Methods, compositions, and drug 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 and priority of U.S. Provisional Application No. 62 / 411,783, filed on 24 October 2016, the contents of which are incorporated herein by reference in their entirety.

[0002] This disclosure relates to methods and compositions for treating, preventing, and / or delaying the onset or progression of, or mitigating conditions associated with elevated levels of interferon-gamma (IFNγ, IFN-gamma), such as 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. This 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. IFNγ exhibits antiproliferative and immunomodulatory activity, binding to the IFNγ-R heterodimeric receptor on the majority of primary cells in the immune system and inducing a cascade of inflammatory events. The immunomodulatory activity of IFNγ is known to have beneficial effects in several clinical conditions. However, there are many clinical situations in which IFNγ activity is known to have adverse effects. For example, autoimmune diseases are associated with high levels of IFNγ in the blood and affected tissues of autoimmune patients. IFNγ activity has also been linked to disease conditions such as cachexia and septic shock.

[0004] IFNγ is involved in several disorders, and anti-IFNγ agents are currently under development as therapeutic agents. [Overview of the project] [Means for solving the problem]

[0005] In various embodiments, the present invention provides a number of variable-dose treatment regimens for treating diseases, disorders, or conditions 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 dose and a second dose of an antibody that binds to interferon-gamma (IFNγ) to a subject. The subject may be an adult or a pediatric subject. The first dose is 1.0 or 3.0 mg per kg of body weight of the subject, and the second dose is 3.0, 6.0 or 10.0 mg per kg of body weight of the subject. Optionally, a third dose of 1.0 mg per kg of body weight of the subject may be administered.

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

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

[0009] In yet another embodiment, the present invention provides a method for treating a condition in a human subject by intravenously administering a first dose and a second dose of an antibody conjugated to interferon-gamma (IFNγ) to the subject. The subject is an adult subject or a pediatric subject. The condition is graft rejection, such as parenchymal organ transplant failure or acute bone marrow 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 performed on a subject after receiving CART cell therapy. The first dose is 1.0 to 10 mg per kg of the subject's body weight, and the second dose is 1.0 to 10 mg per kg of the subject's body weight. For example, the second dose is 1.0, 3.0, 6.0, or 10.0 mg per kg. The second dose is preferably higher or lower than the first dose. Depending on the circumstances, a third dose of 1.0 to 10 mg per kg of the subject's body weight may be administered. For example, the first, second, or third doses may be 1.0, 3.0, 6.0, or 10.0 mg per kg of the subject's body weight.

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

[0011] The dosage of the antibody is administered within 1 hour, 6 hours or 12 hours.

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

[0013] The dosage of the antibody is administered as a single injection.

[0014] The antibody is administered as monotherapy or in combination therapy.

[0015] Optionally, the method of the present invention further comprises the step of administering dexamethasone immediately before dosing with the antibody. Dexamethasone is administered at a dose of at least 10 mg / m 2 or at least 5 mg / m 2 of the dose.

[0016] The subject has not previously received treatment for HLH.

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

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

[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, wherein the concentration of the antibody is 5 mg / ml or 25 mg / ml and the pH of the solution is between 5.8 and 6.2. The antibody is solubilized in the solution, and thus the solution is clear, colorless and free of precipitate.

[0020] In another aspect, the present 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, wherein the concentration of the antibody is 25 mg / ml and the pH of the solution is between 5.8 and 6.2. The antibody is solubilized in the solution, and thus 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, wherein the concentration of the antibody is 5 mg / ml and the pH of the solution is between 5.8 and 6.2. The antibody is solubilized in the solution, and thus the solution is clear, colorless and free of precipitate

[0022] Antibodies that bind to interferon-gamma (IFNγ) include: Variable heavy chain complementarity determination region 1 (VH CDR1) containing the amino acid sequence of SYAMS (SEQ ID NO: 1); Variable heavy chain complementarity determination region 2 (VH CDR2) containing the amino acid sequence of AISGSGGSTYYADSVKG (SEQ ID NO: 2); and Variable heavy chain complementarity determination region 3 (VH CDR3) containing the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO: 3); Variable light chain complementarity determination region 1 (VL CDR1) containing the amino acid sequence of TRSSSGSIASNYVQ (SEQ ID NO: 4); Variable light chain complementarity determination region 2 (VL CDR2) containing the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and Variable light chain complementarity determination region 3 (VL CDR3) containing the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6). For example, the antibody includes a heavy chain variable amino acid sequence which is the amino acid sequence of SEQ ID NO: 47, and a light chain variable amino acid sequence which is the amino acid sequence of SEQ ID NO: 48. Any of the above embodiments or aspects can be combined with any other embodiments or aspects. Unless otherwise defined, all scientific and technical terms used herein have the same meaning as those commonly understood by those skilled in the art in which the invention relates. Methods and materials similar to or equivalent to those described herein may be used in carrying out the invention, but suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are implicitly incorporated by reference in their entirety. In case of any conflict, this specification, including definitions, shall prevail. Furthermore, the materials, methods, and examples described herein are merely illustrative and not intended to be limiting.

[0023] Other features and advantages of the present invention will become apparent from and will be included in the following detailed description and claims. [Brief explanation of the drawing]

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

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

[0028] [Figure 5] Figure 5 is a graph showing that IFNγ was sufficiently neutralized, as indicated by the undetectable level of IFNγ-induced chemokines.

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

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

[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 between active MAS and active sJIA without MAS at the time of sampling (Act sJIA). 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), as well as changes in serum levels of IFNγ, CXCL9, CXCL10, and CXCL11 (Figure 9B), in one patient who experienced 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 correlations between IFNγ and CXCL9 levels and ferritin levels, neutrophil and platelet counts, as well as LDH and ALT levels, in patients with active MAS at sampling (red circles) and patients with active sJIA without MAS at 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 correlations between IFNγ and CXCL9 levels and ferritin levels, neutrophil and platelet counts, as well as LDH and ALT levels, in patients with active MAS at sampling (red circles) and patients with active sJIA without MAS at sampling (black triangles). The Spearman correlation coefficient (Rs) and significance level (p) for each correlation are shown in Table 3.

[0034] [Figure 11-1] Figures 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 IFNγ levels and CXCL9 and CXCL10 levels 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] Figures 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 IFNγ levels and CXCL9 and CXCL10 levels 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]Figure 12 is a schematic diagram of the screening, treatment, and follow-up portions of the test shown in Example 7.

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

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

[0038] [Figure 15] Figure 15 shows a series of graphs and tables illustrating the effect of NI-0501 administration on platelet count in patients.

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

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

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

[0042] [Figure 19] Figure 19 is a schematic diagram of the screening, treatment, and follow-up portions of the test shown in Example 8. [Modes for carrying out the invention]

[0043] The compositions and methods presented herein utilize a fully human IgG1 anti-interferon-gamma (IFNγ) monoclonal antibody (mAb), referred herein to as NI-0501, which binds to and neutralizes IFNγ. NI-0501 binds to both the soluble and receptor (IFNγR1) forms of IFNγ. As NI-0501 is human IgG1, it retains the properties of this immunoglobulin isotype, including its ability to associate with the Fcγ receptor and bind to complement. IFNγ is one of the most potent and multifaceted 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 produce a variety of physiological and cellular responses. Over the past 20 years, numerous studies have linked IFNγ to the pathogenesis and maintenance of inflammatory diseases (e.g., Billiau A, "Interferon-gamma: biology and role in pathogenesis," Adv. Immunol., 1996; Vol. 62: pp. 61-130; Schoenborn JR, Wilson CB, "Regulation of interferon-gamma during innate and adaptive immune responses," Adv. Immunol., 2007; Vol. 96: pp. 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; Vol. 226: pp. 29-40. IFNγ is produced primarily by natural killer (NK) and natural killer T (NKT) cells as part of the innate immune response, when antigen-specific immunity is generated by CD4 Th1 and CD8 cytotoxic T lymphocyte (CTL) effector T cells.

[0044] The compositions and methods presented herein are useful in the treatment of diseases, disorders, and conditions associated with elevated IFNγ levels. 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 parenchymal organ transplant failure and acute bone marrow graft rejection. Furthermore, the compositions and methods are also useful in the treatment or mitigation of side effects of CAR-T cell therapy.

[0045] HLH is a syndrome characterized by severe immune system dysfunction or the absence of cytotoxic function by NK and CD8+ T cells, accompanied by marked activation of the immune system. HLH includes primary (hereditary / familial) HLH and secondary HLH, both of which are clinically explained by immune system dysregulation leading to severe hypercytokinemia with adverse consequences for various tissues and organs (Henter JI, Elinder G, Soder O, et al., "Hypercytokinemia in familial hemophagocytic lymphohistiocytosis," Blood, 1991; vol. 78: pp. 2918-2922). The 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. Most cases of primary HLH occur in infancy and early childhood, with an estimated prevalence of 1 case per 50,000 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; Vol. 80: pp. 428-435). The disease is always fatal, and without treatment, the median survival time is less than two months after symptom onset (Janka GE. Familial hemophagocytic lymphohistiocytosis., Eur. J. Pediatr., 1983; Vol. 140: pp. 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; Vol. 10 (No. 2): pp. 197-203).

[0047] The cytotoxic dysfunction present in HLH leads to hypercytokinemia and hemophagocytosis, which in turn trigger all the typical symptoms of HLH (Dhote R, Simon J, Papo T et al., Reactive hemophagocytic syndrome in adult systemic disease: report of 26 cases and literature review., Arthritis Rheum., 2003; Vol. 49: pp. 633-639; Risdall RJ, McKenna RW, Nesbit ME et al., Virus-associated hemophagocytic syndrome: a benign histiocytic proliferation distinct From malignant histiocytosis, Cancer, 1979; Vol. 44: pp. 993-1002; and Risdall RJ, Brunning RD, Hernandez JI, Gordon DH. Bacteria-associated hemophagocytic syndrome. Cancer, 1984; Vol. 54: pp. 2968-2972). Typical symptoms of HLH include, for example, prolonged fever, splenomegaly, hepatomegaly, cytopenia, hyperferritinemia, hypertriglyceridemia, hypofibrinogenemia, hemophagocytosis, hypercytokinemia, and / or lymphohistiocytic infiltration, myelodysplasia, 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 disease, or neoplasm, in which case it is called secondary HLH. Secondary HLH presents with the same signs and symptoms as the primary form and can be equally severe. Current treatment for secondary HLH aims to address the underlying cause. This is certainly the case with HLH caused by infections such as leishmaniasis. Notably, the presence of certain infections, particularly viral infections such as those caused by CMV or EBV, is very often the trigger for the manifestation of the primary form of HLH. This finding is further supported by evidence that infection with lymphocytic choriomeningitis virus (LCMV) is necessary for the development of primary HLH in animal models of the disease (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: pp. 735-743; Pachlopnik SJ, Ho CH, Chretien F et al., Neutralization of IFNgamma defeats hemophagocytosis in LCMV-infected perforin- and Rab27a-deficient mice., EMBO Mol. Med., 2009; Vol. 1: pp. 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; Vol. 121: pp. 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 neoplasms, particularly hematological malignancies, the severity of the patient's condition often 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 with rheumatic diseases such as systemic juvenile idiopathic arthritis (sJIA) and systemic lupus erythematosus (SLE) is often referred to by rheumatologists as macrophage activation syndrome (MAS) and may precede the onset 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 a very severe and life-threatening condition, but usually resolves with appropriate treatment consisting of corticosteroids and cyclosporine in most cases. However, in approximately 15% of patients who develop 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] While primary HLH is generally understood to be a childhood disorder, HLH can occur in adults, and increased awareness suggests it may be more frequent than previously understood. In the majority of adult patients, the disorder arises between malignant tumors (primarily non-Hodgkin lymphoma), infections, autoinflammatory or autoimmune diseases, and iatrogenic immunodeficiency.

[0053] Currently, there are no approved medications for treating HLH. However, guidelines for managing HLH patients have been established by experts in this field (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; Vol. 48: pp. 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; Vol. 100: pp. 2367-2373; and Jordan MB, Allen CE, Weitzman S, Filipovich AH, McClain KL. How I treat hemophagocytic Lymphohistiocytosis. Blood, 2011; Vol. 118: pp. 4041-4052).

[0054] The management of primary HLH patients currently includes the following steps (Henter et al., Blood Cancer, 2007): (i) an 8-week induction therapy with a combination of corticosteroids and immunosuppressants (e.g., etoposide, CsA, alemtuzumab, antithymocyte globulin); (ii) maintenance therapy until transplantation; and (iii) transplantation in all patients in whom a gene deficiency has been identified, and ultimately in cases of very severe HLH without disease-related mutations.

[0055] The primary goal of induction therapy is to suppress the life-threatening inflammatory processes that characterize 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; vol. 129: pp. 622-630). Transplantation is the only curative treatment for HLH associated with highly penetrating gene 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; Vol. 118: pp. 4577-4584).

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

[0058] In recent years, there has been increasing evidence of the central role of IFNγ in the development of HLH (Henter JI, Elinder G, Soder O et al., Hypercytokinemia in familial hemophagocytic lymphohistiocytosis., Blood, 1991; Vol. 78: pp. 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; Vol. 104: pp. 735-743; Pachlopnik SJ, Ho CH, Chretien F et al., Neutralization of IFNgamma defeats heemophagocytosis in LCMV-infected perforin- and Rab27a-deficient mice., EMBO Mol. Med., 2009; Vol. 1: pp. 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; Vol. 121: pp. 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] All gene mutations that characterize primary forms of HLH affect proteins involved in the same process, ultimately impairing cytotoxic activity. The first mutation identified in HLH patients was perforin mutation.

[0060] Perforin knockout (KO) mice are considered a relevant model for human diseases. Indeed, when these mice are infected with LCMV, they exhibit all the distinctive diagnostic, as well as many of the distinctive clinical and laboratory features of the human disease, and die if left untreated. For these reasons, perforin KO mice are used to study the pathophysiology of HLH. The HLH-like pathology that occurs in perforin KO mice is dependent on the production of CD8+ T cells and IFNγ in response to antigen stimulation.

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

[0062] Two models of secondary HLH are being investigated in connection with the NI-0501 development program. In one model, repeated administration of CpG (which induces TLR9 stimulation) is used to mimic chronic severe hyperstimulation in healthy mice (i.e., those with normal genes for the cytotoxic pathway) as a model of HLH secondary to infection. These mice do not necessarily die, but develop the typical clinical and laboratory features of HLH. Neutralizing IFNγ with anti-IFNγ antibody administration reverses the clinical and laboratory features of the disease. Interestingly, in this model, it has been demonstrated that administration of anti-IFNγ antibody leads to sufficient neutralization of the effects of IFNγ in relevant target tissues such as the liver and spleen (manuscript in preparation).

[0063] To investigate the physiological pathology of secondary HLH occurring in the context of rheumatic disease, an animal model was generated using IL-6 transgenic mice that express high levels of IL-6, similar to those observed in patients with sJIA, the rheumatic disease most frequently associated with secondary HLH. When induced with Toll-like receptor (TLR) ligands, these mice died with many of the characteristics of 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; vol. 64: pp. 1680-1688). In these mice, neutralization of IFNγ by administration of an anti-IFNγ antibody significantly improved survival and restored laboratory parameters (Prencipe G et al., in preparation for publication).

[0064] The high circulating IFNγ levels in patients with primary HLH further highlight the importance of IFNγ in HLH (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), with 53.5% having levels above 1000 pg / mL. It has also been reported that IFNγ levels rise rapidly in the early stages and can decrease from >5000 pg / mL to normal within 48 hours of effective treatment of HLH.

[0065] In a recent observational study of patients with secondary forms of HLH, high levels of IFNγ were demonstrated in both patients with HLH secondary to infection and those with HLH occurring in the context of sJIA. Levels of three chemokines known to be induced by IFNγ—CXCL9, CXCL10, and CXCL11—were also significantly elevated. Notably, levels of IFNγ, and the levels of these three IFNγ chemokines, were found to be significantly correlated with disease severity parameters, such as ferritin, platelet count, and transaminase (Bracaglia et al., submitted).

[0066] Hypercytokinemia and tissue infiltration by activated lymphocytes and histiocytes are involved in all HLH symptoms and depend on increased CD8+ T cell activity and high IFNγ levels; therefore, neutralizing IFNγ constitutes a rational therapeutic approach. In fact, 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, as well as data from observations conducted in both patients with primary and secondary HLH, which confirm the crucial role that IFNγ plays in the pathogenesis of this disease, neutralization of IFNγ provides a robust rationale for developing targeted therapies for HLH that must be effective with no or limited toxicity.

[0068] This disclosure also provides compositions and methods useful in identifying or accurately identifying patient populations affected by a disorder, wherein patients have elevated levels of CXCL9 alone or in combination with one or more additional interferon-gamma (IFNγ) related biomarkers. In particular, this 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 series of pieces of evidence from animal models demonstrate the central pathogenic role of IFNγ in primary hemophagocytic lymphohistiocytosis (HLH). High levels of IFNγ are also found in humans with HLH. Previous reports have shown high levels of IFNγ, along with three IFNγ-related chemokines, CXCL9, CXCL10, and CXCL11, in patients with active MAS, a form of secondary HLH occurring in the context of systemic juvenile idiopathic arthritis (sJIA) (e.g., Bracaglia C., Caiello). See I, De Graaf K. et al., Pediatric Rheumatology, 2014, Vol. 12 (Supplement 1): O3. Indirect evidence in mice suggests that IFNγ is mostly produced in peripheral tissues and may have relatively low blood concentrations.

[0070] The term macrophage activation syndrome (MAS) refers to a severe, potentially fatal complication of chronic inflammatory rheumatic disease. MAS commonly occurs in the context of systemic juvenile idiopathic arthritis (sJIA), with 10–20% of patients developing the syndrome during the course of the disease. MAS can also occur, though less frequently, in systemic lupus erythematosus, Kawasaki disease, and other autoimmune and autoinflammatory disorders. In sJIA, MAS generally occurs during the disease activity phase, including at the onset of the disease. Infection triggers can be identified in a high percentage of patients. Typical features of MAS include fever, splenomegaly, hemorrhage, and signs of hepatic, central nervous system, and renal involvement, which can lead to multiple organ failure. Laboratory abnormalities include decreased white blood cell count, platelet count, and hemoglobin levels, hypertransaminasemia, a 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., Vol. 13 (No. 4): pp. 289-2898). 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, Vol. 66 (No. 11): pp. 3160-3199; Hashkes, PJ et al., Mortality outcomes in pediatric rheumatology in the US., Arthritis Rheumatol, 2010, Vol. 62 (No. 2): pp. 599-608).A better understanding of disease pathogenesis could lead to significant improvements in the management and outcomes of MAS, along with the identification of new therapeutic targets and the potential development of targeted therapies.

[0071] MAS shares most of the clinical features and laboratory abnormalities of hemophagocytic lymphohistiocytosis (HLH) and is actually currently classified within secondary or reactive HLH (sec-HLH) (Jordan, MB et al., How I treat hemophagocytic lymphohistiocytosis., Blood, 2011, vol. 118 (no. 15): pp. 4041-452). Primary HLH (p-HLH) is caused by mutations in genes encoding proteins involved in granular exocytosis, generally leading to incomplete cytotoxic activity of CD8+ lymphocytes and NK cells, including PRF1, UNC13D, STXBP2, STX11, RAB27A, and XIAP. According to the current classification, HLH is defined as secondary or reactive if there is no 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 is commonly referred to as MAS. The genetic basis for the development of MAS is gradually being elucidated, and several studies have shown a link between MAS, and sec-HLH in general, and heterozygosity for low-penetration variants or mutations of the same causative gene 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, vol. 66 (no. 12): pp. 3486-3495; 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, Vol. 49 (No. 3): pp. 441-449.; Zhang, K. et al., Macrophage activation syndrome in patients with systemic juvenile idiopathic arthritis is associated with MUNC 13-4 polymorphisms., Arthritis Rheum, 2008, Vol. 58 (No. 9): pp. 2892-2896; and Zhang, M. et al., Genetic defects in cytolysis in macrophage activation syndrome. Curr Rheumatol Rep, 2014, Vol. 16 (No. 9): p. 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 (Supplement 1): p. 53). The similarities in these genetic backgrounds between p-HLH and MAS further support a shared pathogenic mechanism.

[0072] Studies in patients with p-HLH and in mouse models of p-HLH support the hypothesis that incomplete cytotoxic activity and abnormal antigen-presenting cell (APC)-CD8+ T cell crosstalk lead to incomplete silencing of the immune response and abnormal T cell activation. This results in uncontrolled immune activation and production of pro-inflammatory cytokines by T lymphocytes and macrophages, leading to organ damage. Animal models of p-HLH conducted in perforin-deficient mice and Rab27-deficient mice demonstrate the crucial role of interferon-gamma (IFNγ) produced by activated CD8+ T cells. In perforin-deficient mice, neutralization of IFNγ leads to survival from another lethal syndrome, along with a 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, Vol. 104 (No. 3): pp. 735-743; Pachlopnik Schmid, J. et al., Neutralization of IFNgamma defeats heemophagocytosis in LCMV-infected perforin- and Rab27a-deficient mice., EMBO Mol Med, 2009, Vol. 1 (No. 2): pp. 112-124). In Rab27-deficient mice, where the disease does not lead to death, neutralization of IFNγ results in a significant improvement in the involvement of peripheral organs, 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, vol. 148 (no. 2): pp. 301-301; 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 diagnostic and prognostic significance of a specific Th1 / Th2 cytokine pattern in children with hemophagocytic syndrome. Br J Haematol, 2008, vol. 143 (no. 1): pp. 84-91; Xu, XJ et al., Diagnostic accuracy of a specific cytokine pattern in hemophagocytic lymphohistiocytosis in children., J Pediatr, 2012, vol. 160 (no. 6): pp. 984-90 e1). Therefore, it is not necessarily based on the presence of gene mutations. It should be noted that these studies included a fluctuating but significant proportion of patients without demonstrable genetic causes (Ibid).

[0073] To search for biomarkers of in vivo IFNγ production, the studies presented herein were designed to evaluate the correlation of serum levels of IFNγ and serum levels of 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, where approximately 37% (20 out of 54) had MAS at the time of sampling. The relationship between circulating levels and disease activity parameters was evaluated, as well as the correlation between IFNγ levels and CXCL9, CXCL10, and CXCL11 levels. In some embodiments, the biomarker is total IFNγ level, which is useful as a pharmacodynamic biomarker.

[0074] As demonstrated herein, IFNγ levels and the levels of 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, disease severity laboratory parameters such as ferritin, neutrophils, platelets, alanine aminotransferase, and lactate dehydrogenase were significantly correlated with IFNγ and CXCL9, less significantly correlated with CXCL10 and CXCL11, and 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, less significantly correlated with CXCL10 levels, and 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, February 2011; Vol. 89 (No. 2): pp. 207-2015), the findings disclosed herein demonstrate that CXCL9 is a biomarker of IFNγ production in MAS.

[0076] The studies presented herein 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 associated with 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] This disclosure provides compositions and methods for treating, preventing, and / or delaying the onset or progression of symptoms associated with inflammatory disorders associated with graft rejection, such as transplant failure, graft rejection, and / or parenchymal organ transplant failure, and acute bone marrow graft rejection, using neutralizing anti-IFNγ antibodies or their antigen-binding fragments. This disclosure provides compositions and methods for treating, inhibiting, delaying the progression of, or instead improving the symptoms of graft-versus-host disease (GvHD) in subjects who have received or are receiving a transplant or a series of transplants containing biomaterials. This disclosure provides compositions and methods for extending the survival of transplanted biomaterials using neutralizing anti-IFNγ antibodies or their antigen-binding fragments. This disclosure provides compositions and methods for treating, preventing, and / or delaying the onset or progression of paraneoplastic cerebellar degeneration, hemorrhagic fever, sarcoidosis, adult-onset Still's disease, or symptoms associated with CART-T cell therapy, using a neutralizing anti-IFNγ antibody or its antigen-binding fragment.

[0078] The compositions and methods presented herein are useful, for example, in the transplantation of any biological material, including cells, tissues, bone marrow, and / or organs, including, in non-limiting examples, the heart, kidneys, pancreas, liver, and / or intestines. In some embodiments, the biological material to be transplanted 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 biological material to be transplanted is one or more hepatocytes or derived therefrom.

[0079] The compositions and methods presented herein involve administering NI-0501 to subjects in need to treat, prevent, and / or delay the onset or progression of symptoms associated with graft failure, graft rejection, and / or inflammatory disorders associated with graft rejection. In some embodiments, graft rejection, also referred to herein as graft failure, is acute. In some embodiments, graft rejection is hyperacute.

[0080] The neutralizing anti-IFNγ antibody of the present invention includes, 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 comprising the complementarity-determining regions (CDRs) as defined by Chothia et al., 1989 and E.A. Kabat et al., 1991 are highlighted in the underlined and italicized text below (see Chothia, C et al., Nature, Vol. 342: pp. 877-883 (1989); Kabat, E. et al., Sequences of Protein of immunological interest, 5th edition, US Department of Health and Human Services, US Government Printing Office (1991)). Table 1A. VH CDR sequences derived from antibody clones that bind to and neutralize IFNγ. [Table 1A-1] [Table 1A-2] Table 1B. VL CDR sequences derived from antibody clones that bind to and neutralize IFNγ. [Table 1B-1] [Table 1B-2]

[0081] An exemplary antibody of the present invention is, for example, the anti-IFNγ antibody described in PCT Publication WO2006 / 109191, the entirety of which is incorporated herein by reference.

[0082] An exemplary antibody of the present invention is, for example, an antibody that binds to human IFNγ, referred to herein as NI-0501. The heavy chain, light chain, variable heavy (VH) chain, 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, which is incorporated herein by reference in its entirety. 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"), and ARC1.2R3P2_D6 ("D6"). Examples of antibodies include those designated as 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 to an IgG isotype. In some embodiments, the IFNγ antibody is formatted to an IgG1 isotype.

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

[0086] The compositions and methods are useful in treating any of the various disorders related to interferon-gamma (IFNγ) expression and / or activity, including abnormal IFNγ expression and / or activity. The compositions and methods of this disclosure are useful in treating hemophagocytic lymphohistiocytosis (HLH). HLH is a rare, severe, and life-threatening pathological immune-activating disorder characterized by clinical signs and symptoms of extreme inflammation (fever, splenomegaly, cytopenia, coagulation disorders) leading to the development of an abnormal immune-mediated pathology that can ultimately lead to 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, vol. 78: pp. 2918-2922). HLH includes primary (hereditary / familial) HLH and secondary HLH.

[0087] Primary HLH is a heterogeneous autosomal recessive genetic disorder that mostly occurs in infancy and early childhood, with an estimated prevalence of 1 in 50,000 births in Europe (Janka GE: Familial hemophagocytic lymphohistiocytosis., Eur. J. Pediatr., 1983, Vol. 140: pp. 221-230). The disease is always fatal, and without treatment, the median survival time is less than two months after the onset of symptoms (Filipovich AH: Hemophagocytic lymphohistiocytosis (HLH) and related disorders., Hematology Am Soc Hematol Educ Program, 2009: pp. 127-131).

[0088] All gene defects in primary HHL affect genes involved in the cytotoxic pathway of NK cells and / or cytotoxic lymphocytes, which are necessary for eliminating activated macrophages and encode proteins related to perforin synthesis, cytolytic granule maturation, granule exocytosis, and releasing 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 Supplement): pp. 82-89). In approximately 20-40% of patients with primary HLH, the cytotoxic dysfunction that characterizes 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 approximately 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. Furthermore, certain immunodeficiency syndromes, such as Glycerin syndrome type 2 (GS-2) and Chediak-Higashi syndrome (CHS), are frequently associated with HLH (Janka GE, Lehmberg K: Hemophagocytic lymphohistiocytosis: pathogenesis and treatment, Hematology Am Soc Hematol Educ). Program, 2013, Vol. 2013: pp. 605-611).

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

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

[0091] MAS is a severe, potentially life-threatening complication of rheumatic disease, caused by excessive activation and proliferation of T lymphocytes and macrophages. This uncontrolled increase in immune cells leads to marked hypercytokinemia, as well as a hyperinflammatory state accompanied by fever, cytopenia, hepatosplenomegaly, liver dysfunction, coagulation abnormalities, and hyperferritinemia, which 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, Vol. 66: pp. 145-159). Due to its strong clinical and pathological similarity to HLH, MAS is classified as a secondary or acquired form of HLH. In fact, recent studies have demonstrated that the majority of patients with MAS have impaired NK and perforin function tests, and that a significant number of MAS patients exhibit polymorphic 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, Vol. 16: p. 439).

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

[0093] Because MAS is potentially fatal, timely diagnosis and immediate therapeutic intervention are essential for the proper management of this disease. Reported mortality rates for MAS reach 20–30%, and it remains a major source of mortality in pediatric rheumatology. 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 have been proposed for diagnosing MAS in patients with sJIA. 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, Vol. 48: pp. 124-131), which were mainly developed for primary (hereditary) forms of HLH, are sometimes recommended. However, these have some limitations and do not apply to patients with sJIA. For example, these patients often have elevated white blood cell and platelet counts, as well as elevated serum fibrinogen levels, as part of the sJIA inflammatory response, so criteria such as cytopenia and hypofibrinogenemia below the threshold required by HLH-2004 become apparent only 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, Vol. 66: pp. 145-159). A significant proportion of patients with MAS may not have hemophagocytosis at the time of examination (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, Vol. 66: pp. 3160-3169). Furthermore, hemophagocytosis, NK cell activity, and sCD25 are not routinely evaluated in relation to MAS.

[0095] An alternative approach is based on the application of the preliminary diagnostic guidelines (PDG) for MAS complicating sJIA, which were developed through an analysis comparing a cohort of patients with MAS with a group of patients with sJIA1 relapses.

[0096] Recently, the HLH-2004 diagnostic guidelines and preliminary diagnostic guidelines for sJIA-associated MAS were compared in a large patient population for their ability to differentiate between sJIA / MAS, 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, vol. 66: pp. 2871-2880). Despite some limitations due to its retrospective nature, this study appears to demonstrate that the preliminary MAS guidelines achieve the best balance of sensitivity and specificity, and the best agreement with diagnoses made by treating physicians. The sensitivity of the HLH-2004 reference set was <30%. Nevertheless, the proportion of patients meeting each of the single criteria for PDG varies considerably, and some clinical features (e.g., CNS dysfunction and bleeding) may manifest later in MAS, which has been reported to lead to lower sensitivity 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] A diagnostic score (HScore) was recently developed and validated in a retrospective cohort of 312 patients, of whom 162 were diagnosed with 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, Vol. 66: pp. 2613-2620).

[0098] Nine variables (three clinical variables [i.e., known underlying immunosuppression, high temperature, and organ enlargement], five biological variables [i.e., triglycerides, ferritin, serum glutamate oxaloacetate transaminase, fibrinogen levels, and cytopenia], and one cytological variable [i.e., hemophagocytic features in bone marrow aspirate]) were preserved by the HS score, and the probability of having hemophagocytic syndrome ranged from <1% for HS score ≤ 90 to >99% for HS score ≥ 250.

[0099] Until a final consensus is reached on validated diagnostic criteria for MAS, clinical diagnosis by specialists remains crucial in distinguishing MAS from overlapping conditions such as SJIA relapses or sepsis-like syndromes.

[0100] Currently, there are no approved drugs for the treatment of MAS. High-dose glucocorticoids are typically the first-line treatment for MAS. For patients who do not respond to glucocorticoids, cyclosporine A (CsA) is suggested 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, etoposide administration is also being considered in patients who have failed high-dose glucocorticoid therapy. However, the potential toxicity of the drug remains a major concern. Another current first-line treatment for HLH is dexamethasone. However, treatments such as etoposide and / or dexamethasone are myelosuppressive and / or broadly immunosuppressive. Currently, there is no standard treatment for second-line HLH, and treatments such as alemtuzumab / ATG are severely immunosuppressive, and survival is considered very poor with these treatments.

[0102] The usefulness of biologics inhibiting the IL-1, IL-6R, or TNFα pathways in the treatment of macrophage activation syndrome (MAS) remains unclear. While biologics inhibiting these pathways have been reported to be effective in isolated cases, the efficacy of these treatments in patients who develop MAS in the context of these therapies remains unknown (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, vol. 7: pp. 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, Vol. 367: pp. 2396-2406), and reports of patients who did not respond to these treatments, indicate that inhibition of IL-1, IL-6R, or TNFα does not provide complete protection from MAS development or effective treatment of end-stage syndrome.

[0103] A large-scale retrospective multicenter study investigated the clinical, laboratory, and histopathological characteristics of MAS / sJIA, as well as current treatments and outcomes, 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, vol. 66: pp. 3160-3169). At the onset of the disease, MAS occurred in the context of active sJIA in approximately half of the patients, or during sJIA relapse in 30%. Infection triggers were identified in one-third of the patients. Among the 24 patients in whom infection types were reported, EBV was the most common causative factor (25%). In 11 patients (3.8%), MAS was thought to be related to treatment side effects, and of these, 8 were accompanied by biological agents targeting the IL-6 (N=4), IL-1 (N=3), or TNFα (N=1) pathways. Almost all patients were given glucocorticoids. Cyclosporine, biological agents, and etoposide were given to 61%, 15%, and 12% of patients, respectively.

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

[0105] The compositions and methods, including the NI-0501 composition of this disclosure, are advantageous over current treatments for primary and secondary HLH.

[0106] MAS and HLH are characterized by persistent immune cell activation and a cytokine storm of pro-inflammatory cytokines, accompanied by the 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 heemophagocytosis in LCMV-infected perforin- and Rab27a-deficient mice. EMBO Mol Med, 2009, Vol. 1: pp. 112-124; Zoller EE, Lykens JE, Terrell CE, Alberti J et al.: Hemophagocytosis causes a consumptive anemia of inflammation. J. Exp. Med., 2011, Vol. 208: pp. 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. Evidence is accumulating to support the central role of IFNγ in the development of both conditions (Clin.Invest., 2011, Vol. 121: pp. 2264-2277).

[0107] Regarding primary HLH, perforin knockout mice are considered a relevant model because, upon infection with LCMV, they develop all the unique diagnostic and clinical features of the human disease, as well as many of the unique clinical and laboratory features. HLH-like disease occurring in mice is dependent on CD8+ T cells and IFNγ produced in response to antigen stimulation (Imashuku S, Hibi S, Fujiwara F, Todo S: Hyper-interleukin (IL)-6-naemia in haemophagocytic lymphohistiocytosis., Br. J. Haematol., 1996, Vol. 93: pp. 803-807). It has been demonstrated that neutralization of high circulating levels of IFNγ with anti-IFNγ antibody not only reverses clinical and laboratory abnormalities but also dramatically improves survival. Conversely, 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; 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: pp. 984-90e1). The high circulating IFNγ levels found in these patients further reinforce the importance of IFNγ in HLH (Henter JI, Elinder G, Soder O, Hansson M et al.: Hypercytokinemia in familial hemophagocytic lymphohistiocytosis., Blood, 1991, Vol. 78: pp. 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, Vol. 160: pp. 984-90e1). 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), with 53.5% having levels above 1000 pg / mL. It was also reported that IFNγ levels rose rapidly in the early stages and could decrease 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 under the auspices of the NI-0501 Development Program. First, in a mouse model mimicking infection-driven HLH, repeated administration of CpG induced hypercytokinemia via TLR9 activation, leading to clinical (e.g., weight loss, splenomegaly) and laboratory (e.g., cytopenia, hyperferritinemia) features of HLH33. Neutralization of IFNγ by administration of anti-IFNγ antibodies reversed the clinical and laboratory features of the disease. Neutralization of IFNγ was 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 greater than the measured amount of IFNγ in the blood, suggesting a greater likelihood of reflecting IFNγ production in the tissues. Following TLR9 stimulation, two IFNγ-inducible chemokines (CXCL9 and CXCL10) were upregulated in both the blood and liver, and a significant correlation was observed between serum IFNγ levels and serum concentrations of CXCL9 and CXCL10. Neutralization of IFNγ induced a significant decrease in serum CXCL9 and CXCL10 levels, as well as a significant decrease in 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, we tested an animal model of IL-6 transgenic mice expressing high levels of IL-6, as it mimics the condition of patients with sJIA, a rheumatic disease most frequently associated with secondary forms of HLH. Induction with Toll-like receptor (TLR) ligands resulted in increased lethality, increased inflammatory cytokine production, and hyperactivation of inflammatory signaling pathways. Furthermore, these mice exhibited decreased platelet and neutrophil counts, and elevated sCD25, ferritin, and LDH levels, similar to many of the features commonly found 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, Vol. 64: pp. 1680-1688). In these mice, neutralization of IFNγ by administration of anti-IFNγ antibody significantly improved survival and restored laboratory parameters (Prencipe G et al., manuscript in preparation).

[0110] Similar evidence has recently been gathered in observational studies conducted in patients with secondary forms of HLH, either secondary to infection or of unknown cause (pHLH was ruled out due to normal cytotoxic activity, absence of known gene mutations causing pHLH, and no family history) or in patients with MAS occurring in the context of sJIA.

[0111] Serum samples were analyzed in 14 patients with secondary HLH (of which the underlying infection could be identified in 7 patients) during active, terminal disease and disease remission. During the active phase, levels of IFNγ, CXCL9, and CXCL10 were significantly higher compared to 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 were significantly correlated with CXCL9 levels (p=0.0018) and less significantly correlated with CXCL10 levels (p=0.014). Levels of IFNγ and chemokines (particularly CXCL9) significantly correlate with disease severity parameters 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 for the 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 observed in patients with MAS occurring 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. IL-6 levels 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, particularly for CXCL9, with the median level being approximately 15 times higher in patients with active sJIA without MAS (13392 vs. 837 pg / mL; p=0.005). Notably, only in patients with MAS did a significant correlation between CXCL9 levels and generally abnormal parameters such as ferritin (p=0.041), neutrophil (p=0.010), platelet (p=0.022) counts, ALT (p=0.044), and LDH (p=0.013) emerge. IFNγ levels also correlated with disease severity parameters, with the exception of LDH, for which statistical significance was not achieved (Bracaglia et al., manuscript in preparation).

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

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

[0115] The rationale for identifying this patient population as one that would benefit from treatment with NI-0501, and for evaluating the efficacy of NI-0501 in MAS / sHLH, is based on several factors. First, preclinical data obtained in animal models associated with MAS in sJIA showed that IFNγ neutralization significantly improved survival and reversed changes in laboratory parameters. Second, observational data from patients with MAS / sHLH demonstrate the presence of high levels of IFNγ, and more importantly, extremely elevated levels of IFNγ-inducible chemokines, CXCL9, CXCL10, and CXCL11. Third, in patients with MAS / sHLH, concentrations of IFNγ and CXCL9 are significantly correlated with disease parameters such as ferritin, platelet count, and transaminase. Next, in previous trials where all administered infusions were well-tolerated, a favorable tolerability profile and no relevant safety concerns were observed in pHLH patients. This confirms observations made in healthy volunteers, no infections caused by pathogens for which IFNγ neutralization is known to be beneficial were reported, and any infections that occurred in some pHLH patients were not related to treatment with NI-0501, but rather to their immune status, duration of illness, and previous or concurrent treatments. Fifth, preliminary data from previous clinical trials showed favorable effects on disease parameters, with visible effects occurring within day one 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 enabled 10 out of 18 evaluable patients at cutoff to be transferred to HSCT. Next, evidence from PK modeling and simulation techniques demonstrates a predictable pharmacokinetic profile for NI-0501, and that IFNγ neutralization is achieved and maintained. Finally, in the event that the disease is not adequately controlled with NI-0501, conventional treatment (e.g., CsA) can be initiated immediately without requiring a drug-free period.

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

[0117] Therefore, NI-0501 is an innovative and effective treatment approach in managing this severe, life-threatening complication of rheumatic disease, with potentially limited side effects associated with long-term, high-dose glucocorticoid treatment. Administration of anti-IFNγ antibody

[0118] It will be understood that the administration of the therapeutic substance according to the present invention will be carried out with appropriate carriers, excipients, and other agents incorporated into the formulation to provide improvements in transport, delivery, and tolerability. Numerous suitable formulations can be found in prescription collections known to all pharmacists: Remington's Pharmaceutical Sciences (15th edition, Mack Publishing Company, Easton, PA (1975), particularly Chapter 87 by Blaug, Seymour. Examples of these formulations include powders, pastes, ointments, gels, waxes, oils, lipids, vesicle-containing lipids (cationic or anionic) (such as Lipofectin®), DNA conjugates, anhydrous absorbent pastes, oil-in-water emulsions and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. Any of the aforementioned mixtures may be appropriate in the treatments and therapies according to the present invention, provided that the active ingredients in the formulation are not inactivated by the formulation and the formulation is physiologically compatible with and tolerable to the route of administration. For further 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 (No. 2): pp. 210-218 (2000), Wang W., "Lyophilization and development of solid protein pharmaceuticals.”, Int. See also J. Pharm., Vol. 203 (Nos. 1-2): pp. 1-60 (2000), Charman WN, "Lipids, lipophilic drugs, and oral drug delivery—some emerging concepts," J Pharm Sci., Vol. 89 (No. 8): pp. 967-978 (2000), Powell et al., "Compendium of excipients for parenteral formulations," PDA J Pharm Sci Technol., Vol. 52: pp. 238-311 (1998), and the citations within them.

[0119] The efficacy of the treatment is determined in relation to any known method for diagnosing or treating a specific immune-related disorder. The alleviation of one or more symptoms of an immune-related disorder demonstrates that the antibody confers clinical utility.

[0120] 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 having high specificity and high affinity for its target antigen, is administered to a subject and generally has an effect due to binding to the target. By administering an antibody, the signaling function of the target can be inhibited, blocked, or interfered with. By administering an antibody, the binding of the target to its naturally bound endogenous ligand can be inhibited, blocked, or interfered with.

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

[0122] The antibodies or fragments thereof of the present invention can be administered in the form of pharmaceutical compositions 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 (edited by 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 Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, delivered in New York.

[0123] The formulation may contain more than one active compound, for example, anti-IFNγ antagonists, preferably having complementary activity that does not adversely affect each other, as needed for the specific 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 inhibitors. Such molecules are appropriately present in combination in amounts effective for the intended purpose.

[0124] In one embodiment, an active compound, such as an anti-IFNγ antagonist, is administered in combination therapy, i.e., in combination with one or more additional agents useful for treating a pathological condition or disorder. In this context, the term “combined” means that the agents are administered substantially simultaneously, concurrently, or sequentially. When administered sequentially, it is preferable that the first of the two compounds is still detectable at an effective concentration at the treatment site at the start of administration of the second compound.

[0125] For example, a combination therapy may include one or more neutralizing anti-IFNγ antibodies of the present invention, co-formulated and / or administered concurrently 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 agents or cell proliferation inhibitors, as described in more detail below. Such a combination therapy can advantageously utilize the administered therapeutic agents at lower doses and thus avoid the toxicity 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 before administration of NI-0501. In some embodiments, the additional agent includes at least etoposide. In some embodiments, the subject receives etoposide before administration of NI-0501.

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

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

[0129] In some embodiments, one or more additional agents include analgesia, transfusion of blood products, electrolyte and glucose infusions, antibiotics, antifungal and antiviral treatments, and / or general supportive care.

[0130] When using antibody fragments, minimal inhibitory fragments that specifically bind to the binding domain of the target protein and / or minimal inhibitory fragments that interfere with or instead antagonize IFNγ signaling are preferred. For example, based on the variable region sequence of the antibody, peptide molecules that retain the ability to bind to the target protein sequence can be designed. Such peptides can be synthesized chemically and / or produced by recombinant DNA technology (see, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, Vol. 90: pp. 7889-7893 (1993)). The formulation may also contain more than one active compound, preferably having complementary activity that does not adversely affect each other, as required for the specific indication being treated. Alternatively or in addition, the composition may include agents that enhance its function, such as cytotoxic agents, cytokines, chemotherapeutic agents, or growth inhibitors. Such molecules are appropriately present in combination in amounts effective for the intended purpose. Medication regimen

[0131] The present invention further provides drug regimens for treating, preventing, and / or delaying the onset or progression of symptoms associated with elevated IFN-γ levels. The drug regimens are regimens with a number of variable doses. An antibody that binds to interferon-gamma (IFNγ) in doses of 1.0 to 10 mg per kg of body weight of the subject. 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] Numerous variable-dose regimens for treating primary hemophagocytic lymphohistiocytosis (HLH) in human subjects include an induction dose or first dose and a treatment dose or second dose of an anti-IFN-γ monoclonal antibody. Subjects are adult or pediatric subjects.

[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 as a single dose. Alternatively, the first dose is administered more than once over the induction period.

[0135] The second dose is administered over one or more treatment periods. The second dose is administered over the first treatment period. During the first treatment period, the second dose is administered every three days after the first dose. The first treatment period lasts about one week, two weeks, three weeks, four weeks, five weeks, six weeks, or longer. The first treatment period is preferably two weeks. If necessary, the second dose is administered over a second treatment period following the completion of the first treatment period. During the second treatment period, the second dose is administered twice a week. The second treatment period lasts 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, or 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 may be 1.0, 3.0, or 6.0 mg per kg of body weight. The third dose is administered over the remainder of the first and second treatment periods. Alternatively, the third dose is administered over a third treatment period. In some embodiments, the third dose is referred to as the maintenance dose, and the third treatment period is referred to as the maintenance period.

[0137] Numerous variable-dose regimens for treating secondary hemophagocytic lymphohistiocytosis (HLH) in human children include 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 per kg of body weight. The second dose is 3.0 mg per kg of body weight. 10.0 mg per kg of body weight.

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

[0140] The second dose is administered over one or more treatment periods. The second dose is administered over the first treatment period. During the first treatment period, the second dose is administered every three days after the first dose. The first treatment period lasts about one week, two weeks, three weeks, four weeks, five weeks, six weeks, or longer. The first treatment period is preferably two weeks. If necessary, the second dose is administered over a second treatment period following the completion of the first treatment period. During the second treatment period, the second dose is administered twice a week. The second treatment period lasts 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, or 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 per kg of body weight. The third dose is administered over the remainder of the first and second treatment periods. Alternatively, the third dose is administered over a third treatment period. In some embodiments, the third dose is referred to as the maintenance dose, and the third treatment period is referred to as the maintenance period.

[0142] Numerous variable-dose regimens for treating secondary hemophagocytic lymphohistiocytosis (HLH) in adult humans include 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 as a single dose. Alternatively, the first dose may be administered more than once over the induction period.

[0144] The second dose is administered over one or more treatment periods. The second dose is administered over the first treatment period. During the first treatment period, the second dose is administered every three days after the first dose. The first treatment period lasts about one week, two weeks, three weeks, four weeks, five weeks, six weeks, or longer. The first treatment period is preferably two weeks. If necessary, the second dose is administered over a second treatment period following the completion of the first treatment period. During the second treatment period, the second dose is administered twice a week. The second treatment period lasts 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, or 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 may be 1.0, 3.0, or 6.0 mg per kg of body weight. The third dose is administered over the remainder of the first and second treatment periods. Alternatively, the third dose is administered over a third treatment period. In some embodiments, the third dose is referred to as the maintenance dose, and the third treatment period is referred to as the maintenance period.

[0146] Numerous variable-dose regimens for treating conditions in human subjects include an induction dose or first dose and a treatment dose or second dose of anti-IFN-g monoclonal antibody. Subjects are adult or pediatric subjects. Conditions are associated with elevated IFN-g levels. Conditions include parenchymal organ transplant failure or acute bone marrow graft rejection, graft-versus-host disease and other graft rejection, paraneoplastic cerebellar degeneration, hemorrhagic fever, sarcoidosis, and adult-onset Still's disease. In other embodiments, the drug regimen is administered to subjects after CAR-T cell therapy.

[0147] The first dose is 1.0 to 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 to 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 as a single dose. Alternatively, the first dose is administered more than once over the induction period.

[0149] The second dose is administered over one or more treatment periods. The second dose is administered over the first treatment period. During the first treatment period, the second dose is administered every three days after the first dose. The first treatment period lasts about one week, two weeks, three weeks, four weeks, five weeks, six weeks, or longer. The first treatment period is preferably two weeks. If necessary, the second dose is administered over a second treatment period following the completion of the first treatment period. During the second treatment period, the second dose is administered twice a week. The second treatment period lasts 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, or 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 may be 1.0, 3.0, 6.0, or 10.0 mg per kg of body weight. The third dose is administered over the remainder of the first and second treatment periods. Alternatively, the third dose is administered over a third treatment period. In some embodiments, the third dose is referred to as the maintenance dose, and the third treatment period is referred to as the maintenance period.

[0151] In the various variable dosing regimens according to the present invention, the first and second periods for the second dose can be adjusted from time to time, for example, based on the subject's health condition. 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 several variable dosing regimens according to the present invention, the dose is administered over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours. Administration over 1 hour is preferred. The duration of dose infusion is based on several factors known in the art, such as age, weight, physical condition, or adverse reactions to the drug. The duration of dose infusion is generally a rapid infusion rate tolerable by the subject (i.e., without causing adverse reactions).

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

[0154] In some cases, subjects are administered dexamethasone immediately before antibody administration. The dexamethasone dose is at least 10 mg / m². 2 It is administered at a dose of 5 mg / m². Alternatively, dexamethasone is administered at least 5 mg / m². 2 It is administered in the following dosage.

[0155] In some embodiments, the subjects have not previously received treatment for HLH. Pharmaceutical composition

[0156] The antibodies or soluble chimeric polypeptides of the present invention (also referred to herein as “active compounds”), as well as their derivatives, fragments, analogs, and homologs, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions generally comprise an antibody or soluble chimeric polypeptide and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” includes any solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic agents, and absorption retarders suitable for pharmaceutical administration. Suitable carriers are described in Remington’s standard reference text in the art. This is described in the latest edition of Pharmaceutical Sciences, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, physiological saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as liposomes and non-volatile oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active compound, their use in the composition is intended. Supplementary active compounds may also be incorporated into the composition.

[0157] The pharmaceutical compositions of the present invention are formulated to suit their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. 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 oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial 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 acetic acid, citrate, or phosphoric acid; and agents for adjusting tonicity, such as sodium chloride or dextrose. pH can be adjusted using an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be sealed in glass or plastic ampoules, disposable syringes, or multi-dose vials.

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

[0159] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (if water-soluble) or dispersants and sterile powders for the immediate preparation of sterile injection 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 fluid enough to allow for easy syringe passage. The composition must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Reasonable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, such as sugars, polyhydric alcohols such as mannitol and sorbitol, and sodium chloride in the composition. By including absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition, sustained absorption of the injectable composition can be achieved.

[0160] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound in a suitable solvent, along with one or a combination of the components listed above as needed, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary components from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preparation method is vacuum drying and freeze-drying, where the powder of the active component and any additional desired components is obtained from a pre-sterilized filtered solution.

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

[0162] The pH of the injectable pharmaceutical composition is between approximately 5.8 and 6.2. Preferably, the pH of the injectable pharmaceutical composition is 6.0.

[0163] In some embodiments, an anti-IFN-γ antibody such as NI-0501 is formulated as follows (per 1 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 unit doses. These unit doses are packaged in containers. These containers may be glass or plastic. They may be syringes, vials, infusion bottles, ampoules, or carpoules. Containers may hold volumes of 1 to 25 ml. For example, containers may hold volumes of 2, 5, 10, or 20 ml.

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

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

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

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

[0169] Oral compositions generally contain an inert diluent or edible carrier. These can be encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, lozenges, 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 applied orally, swirled in the mouth, and then coughed up or swallowed. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following ingredients or compounds of similar properties: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose, disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; lubricants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavorings such as peppermint, methyl salicylate, or orange flavoring.

[0170] For administration by inhalation, the compound is delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, such as a gas like carbon dioxide, or from a nebulizer.

[0171] Systemic administration may be by mucosal or transdermal means. For mucosal or transdermal administration, a suitable penetrating agent is used in the formulation for the barrier to permeation. Such penetrating agents are generally known in the art and include, for example, surfactants, bile salts, and fusidic acid derivatives for mucosal administration. Mucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, plasters, gels, or creams as is generally known in the art.

[0172] The compound can also be prepared in the form of suppositories (e.g., using conventional suppository bases such as cocoa butter and other glycerides) or retained enemas for rectal delivery.

[0173] In one embodiment, the active compound is prepared using a sustained-release formulation that includes a carrier, such as an implant and a microcapsule encapsulation delivery system, to protect the compound from rapid elimination from the body. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyacid anhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. The materials can also be commercially obtained from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (containing liposomes targeted to infected cells having 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, as described, for example, in U.S. Patent No. 4,522,811.

[0174] Oral or parenteral compositions are particularly advantageous to be formulated into unit dosage forms for ease of administration and uniformity of dosage. A unit dosage form, as used herein, refers to a physically distinct unit suitable as a unit dose to a subject to be treated, each unit containing a predetermined quantity of the active compound calculated to produce the desired therapeutic effect in conjunction with the required pharmaceutical carrier. The details of the unit dosage forms of the present invention are defined and directly depend upon the unique properties of the active compound, the specific therapeutic effect to be achieved, and the technically specific limitations of the formulation of such active compounds for treating an individual.

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

[0176] The levels of CXCL9 and other biomarkers are detected using one of a variety of standard detection techniques. Detectors can be used to detect the presence of a given target (or its protein fragment) in a sample. In some embodiments, the detector contains a detectable label. In some embodiments, the detector is an antibody (or a fragment thereof) or a probe. In some embodiments, the drug or probe is labeled. The term “labeling” is intended to encompass, with respect to probes or antibodies, 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 directly labeled reagent. Examples of indirect labeling include the detection of a primary antibody using a fluorescently labeled secondary antibody and the biotin-end labeling of a DNA probe so that it can be detected with fluorescently labeled streptavidin.

[0177] The term “biological sample” includes tissues, cells, and bodily fluids isolated from a subject, as well as tissues, cells, and fluids present within the subject. Therefore, the use of the term “biological sample” includes blood, and fractions or components of blood, including serum, plasma, or lymph. Bodily fluids may be fluids 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, cerebrospinal fluid, pleural fluid, respiratory fluid, intestinal fluid, and genitourinary fluid, saliva, organ system fluids, ascites, tumor cystic fluid, amniotic fluid, and combinations thereof. Biological samples also include all experimentally isolated fractions of the aforementioned fluids. Biological samples also include solutions or mixtures containing homogenized solid materials, such as feces, tissues, and biopsy samples. The detection methods of the present invention can be used to detect analytes such as mRNA, proteins, or genomic DNA in biological samples in vitro and 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 blotting, immunoprecipitation, and immunofluorescence. In vitro techniques for detecting analyte genomic DNA include Southern hybridization. Procedures for performing immunoassays are described, for example, in "ELISA: Theory and Practice: Methods in Molecular Biology," Vol. 42, JR 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.Furthermore, an in vivo technique for detecting analyte proteins involves introducing labeled anti-analyte protein antibodies into the subject. For example, the antibody may 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 this invention shall have meanings generally understood by those skilled in the art. Furthermore, unless the context requires otherwise, singular terms shall include plurals and plural terms shall include singulars. In general, the nomenclature and techniques used in connection with cell and tissue cultures, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are 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 carried out according to the manufacturer's specifications, as commonly practiced in the art, or as described herein. The aforementioned techniques and procedures are generally carried out according to conventional methods well known in the art, and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). The nomenclature, testing procedures, and techniques used in relation to analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are well known and commonly used in the art. Standard techniques are used in chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, as well as in patient care.

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

[0180] The term “multiple variable doses” encompasses various doses of anti-IFN-gamma antibody administered to a subject for therapeutic treatment. A “multiple variable dose regimen” or “multiple variable dose treatment” describes a treatment schedule based on the administration of varying amounts of anti-IFN-gamma antibody at different points throughout the course of treatment. In one embodiment, the present invention describes a multiple variable dose treatment method comprising an induction phase and a treatment phase, in which 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 present invention describes a multiple variable dose treatment method comprising an induction phase, a first treatment phase, and a second treatment phase, in which 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. Preferably, the dose is the same in the first and second treatment phases.

[0181] The terms “induction phase” or “loading phase,” as used herein, refer to a period of treatment involving the administration of an IFN-gamma antibody to a subject in order 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 for which IFN-gamma is harmful.

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

[0183] The terms “induction dose” and “loading dose” are used interchangeably herein and refer to the first dose of anti-IFN-gamma antibody, which is greater or less than the maintenance or treatment dose. The induction dose may be a single dose or a set of doses. The induction dose is often used to bring the drug in the body to a steady-state level and can also be used to rapidly achieve maintenance drug levels. Following the induction dose, a smaller or larger dose of anti-IFN-gamma antibody, i.e., the treatment dose, is administered. The induction dose is administered during the induction phase of treatment.

[0184] The terms “treatment phase” or “maintenance phase,” as used herein, refer to a period of treatment involving the administration of an anti-IFN-gamma antibody to a subject in order to maintain a desired therapeutic effect. The treatment phase follows the induction phase and is therefore initiated once a threshold level has been achieved. There may be more than one treatment phase, which are shortened or extended to maintain a particular threshold or to achieve a desired clinical response. Preferably, during the treatment phase, the substance is administered every three days after the induction dose (e.g., the initial dose). Alternatively, the substance is administered once or twice a week.

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

[0186] A “dosage regimen” or “medication regimen” includes a treatment regimen based on a determined set of doses.

[0187] As used herein, the term "dosing" refers to administering a substance (e.g., an anti-IFN-gamma antibody) to achieve a therapeutic objective (e.g., treatment of an IFN-gamma-related disorder).

[0188] The terms “once-two-weeks dosing regimen,” “once-two-weeks dosing,” and “once-two-weeks 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 IFN-gamma-related disorder). A once-two-weeks dosing regimen does not necessarily include a once-weekly dosing regimen.

[0189] The term "combination," as in the phrase "combination of a first drug and a second drug," includes the simultaneous administration of a first drug and a second drug, which may, for example, be dissolved or mixed in the same pharmaceutically acceptable carrier, or the second drug may be administered following the administration of the first drug, or the first drug may be administered following the administration of the second drug. Accordingly, the present invention includes methods for combination therapeutic treatment and combination pharmaceutical compositions.

[0190] The term "simultaneous" as in the phrase "simultaneous therapeutic treatment" includes administering a drug in the presence of a second drug. Simultaneous therapeutic treatment methods include methods of simultaneously administering a first, second, third, or additional drug. Simultaneous therapeutic treatment methods also include methods of administering a first or additional drug in the presence of a second or additional drug, where the second or additional drug may have been administered previously, for example. Simultaneous therapeutic treatment methods may be performed stepwise by different practitioners. For example, one practitioner may administer a first drug to a subject, and a second practitioner may administer a second drug to the subject, and the administration steps may be performed simultaneously or nearly simultaneously, or at different times, as long as the first drug (and additional drug) has been administered in the presence of the second drug (and additional drug). The practitioner and the subject may be the same entity (e.g., a human).

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

[0192] The compositions and methods presented herein, NI-0501, are formulated as a sterile concentrate for injection (per 1 mL). In some embodiments, NI-0501 is formulated as follows: 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 between 5.8 and 6.2. In some embodiments, NI-0501 is formulated as follows: 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.

[0193] The compositions and methods presented herein involve administering NI-0501 to subjects in need to treat, prevent, and / or delay the onset or progression of symptoms associated with HLH. In some embodiments, NI-0501 is administered to subjects in need by IV infusion at an initial dose of 1 mg / kg over 1 hour. In certain patient populations, e.g., low-weight and / or very young patients, the IV infusion may last longer than 1 hour, e.g., at least 90 minutes, at least 2 hours, or at least 3 hours or longer.

[0194] In some embodiments, NI-0501 is administered to subjects requiring it in an initial dose of 1 mg / kg over 1 hour via at least one additional IV infusion after the initial IV infusion. In some embodiments, at least one additional IV infusion is a higher dose than the initial dose of 1 mg / kg. In some embodiments, the dose of at least one additional IV infusion is 3 mg / kg. In some embodiments, at least one additional IV infusion is administered at least 3 days 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 days, 6 days, 9 days, 12 days, and 15 days after the initial IV infusion. In some embodiments, at least one additional IV infusion is administered 3 days, 6 days, 9 days, 12 days, and 15 days after the initial IV infusion.

[0195] In some embodiments, NI-0501 is administered to subjects requiring it at an initial dose of 1 mg / kg over 1 hour in a series of additional IV infusions following the initial IV infusion, where the series of additional IV infusions includes at least one series of bi-weekly IV infusions. In some embodiments, at least one series of bi-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 bi-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, 4 weeks, 5 weeks, 6 weeks, 7 weeks, and 8 weeks after the initial IV infusion.

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

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

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

[0199] In some embodiments, infusions are administered every three days after the initial dose until 15 days after the initial dose. In some embodiments, infusions are administered every three days after the initial dose 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 infusion dose is increased to 3 mg / kg at any point after the initial dose. In some embodiments, after at least two infusions at 3 mg / kg, the dose of NI-0501 is increased to 6 mg / kg over up to four infusions.

[0200] The compositions and methods presented herein involve administering NI-0501 to subjects in need to treat, prevent, and / or delay the onset or progression of symptoms associated with HLH. In some embodiments, NI-0501 is administered to subjects in need by IV infusion at an initial dose of 1 mg / kg over 1 hour. In some embodiments, infusions are administered every 3 days after the initial dose until 15 days after the initial dose. In some embodiments, infusions are administered every 3 days after the initial dose 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 dose of the infusion is increased to 3 mg / kg at any point after the initial dose. In some embodiments, after at least two infusions at 3 mg / kg, the dose of NI-0501 is increased to 6 mg / kg over up to four infusions.

[0201] The compositions and methods presented herein involve administering NI-0501 to a subject in need to treat, prevent, and / or delay the onset or progression of symptoms associated with HLH. In some embodiments, NI-0501 is administered to a subject in need by IV infusion at a dose greater than 6 mg / kg. In some embodiments, NI-0501 is administered to a subject in need by IV infusion at a second dose greater than 6 mg / kg after the initial 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 over a week. In some embodiments, the second dose is 10 mg / kg and is repeated daily over a two-week period. In some embodiments, the second dose is 10 mg / kg and is repeated daily over a longer period than two weeks.

[0202] In some embodiments, NI-0501 is administered to subjects in need to treat, prevent, and / or delay the onset or progression of symptoms associated with secondary HLH. In some embodiments, NI-0501 is administered to subjects in need to treat, prevent, and / or delay the onset or progression of symptoms associated with secondary HLH in the background of sJIA. In some embodiments, NI-0501 is administered to subjects in need 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 3 mg / kg every 3 days for at least 4 weeks (i.e., up to SD27).

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

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

[0205] In the compositions and methods presented herein, NI-0501 is administered to a subject that needs it to treat, prevent, and / or delay the onset or progression of, or mitigate, symptoms associated with HLH, where the subject is being administered dexamethasone as background. In some embodiments, the subject is a naïve patient with respect to treatment (i.e., has not previously received treatment for HLH) and is administered dexamethasone at a dose of at least 10 mg / m 2 . In some embodiments, the subject is receiving NI-0501 as a second-line HLH treatment and is administered dexamethasone at a dose in the range of 10 mg / m 2 to 5 mg / m 2 . In some embodiments, the subject is receiving NI-0501 as a second-line HLH treatment and is administered dexamethasone at a dose of at least 5 mg / m 2 . In some embodiments, the subject is receiving NI-0501 as a second-line HLH treatment and is administered dexamethasone at a dose less than 5 mg / m 2 .

[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 therapeutic agents, anti-inflammatory agents, and / or immunosuppressants, in non-limiting examples. 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 administered simultaneously. Alternatively, NI-0501 and the additional agent are kept separate, for example, each is formulated into a separate therapeutic composition and administered simultaneously, or administered at different times during the treatment regimen. For example, NI-0501 is administered before the administration of the additional agent, NI-0501 is administered after the administration of the additional agent, or NI-0501 and the additional agent are administered alternately. As described herein, NI-0501 and additional drugs are administered in single or multiple doses.

[0207] In some embodiments, NI-0501 and additional drugs(s) are administered simultaneously. For example, NI-0501 and additional drugs(s) may be formulated into a single composition, or administered as two or more separate compositions. In some embodiments, NI-0501 and additional drugs(s) are administered sequentially, or NI-0501 and additional drugs are administered at different times during the 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 before administration of NI-0501. In some embodiments, the additional agent includes at least etoposide. In some embodiments, the subject receives etoposide before administration of NI-0501.

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

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

[0211] In some embodiments, one or more additional agents include analgesia, transfusion 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 symptoms associated with transplant failure, graft rejection, and / or inflammatory disorders associated with graft rejection, or in mitigating them. The compositions and methods presented herein are useful in treating, inhibiting, delaying the progression of, or instead improving the symptoms of graft-versus-host disease (GvHD) in subjects who have received or are receiving a transplant or a series of transplants containing biomaterials. The compositions and methods presented herein are useful in extending the survival of transplanted biomaterials.

[0213] The compositions and methods presented herein are useful, for example, in the transplantation of any biological material including, for example, cells, tissues, bone marrow, and / or organs, or at least a portion of organs, including, in non-limiting examples, the heart, kidneys, pancreas, liver, and / or intestines. In some embodiments, the biological material to be transplanted 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 biological material to be transplanted is one or more hepatocytes or derived therefrom.

[0214] The compositions and methods presented herein involve administering NI-0501 to subjects in need to treat, prevent, and / or delay the onset or progression of symptoms associated with graft failure, graft rejection, and / or inflammatory disorders associated with graft rejection. In some embodiments, graft rejection, also referred to herein as graft failure, is acute. In some embodiments, graft rejection is hyperacute.

[0215] In the compositions and methods presented herein, NI-0501 is formulated as a sterile concentrate for injection (per 1 mL). In some embodiments, NI-0501 is formulated as follows: 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 between 5.8 and 6.2. In some embodiments, NI-0501 is formulated as follows: 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.

[0216] In some embodiments, graft rejection is chronic. In some embodiments, NI-0501 is administered to the subject requiring it by IV infusion at an initial dose of 1 mg / kg over 1 hour. In certain patient populations, e.g., low-weight and / or very young patient populations, the IV infusion may last longer than 1 hour, e.g., at least 90 minutes, at least 2 hours, or at least 3 hours or longer.

[0217] In some embodiments, NI-0501 is administered to subjects requiring it in an initial dose of 1 mg / kg over 1 hour via at least one additional IV infusion after the initial IV infusion. In some embodiments, at least one additional IV infusion is a higher dose than the initial dose of 1 mg / kg. In some embodiments, the dose of at least one additional IV infusion is 3 mg / kg. In some embodiments, at least one additional IV infusion is administered at least 3 days 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 days, 6 days, 9 days, 12 days, and 15 days after the initial IV infusion. In some embodiments, at least one additional IV infusion is administered 3 days, 6 days, 9 days, 12 days, and 15 days after the initial IV infusion.

[0218] In some embodiments, NI-0501 is administered to subjects requiring it at an initial dose of 1 mg / kg over 1 hour in a series of additional IV infusions following the initial IV infusion, where the series of additional IV infusions includes at least one series of bi-weekly IV infusions. In some embodiments, at least one series of bi-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 bi-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, 4 weeks, 5 weeks, 6 weeks, 7 weeks, and 8 weeks after the initial IV infusion.

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

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

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

[0222] In some embodiments, infusions are administered every three days after the initial dose until 15 days after the initial dose. In some embodiments, infusions are administered every three days after the initial dose 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 infusion dose is increased to 3 mg / kg at any point after the initial dose. In some embodiments, after at least two infusions at 3 mg / kg, the dose of NI-0501 is increased to 6 mg / kg over up to four infusions.

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

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

[0225] In some patients with or suspected of having 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. Therefore, IFNγ activity is blocked or inhibited by the use of anti-IFNγ antagonists that interfere with, inhibit, reduce, or instead antagonize IFNγ signaling, such as neutralizing anti-IFNγ antibodies or other polypeptide-based therapeutics, peptide-based therapeutics, small molecule inhibitors, nucleic acid-based therapeutics and their derivatives. Accordingly, compositions and methods are useful in treating, delaying the progression of, or instead improving the symptoms of disorders that depend on, are driven by, are associated with, or are instead affected by, abnormal IFNγ expression and / or activity, such as elevated levels, abnormal 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 their derivatives to patients exhibiting elevated levels of CXCL9 and / or other biomarker expression. Patients who are likely candidates for treatment with an anti-IFNγ antagonist, such as a neutralizing anti-IFNγ antibody described herein, are identified by detecting levels 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 an anti-IFNγ antagonist, which may include any of the neutralizing anti-IFNγ antibodies or other polypeptide-based therapies, peptide-based therapies, small molecule inhibitors, nucleic acid-based therapies and their derivatives 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 derived from a patient who does not have or is suspected of having primary or secondary HLH or an 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 elevation.

[0228] The detected levels of CXCL9 alone or in combination with one or more other IFNγ-related biomarkers are useful for accurately identifying or alternatively stratifying a patient population. In some embodiments, the detected levels are used to determine the dosage of anti-IFNγ antagonist to be administered to a given patient. In some embodiments, the detected levels are used to categorize or alternatively stratify a patient population. For example, patients can be classified as having "severe" or high-grade MAS, or conversely, not having severe or low-grade MAS, based on the detected level of CXCL9.

[0229] The sample is, for example, blood or a blood component, such as serum, plasma. In some embodiments, the sample is another body fluid, such as, by way of non-limiting example, urine, synovial fluid, bronchoalveolar fluid, cerebrospinal fluid, bronchoalveolar lavage (BAL), and / or saliva. In some embodiments, the biological sample is CSF. In some embodiments, the biological sample is CSF from an HLH patient.

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

[0231] Disorders for which the compositions and methods of the present invention are useful include any disorder having an abnormality, such as an increase, in IFNγ expression and / or activity, particularly HLH, MAS, and / or sJIA, including secondary HLH.

[0232] By way of non-limiting example, the methods and compositions presented 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 abnormal IFNγ activity and / or expression.

[0233] If 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 an immunologically active (e.g., antigen-binding) fragment thereof. Suitable neutralizing anti-IFNγ antibodies include any of the anti-IFNγ antibodies described herein.

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

[0236] In some embodiments, the anti-IFNγ antibody or its immunologically active fragment is a heavy chain comprising a combination of VH CDR1, VH CDR2, and VH CDR3 sequences, wherein the combination is one of the three heavy chain CDR sequences shown in row 1A (VH CDR1, VH It contains a heavy chain, which is a combination of CDR2 and VH CDR3.

[0237] In some embodiments, the anti-IFNγ antibody or its immunologically active fragment is a light chain comprising a combination of VL CDR1, VL CDR2, and VL CDR3 sequences, wherein the combination is one of the three light chain CDR sequences shown in row 1B (VL CDR1, VL CDR3). It includes a light chain, which is a combination of CDR2 and VL CDR3.

[0238] In some embodiments, the anti-IFNγ antibody or its immunologically active fragment is a heavy chain comprising a combination of VH CDR1, VH CDR2, and VH CDR3 sequences, wherein the combination is one of the three heavy chain CDR sequences shown in row 1A (VH CDR1, VH The heavy chain is a combination of CDR2 and VH CDR3, and the light chain includes a combination of VL CDR1, VL CDR2, and VL CDR3 sequences, wherein the combination is a combination of the three light chain CDR sequences (VL CDR1, VL CDR2, VL CDR3) shown in row 1B.

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

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

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

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

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

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

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

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

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

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

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

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

[0251] In some embodiments, the anti-IFNγ antibody or an immunologically active fragment thereof comprises an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or greater 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 its immunologically active fragment contains an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99%, or greater 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 its immunologically active fragment includes an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99%, or greater 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 greater 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 its immunologically active fragment contains 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 its immunologically active fragment contains 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 its immunologically active fragment includes 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, an anti-IFNγ antibody or an immunologically active fragment thereof is administered in a therapeutically effective dose. The therapeutically effective dose of the antibody of the present invention generally refers to the amount necessary to achieve a therapeutic objective. This therapeutic objective may, in certain cases, be the binding interaction between the antibody and its target antigen that interferes with the function of the target. A general range for therapeutically effective dosing of the antibody or antibody fragment of the present invention may, in non-limiting examples, range from about 0.1 mg per kg of body weight to about 50 mg per kg of body weight. A typical dosing frequency may range, for example, from twice a day to once a week.

[0257] In some embodiments, an anti-IFNγ antibody or an immunologically active fragment thereof is administered in an initial dose, i.e., a loading dose, ranging from about 0.5 mg / kg to about 2 mg / kg, for example, 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, an anti-IFNγ antibody or an immunologically active fragment thereof is administered in an initial dose of about 1.0 mg / kg.

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

[0259] In some embodiments, one or more maintenance doses comprise at least two or more doses, each maintenance dose being the same. 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, one or more maintenance doses comprise at least two or more doses, and each maintenance dose is not the same. In some embodiments, two or more maintenance doses are administered in escalating doses. In some embodiments, two or more maintenance doses are administered in tapering doses.

[0261] In some embodiments, one or more maintenance doses comprise at least two or more doses, each maintenance dose administered at cyclical time intervals. In some embodiments, two or more doses are administered at escalating time intervals. In some embodiments, two or more doses are administered at tapering time intervals.

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

[0263] The pharmaceutical compositions according to the present invention may include the anti-IFNγ antibody and carrier of the present invention. These pharmaceutical compositions may be included in kits, such as diagnostic kits.

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

[0265] The present invention will be further illustrated in the following embodiments, and the scope of the present invention as described in the claims will not be limited thereto. [Examples]

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

[0267] In 20 patients with sJIA who had MAS at the time of sampling (n=54), circulating levels of IFNγ, CXCL9, CXCL10, CXCL11, and IL-6 were measured using the Luminex multiplex assay. The association between these circulating levels and disease activity parameters was evaluated along with the correlation between IFNγ levels and CXCL9, CXCL10, and CXCL11 levels.

[0268] In active MAS, IFNγ levels and levels of three IFNγ-related chemokines (CXCL9, CXCL10, and CXCL11) were significantly elevated compared to active sJIA without MAS at the time of sampling (all p-values ​​< 0.005). In active MAS, laboratory parameters of disease severity (ferritin, neutrophils, platelets, alanine aminotransferase, and lactate dehydrogenase) were significantly correlated with IFNγ and CXCL9, and less significantly with CXCL10 and CXCL11, but showed no correlation with IL-6 levels. As shown in Table 7 below, there was no significant correlation between laboratory parameters and cytokine levels in patients with active sJIA without MAS. In active MAS, IFNγ levels were significantly correlated with CXCL9 levels (r=0.69; r 2 (r=0.47; p=0.001), and correlated less with the level of CXCL10 (r=0.53; r 2 (r=-0.04; p=0.886), there was no correlation with the level of CXCL11 (r=-0.04; p=0.886). Table 7. Correlation between disease activity laboratory parameters and IFNγ, CXCL9, CXCL10, CXCL11, and IL-6 in patients with MAS and patients with active sJIA. [Table 7]

[0269] High levels of IFNγ and CXCL9 in patients with active MAS are significantly correlated with laboratory parameters of disease severity. IFNγ and CXCL9 are closely correlated in patients with active MAS. 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, February 2011; Vol. 89 (No. 2): pp. 207-2015), these findings demonstrate that CXCL9 is a biomarker for IFNγ production in MAS. (Example 2) Correlation between CXCL9 levels and IFNγ 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 treated with NI-0501 antibody, and from patients who received NI-0501 antibody on a humanitarian basis.

[0271] As shown in Figure 1, serum levels of CXCL9 and IFNγ were measured in samples from six patients with primary HLH and three patients on humanitarian use, respectively, using Luminex and Meso Scale Discovery (MSD) techniques. Correlation was performed between CXCL9 concentration and total IFNγ concentration. Statistical analysis was performed, and p-values ​​were obtained using Spearman's test.

[0272] As shown in Figure 2, pre-administration serum levels of CXCL9 and IFNγ were measured using Luminex and MSD techniques in samples obtained from six patients with primary HLH and three patients on humanitarian use. Correlation was performed between CXCL9 concentration and total IFNγ concentration. Statistical analysis was performed, and p-values ​​were obtained using Spearman's test. (Example 3) Correlation between CXCL9 levels and IFNγ levels in patients with secondary hemophagocytic lymphohistiocytosis (HLH)

[0273] The studies presented herein are based on observational studies in secondary HLH patients administered NI-0501 antibody, and on patients who received NI-0501 antibody on a humanitarian basis.

[0274] Specifically, these are patients with systemic juvenile idiopathic arthritis (sJIA) that has developed macrophage activation syndrome (MAS, a form of secondary HLH). In 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 Figures 3A and 3B, serum levels of CXCL9 and IFNγ were measured by multiplex assay using Luminex technology from samples obtained 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. Statistical analysis was conducted, and p-values ​​were obtained using Spearman's 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 in patients with MAS secondary to sJIA and patients with active sJIA at the time of sampling. Correlations were performed between IFNγ or CXCL9 levels and ferritin, platelet count, neutrophil count, or ALT (alanine aminotransferase). Statistical analysis was performed, and p-values ​​were obtained using Spearman's test. (Example 4) Correlation between CXCL9 levels and IFNγ levels in patients with severe hemophagocytic lymphohistiocytosis (HLH)

[0277] The studies presented herein are from patients who received the NI-0501 antibody on a humanitarian basis. These patients presented with symptoms of NLRC4-associated disease and severe hemophagocytic lymphohistiocytosis (HLH). It has recently been reported that mutations in the NLRC4 gene cause relapsing macrophage activation syndrome and increased production of IL-18, which is known to induce IFNγ.

[0278] Patients in this study had the following characteristics: they presented at 20 days of age with fever, rash, marked hepatosplenomegaly, pancytopenia, hypofibrinogenemia, hypertriglyceridemia, and markedly elevated ferritin and sCD25. This was followed by multiple organ failure requiring admission to the ICU. The HLH diagnosis was based on 6 of the 8 HLH-2004 criteria. Genes causing primary HLH (PRF1, UNC13D, STXBP2, STX11, RAB27A, XIAP) and functional tests (perforin expression, degranulation, and cytotoxicity) were negative. High-dose IV glucocorticoids and IV cyclosporine A accompanied by progressive improvement in general condition and laboratory abnormalities. HLH reactivation, rapid deterioration of general condition, and new ICU admission were induced by infection (Candida Albicans and Klebsiella Pneumoniae sepsis). Treatment with etoposide and / or ATG was not considered due to the presence of active infection in subjects who were already immunocompromised.

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

[0280] Dexamethasone (13.6 mg / m²) 2 Treatment with NI-0501 on a humane basis was initiated against a background of oral cyclosporine A (6 mg / kg) and iv-cyclosporine A. NI-0501 was administered every 3 days according to pharmacokinetics, and then every 7 days thereafter. No infusion reactions were observed. NI-0501 was well tolerated. The clinical features and laboratory abnormalities of HLH gradually improved. The active, ongoing infection was rapidly cleared. After 5 months of treatment, the patient remained in excellent condition. The patient was given oral cyclosporine A (6 mg / kg) and prednisone (0.3 mg / kg, 0.9 mg / m³). 2I continued receiving a drug equivalent to dexamethasone. All HLH parameters normalized.

[0281] The subjects continued to exhibit episodes of unexplained inflammation. Analysis of NLRC4 revealed a novel missense mutation (T337N). Elevated serum IL-18 levels were demonstrated, confirming the association with the NLRC4 mutation. High levels of IFNγ complexed with NI-0501 demonstrated high IFNγ production. IFNγ was sufficiently neutralized, as indicated by the undetectable levels of IFNγ-inducible chemokines (Figure 5 and Table 8). Circulating levels of IL-18 remained persistently elevated.

[0282] Therefore, this study demonstrates that in patients with severe refractory HLH (caused by NLRC4 mutations), blocking IFNγ with NI-0501 is well-tolerated, without safety concerns, allows control of all HLH characteristics, enables rapid glucocorticoid tapering, and facilitates the resolution of ongoing active infections. (Example 5) Targeted treatment method for hemophagocytic lymphohistiocytosis (HLH) using NI-0501

[0283] The studies presented herein are from a pilot Phase 2 trial in children with primary HLH. Primary HLH (pHLH) is a rare immunomodulatory disorder that is invariably fatal if left untreated. pHLH is driven by pathological immune activation, leading to the development of fever, splenomegaly, cytopenia, and coagulation disorders, which can result in 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 from observational studies in patients with HLH, high IFNγ production is considered a critical factor driving disease development. Immunochemotherapy, primarily etoposide-based regimens, is currently the only pharmacological approach to control HLH and lead patients to curative allogeneic hematopoietic stem cell transplantation (alloHSCT). Despite recent attempts to further enhance treatment regimens, mortality and morbidity remain high, partly due 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 targeting method for controlling HLH.

[0285] Methods: An open-label phase 2 trial 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 every 3 days at an initial dose of 1 mg / kg, with dexamethasone 5-10 mg / m² as the initial background dose in each patient. 2 PK data and / or clinical response were used as a guide for possible dose increases. Treatment duration ranged from 4 to 8 weeks. The ability to transition to allo-HSCT, relevant HLH disease parameters, and 8-week survival were evaluated.

[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 conventional treatment received NI-0501 as a second-line treatment. One patient was treated with NI-0501 as a first-line treatment. Nine patients had known HLH gene defects (3 with FHL2, 2 with FHL3, 2 with GS-2, 1 with XLP1, and 1 with XLP2). The majority of patients were in the severe end-stage of the HLH spectrum, had impaired general condition, and had significant toxicity derived from previous HLH treatment. Of the 13 patients, 12 had elevated ferritin levels, 8 had elevated sCD25 levels, 10 had cytopenia, 8 had splenomegaly, and 9 had hypofibrinogenemia and hypertriglyceridemia. Hepatic 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), and a satisfactory response was achieved in 9 out of 13 patients. Six patients proceeded to HSCT. Two patients with good HLH control are scheduled to proceed to HSCT once a suitable donor is identified. One patient (whose disease control was achieved with first-line NI-0501) is not yet scheduled for HSCT, considering the absence of a causative HLH gene mutation. Eleven out of 13 patients were alive at 8 weeks. CNS signs and symptoms resolved in two evaluable patients. During the first four weeks of NI-0501 treatment, 50% of patients were able to reduce their dexamethasone dose by more than 50%.

[0288] The evaluation of biomarkers, particularly CXCL9, a chemokine known to be subtly induced by IFNγ, not only enabled the demonstration of complete IFNγ neutralization but also appears to be a novel parameter for diagnosing HLH, correlating with IFNγ production (Figures 7A and 7B).

[0289] NI-0501 was well-tolerated, and no safety concerns were identified. No infections for which IFNγ neutralization is known to be advantageous were reported, and no infections occurred in patients who had not received prior chemotherapy. At least one SAE was reported in seven patients, all of which were assessed by the DMC as not being related to NI-0501 administration. No unexpected events attributable to "off-target" effects of NI-0501 (e.g., myelotoxicity, hemodynamic effects) were observed.

[0290] Conclusion: Targeted neutralization of IFNγ with NI-0501 provides an innovative and potentially less toxic approach to managing HLH. The results of this trial demonstrate that NI-0501 is a safe and effective treatment option for patients with primary HLH who have poorly responded to or are intolerant to conventional treatments. Furthermore, treatment with NI-0501 was not associated with any of the typical short-term 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 is expected to achieve similar significant clinical utility. (Example 6) Elevated circulating levels of interferon-γ and interferon-inducible chemokines characterize patients with macrophage activation syndrome complicating systemic JIA.

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

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

[0293] As detailed below, circulating levels of IFNγ and IFN-inducible chemokines were significantly elevated during MAS, also referred to herein as active MAS, and sec-HLH. IFNγ and IFN-inducible chemokine levels were significantly higher in patients with MAS compared to patients with active sJIA without MAS. In the latter group, IFNγ and IFNγ-inducible chemokines were comparable to those in 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, were significantly correlated with IFNγ and CXCL9 levels. In a mouse model of MAS, serum ferritin levels were significantly correlated with CXCL9 mRNA levels in the liver and spleen.

[0294] Therefore, the following studies demonstrate that IFNγ plays a central role in MAS, based on its correlation with high levels of IFNγ and IFN-inducible chemokines, and with the severity of laboratory abnormalities, particularly CXCL9 and 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 at three Pediatric Rheumatology Centres: the Ospedale Pediatrico Bambino Gesu in Rome, the Istituto Giannina Gaslini in Genoa, and the Cincinnati Children's Hospital Medical Centre. Fifty-four patients with sJIA meeting the ILAR classification criteria for systemic arthritis (age at onset 7.9 years, interquartile range 4.6–13.6 years; 48% female) were examined (Petty, RE et al., International League of Associations for Rheumatology classification of juvenile idiopathic arthritis: revised 2nd edition, Edmonton, 2001, J Rheumatol, 2004, Vol. 31 (No. 2): pp. 390–392). For 20 SJIA patients, samples were collected during episodes of active, end-stage MAS diagnosed by physicians treating patients at each of the three centers. Inductive analysis showed that 17 of these 20 episodes (85%) met the newly proposed MAS classification criteria (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 (Supplement 1): O1). Samples were available from 28 patients with active SJIA without evidence of MAS. From 35 SJIA patients (both those with and without a history of MAS), 35 samples were available during clinically inactive periods of illness as defined by 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] Since IFNγ has been shown to be elevated in patients with sec-HLH (excluding rheumatic diseases), samples were also collected from 11 patients with sec-HLH (age at onset 8.6 years, interquartile range 4.1–12.9 years; 36% female) at Ospedale Pediatrico Bambino Gesu and used as positive controls. All sec-HLH patients met the 2004-HLH diagnostic guidelines (Henter, JI et al., HLH-2004: Diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer, 2007, Vol. 48 (No. 2): pp. 124–31), with 6 patients meeting five criteria and 5 patients meeting four criteria. It should be noted that sCD25 levels in U / ml units were unavailable because this study is not routinely conducted at the facilities where these patients were mobilized. Primary HLH was ruled out based on the absence of a 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 contributed one sample obtained during active disease.

[0297] Regarding diagnosis, clinical and laboratory characteristics of all patients at the time of sampling were collected by the principal investigator at each center in a centralized web database. Of the 20 MAS patients sampled during active disease, 6 had received no treatment at the time of sampling, and the remaining 14 patients had already received one of the MAS-specific treatments, including glucocorticoid pulse, cyclosporine A, anakinra, or cyclophosphamide. Of the 11 patients with active disease and sec-HLH, 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 this study. Written informed consent was obtained from all participants.

[0298] Cytokine quantification. Levels of IL-6, IL-1β, IFNγ, CXCL9, CXCL10, and CXCL11 were analyzed using 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 standard material, blank, and quality control samples were added in two strips to a Milliplex MAP 96-well plate, followed by the addition of 25 μl of serum matrix. 25 μl of assay buffer was added to each sample well, followed by the addition of 25 μl of sample. Samples were added in two or three strips depending on the available sample volume. Plates were measured using a Luminex 200® system (Luminex Corp.). Raw data was acquired using x PONENT software version 3.1 (Luminex Corp.), and the data was analyzed using Milliplex Analyst software version 3.5.5.0 (Millipore). Subsequently, the raw data obtained from Milliplex Analyst software was further analyzed using 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 MAS-like syndrome induced by administration of TLR ligands, have been previously described (Strippoli, 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, Vol. 64 (No. 5): pp. 1680-1688). Mice were maintained under conditions free from specific pathogens and handled in accordance with national police procedures. The experimental protocol was approved by the local ethics committee. All experiments were performed on mice between 10 and 14 weeks of age. Mice were intraperitoneally administered a single dose of lipopolysaccharide (LPS, E. coli serotype 055:B5; Sigma-Aldrich) at a dose of 5 μg per g of body weight. Mice were sacrificed 30 hours later. Total RNA was extracted from spleen and liver tissue 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) along with mouse Cxcl9 and Cxcl10 gene expression assays (Applied Biosystems). Gene expression data were normalized using mouse HPrt (Applied Biosystems). The data were divided into two sets. -Δct It is expressed as an arbitrary unit (AU) determined by the method. Serum ferritin concentrations were determined using a commercially available ELISA kit (ALPCO Diagnostics) according to the manufacturer's instructions.

[0300] Statistical analysis was performed using GraphPad Prism 5 software. Continuous variables (quantitative demographic data, clinical data, and laboratory data) were expressed as medians and interquartile ranges (IQRs) and compared using the Mann-Whitney U test. Two paired groups were compared using the Wilcoxon signed-rank test without assuming that the distribution of before-and-after differences followed a Gaussian distribution. The relationship with laboratory parameters was evaluated using Spearman rank correlation. A p-value < 0.05 was considered statistically significant.

[0301] Results: Elevated levels of IFNγ and IFNγ-inducible chemokines in patients with MAS. When patients with active sJIA without MAS at the time of sampling were compared with patients sampled during clinically inactive disease, as predicted (de Benedetti, F. et al., Correlation of serum interleukin-6 levels with joint involvement and thrombocytosis in systemic juvenile rheumatoid arthritis., Arthritis Rheum, 1991, Vol. 34 (No. 9): pp. 1158-1163), IL-6 levels were found to be significantly higher in patients with active sJIA compared to patients with clinically inactive disease (p<0.01). As reported in several previous studies on active sJIA, serum IL-1β levels were below the detection limit in the majority of patients, regardless of disease activity. It is noteworthy that there was no difference in IFNγ and the levels of the three IFNγ-inducible chemokines between patients with clinically active sJIA and patients with clinically inactive disease.

[0302] When patients with MAS at the time of sampling were compared to patients with active sJIA without MAS at the time of sampling, IL-1β and IL-6 levels were similar, suggesting that the levels of these two cytokines, known to play a central role in active sJIA, do not increase 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 to patients with active sJIA without MAS at the time of sampling. Levels of three IFNγ-related chemokines, CXCL9, CXCL10, and CXCL11, were also significantly higher in patients with active MAS compared to patients with active sJIA without MAS at the time of sampling. This difference was particularly evident with respect to CXCL9, where the median level was approximately 15 times higher in patients with MAS compared to patients with active sJIA without MAS.

[0303] In patients with sec-HLH, IFNγ levels and levels of three IFNγ-related chemokines were significantly elevated. The levels of IFNγ and IFNγ-related chemokines were almost indistinguishable from those in patients with MAS, and the difference was not statistically significant. Incidentally, levels of IFNγ and the three IFNγ-inducible chemokines in patients with active MAS and active sec-HLH were comparable to those in untreated and already 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 active sJIA without MAS. Consistent with the results obtained in the cross-sectional analysis, 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, and it was demonstrated that IFNγ and IFNγ-inducible chemokine levels returned to normal once the clinical symptoms of MAS subsided. For example, one patient in this study experienced three episodes of MAS, and serum samples were obtained between these episodes as well as during disease periods without MAS at the time of sampling. In this patient, elevated levels of IFNγ and three IFNγ-related chemokines were observed only during MAS episodes, further confirming the relationship between increased production of IFNγ and the three IFNγ-inducible chemokines and active MAS (Figures 9A-9B).

[0305] Levels of IFNγ and IFNγ-related chemokines correlate with laboratory abnormalities in MAS. Next, the correlation between levels of IFNγ and three IFNγ-inducible chemokines and laboratory parameters of MAS at the time of sampling was investigated. In patients with active sJIA without MAS, levels of IFNγ and the three IFNγ-inducible chemokines were not associated with laboratory parameters of MAS, with one exception: levels of CXCL9, CXCL10, and CXCL11 correlated with ALT levels ranging from 0.17 to 0.25. 2A weak correlation was found (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 s-JIA without MAS. No significant correlation was found between the laboratory features of MAS and IL-1 and IL-6 in patients with MAS at the time of sampling. In contrast, in patients with MAS at the time of sampling, levels of IFNγ and IFNγ-inducible chemokines were associated with elevated ferritin levels, neutrophil and platelet counts, as well as elevated LDH and ALT, all of which are generally abnormal in patients with MAS (Table 2). The correlation with laboratory abnormalities was particularly clear 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 mentioned above, these correlations were not present in patients with active s-JIA without MAS at the time of sampling. One patient in this group had remarkably high levels of IFNγ (336.2 pg / ml), CXCL9 (549400 pg / ml), and CXCL10 (35066 pg / ml). This patient had particularly severe MAS and was admitted to the intensive care unit with severe central nervous system complications. This finding further supports the hypothesis that there is a strong association between IFNγ and CXCL9 levels and disease severity. Taken together, these results suggest that increased production of IFNγ and IFNγ-related chemokines is a characteristic of active MAS that strongly correlates with the severity of laboratory abnormalities in MAS. [Table 2-1] [Table 2-2] [Table 3]

[0306] Correlation between IFNγ and IFNγ-inducible chemokine levels in patients with MAS. To further characterize the relationship between IFNγ and three IFNγ-inducible chemokines in patients with MAS, the correlation between IFNγ levels and the levels of each individual chemokine was evaluated. In particular, CXCL9 appears to be primarily and specifically induced by IFNγ, but CXCL10 and CXCL11 are also induced by type I interferon. Consistent with this, in patients with active MAS, the circulating level of IFNγ was significantly correlated with CXCL9 (r=0.693;r 2 (=0.48; p=0.001), 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-inducible chemokines correlate with disease activity in a mouse model of MAS. To further investigate the association between IFNγ-inducible chemokine production and MAS, the expression of these chemokines in target tissues (liver and spleen) was examined in a mouse model of MAS. In this model, the clinical and laboratory features of MAS were induced by mimicking acute infection using TLR4 agonist lipopolysaccharide (LPS) against a background of high levels of IL-6 in IL-6 transgenic mice (Strippoli et al., Arthritis Rheum, 2012). This method replicates 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 ferritin levels significantly correlated with CXCL9 expression levels in the spleen and liver, as well as CXCL10 expression levels in the liver, demonstrating 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 show a clear correlation between increased IFNγ production, increased CXCL9 expression, and to a lower degree CXCL10 expression, as well as laboratory abnormalities in MAS.

[0308] Studies in both patient and animal models of p-HLH have demonstrated the central role of IFNγ in disease pathogenesis. However, the role of IFNγ in sec-HLH, including MAS in the context of sJIA, remains unclear. This study definitively demonstrates the presence of high levels of IFNγ and IFNγ-inducible chemokines 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 the three IFNγ-related chemokines were comparable between patients with active sJIA and patients with clinically inactive disease. This result contradicts the pathogenic role of IFNγ in sJIA and is actually consistent with several findings by other authors. Three gene expression studies have failed to identify 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, Vol. 201 (No. 9): pp. 1479-1486. ​​After ex vivo stimulation of PBMCs, the number of IFNγ-producing cells in patients with active sJIA was similar to that of the control (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, Vol. 6 (No. 5): e20014. Consistently, neither patients with active nor inactive SJIA showed 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, Vol. 66 (No. 5): pp. 589-5898). CXCL9 and CXCL10 are almost undetectable in the synovial tissue of sJIA patients, but these chemokines can be found at high levels in the synovial tissue of patients with minor or polyarticular JIA, supporting the idea that IFNγ does not play a role in joint inflammation in sJIA (Sikora, KA et al., The limited role of interferon-gamma in systemic juvenile idiopathic Arthritis cannot be explained by cellular hyporesponsiveness., Arthritis Rheum, 2012, Vol. 64 (No. 11): pp. 3799-808. Recent data from mice have shown that immunostimulation of IFNγ knockout mice using Freund's complete adjuvant induces a systemic inflammatory syndrome including characteristics of sJIA, thus 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 Freund's complete adjuvant: regulation by interferon-gamma. Arthritis Rheumatol, 2014, Vol. 66 (No. 5): pp. 1340-1351.

[0309] In stark contrast, this study showed significantly higher levels of IFNγ and IFNγ-related chemokines in patients with active MAS at sampling compared to patients with active sJIA without MAS at sampling. This was also confirmed in individual patients using stepwise samples obtained both during active MAS and during active sJIA without MAS. Incidentally, this study found no significant elevation in IL-6 or IL-1β levels, nor any correlation with MAS laboratory parameters, in patients sampled during MAS, thus suggesting that these cytokines are not decisively involved in the pathogenic mechanism of sJIA (De Benedetti, F. et al., Randomized trial of tocilizumab in systemic juvenile idiopathic arthritis., N Engl J Med, 2012, vol. 367 (no. 25): pp. 2385-2395; Ruperto, N. et al., Two randomized trials of canakinumab In systemic juvenile idiopathic arthritis, N Engl J Med, 2012, Vol. 367 (No. 25): pp. 2396-406, it is suggested that this may not be significant in maintaining MAS. This finding of elevated IFNγ and IFNγ-related chemokines is consistent with several previous findings. Shimizu et al. reported that levels of neopterin, a catabolic product of guanosine triphosphate synthesized by human macrophages in response to IFNγ stimulation, were higher in patients with MAS during sJIA compared to 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, Vol. 49 (No. 9): pp. 1645-1653. 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 low levels of IFNγ and CXCL10 (Put et al., Rheumatology, 2015).

[0310] Interestingly, this study found not only significantly elevated levels of IFNγ and IFNγ-related chemokines, but also that these levels, particularly CXCL9 levels, correlated tightly with the laboratory characteristics of MAS, thus demonstrating a link to disease severity. This study further supports the association with disease severity in one patient with severe disease involving multiple organ failure and central nervous system complications accompanied by generalized seizures requiring prolonged intensive care unit hospitalization, where significantly elevated levels of IFNγ, CXCL9, and CXCL10 were found.

[0311] In patients with MAS, CXCL9 was found to have the strongest correlation with IFNγ levels among three IFNγ-inducible chemokines. This finding is consistent with the established concept that CXCL9 production is specifically and uniquely 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, CXCL3 ligands: redundant, collaborative and antagonistic functions., Immunol Cell Biol, 2011, vol. 89(2): pp. 207-2015). This suggests that CXCL9 levels may serve as a highly sensitive and specific biomarker for MAS activity. Indeed, using a mouse model of MAS that mimics MAS induction by infectious stimuli against a background of high IL-6 levels (Strippoli et al., Arthritis Rheum, 2012), this study also found that CXCL9 expression levels in the liver and spleen were significantly correlated with circulating ferritin levels. Regarding CXCL10 expression levels, this correlation was present only in the liver and not in the spleen. This is further supported by findings in patients with MAS, where CXCL9 levels strongly correlated with all MAS laboratory parameters. Taken together, these findings in humans and mice demonstrate that CXCL9 strongly correlates with the characteristics of MAS and IFNγ production, further supporting the hypothesis that excessive IFNγ production plays a major pathogenic role in MAS. These findings are consistent with immunohistochemical data generated by Put et al. using stepwise lymph node biopsy material from the same SJIA patients, obtained during active sJIA without MAS and during MAS.Put et al. reported that CXCL10 and indoleamine 2,3-dioxygenase, both IFNγ-inducible proteins, were detected at high levels by immunohistochemical analysis in tissues obtained during MAS, but were not detected in tissues obtained during active sJIA without MAS (Put et al., Rheumatology, 2015).

[0312] These results in MAS and sec-HLH, combined with available literature findings in patients with p-HLH, support the hypothesis that elevated IFNγ and IFNγ-related chemokines, particularly CXCL9, are a distinctive 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, Vol. 46 (No. 10): pp. 1140-116), suggesting that IFNγ overproduction can occur even in HLH situations induced solely by inflammasome dysregulation.

[0313] Data from both perforin knockout and Rab27a knockout mice, animal models of p-HLH, clearly demonstrate the pathogenic role of IFNγ. Similarly, recent data from a TLR9-induced model of HLH, a model of HLH secondary to infection, have shown a major role in increased IFNγ production (Behrens, EM et al., Repeated TLR9 stimulation results in macrophage activation syndrome-like disease in mice., J Clin Invest, 2011, vol. 121(no. 6): pp. 2264-77 and (Bautois et al., in progress)). Additional studies have recently demonstrated that treatment with anti-IFNγ antibodies in the above mouse models of MAS led to increased survival and reversal of the clinical and laboratory characteristics of MAS (Prencipe et al., in progress). Taken together, these results from animal studies and these findings provide a rationale for IFNγ neutralization as a therapeutic approach for MAS. (Example 7) Safety, tolerability, pharmacokinetics, and efficacy evaluation of multiple intravenous doses of anti-interferon-gamma anti-IFNγ 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) administrations of the 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 appropriate NI-0501 therapeutic dose regimens 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 to IFNγ derived from non-human primate species, including rhesus monkeys and cynomolgus monkeys, but not to IFNγ derived from dogs, cats, pigs, rabbits, rats, or mice. Toxicity and safety studies in cynomolgus monkeys have shown no off-target toxicity attributable to NI-0501 administration, that once-weekly administration of NI-0501 is well-tolerated, does not require antibiotic prophylaxis, and that no abnormal histopathological or behavioral findings were observed during these previous studies.

[0316] Since NI-0501 can bind to free IFNγ and IFNγR1-bound IFNγ, we conducted tests to investigate the potential of NI-0501 to mediate ADCC and CDC activity in the presence of the target. Absence of ADCC activity was demonstrated, and no induction of CDC activity was observed.

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

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

[0319] In 14 out of 20 subjects (70%), a total of 41 adverse events (AEs) were observed after the initiation of drug infusion, 10 of which were reported by 4 subjects who received placebo. 36 (87.8%) of the AEs were of mild severity, and 5 (12.2%) were of moderate severity. No severe or life-threatening AEs were reported. Of the 14 subjects who experienced AEs, 23 (56.1%) of 10 were reported to be drug-related (or at least reasonably likely). The vast majority of AEs were solitary, and no trend was observed that was associated with escalating NI-0501 dosage. All NI-0501 infusions were successful.

[0320] In summary, NI-0501 infusion was well-tolerated, and no serious or unexpected off-target safety or immunogenicity concerns were observed during the 8-week monitoring period after drug infusion.

[0321] Phase 2 / 3 Clinical Trial Materials and Methods: These trials will be conducted in patients with primary HLH. The trials will be divided into three parts: screening, treatment, and follow-up. An overview is shown in Figure 12.

[0322] In these trials, eligible patients include those who are naive to HLH treatment (also referred to herein as “first-line patients”) or those who may have already received conventional HLH treatment and, for example, have not achieved an adequate response by the treating physician or have shown signs of intolerance (also referred to herein as “second-line patients”). Patients receiving NI-0501 after failure of conventional HLH treatment or intolerance to it constitute the central cohort of trials to demonstrate the efficacy of NI-0501 as a second-line treatment for primary HLH. Patients naive to treatment are enrolled to collect efficacy and safety data in first-line settings.

[0323] The following patients will be excluded from this study: patients with a diagnosis of secondary HLH as a result of a proven rheumatic or neoplasm; patients who have been previously treated with any T-cell depletion agent (e.g., anti-thymocyte globulin (ATG), anti-CD52 therapy, etc.) or any other biological agent within two weeks prior to screening, or within five times the defined half-life of those agents (with the exception of rituximab in the case of proven B-cell EBV infection); patients with active mycobacteria, histoplasma Patients with Capsulatum, Shigella, Salmonella, Campylobacter, and Leishmania infections; patients with evidence of a past history of tuberculosis or latent tuberculosis; patients who test positive for HIV antibodies, hepatitis B surface antigen, or hepatitis C antibodies; patients with malignant tumors; patients with other complications or malformations that severely affect cardiovascular, pulmonary, hepatic, or renal function; patients with a history of hypersensitivity or allergy to any component of the test regimen; patients who have received a live or attenuated live vaccine (including BCG) within 12 weeks prior to screening; and / or pregnant or lactating women.

[0324] The studies presented herein utilize the anti-interferon-gamma antibody NI-0501, a fully human IgG1 monoclonal antibody (mAb) using human IFNγ as the test subject. NI-0501 is provided as a sterile concentrate for injection (per 1 mL), as shown in Table 4 below. [Table 4]

[0325] In these trials, NI-0501 will be administered via IV infusion at an initial dose of 1 mg / kg over 1 hour. This dose is expected to inhibit at least 99% of the effects of IFNγ over 3 days in patients with baseline IFNγ concentrations lower than or equal to 3400 pg / mL. Infusions will be administered every 3 days until day 15 of the trial (SD15) (infusion #6), and then twice weekly thereafter. The NI-0501 dose may be increased up to 3 mg / kg at any point during the trial according to predetermined criteria guided by clinical and laboratory responses in each patient (as shown in Table 5 below). After at least two infusions at 3 mg / kg, if, at re-evaluation, the same clinical and laboratory criteria that determined the patient's eligibility for 3 mg / kg of NI-0501 still apply, the NI-0501 dose may be increased up to 6 mg / kg over a maximum of four infusions, with regular monitoring of clinical and laboratory HLH parameters. Based on the progress of these parameters, the dose of NI-0501 may be i) reduced back to 3 mg / kg, or ii) kept at 6 mg / kg with additional IV infusions if excessively high IFNγ production, and therefore rapid elimination of NI-0501, is indicated by evidence of PK and PD (or increased beyond 6 mg / kg). Dose increases may be made at any point during the study, provided that the clinical and laboratory criteria described herein are met. [Table 5-1] [Table 5-2]

[0326] As shown in Figure 12, these studies administered NI-0501 over an 8-week period, dividing the treatment period into two separate periods: Treatment Period 1 and Treatment Period 2.

[0327] After administering NI-0501 for eight weeks, pre-transplant conditioning can be initiated in preparation for hematopoietic stem cell transplantation (HSCT). If transplantation is feasible based on the patient's condition and donor availability, the predicted duration of conditioning can be shortened, but should not be less than four weeks. If a suitable donor has not been identified by week eight, or if the transplant schedule needs to be delayed for reasons unrelated to NI-0501 administration, conditioning with NI-0501 can be continued in the context of a long-term follow-up study, provided that a favorable benefit / risk for the patient has been established.

[0328] In these trials, NI-0501 is administered against a background of dexamethasone, which can be gradually tapered depending on the patient's condition. For patients sensitive to treatment, 10 mg / m² is administered. 2 NI-0501 is administered against a background of dexamethasone. Patients receiving NI-0501 as a second-line HLH treatment should receive at least 5 mg / m² of dexamethasone. 2 The patient should be administered at the specified dose, or, if higher, at the same dose administered before screening. The patient must have received dexamethasone from SD-1.

[0329] Dexamethasone can be gradually reduced according to the patient's condition and at the discretion of the treating physician. The tapering scheme can be chosen by the treating physician as long as the dexamethasone dose at each step does not exceed half and the frequency of changes does not exceed weekly.

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

[0331] As recommended in the HLH treatment guidelines, patients will receive prophylactic treatment for Pneumocystis jiroveci, fungal, and herpes zoster virus infections from the day before initiating treatment with NI-0501 until the end of the study. Patients will receive prophylactic treatment from the day before initiating treatment with NI-0501 (i.e., SD-1) until the end of the study. For example, to prevent Pneumocystis jiroveci, patients will receive, for example, 750 mg / m² per day orally in equally divided doses twice daily for three consecutive days per week. 2 Sulfamethoxazole 150 mg / m² per day 2 It can be administered with trimethoprim. To prevent fungal infections, patients may receive, for example, fluconazole 12 mg / kg daily, up to a maximum daily dose of 400 mg. To prevent HZ virus infections, patients may receive, for example, acyclovir, 200 mg four times daily for children over 2 years of age, and 100 mg four times daily for children under 2 years of age. These treatments should be administered orally whenever possible, otherwise intravenously.

[0332] Patients may also receive any of the following combination therapies: cyclosporine A, intrathecal methotrexate and glucocorticosteroids, and others. If cyclosporine A (CsA) is already being administered to a patient prior to screening, it may be continued. CsA may be discontinued at any time. Once NI-0501 administration is initiated, CsA will not be newly introduced during the course of the study.

[0333] If the patient is receiving intrathecal methotrexate and glucocorticoids at the start of treatment with NI-0501, this treatment should be continued as needed. If CNS symptoms appear before the start of treatment with NI-0501, treatment with intrathecal methotrexate and glucocorticoids must be initiated before the first dose of NI-0501.

[0334] IV immunoglobulin (IVIG) is permitted only as a supplemental treatment in cases of demonstrated immunoglobulin deficiency. For example, if supplementation is justified by demonstrated immunoglobulin deficiency, IVIG may be administered at a dose of 0.5 g / kg every four weeks or more to maintain adequate IgG levels. Any prior infusions within four weeks prior to screening, as well as any infusions during treatment with NI-0501, are permitted.

[0335] Analgesia, transfusion of blood products, electrolyte and glucose infusions, antibiotics, antifungal and antiviral treatments, and general supportive care are acceptable. If HLH improvement is not sustained or limited when the maximum NI-0501 dose level is achieved, additional HLH treatment may be acceptable. As used herein, "not sustained HLH improvement" means that the patient is unable to maintain at least a 50% improvement from baseline with respect to three HLH parameters (see Table 6 below). The lack of HLH improvement must be demonstrated by at least two consecutive measurements. As used herein, "limited HLH improvement" means that the change from baseline is less than 50% in at least three HLH clinical and laboratory criteria. Etoposide should be administered as additional HLH treatment unless there is clear evidence of a lack of response to or intolerance to the drug from prior medical history.

[0336] The following treatments should not be used concurrently with NI-0501 administration: etoposide, T-cell depletion agents, or any other biological agents, generally not permitted except for: G-CSF in cases of prolonged neutropenia; rituximab in cases of demonstrated B-cell EBV infection; and additional HLH treatment when HLH improvement is not sustained or limited (as defined herein) at the maximum NI-0501 dose level. Etoposide should be administered unless there is clear evidence of a lack of response to or intolerance to the drug from prior medical history. Vaccination with live or attenuated vaccines (including BCG) should be avoided throughout the entire study, including the 4-week follow-up period. If NI-0501 concentrations remain at therapeutic levels after the end of the study, the vaccination-free period should be extended until measurable concentrations of NI-0501 are no longer detectable.

[0337] The progression of disease-characterizing clinical signs (fever, splenomegaly, CNS symptoms) and laboratory parameters (CBC, fibrinogen, ferritin, sCD25 levels) is used to assess the realization of the response and the time to response. The primary efficacy endpoint is the overall response rate, i.e., the realization of a complete or partial response or improvement in HLH at end of treatment (EoT), as defined in Table 6 below. Secondary efficacy endpoints include time to response at any point in the study; durability of the response, i.e., maintenance of the response to and beyond the end of time (EoT) of the response achieved at any point in the study (including data collected in any long-term follow-up studies); the number of patients who can reduce glucocorticoids by 50% or more of the baseline dose; the number of patients who can proceed to HSCT if deemed necessary; survival at week 8 (or EoT) and end of the study; serum concentrations of NI-0501 to determine the NI-0501 pharmacokinetic (PK) profile; determination of pharmacodynamic (PD) effects, including circulating total IFNγ and its neutralization markers, i.e., levels of 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 particular attention paid to infections; progress of laboratory parameters such as complete blood cell count (CBC), focusing on red blood cells (hemoglobin), neutrophils, and platelets, liver function tests, kidney function tests, and coagulation; the number of patients discontinued 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) is assessed using an exact one-sided binomial test at the 0.025 level. Time to response, response durability, and survival are shown using Kaplan-Meier curves, and medians are calculated where available. 95% confidence intervals are calculated for the medians of each of these endpoints. Additional endpoints based on binary outcomes, including the number of patients who reduce glucocorticoids by 50% or more, and the number of patients who can proceed to HSCT, are converted to percentages, and relevant 95% confidence intervals are calculated. Statistical significance by p-value is obtained only for the primary endpoint. All other endpoints are considered supporting to the primary endpoint, and consequently, no formal hierarchy of endpoints is shown.

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

[0341] Of the 10 patients who underwent hematopoietic stem cell transplantation (HSCT), engraftment was successful in all; one patient required a boost with CD34 stem cells due to a mixed chimeric phenomenon at post-HSCT D+145. One patient experienced secondary transplant failure, followed by reactivation of HLH. This patient died at post-HSCT D+68 due to acute respiratory failure and bacterial infection. Another patient died at post-HSCT D+47 (septic shock in the context of severe GvHD). Mild GvHD was reported in three other patients, and has resolved / is resolving.

[0342] In 8 out of 10 transplant recipients, the neutralization of serum concentrations of NI-0501 at HSCT was measured, as reflected in CXCL9 (a chemokine subtly induced by IFNγ) levels falling below the quantifiable limit. Therefore, these data suggest that NI-0501 can be administered without the short- or long-term toxicity reported with etoposide-based regimens. This translates to a reduced risk of allo-HSCT-related complications.

[0343] These data demonstrate that treatment with NI-0501 can improve and / or eliminate relevant clinical and laboratory abnormalities in HLH, including CNS signs and symptoms. The response to NI-0501 is independent of the presence and type of causative mutations and / or the presence and type of infection triggers. NI-0501 was well-tolerated. No safety concerns have arisen to date (e.g., no myelotoxicity, no widespread immunosuppression). No infections caused by pathogens known to be promoted by IFNγ neutralization have been observed. IFNγ neutralization with NI-0501 could provide an innovative targeted approach for 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) presenting with macrophage activation syndrome / secondary HLH (MAS / sHLH).

[0344] The trial 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 trial to evaluate the PK profile and dosing strategy of NI-0501 and to pre-assess the benefit / risk of NI-0501 in this patient population; and (ii) a pivotal trial to demonstrate the efficacy and safety of NI-0501 (the trial will continue once the dosing regimen and positive benefit / risk profile of NI-0501 are confirmed). An overview of this trial design is shown in Figure 19.

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

[0346] The study population included sJIA patients with MAS / sHLH who showed an inadequate response to high-dose glucocorticoid treatment. Inclusion criteria included: (i) sex: male and female; (ii) age: <16 years at the 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 criterion: platelet count ≤ 262 × 10⁻¹⁰ 9 / L, WBC number ≦4.0×10 9 (b) Clinical criteria: hepatomegaly, manifestation of hemorrhagic disorders, and / or CNS dysfunction; (iv) patients showing an inadequate response to high-dose iv-glucocorticoid treatment (including, but not limited to, pulses of 30 mg / kg mPDN for 3 consecutive days) for at least 3 days in accordance with local standard of care; (v) high-dose iv-glucocorticoids should not be reduced below 2 mg / kg mPDN per day, equivalent to two separate daily doses of up to 60 mg per day. If the patient's condition and / or laboratory parameters rapidly deteriorate, this may be included within 3 days of initiation of high-dose iv-glucocorticoids; (vi) patient consent (or consent of legally authorized representative(s)); and (vii) acceptance of contraceptive measures if the patient is past puberty.

[0347] Exclusion criteria include: (i) a diagnosis of suspected or confirmed primary HLH or HLH resulting from a neoplasm; (ii) patients treated with anakinra, tocilizumab, canakinumab, TNF inhibitors, rituximab, or any other biological agent within five times the defined half-life of those agents; (iii) active mycobacteria (typical and atypical), histoplasma (iv) Infection with Capsulatum, Shigella, Salmonella, Campylobacter, or Leishmania; (v) Evidence of latent tuberculosis; (v) Positive serological testing for HIV antibodies; (vi) Presence of malignant tumors; (vii) Patients with other complications or malformations that severely affect cardiovascular, pulmonary, CNS, hepatic, or renal function, which may, in the opinion of the attending physician, be likely to respond to treatment and / or significantly affect the assessment of the safety of NI-0501; (viii) History of hypersensitivity or allergy to any component of the study regimen; (ix) Having received the BCG vaccine within 12 weeks prior to screening; (x) Having received another live or attenuated live vaccine within 6 weeks prior to screening; and / or (xi) Female patients who are pregnant or lactating.

[0348] Dosage 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 by infusion over 1 hour at an initial dose of 6 mg / kg at SD0. Treatment with NI-0501 is continued at a dose of 3 mg / kg every 3 days for 4 weeks (i.e., up to SD27). Once 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 a further 4 weeks (i.e., up to SD56) as maintenance treatment 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 (i.e., exceptionally high IFNγ production is signaled), the dose of NI-0501 can be increased to 10 mg / kg, guided by clinical and PK evidence. This dose increase will only be approved after the benefit / risk profile in that individual patient has been carefully evaluated.

[0349] In Part 2, the trial will continue once the proposed drug regimen is deemed valid and the positive benefits / risks of NI-0501 are demonstrated. Minor modifications to the drug regimen may be applied as needed, based on the evidence obtained in Part 1.

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

[0351] Sample size: Part 1 will enroll at least 5 evaluable patients. Part 2 will enroll at a later stage of the study at least 10 more evaluable patients, for a total of 15 evaluable patients. A sample size of 15 is not formally justified given the rare orphan nature of the disease and the lack of any approved treatments. Nevertheless, based on the assumption that at least 50% of patients will respond inadequately to systemic glucocorticoids alone, i.e., that 50% of patients receiving glucocorticoids will achieve MAS remission by 8 weeks after the start of treatment, this study has 70% power for improvement from 50% to 77% using a one-sided significance level of 5%.

[0352] Definition of trial duration and trial end: The trial duration is 8 weeks for each patient (plus a maximum of 1 week of screening). Trial end is defined as the last visit of the last patient. All patients who have received at least one dose of NI-0501 are required to participate in the NI-0501-05 trial for long-term follow-up.

[0353] Study endpoints: In Part 1 (Pilot) of the study, the following will be evaluated to confirm the medication 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 progression of distinct features of MAS, such as cytopenia, hepatic dysfunction, and coagulation disorders, at 2, 4, 6, and 8 weeks after initiation of NI-0501; and (v) the dose and duration of treatment with NI-0501. In the pivotal part 2 of the study, the efficacy endpoints are as follows: (a) the primary efficacy endpoint: the number of patients who achieve MAS remission by 8 weeks after the initiation of treatment with NI-0501; and (b) the secondary efficacy endpoints: time to MAS remission; time to first response as assessed by the principal investigator; the number of patients who can tape down glucocorticoids to the same (or lower) dose they were receiving before MAS developed at any point during the study; time to glucocorticoid tapering; survival at the end of the study; and the number of patients who discontinued the study due to lack of efficacy. In the pivotal part 2 of the study, the endpoints for the safety study are as follows: (a) Incidence, severity, causality, and outcome of AEs (serious and non-serious), with particular attention paid to infections; progression of laboratory parameters, particularly CBC (focusing on hemoglobin, neutrophils, and platelets), LFT, and coagulation parameters; number of patients who discontinued the study for safety reasons; and levels of circulating antibodies against NI-0501 (if any) to determine immunogenicity (ADA).

[0354] Pharmacokinetics and pharmacodynamics are assessed by the following PK profiles of NI-0501: levels of circulating free IFNγ before administration 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); correlations between chemokine levels (CXCL9, CXCL10) and levels of free NI-0501, free IFNγ (pre-administration), and total IFNγ; correlations between chemokine and total IFNγ levels and laboratory parameters of MAS severity, e.g., ferritin, platelet count, LFT (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 illustrate, not limit, 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 method for treating primary hemophagocytic lymphohistiocytosis (HLH) in human subjects requiring treatment for HLH, comprising multiple variable doses, wherein the subject a) an antibody that binds to interferon-gamma (IFNγ) in a first dose of 1.0 or 3.0 mg per kg of body weight of the subject, to be administered to the subject within 12 hours; and b) Within 12 hours, administer a second dose of the antibody at 3.0, 6.0, or 10.0 mg per kg of body weight of the subject. The step includes administering intravenously, A method comprising the antibody having a variable heavy chain complementarity determination region 1 (VH CDR1) containing the amino acid sequence of SYAMS (SEQ ID NO: 1); a variable heavy chain complementarity determination region 2 (VH CDR2) containing the amino acid sequence of AISGSGGSTYYADSVKG (SEQ ID NO: 2); and a variable heavy chain complementarity determination region 3 (VH CDR3) containing the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO: 3); a variable light chain complementarity determination region 1 (VL CDR1) containing the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO: 4); a variable light chain complementarity determination region 2 (VL CDR2) region containing the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and a variable light chain complementarity determination region 3 (VL CDR3) region containing the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6). (Item 2) The method according to item 1, wherein the antibody comprises a heavy chain variable amino acid sequence which is the amino acid sequence of SEQ ID NO: 47, and a light chain variable amino acid sequence which is the amino acid sequence of SEQ ID NO: 48. (Item 3) The method according to item 1, wherein the dose of the antibody is administered within 6 hours. (Item 4) The method according to item 3, wherein the dose of the antibody is administered within one hour. (Item 5) The method according to item 1, wherein the second dose is administered every three days thereafter over a first treatment period. (Item 6) The method according to item 5, wherein the second dose is administered over a second treatment period following the completion of the first treatment period of 1, 2, 3, 4, 5, 6, 7, or 8 weeks after the first dose. (Item 7) The method according to item 6, wherein the second treatment period is twice a week. (Item 8) The method according to item 1, further comprising the step of administering a third dose of the antibody, 1.0 mg per kg of body weight of the subject, to the subject within 12 hours. (Item 9) The method according to item 1, wherein the dose of the antibody is administered as a single injection. (Item 10) The method according to item 1, wherein the antibody is administered as monotherapy or in combination therapy. (Item 11) The method according to item 1, wherein the subject is an adult subject or a child subject. (Item 12) A method for treating secondary hemophagocytic lymphohistiocytosis (HLH) in human pediatric subjects requiring treatment for HLH, comprising multiple variable doses, wherein the subject a) an antibody that binds to interferon-gamma (IFNγ) in a first dose of 6.0 mg per kg of body weight of the subject, to be administered to the subject within 12 hours; and b) Within 12 hours, the subject receives a second dose of the antibody at a rate of 3.0 mg per kg of body weight. The step includes administering intravenously, A method comprising the antibody having a variable heavy chain complementarity determination region 1 (VH CDR1) containing the amino acid sequence of SYAMS (SEQ ID NO: 1); a variable heavy chain complementarity determination region 2 (VH CDR2) containing the amino acid sequence of AISGSGGSTYYADSVKG (SEQ ID NO: 2); and a variable heavy chain complementarity determination region 3 (VH CDR3) containing the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO: 3); a variable light chain complementarity determination region 1 (VL CDR1) containing the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO: 4); a variable light chain complementarity determination region 2 (VL CDR2) region containing the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and a variable light chain complementarity determination region 3 (VL CDR3) region containing the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6). (Item 13) The method according to item 12, wherein the dose of the antibody is administered within 6 hours. (Item 14) The method according to item 13, wherein the dose of the antibody is administered within one hour. (Item 15) The method according to item 12, wherein the second dose is administered every three days thereafter over a first treatment period. (Item 16) The method according to item 15, wherein the second dose is administered over a second treatment period following the completion of the first treatment period. (Item 17) The method according to item 16, wherein the second treatment period is twice a week. (Item 18) The method according to item 12, further comprising the step of administering an additional dose of the antibody at 6.0 mg per kg of body weight of the subject within 12 hours after the second dose. (Item 19) The method according to item 12, wherein the dose of the antibody is administered as a single injection. (Item 20) The method according to item 12, wherein the antibody is administered as monotherapy or in combination therapy. (Item 21) A method for treating secondary hemophagocytic lymphohistiocytosis (HLH) in adult human subjects requiring treatment for HLH, comprising multiple variable doses, wherein the subject a) an antibody that binds to interferon-gamma (IFNγ) in a first dose of 3 mg or 6.0 mg per kg of body weight of the subject, to be administered to the subject within 12 hours; and b) Within 12 hours, administer a second dose of the antibody no more than 10.0 mg per kg of body weight of the subject. The step includes administering intravenously, A method comprising the antibody having a variable heavy chain complementarity determination region 1 (VH CDR1) containing the amino acid sequence of SYAMS (SEQ ID NO: 1); a variable heavy chain complementarity determination region 2 (VH CDR2) containing the amino acid sequence of AISGSGGSTYYADSVKG (SEQ ID NO: 2); and a variable heavy chain complementarity determination region 3 (VH CDR3) containing the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO: 3); a variable light chain complementarity determination region 1 (VL CDR1) containing the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO: 4); a variable light chain complementarity determination region 2 (VL CDR2) region containing the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and a variable light chain complementarity determination region 3 (VL CDR3) region containing the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6). (Item 22) The method according to item 21, wherein the dose of the antibody is administered within 6 hours. (Item 23) The method according to item 22, wherein the dose of the antibody is administered within one hour. (Item 24) The method according to item 22, wherein the second dose is administered every three days thereafter over a first treatment period. (Item 25) The method according to item 24, wherein the second dose is administered over a second treatment period following the completion of the first treatment period. (Item 26) The method according to item 25, wherein the second treatment period is twice a week. (Item 27) The method according to item 21, further comprising the step of administering an additional dose of the antibody at 1.0, 3.0, or 6.0 mg per kg of body weight of the subject within 12 hours after the second dose. (Item 28) The method according to item 21, wherein the dose of the antibody is administered as a single injection. (Item 29) The method according to item 21, wherein the antibody is administered as monotherapy or in combination therapy. (Item 30) A method for treating a condition in a human subject requiring treatment of a condition, comprising multiple variable doses, wherein the subject a) A first dose of an antibody that binds to interferon-gamma (IFNγ), to be administered to the subject within 12 hours; and b) Within 12 hours, a second dose of the antibody equal to the body weight of the subject. The step includes administering intravenously, A method comprising the antibody having a variable heavy chain complementarity determination region 1 (VH CDR1) containing the amino acid sequence of SYAMS (SEQ ID NO: 1); a variable heavy chain complementarity determination region 2 (VH CDR2) containing the amino acid sequence of AISGSGGSTYYADSVKG (SEQ ID NO: 2); and a variable heavy chain complementarity determination region 3 (VH CDR3) containing the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO: 3); a variable light chain complementarity determination region 1 (VL CDR1) containing the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO: 4); a variable light chain complementarity determination region 2 (VL CDR2) region containing the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and a variable light chain complementarity determination region 3 (VL CDR3) region containing the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6). (Item 31) The method according to item 30, wherein the antibody comprises a heavy chain variable amino acid sequence which is the amino acid sequence of SEQ ID NO: 47, and a light chain variable amino acid sequence which is the amino acid sequence of SEQ ID NO: 48. (Item 32) The method according to item 30, wherein the dose of the antibody is administered within 6 hours. (Item 33) The method according to item 32, wherein the dose of the antibody is administered within one hour. (Item 34) The method according to item 30, wherein the second dose is administered every three days thereafter over a first treatment period. (Item 35) The method according to item 34, wherein the second dose is administered over a second treatment period following the completion of the first treatment period. (Item 36) The method according to item 35, wherein the second treatment period is twice a week. (Item 37) The method according to item 30, wherein the first dose is 1.0 to 10 mg per kg of body weight of the subject. (Item 38) The method according to item 30, wherein the dose is 1.0 to 10 mg per kg of body weight of the subject, and the second dose is lower or higher than the first dose. (Item 39) The method according to item 30, further comprising the step of administering a third dose of the antibody, in a dose of 1.0 to 10 mg per kg of body weight of the subject, to be administered to the subject within 12 hours. (Item 40) The method according to item 30, wherein the dose of the antibody is administered as a single injection. (Item 41) The method according to item 30, wherein the antibody is administered as monotherapy or in combination therapy. (Item 42) The method according to item 30, wherein the subject is an adult subject or a child subject. (Item 43) The method according to item 30, wherein the aforementioned condition is graft rejection. (Item 44) The method according to item 43, wherein the graft rejection is parenchymal organ transplant failure or acute bone marrow graft rejection. (Item 45) The method according to item 30, wherein the condition is graft-versus-host disease, paraneoplastic cerebellar degeneration, hemorrhagic fever, sarcoidosis, or adult-onset Still's disease. (Item 46) The method described in item 30, which is performed on subjects after they have received CART cell therapy. (Item 47) The method according to any one of the preceding items, wherein the subject is administered dexamethasone immediately before administering the antibody. (Item 48) The aforementioned dexamethasone is at least 10 mg / m² 2 The method described in item 30, administered in the specified dose. (Item 49) The aforementioned dexamethasone is at least 5 mg / m² 2 The method described in item 30, administered in the specified dose. (Item 50) The method described in any one of the preceding items, wherein the subject has not previously received treatment for HLH. (Item 51) The method according to any one of the preceding items, further comprising the step of administering at least a second drug to the subject. (Item 52) The method according to item 523, wherein the second agent is a therapeutic agent, an anti-inflammatory agent, and / or an immunosuppressant. (Item 53) Per 1 mL: a) Fully human anti-interferon-gamma (IFNγ) monoclonal antibody 5 mg or 25 mg; and b) 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 between 5.8 and 6.2 An injectable pharmaceutical preparation containing [the specified ingredient]. (Item 54) The formulation according to item 53, wherein the antibody comprises a variable heavy chain complementarity determination region 1 (VH CDR1) containing the amino acid sequence of SYAMS (SEQ ID NO: 1); a variable heavy chain complementarity determination region 2 (VH CDR2) containing the amino acid sequence of AISGSGGSTYYADSVKG (SEQ ID NO: 2); and a variable heavy chain complementarity determination region 3 (VH CDR3) containing the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO: 3); a variable light chain complementarity determination region 1 (VL CDR1) containing the amino acid sequence of TRSSSGSIASNYVQ (SEQ ID NO: 4); a variable light chain complementarity determination region 2 (VL CDR2) region containing the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and a variable light chain complementarity determination region 3 (VL CDR3) region containing the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6). (Item 55) The formulation according to item 54, wherein the antibody comprises a heavy chain variable amino acid sequence which is the amino acid sequence of SEQ ID NO: 47, and a light chain variable amino acid sequence which 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, wherein the antibody concentration is 5 mg / ml or 25 mg / ml and the pH of the solution is between 5.8 and 6.2. (Item 57) The antibody is solubilized in the solution, and therefore the solution is clear, colorless, and free of precipitate, in the unit dose vial described in item 56. (Item 58) A unit dose vial according to item 56, wherein the antibody comprises a variable heavy chain complementarity determination region 1 (VH CDR1) containing the amino acid sequence of SYAMS (SEQ ID NO: 1); a variable heavy chain complementarity determination region 2 (VH CDR2) containing the amino acid sequence of AISGSGGSTYYADSVKG (SEQ ID NO: 2); and a variable heavy chain complementarity determination region 3 (VH CDR3) containing the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO: 3); a variable light chain complementarity determination region 1 (VL CDR1) containing the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO: 4); a variable light chain complementarity determination region 2 (VL CDR2) containing the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and a variable light chain complementarity determination region 3 (VL CDR3) containing the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6). (Item 59) A unit dose vial according to item 58, wherein the antibody comprises a heavy chain variable amino acid sequence which is the amino acid sequence of SEQ ID NO: 47, and a light chain variable amino acid sequence which 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, wherein the antibody concentration is 25 mg / ml and the pH of the solution is between 5.8 and 6.2. (Item 61) The antibody is solubilized in the solution, and therefore the solution is clear, colorless, and free of precipitate, as described in item 60, in a unit dose vial. (Item 62) A unit dose vial according to item 60, wherein the antibody comprises a variable heavy chain complementarity determination region 1 (VH CDR1) containing the amino acid sequence of SYAMS (SEQ ID NO: 1); a variable heavy chain complementarity determination region 2 (VH CDR2) containing the amino acid sequence of AISGSGGSTYYADSVKG (SEQ ID NO: 2); and a variable heavy chain complementarity determination region 3 (VH CDR3) containing the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO: 3); a variable light chain complementarity determination region 1 (VL CDR1) containing the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO: 4); a variable light chain complementarity determination region 2 (VL CDR2) containing the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and a variable light chain complementarity determination region 3 (VL CDR3) containing the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6). (Item 63) A unit dose vial as described in item 62, wherein the antibody comprises a heavy chain variable amino acid sequence which is the amino acid sequence of SEQ ID NO: 47, and a light chain variable amino acid sequence which 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, wherein the antibody concentration is 5 mg / ml and the pH of the solution is between 5.8 and 6.2. (Item 65) A unit dose vial as described in item 64, wherein the antibody is solubilized in the solution, and therefore the solution is clear, colorless, and free of precipitate. (Item 66) A unit dose vial according to item 64, wherein the antibody comprises a variable heavy chain complementarity determination region 1 (VH CDR1) containing the amino acid sequence of SYAMS (SEQ ID NO: 1); a variable heavy chain complementarity determination region 2 (VH CDR2) containing the amino acid sequence of AISGSGGSTYYADSVKG (SEQ ID NO: 2); and a variable heavy chain complementarity determination region 3 (VH CDR3) containing the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO: 3); a variable light chain complementarity determination region 1 (VL CDR1) containing the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO: 4); a variable light chain complementarity determination region 2 (VL CDR2) containing the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and a variable light chain complementarity determination region 3 (VL CDR3) containing the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6). (Item 67) A unit dose vial according to item 66, wherein the antibody comprises a heavy chain variable amino acid sequence which is the amino acid sequence of SEQ ID NO: 47, and a light chain variable amino acid sequence which is the amino acid sequence of SEQ ID NO: 48.

Claims

1. An injectable pharmaceutical preparation comprising 25 mg per 1 mL of fully human anti-interferon-gamma (IFNγ) monoclonal antibody, L-histidine, L-histidine monohydrochloride monohydrate, sodium chloride (NaCl), and polysorbate 80, The pH of the aforementioned formulation is between 5.8 and 6.

2. A pharmaceutical preparation for injection in which the antibody comprises a variable heavy chain complementarity determination region 1 (VH CDR1) containing the amino acid sequence of SYAMS (SEQ ID NO: 1); a variable heavy chain complementarity determination region 2 (VH CDR2) containing the amino acid sequence of AISGSGGGSTYYADSVKG (SEQ ID NO: 2); and a variable heavy chain complementarity determination region 3 (VH CDR3) containing the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO: 3); a variable light chain complementarity determination region 1 (VL CDR1) containing the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO: 4); a variable light chain complementarity determination region 2 (VL CDR2) containing the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and a variable light chain complementarity determination region 3 (VL CDR3) containing the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6).

2. The injectable pharmaceutical preparation according to claim 1, wherein the antibody comprises a heavy chain variable amino acid sequence which is the amino acid sequence of SEQ ID NO: 47 and a light chain variable amino acid sequence which is the amino acid sequence of SEQ ID NO:

48.

3. The injectable pharmaceutical preparation according to claim 1, wherein the preparation comprises 1.55 mg of L-histidine, 3.14 mg of L-histidine monohydrochloride monohydrate, 7.31 mg of sodium chloride (NaCl), and 0.05 mg of polysorbate 80.

4. The injectable pharmaceutical preparation according to any one of claims 1 to 3, wherein the injectable pharmaceutical preparation is for use in the treatment of symptoms of primary hemophagocytic lymphohistiocytosis (HLH), secondary HLH, or macrophage activation syndrome (MAS).

5. A unit dose vial containing a 20 ml injection-appropriate solution of fully human anti-interferon-gamma (IFNγ) monoclonal antibody, The concentration of the antibody is 25 mg / ml, and the pH of the solution is between 5.8 and 6.

2. A unit dose vial of the antibody comprising: variable heavy chain complementarity determination region 1 (VH CDR1) containing the amino acid sequence of SYAMS (SEQ ID NO: 1); variable heavy chain complementarity determination region 2 (VH CDR2) containing the amino acid sequence of AISGSGGGSTYYADSVKG (SEQ ID NO: 2); and variable heavy chain complementarity determination region 3 (VH CDR3) containing the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO: 3); variable light chain complementarity determination region 1 (VL CDR1) containing the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO: 4); variable light chain complementarity determination region 2 (VL CDR2) containing the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and variable light chain complementarity determination region 3 (VL CDR3) containing the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6).

6. A unit dose vial containing a fully human anti-interferon-gamma (IFNγ) monoclonal antibody solution suitable for 10 ml injection, The concentration of the antibody is 25 mg / ml, and the pH of the solution is between 5.8 and 6.

2. A unit dose vial of the antibody comprising: variable heavy chain complementarity determination region 1 (VH CDR1) containing the amino acid sequence of SYAMS (SEQ ID NO: 1); variable heavy chain complementarity determination region 2 (VH CDR2) containing the amino acid sequence of AISGSGGGSTYYADSVKG (SEQ ID NO: 2); and variable heavy chain complementarity determination region 3 (VH CDR3) containing the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO: 3); variable light chain complementarity determination region 1 (VL CDR1) containing the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO: 4); variable light chain complementarity determination region 2 (VL CDR2) containing the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and variable light chain complementarity determination region 3 (VL CDR3) containing the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6).

7. The unit dose vial according to claim 5 or 6, wherein the antibody is solubilized in the solution, and therefore the solution is clear, colorless, and free from precipitate.

8. The unit dose vial according to claim 5 or 6, wherein the antibody comprises a heavy chain variable amino acid sequence which is the amino acid sequence of SEQ ID NO: 47, and a light chain variable amino acid sequence which is the amino acid sequence of SEQ ID NO:

48.

9. The unit dose vial according to claim 5 or 6, wherein the solution further comprises L-histidine, L-histidine monohydrochloride monohydrate, sodium chloride (NaCl), and polysorbate 80.

10. The unit dose vial according to claim 9, wherein the solution comprises 1.55 mg of L-histidine, 3.14 mg of L-histidine monohydrochloride monohydrate, 7.31 mg of sodium chloride (NaCl), and 0.05 mg of polysorbate 80.

11. The unit dose vial according to claim 5 or 6, wherein the unit dose vial is for use in the treatment of symptoms of primary hemophagocytic lymphohistiocytosis (HLH), secondary HLH, or macrophage activation syndrome (MAS).

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