This study found significantly elevated levels of IFNγ as well as CXCL9 and CXCL10 in one patient with severe disease with multiple organ failure and central nervous system complications accompanied by generalized seizures requiring prolonged intensive care admission, further supporting the link with disease severity.

[0311] In patients with MAS, of the three IFNγ-inducible chemokines, CXCL9 was found to have the strongest correlation with IFNγ levels. This finding is consistent with the established concept that CXCL9 production appears to be specifically and exclusively induced by IFNγ, in contrast to the production of CXCL10 and CXCL11, which can also be induced by type I interferons (Groom, JR and AD Luster, CXCR3 ligands: redundant, collaborative and antagonistic functions. Immunol Cell Biol, 2011, 89(2):207-15). This suggests that CXCL9 levels may serve as a sensitive and specific biomarker for MAS activity. Indeed, by using a mouse model of MAS that mimics the induction of MAS by infectious stimuli in the context of high IL-6 levels (Strippoli et al., Arthritis Rheum, 2012), this study also found that the expression levels of CXCL9 in the liver and spleen significantly correlated with circulating levels of ferritin. For CXCL10 expression levels, this correlation was only present for liver, but not for spleen levels. This is also supported by findings in patients with MAS, where CXCL9 levels were strictly correlated with all laboratory parameters of MAS. Taken together, these findings in humans and mice indicate that CXCL9 strongly correlates with MAS features and IFNγ production, further supporting the hypothesis that excessive production of IFNγ plays a major pathogenic role in MAS. These findings are also consistent with immunohistochemical data generated by Put et al. using staged lymph node biopsies from the same SJIA patients, obtained during active sJIA without MAS, as well as during MAS.Put et al. reported that CXCL10 and indoleamine 2,3-dioxygenase, both IFNγ-inducible proteins, were detected at high levels by immunohistochemistry in tissue obtained during MAS but not in tissue obtained during active sJIA without MAS (Put et al., Rheumatology, 2015).

[0312] These results in MAS and sec-HLH, together with the findings available in the literature in patients with p-HLH, support the hypothesis that elevated IFNγ and IFNγ-related chemokines, especially CXCL9, are a unique feature of HLH, regardless of the underlying cause. In this regard, it is interesting to note that high levels of CXCL9 were detected in patients with recurrent MAS induced by NLRC4 gain-of-function mutations (Canna, SW, et al., An activating NLRC4 inflammasome mutation causes autoinflammation with recurrent macrophage activation syndrome., Nat Genet, 2014, 46(10):1140-6), suggesting that IFNγ overproduction may occur even in the context of HLH induced solely by inflammasome dysregulation.

[0313] Data in both animal models of p-HLH, perforin knockout mice and Rab27a knockout mice, clearly demonstrate a pathogenic role for IFNγ. Similarly, recent data in the TLR9-inducible model of HLH, a model of HLH secondary to infection, also indicate a key role for increased IFNγ production (Behrens, EM et al., Repeated TLR9 stimulation results in macrophage activation syndrome-like disease in mice. J Clin Invest 2011, 121(6):2264-77 and (Bautois et al., in progress). Additional studies have recently demonstrated that treatment with anti-IFNγ antibodies in the above mouse model of MAS led to increased survival and reverted clinical and laboratory features of MAS (Prencipe et al., in progress). Taken together, the results of this study and these findings in animals provide a rationale for IFNγ neutralization as a therapeutic approach in MAS. Example 7 Safety, tolerability, pharmacokinetics, and efficacy evaluation of multiple-dose intravenous anti-interferon gamma anti-IFN-γ monoclonal antibody in pediatric patients with primary hemophagocytic lymphohistiocytosis (HLH)

[0314] The studies presented herein were designed to determine the safety and tolerability profile of multiple intravenous (IV) administration of an anti-IFNγ antibody, referred to herein as NI-0501; to determine the efficacy and benefit / risk profile of NI-0501 in HLH patients; to describe the pharmacokinetic (PK) profile of NI-0501 in HLH patients; to define a reasonable NI-0501 treatment dose regimen for HLH; and to evaluate the immunogenicity of NI-0501.

[0315] Preclinical Studies: Previous studies have demonstrated that NI-0501 exhibits similar binding affinity and blocking activity against IFNγ from non-human primate species, including rhesus and cynomolgus monkeys, but not against IFNγ from dogs, cats, pigs, rabbits, rats or mice. Toxicity and safety studies in cynomolgus monkeys have demonstrated that there is no off-target toxicity attributable to administration of NI-0501, weekly dosing of NI-0501 is well tolerated and does not require antibiotic prophylaxis, and no abnormal histopathological or behavioral findings were observed during these previous studies.

[0316] Because NI-0501 can bind to free IFNγ and IFNγR1-bound IFNγ, studies were performed to investigate the potential of NI-0501 to mediate ADCC and CDC activity in the presence of target. No ADCC activity was demonstrated, and no induction of CDC activity was observed.

[0317] Phase I Clinical Trial: A Phase 1 randomized, double-blind, placebo-controlled, single ascending dose study in 20 healthy adult volunteers investigating the safety, tolerability, and pharmacokinetic profile of a single intravenous (IV) dose of NI-0501. During this study, 6 subjects received placebo and 3, 3, 4, and 4 subjects (14 subjects total) received NI-0501 at doses of 0.01, 0.1, 1, and 3 mg / kg, respectively.

[0318] PK analysis of NI-0501 revealed a predictive profile for an IgG1 with a long half-life (approximately 22 days), slow clearance (≦0.007 L / h) and low volume of distribution (mean <6 L).

[0319] A total of 41 adverse events (AEs) were observed after the start of drug infusion in 14 of 20 subjects (70%), of which 10 were reported by 4 subjects receiving placebo. Thirty-six (87.8%) AEs were of mild intensity and 5 (12.2%) were of moderate intensity. No severe or life-threatening AEs were reported. Twenty-three AEs (56.1%) in 10 of 14 subjects who experienced an AE were reported as drug-related (at least plausibly). The majority of AEs were isolated, and no trends related to the escalating NI-0501 dose were observed. All NI-0501 infusions were uneventful.

[0320] In summary, NI-0501 infusions were well tolerated, and effects observed during 8 weeks of monitoring following drug infusion did not reveal any serious or unexpected off-target safety or immunogenicity concerns.

[0321] Phase 2 / 3 Clinical Trials Materials and Methods: These studies are performed on primary HLH patients. The studies are divided into three parts: screening, treatment, and follow-up. An overview is shown in Figure 12.

[0322] In these studies, suitable patients include patients who are naïve to HLH treatment (also referred to herein as "first-line patients"), or patients who may have already received conventional HLH treatment and, for example, have not responded adequately to or have shown signs of intolerance according to the treating physician (also referred to herein as "second-line patients"). Patients who receive NI-0501 after failing or showing intolerance to conventional HLH treatment are the core cohort of the study to demonstrate the efficacy of NI-0501 as a second-line treatment for primary HLH. Treatment-naïve patients are enrolled for the collection of efficacy and safety data in the first-line setting.

[0323] The following patients will be excluded from this study: patients with a diagnosis of secondary HLH as a result of proven rheumatic or neoplastic disease; patients who have been previously treated with any T-cell depleting agent (e.g., antithymocyte globulin (ATG), anti-CD52 therapy, etc.) during the 2 weeks prior to screening or with any other biologic within 5 of their defined half-lives (with the exception of rituximab in cases of documented B-cell EBV infection); patients with active mycobacteria, Histoplasma Patients with Salmonella Capsulatum, Shigella, Salmonella, Campylobacter, and Leishmania infections; patients with evidence of tuberculosis or a past history of latent tuberculosis; patients with positive serology for HIV antibodies, hepatitis B surface antigen, or hepatitis C antibodies; patients with existing malignancies; patients with another comorbidity or malformation severely affecting cardiovascular, pulmonary, hepatic, or renal function; patients with a history of hypersensitivity or allergy to any component of the study regimen; patients who have received a live or live-attenuated (including BCG) vaccine within the previous 12 weeks after screening; and / or pregnant or lactating female patients.

[0324] The studies presented herein use the anti-interferon gamma antibody NI-0501, a fully human IgG1 monoclonal antibody (mAb) directed against human IFNγ. NI-0501 is provided as a sterile concentrate for injection (per mL) as shown in Table 4 below. [Table 4]

[0325] In these studies, NI-0501 is administered at an initial dose of 1 mg / kg over 1 hour by IV infusion. This dose is expected to inhibit at least 99% of the effects of IFNγ over 3 days in patients with baseline IFNγ concentrations less than or equal to 3400 pg / mL. Infusions are administered every 3 days until study day 15 (SD15) (infusion #6) and then twice weekly thereafter. The NI-0501 dose can be increased up to 3 mg / kg at any time during the study according to predefined criteria guided by clinical and laboratory response in each patient (as described in Table 5 below). After a minimum of two infusions at 3 mg / kg, if, upon reassessment, the same clinical and laboratory criteria that qualified the patient to receive NI-0501 at 3 mg / kg are still found to apply, the dose of NI-0501 can be increased up to 6 mg / kg over up to four infusions with regular monitoring of clinical and laboratory HLH parameters. Based on the evolution of these parameters, the dose of NI-0501 can be i) decreased back to 3 mg / kg, or ii) maintained at 6 mg / kg with additional IV infusions (or increased above 6 mg / kg) if PK and PD evidence indicates excessively high IFNγ production and thus rapid NI-0501 clearance. Dose escalation can occur at any time during the study if the clinical and laboratory criteria described herein are met. [Table 5-1] [Table 5-2]

[0326] As shown in FIG. 12, in these studies, NI-0501 is administered for 8 weeks, with the treatment period divided into two separate periods: Treatment Period 1 and 2.

[0327] After 8 weeks of administration of NI-0501, conditioning treatment can be initiated in preparation for hematopoietic stem cell transplantation (HSCT). If patient condition and donor availability permit transplantation, the expected duration of treatment can be shortened, but not to less than 4 weeks. If a suitable donor has not been identified by week 8 or the transplant schedule needs to be delayed for reasons unrelated to administration of NI-0501, treatment with NI-0501 can be continued in the context of a long-term follow-up study, provided a favorable benefit / risk profile has been established for the patient.

[0328] In these studies, NI-0501 will be administered on a background of dexamethasone, which can be tapered depending on the patient's condition. Treatment-naïve patients will receive 10 mg / m 2 NI-0501 will be administered on a background of dexamethasone. Patients receiving NI-0501 as a second-line HLH treatment will be administered dexamethasone at a dose of at least 5 mg / m 2 Patients should receive dexamethasone at a dose of 0.01 mg / kg / day or, if higher, at the same dose given prior to screening. Patients should have received dexamethasone from SD-1.

[0329] Dexamethasone can be tapered according to the patient's condition and at the discretion of the treating physician. The tapering scheme can be selected by the treating physician, provided that the dexamethasone dose is not reduced by more than half at each step and the frequency of change is not more than weekly.

[0330] If the disease worsens after tapering of dexamethasone, the dose of dexamethasone can be increased and maintained until a satisfactory response is achieved as determined by the treating physician.

[0331] As recommended in HLH treatment guidelines, patients will receive prophylactic treatment against Pneumocystis jiroveci, fungal, and varicella zoster virus infections from the day before treatment with NI-0501 is initiated until the end of the study. Patients will receive prophylactic treatment from the day before treatment with NI-0501 is initiated (i.e., SD-1) until the end of the study. For example, for prevention of Pneumocystis jiroveci, patients will receive, for example, 750 mg / m2 per day given orally in equally divided doses twice daily for three consecutive days per week. 2 of sulfamethoxazole at 150 mg / m per day 2 For fungal infection prevention, patients may receive, for example, fluconazole 12 mg / kg daily, up to a daily dose of 400 mg. For HZ virus prevention, patients may receive, for example, acyclovir, 200 mg four times a day for children over 2 years old, and 100 mg four times a day for children under 2 years old. These treatments are given orally whenever possible, otherwise intravenously.

[0332] Patients may also receive any of a variety of concomitant therapies, such as, for example, cyclosporine A, intrathecal methotrexate and glucocorticosteroids, and others. If cyclosporine A (CsA) is already being administered to the patient prior to screening, it may be continued. CsA may be discontinued at any time. CsA will not be introduced de novo during the course of the study once NI-0501 administration has commenced.

[0333] If a patient is receiving intrathecal methotrexate and glucocorticoids at the initiation of treatment with NI-0501, this treatment will be continued as needed. If the emergence of CNS symptoms occurs prior to the initiation of treatment with NI-0501, treatment with intrathecal methotrexate and glucocorticoids must be initiated prior to the first dose of NI-0501.

[0334] IV immunoglobulin (IVIG) is only acceptable as replacement treatment in cases of documented immunoglobulin deficiency. For example, if replacement is warranted due to documented immunoglobulin deficiency, IVIG can be given at a dose of 0.5 g / kg every 4 weeks or more frequently to maintain adequate IgG levels. Any previous infusions within 4 weeks prior to screening are acceptable, as well as any infusions during treatment with NI-0501.

[0335] Analgesic treatment, transfusion of blood products, electrolyte and glucose infusion, antibiotic, antifungal and antiviral treatment and general supportive care are permitted. If HLH improvement is not sustained or limited when the maximum NI-0501 dose level is achieved, additional HLH treatment may be permitted. As used herein, non-sustained HLH improvement refers to the patient's inability to maintain at least 50% improvement from baseline for the three HLH parameters (see Table 6 below). Lack of HLH improvement must be documented by at least two consecutive measurements. As used herein, limited HLH improvement refers to less than 50% change from baseline in at least three HLH clinical and laboratory criteria. Etoposide should be administered as additional HLH treatment unless previous medical history leads to clear evidence of lack of response or intolerance to the drug.

[0336] The following treatments should not be used concomitantly with NI-0501 administration: etoposide, T-cell depleting agents, or any other biologics are generally not tolerated, except for: G-CSF, in cases of prolonged neutropenia; rituximab, in cases of documented B-cell EBV infection; and additional HLH treatments, where HLH improvement at the highest NI-0501 dose level is not sustained or is limited (as defined herein). Etoposide should be administered unless prior medical history leads to clear evidence of lack of response or intolerance to the drug. Vaccination with live or attenuated (including BCG) vaccines must be avoided throughout the study, including the 4-week follow-up period. If NI-0501 concentrations remain at therapeutic levels after study completion, the period without vaccination should be extended until measurable concentrations of NI-0501 are no longer detectable.

[0337] Clinical signs characterizing the disease (fever, splenomegaly, CNS symptoms) and evolution of laboratory parameters (CBC, fibrinogen, ferritin, sCD25 levels) will be used to assess achievement of response and time to response. Primary efficacy endpoints include overall response rate, i.e., achievement of either complete or partial response or HLH improvement at the end of treatment (EoT), as defined in Table 6 below. Secondary efficacy endpoints include time to response at any time during the study; durability of response, i.e., maintenance of the response achieved at any time during the study up to and past the EoT (including data collected in any long-term follow-up studies); the number of patients who can reduce glucocorticoids by 50% or more from the baseline dose; the number of patients who can proceed to HSCT, if deemed necessary; survival at 8 weeks (or EoT) and end of study; serum concentrations of NI-0501 to determine the NI-0501 pharmacokinetic (PK) profile; determination of pharmacodynamic (PD) effects, including levels of circulating total IFNγ and markers of its neutralization, i.e., CXCL9 and CXCL10; and determination of other biomarkers, e.g., sCD25, IL-10. [Table 6-1] [Table 6-2]

[0338] Safety parameters to be collected and evaluated include the incidence, severity, causality and outcome of adverse events (AEs) (serious and non-serious), with special attention to infections; the evolution of laboratory parameters such as complete blood count (CBC), liver tests, renal function tests as well as coagulation, with a focus on red blood cells (hemoglobin), neutrophils and platelets; the number of patients withdrew for safety reasons; and other parameters such as the level of circulating antibodies against NI-0501 (if any) to determine immunogenicity (ADA).

[0339] The primary endpoint (overall response rate) will be assessed using an exact one-sided binomial test at the 0.025 level. Time to response, durability of response and survival time will be presented using Kaplan-Meier curves and medians will be calculated where available. 95% confidence intervals will be calculated around the median for each of these endpoints. Additional endpoints based on binary outcomes, including the number of patients achieving a 50% or greater reduction in glucocorticoids and the number of patients able to proceed to HSCT, will be converted to proportions and associated 95% confidence intervals will be calculated. Statistical significance by p-values ​​will only be obtained for the primary endpoint. All other endpoints will be considered as supportive of the primary endpoint and, as a result, no formal hierarchy of endpoints will be presented.

[0340] Administration of NI-0501 in patients leads to rapid normalization of fever within hours after the first infusion of NI-0501. Figures 13A and 13B show the effect of NI-0501 infusion on body temperature in two patients with body temperature >37.5°C at the start of treatment with NI-0501. Figure 14 is a set of graphs and tables showing the effect of NI-0501 administration in patients on neutrophil counts. Figure 15 is a set of graphs and tables showing the effect of NI-0501 administration in patients on platelet counts. Figure 16 is a set of graphs and tables showing the effect of NI-0501 administration in patients on serum levels of ferritin. Figure 17 is a set of graphs and tables showing the effect of NI-0501 administration in patients on glucocorticoid tapering. Figure 18 is a graph showing that administration of NI-0510 maintained IFNγ neutralization until the time of HSCT. The HLH response to treatment with NI-0501 was also sustained until transplantation. Patients were also evaluated for any CNS complications following administration of NI-0501. A summary of baseline CNS complications and status through the end of treatment (EOT) is shown in Table 11 below. [Table 11]

[0341] Of 10 patients who underwent hematopoietic stem cell transplantation (HSCT), all patients engrafted; one patient required a boost with CD34 stem cells due to mixed chimerism on D+145 after HSCT. One patient experienced secondary graft failure and subsequently reactivated HLH. This patient died on D+68 after HSCT due to acute respiratory failure and bacterial infection. Another patient died on D+47 after HSCT (septic shock in the setting of severe GvHD). In three other patients, mild GvHD was reported and has resolved / is resolving.

[0342] Neutralization of serum concentrations of NI-0501 at the time of HSCT was measured in 8 of 10 transplant recipients, as reflected by levels of CXCL9 (a chemokine exquisitely induced by IFNγ) below the limit of quantification. Thus, these data indicate that NI-0501 can be used without the short-term or long-term toxicities reported for etoposide-based regimens. This translates into a reduced risk of allo-HSCT-related complications.

[0343] These data demonstrate that treatment with NI-0501 can improve and / or resolve relevant clinical and laboratory abnormalities of HLH, including CNS signs and symptoms. Response to NI-0501 is independent of the presence and type of causative mutation and / or the presence and type of infectious trigger. NI-0501 has been well tolerated. No safety concerns have arisen to date (e.g., no bone marrow toxicity, no extensive immunosuppression). No infections caused by pathogens known to be promoted by IFNγ neutralization have been observed. Neutralization of IFNγ by NI-0501 can provide an innovative targeted approach to managing HLH. Example 8 Safety, tolerability, pharmacokinetics, and efficacy of short-term intravenous administration of NI-0501, an anti-interferon gamma (anti-IFNγ) monoclonal antibody, in patients with systemic juvenile idiopathic arthritis (sJIA) with macrophage activation syndrome / secondary HLH (MAS / sHLH)

[0344] The study presented herein is designed to demonstrate the efficacy and safety of NI-0501 for the treatment of MAS / sHLH in patients with sJIA and is divided into two parts: (i) a pilot study to evaluate the PK profile and dosing strategy of NI-0501 and to preliminary evaluate the benefit / risk of NI-0501 in this patient population; and (ii) a pivotal study to demonstrate the efficacy and safety of NI-0501 (study will continue once the dosing regimen and positive benefit / risk profile of NI-0501 are confirmed). An overview of this study design is shown in Figure 19.

[0345] The primary objectives of the pilot study are: (i) to define a reasonable NI-0501 treatment dose regimen for sJIA patients with MAS / sHLH; (ii) to evaluate the benefit / risk profile of NI-0501 in sJIA patients with MAS / sHLH; and (iii) to describe the pharmacokinetic (PK) profile of NI-0501 in sJIA patients with MAS / sHLH. The primary objectives of the pivotal study are to (i) determine the efficacy of NI-0501 in sJIA patients with MAS / sHLH; (ii) evaluate the safety and tolerability profile of short-term intravenous (iv) administration of NI-0501 in sJIA patients with MAS / sHLH; (iii) confirm the positive benefit / risk profile of NI-0501 in sJIA patients with MAS / sHLH; (iv) conduct exploratory evaluation of the chemokines, CXCL9 and CXCL10, as MAS / sHLH diagnostic biomarkers and predictors of response to treatment with NI-0501; and (v) evaluate the immunogenicity of NI-0501 in sJIA patients with MAS / sHLH.

[0346] The study population includes sJIA patients with MAS / sHLH who have shown an inadequate response to high-dose glucocorticoid treatment. Inclusion criteria include: (i) gender: males and females; (ii) age: <16 years at time of sJIA diagnosis; (iii) diagnosis of active MAS / sHLH confirmed by the treating rheumatologist in the presence of at least two of the following laboratory and clinical criteria: (a) laboratory criteria: platelet count ≤ 262 x 10 9 / L, WBC number ≦4.0×10 9 / L, AST level >59U / L, and / or fibrinogen level ≤2.5g / L;(b) clinical criteria: hepatomegaly, hemorrhagic manifestations, and / or CNS dysfunction;(iv) patients showing inadequate response to at least 3 days of high-dose iv glucocorticoid treatment according to local standard of care (including but not limited to pulses of 30mg / kg mPDN for 3 consecutive days);(v) high-dose iv glucocorticoids should not be reduced below 2mg / Kg mPDN per day equivalent to two separate daily doses of up to 60mg per day. May be included within less than 3 days of initiation of high-dose iv glucocorticoids if patient's condition and / or laboratory parameters rapidly deteriorate;(vi) consent of patient (or consent of legally recognized representative(s)); and (vii) tolerance of contraceptive measures if patient has passed puberty.

[0347] Exclusion criteria were: (i) a diagnosis of suspected or confirmed primary HLH or HLH resulting from a neoplastic disease; (ii) patients who had been treated with anakinra, tocilizumab, canakinumab, TNF inhibitors, rituximab or any other biologic agent within five defined half-lives of each; (iii) active mycobacteria (typical and atypical), Histoplasma (iv) evidence of latent tuberculosis; (v) positive serology for HIV antibodies; (vi) presence of malignancy; (vii) patients with another comorbidity or malformation severely affecting cardiovascular, pulmonary, CNS, hepatic or renal function that, in the opinion of the investigator, may significantly affect the likelihood of response to treatment and / or the evaluation of the safety of NI-0501; (viii) history of hypersensitivity or allergy to any component of the study regimen; (ix) receipt of BCG vaccination within 12 weeks prior to screening; (x) receipt of any other live or live attenuated vaccine within 6 weeks prior to screening; and / or (xi) pregnant or lactating female patients.

[0348] Dosing regimen, frequency of administration and duration of treatment: In these studies, NI-0501 is used in the formulation shown in Example 7. In part 1, NI-0501 is administered at SD0 at an initial dose of 6 mg / kg by infusion over 1 hour. Treatment with NI-0501 is continued at a dose of 3 mg / kg every 3 days for 4 weeks (i.e., until SD27). If a complete clinical response (i.e., MAS remission) is achieved, treatment with NI-0501 can be shortened. After 4 weeks, treatment with NI-0501 can be continued for up to another 4 weeks (i.e., until SD56) as maintenance as needed until MAS remission is achieved, and the dose can be reduced to 1 mg / kg and the interval between infusions can be extended to once a week. If the PK profile shows unexpected TMDD (thus signaling exceptionally high IFNγ production), the dose of NI-0501 can be increased to 10 mg / kg, guided by clinical and PK evidence. This dose escalation will only be approved after a careful evaluation of the benefit / risk profile in that individual patient.

[0349] In Part 2, if the proposed dosing regimen is validated and a positive benefit / risk of NI-0501 is demonstrated, the study will continue. If necessary, minor modifications of the dosing regimen may be applied based on the evidence obtained in Part 1.

[0350] Background treatments and concomitant medications: NI-0501 is administered against a background of at least 2 mg / kg methylprednisolone (mPDN), equivalent to a maximum of 60 mg per day (in patients weighing 30 kg or more), which may be tapered during treatment depending on the patient's condition. Patients receive prophylactic treatment for shingles infection, preferably starting the day before (and in any case before starting treatment with NI-0501), until serum NI-0501 levels are no longer detectable. Cyclosporine A (CsA) may be continued if started at least 3 days before starting treatment with NI-0501. CsA dose adjustments may be made to maintain therapeutic levels. CsA may be withdrawn at any time during the study at the discretion of the investigator. CsA may not be introduced once NI-0501 administration has begun. If patients are receiving intrathecal methotrexate and glucocorticoids at the time of starting treatment with NI-0501, this treatment may be continued as needed. Vaccination with live or attenuated (including BCG) vaccines must be avoided throughout the study and, in any case, until serum NI-0501 levels are undetectable. Analgesic treatment, transfusion of blood products, electrolyte and glucose infusions, antibiotic, antifungal and antiviral treatments, and general supportive care are permitted.

[0351] Sample size: In part 1, at least 5 evaluable patients will be enrolled. In part 2, at least 10 evaluable patients will be enrolled during the continuation of the study, achieving a total of 15 evaluable patients. A sample size of 15 has not been formally justified given the rare orphan nature of the disease and the lack of any approved treatment. Nevertheless, based on the assumption that at least 50% of patients respond inadequately to systemic glucocorticoids alone, i.e., 50% of patients receiving glucocorticoids will achieve MAS remission by week 8 after the start of treatment, the study has 70% power to detect an improvement of 50% to 77% using a one-sided significance level of 5%.

[0352] Study Duration and End of Study Definition: The duration of the study will be 8 weeks (plus up to 1 week of screening period) for each patient. End of study will be defined as the last visit of the last patient. All patients who received at least one dose of NI-0501 will be requested to participate in the NI-0501-05 study for long-term follow-up.

[0353] Study Endpoints: Part 1 (pilot) of the study will evaluate the following to validate the dosing regimen in this patient population: (i) the benefit / risk profile of NI-0501; (ii) the PK profile of NI-0501; (iii) levels of chemokines known to be induced by IFNγ (e.g., CXCL9, CXCL10, CXCL11); (iv) the evolution of distinct features of MAS: cytopenias, liver dysfunction and coagulopathy at 2, 4, 6 and 8 weeks after initiation of NI-0501; and (v) dose and duration of treatment with NI-0501. In part 2 (pivotal) of the study, the efficacy study endpoints are as follows: (a): primary efficacy endpoint: number of patients achieving MAS remission by week 8 after initiation of treatment with NI-0501; and (b) secondary efficacy endpoints: time to MAS remission; time to first response according to investigator assessment; number of patients able to taper their glucocorticoids to the same (or lower) dose received before MAS occurred at any time during the study; time to achieve glucocorticoid taper; survival at the end of the study; and number of patients discontinuing the study due to lack of efficacy. In part 2 (pivotal) of the study, safety study endpoints are: (a) incidence, severity, causality and outcome of AEs (serious and non-serious), with special attention to infections; evolution of laboratory parameters, especially CBC (focusing on hemoglobin, neutrophils and platelets), LFTs, and coagulation parameters; number of patients who discontinued the study for safety reasons; and levels of circulating antibodies against NI-0501 to determine immunogenicity (ADA), if any.

[0354] Pharmacokinetics and pharmacodynamics will be assessed by the PK profile of NI-0501, including: levels of circulating free IFNγ pre-dose and total IFNγ (free IFNγ+IFNγ bound to NI-0501) after initiation of NI-0501; levels of chemokines known to be induced by IFNγ (e.g., CXCL9, CXCL10, CXCL11); correlation of chemokine levels (CXCL9, CXCL10) with free NI-0501, free IFNγ (pre-dose) and total IFNγ levels; correlation of chemokine and total IFNγ levels with laboratory parameters of MAS severity, e.g., ferritin, platelet count, LFTs (exploratory analysis); and levels of other potential disease biomarkers (e.g., sCD25, IL-10, IL-6, IL-18, TNFα, neopterin). Other embodiments

[0355] Although the present invention has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative, not limiting, of the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. The present invention provides, for example, the following items: (Item 1) A multiple variable dose method for treating primary hemophagocytic lymphohistiocytosis (HLH) in a human subject in need of such treatment, comprising administering to the subject: a) a first dose of 1.0 or 3.0 mg / kg of the subject's body weight administered within a 12 hour period to the subject, of an antibody that binds interferon gamma (IFNγ); and b) within 12 hours, a second dose of the antibody of 3.0, 6.0, or 10.0 mg / kg of the subject's body weight. administering intravenously to the antibody comprises a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO:1); a variable heavy chain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence of AISGSGGGSTYYADSVKG (SEQ ID NO:2); and a variable heavy chain complementarity determining region 3 (VH CDR3) comprising the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO:3); a variable light chain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO:4); a variable light chain complementarity determining region 2 (VL CDR2) region comprising the amino acid sequence of EDNQRPS (SEQ ID NO:5); and a variable light chain complementarity determining region 3 (VL CDR3) region comprising the amino acid sequence of QSYDGSNRWM (SEQ ID NO:6). (Item 2) 2. The method of claim 1, wherein the antibody comprises a heavy chain variable amino acid sequence having the amino acid sequence of SEQ ID NO: 47 and a light chain variable amino acid sequence having the amino acid sequence of SEQ ID NO: 48. (Item 3) 2. The method of claim 1, wherein the doses of the antibody are administered within 6 hours. (Item 4) 4. The method of claim 3, wherein the dose of the antibody is administered within one hour. (Item 5) 2. The method of claim 1, wherein the second dose is administered every 3 days after the first dose for a first treatment period. (Item 6) 6. The method of claim 5, wherein the second dose is administered for a second treatment period after completion of the first treatment period 1, 2, 3, 4, 5, 6, 7, 8 weeks after the first dose. (Item 7) 7. The method of item 6, wherein the second treatment period is twice weekly. (Item 8) 13. The method of claim 1, further comprising administering to the subject a third dose of 1.0 mg / kg of the subject's body weight within 12 hours. (Item 9) 2. The method of item 1, wherein the dose of the antibody is administered as a single injection. (Item 10) The method of item 1, wherein the antibody is administered as a monotherapy or a combination therapy. (Item 11) 2. The method of claim 1, wherein the subject is an adult subject or a pediatric subject. (Item 12) 1. A multiple variable dose method for treating secondary hemophagocytic lymphohistiocytosis (HLH) in a human pediatric subject in need of such treatment, comprising administering to the subject: a) a first dose of 6.0 mg / kg of the subject's body weight of an antibody that binds interferon gamma (IFNγ) administered to the subject within a 12 hour period; and b) within 12 hours, a second dose of the antibody of 3.0 mg / kg of the subject's body weight administering intravenously to the antibody comprises a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO:1); a variable heavy chain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence of AISGSGGGSTYYADSVKG (SEQ ID NO:2); and a variable heavy chain complementarity determining region 3 (VH CDR3) comprising the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO:3); a variable light chain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO:4); a variable light chain complementarity determining region 2 (VL CDR2) region comprising the amino acid sequence of EDNQRPS (SEQ ID NO:5); and a variable light chain complementarity determining region 3 (VL CDR3) region comprising the amino acid sequence of QSYDGSNRWM (SEQ ID NO:6). (Item 13) 13. The method of claim 12, wherein the doses of the antibody are administered within 6 hours. (Item 14) 14. The method of claim 13, wherein the dose of the antibody is administered within one hour. (Item 15) 13. The method of item 12, wherein the second dose is administered every 3 days after the first dose for a first treatment period. (Item 16) 16. The method of item 15, wherein the second dose is administered for a second treatment period after completion of the first treatment period. (Item 17) 17. The method of item 16, wherein the second treatment period is twice weekly. (Item 18) 13. The method of claim 12, further comprising administering an additional dose of 6.0 mg / kg of the subject's body weight of the antibody within 12 hours after the second dose. (Item 19) 13. The method of claim 12, wherein the dose of the antibody is administered as a single injection. (Item 20) The method of item 12, wherein the antibody is administered as a monotherapy or a combination therapy. (Item 21) 1. A multiple variable dose method for treating secondary hemophagocytic lymphohistiocytosis (HLH) in a human adult subject in need of such treatment, comprising administering to the subject: a) a first dose of 3 mg or 6.0 mg per kg of the subject's body weight administered within a 12 hour period to the subject, of an antibody that binds interferon gamma (IFNγ); and b) within 12 hours, a second dose of the antibody of 10.0 mg / kg or less of the subject's body weight. administering intravenously to the antibody comprises a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO:1); a variable heavy chain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence of AISGSGGGSTYYADSVKG (SEQ ID NO:2); and a variable heavy chain complementarity determining region 3 (VH CDR3) comprising the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO:3); a variable light chain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO:4); a variable light chain complementarity determining region 2 (VL CDR2) region comprising the amino acid sequence of EDNQRPS (SEQ ID NO:5); and a variable light chain complementarity determining region 3 (VL CDR3) region comprising the amino acid sequence of QSYDGSNRWM (SEQ ID NO:6). (Item 22) 22. The method of claim 21, wherein the doses of the antibody are administered within 6 hours. (Item 23) 23. The method of claim 22, wherein the dose of the antibody is administered within one hour. (Item 24) 23. The method of item 22, wherein the second dose is administered every 3 days after the first dose for a first treatment period. (Item 25) 25. The method of item 24, wherein the second dose is administered for a second treatment period after completion of the first treatment period. (Item 26) 26. The method of item 25, wherein the second treatment period is twice weekly. (Item 27) 22. The method of claim 21, further comprising administering an additional dose of 1.0, 3.0 or 6.0 mg / kg of the subject's body weight of the antibody within 12 hours after the second dose. (Item 28) 22. The method of claim 21, wherein the dose of the antibody is administered as a single injection. (Item 29) 22. The method of item 21, wherein the antibody is administered as a monotherapy or a combination therapy. (Item 30) 1. A method for treating a condition in a human subject in need thereof with multiple variable doses, comprising administering to the subject: a) a first dose of an antibody that binds interferon gamma (IFNγ), administered within 12 hours to the subject; and b) within 12 hours, administering a second dose of said antibody to said subject administering intravenously to the antibody comprises a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO:1); a variable heavy chain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence of AISGSGGGSTYYADSVKG (SEQ ID NO:2); and a variable heavy chain complementarity determining region 3 (VH CDR3) comprising the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO:3); a variable light chain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO:4); a variable light chain complementarity determining region 2 (VL CDR2) region comprising the amino acid sequence of EDNQRPS (SEQ ID NO:5); and a variable light chain complementarity determining region 3 (VL CDR3) region comprising the amino acid sequence of QSYDGSNRWM (SEQ ID NO:6). (Item 31) 31. The method of claim 30, wherein the antibody comprises a heavy chain variable amino acid sequence having the amino acid sequence of SEQ ID NO: 47 and a light chain variable amino acid sequence having the amino acid sequence of SEQ ID NO: 48. (Item 32) 31. The method of item 30, wherein the doses of the antibody are administered within 6 hours. (Item 33) 33. The method of claim 32, wherein the dose of the antibody is administered within one hour. (Item 34) 31. The method of item 30, wherein the second dose is administered every three days after the first dose for a first treatment period. (Item 35) 35. The method of claim 34, wherein the second dose is administered for a second treatment period after completion of the first treatment period. (Item 36) 36. The method of claim 35, wherein the second treatment period is twice weekly. (Item 37) 31. The method according to item 30, wherein the first dose is 1.0 to 10 mg per kg of the subject's body weight. (Item 38) 31. The method according to item 30, wherein the dose is 1.0 to 10 mg per kg of the subject's body weight, and the second dose is lower or higher than the first dose. (Item 39) Item 40. The method of item 30, further comprising administering to the subject a third dose of the antibody of 1.0 to 10 mg / kg of the subject's body weight, administered within 12 hours. 31. The method of item 30, wherein the dose of the antibody is administered as a single injection. (Item 41) The method of item 30, wherein the antibody is administered as a monotherapy or a combination therapy. (Item 42) 31. The method of claim 30, wherein the subject is an adult subject or a pediatric subject. (Item 43) 31. The method of item 30, wherein the condition is transplant rejection. (Item 44) 44. The method of item 43, wherein the graft rejection is solid organ transplant disorder, bone marrow acute graft rejection. (Item 45) 31. The method of item 30, wherein the condition is graft-versus-host disease, paraneoplastic cerebellar degeneration, hemorrhagic fever, sarcoidosis, adult-onset Still's disease. (Item 46) The method of item 30, administered to a subject after receiving CART cell therapy. (Item 47) The method of any one of the preceding items, wherein the subject has been administered dexamethasone immediately prior to dosing with the antibody. (Item 48) The dexamethasone is at least 10 mg / m 2 The method of claim 30, wherein the dose is (Item 49) The dexamethasone is at least 5 mg / m 2 The method of claim 30, wherein the dose is (Item 50) The method of any one of the preceding items, wherein the subject has not previously undergone treatment for HLH. (Item 51) The method of any one of the preceding items, further comprising administering to the subject at least a second agent. (Item 52) Item 524. The method of item 523, wherein the second agent is a therapeutic agent, an anti-inflammatory agent, and / or an immunosuppressant. (Item 53) Per mL: a) 5 mg or 25 mg of a fully human anti-interferon gamma (IFNγ) monoclonal antibody; and b) 1.55 mg L-histidine, 3.14 mg L-histidine monohydrochloride monohydrate, 7.31 mg sodium chloride (NaCl), and 0.05 mg polysorbate 80, pH between 5.8 and 6.2 1. An injectable pharmaceutical formulation comprising: (Item 54) 54. The formulation of item 53, wherein the antibody comprises a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO: 1); a variable heavy chain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence of AISGSGGGSTYYADSVKG (SEQ ID NO: 2); and a variable heavy chain complementarity determining region 3 (VH CDR3) comprising the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO: 3); a variable light chain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO: 4); a variable light chain complementarity determining region 2 (VL CDR2) region comprising the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and a variable light chain complementarity determining region 3 (VL CDR3) region comprising the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6). (Item 55) 55. The formulation of claim 54, wherein the antibody comprises a heavy chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO: 47 and a light chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO: 48. (Item 56) A unit dose vial containing 20 ml of a fully human anti-interferon gamma (IFNγ) monoclonal antibody solution suitable for injection, the concentration of the antibody being 5 mg / ml or 25 mg / ml, and the pH of the solution being between 5.8 and 6.2. (Item 57) 57. The unit dose vial of item 56, wherein the antibody is solubilized in the solution, such that the solution is clear, colorless, and without precipitate. (Item 58) 57. The unit dose vial of item 56, wherein the antibody comprises a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO:1); a variable heavy chain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence of AISGSGGGSTYYADSVKG (SEQ ID NO:2); and a variable heavy chain complementarity determining region 3 (VH CDR3) comprising the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO:3); a variable light chain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO:4); a variable light chain complementarity determining region 2 (VL CDR2) region comprising the amino acid sequence of EDNQRPS (SEQ ID NO:5); and a variable light chain complementarity determining region 3 (VL CDR3) region comprising the amino acid sequence of QSYDGSNRWM (SEQ ID NO:6). (Item 59) 59. The unit dose vial of item 58, wherein the antibody comprises a heavy chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO: 47 and a light chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO: 48. (Item 60) A unit dose vial containing 10 ml or 20 ml of a fully human anti-interferon gamma (IFNγ) monoclonal antibody solution suitable for injection, the concentration of the antibody being 25 mg / ml and the pH of said solution being between 5.8 and 6.2. (Item 61) 61. The unit dose vial of item 60, wherein the antibody is solubilized in the solution, such that the solution is clear, colorless, and without precipitate. (Item 62) 61. The unit dose vial of item 60, wherein the antibody comprises a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO:1); a variable heavy chain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence of AISGSGGGSTYYADSVKG (SEQ ID NO:2); and a variable heavy chain complementarity determining region 3 (VH CDR3) comprising the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO:3); a variable light chain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO:4); a variable light chain complementarity determining region 2 (VL CDR2) region comprising the amino acid sequence of EDNQRPS (SEQ ID NO:5); and a variable light chain complementarity determining region 3 (VL CDR3) region comprising the amino acid sequence of QSYDGSNRWM (SEQ ID NO:6). (Item 63) 63. The unit dose vial of claim 62, wherein the antibody comprises a heavy chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO: 47 and a light chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO: 48. (Item 64) A unit dose vial containing 2 ml or 20 ml of a fully human anti-interferon gamma (IFNγ) monoclonal antibody solution suitable for injection, the concentration of the antibody being 5 mg / ml and the pH of said solution being between 5.8 and 6.2. (Item 65) 65. The unit dose vial of item 64, wherein the antibody is solubilized in the solution, such that the solution is clear, colorless, and without precipitate. (Item 66) 65. The unit dose vial of item 64, wherein the antibody comprises a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO:1); a variable heavy chain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence of AISGSGGGSTYYADSVKG (SEQ ID NO:2); and a variable heavy chain complementarity determining region 3 (VH CDR3) comprising the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO:3); a variable light chain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO:4); a variable light chain complementarity determining region 2 (VL CDR2) region comprising the amino acid sequence of EDNQRPS (SEQ ID NO:5); and a variable light chain complementarity determining region 3 (VL CDR3) region comprising the amino acid sequence of QSYDGSNRWM (SEQ ID NO:6). (Item 67) 67. The unit dose vial of item 66, wherein the antibody comprises a heavy chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO: 47 and a light chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO: 48.

Claims

[Claim 1] The invention described in the specification.

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