JP2022187025A - Methods, compositions and dosing regimens for treating or preventing interferon-gamma related indications - Google Patents

Abstract

PROBLEM TO BE SOLVED: 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

COPYRIGHT: (C)2023,JPO&INPIT

Description

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

The present disclosure relates generally to, for example, hemophagocytic lymphohistiocytosis (HLH), hemorrhagic fever, CAR-T cell therapy, transplant failure, graft rejection, graft versus host disease (GvHD), and/or graft rejection. to treat, prevent and/or delay the onset or progression of or alleviate symptoms associated with elevated levels of interferon gamma (IFNγ, IFN-gamma), such as inflammatory disorders associated with to the methods and compositions of The present disclosure also relates to methods and compositions for prolonging survival of implanted biological material.

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

IFNγ has been implicated in several disorders and anti-IFNγ agents are under development as therapeutic agents.

In various aspects, the invention provides multiple variable dose treatment regimens for treating diseases, disorders or conditions associated with elevated IFN-g levels.

In one aspect, the invention provides a method for treating primary hemophagocytic lymphohistiocytosis in humans by intravenously administering to a subject a first dose and a second dose of an antibody that binds interferon gamma (IFNγ). Methods for treating (HLH) are provided. A subject is an adult subject or a pediatric subject. The first dose is 1.0 or 3.0 mg per kg body weight of the subject and the second dose is 3.0, 6.0 or 10.0 mg per kg body weight of the subject. Optionally, a third dose of 1.0 mg per kg body weight of the subject is administered.

In another aspect, the invention provides for the treatment of secondary hemophagocytosis in a human pediatric subject by intravenously administering to the subject a first dose and a second dose of an antibody that binds interferon gamma (IFNγ). Kind Code: A1 Methods for treating lymphohistiocytosis (HLH) are provided. The first dose is 6.0 mg/kg body weight of the subject and the second dose is 3.0 mg/kg body weight of the subject. Optionally, a third dose of 6.0 mg/kg body weight of the subject is administered.

In a further aspect, the invention provides a method for reducing secondary hemophagocytosis in an adult human subject by intravenously administering to the subject a first dose and a second dose of an antibody that binds interferon gamma (IFNγ). A method for treating lymphohistiocytosis (HLH) is provided. The first dose is 3.0 mg/kg body weight of the subject or 6.0 mg/kg body weight of the subject, and the second dose is 10 mg/kg body weight of the subject or less. 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/kg body weight of the subject is administered. For example, the third dose is 1.0, 3.0, or 6.0 mg per kg body weight of the subject.

In yet another aspect, the invention provides a method for treating a condition in a human subject by intravenously administering to the subject a first dose and a second dose of an antibody that binds interferon gamma (IFNγ). provide a way. A subject is an adult subject or a pediatric subject. The condition is graft rejection, such as solid organ transplant injury 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 administered to the subject after receiving CAR T cell therapy. The first dose is 1.0-10 mg/kg body weight of the subject and the second dose is 1.0-10 mg/kg body weight of the subject. 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. Optionally, a third dose of 1.0-10 mg/kg body weight of the subject is administered. For example, the first, second, or third dose is 1.0, 3.0, 6.0 or 10.0 mg per kg body weight of the subject.

Antibodies that bind interferon gamma (IFNγ) have a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO: 1); sex-determining region 2 (VH
CDR2); and variable heavy chain complementarity determining region 3 (VH CDR3) comprising the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO:3); variable light chain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO:4) ); a variable light chain complementarity determining region 2 (VL CDR2) region comprising the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and a variable light chain complementarity determining region 3 (VL CDR3) region comprising the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6). ) region. For example, the antibody comprises a heavy chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO:47 and a light chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO:48.

The dose of antibody is administered within 1 hour, 6 hours or 12 hours.

A second dose is administered over a first treatment period every three days after the first dose. Additionally, a second dose is administered over a second treatment period after completion of the first treatment period. A second treatment period is, for example, twice a week.

The dose of antibody is administered as a single injection.

Antibodies are administered as monotherapy or combination therapy.

Optionally, the methods of the invention further comprise administering dexamethasone immediately prior to administration of the antibody. Dexamethasone is administered at doses of at least 10 mg/m ² or at least 5 mg/m ² .

The subject has not previously received treatment for HLH.

In various aspects, the method further comprises administering at least a second agent to the subject. A second agent is a therapeutic, anti-inflammatory, and/or immunosuppressive agent.

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

In a further aspect, the invention is 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 antibody is 5 mg/ml or 25 mg/ml, and the concentration of the solution is A unit dose vial is provided having a pH between 5.8 and 6.2. The antibody is solubilized in solution, so the solution is clear, colorless and free of precipitate.

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

In yet another aspect, the 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 concentration of the solution is A unit dose vial is provided having a pH between 5.8 and 6.2. The antibody is solubilized in solution, so the solution is clear, colorless and free of precipitate

Antibodies that bind interferon gamma (IFNγ) have: a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO: 1); Complementarity Determining Region 2 (VH CDR2); and Variable Heavy Chain Complementarity Determining Region 3 (VH CDR3) comprising the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO:3); Variable Light Chain Complementation comprising the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO:4) sex determining region 1 (VL CDR1); variable light chain complementarity determining region 2 (VL CDR2) region comprising the amino acid sequence of EDNQRPS (SEQ ID NO:5); and variable light chain complementation region comprising the amino acid sequence of QSYDGSNRWM (SEQ ID NO:6) Includes the sex-determining region 3 (VL CDR3) region. For example, the antibody comprises a heavy chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO:47 and a light chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO:48.
Any of the above aspects or embodiments can be combined with any other aspect or embodiment.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples described herein are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from and encompassed by the following detailed description and claims.

FIG. 1 is a graph showing the correlation between predose 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.

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

Figures 3A and 3B are a series 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 in patients with active sJIA. is a graph of

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

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

FIG. 6 is a graph showing improvement in HLH disease activity during treatment with NI-0501 (2 weeks and end of treatment): platelet count >100×10 ⁹ /L, neutrophil count >1×10 ⁹ . /L, percent of patients with fibrinogen >1.5 g/L and at least a 25% reduction in ferritin.

Figures 7A and 7B are a series of graphs showing the correlation between predose CXCL9 levels and total IFNγ levels 24 hours after NI-0501 infusion. The insets shown in FIG. 7B show examples of individual IFNγ and CXCL9 profiles during treatment with NI-0501.

Figures 8A, 8B, 8C, and 8D show serum IFNγ in individual patients for whom matched samples were available during active MAS and during active sJIA without MAS (Act sJIA) at sampling. FIG. 3 is a series of graphs showing medium and serum levels of CXCL9, CXCL10 and CXCL11. The level of significance (p) was obtained using the Wilcoxon rank test for paired samples.

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

Figures 10A, 10B, 10C, 10D, 10E, 10F, 10G, 10H, 10I, and 10J show a patient with active MAS at sampling (red circles) and a patient with active sJIA without MAS at sampling ( Fig. 4 is a series of graphs showing the correlation of IFNγ and CXCL9 levels with ferritin levels, neutrophil and platelet counts, and with LDH and ALT levels, in black triangles). The Spearman correlation coefficient (Rs) and significance level (p) for each correlation are shown in Table 3. Figures 10A, 10B, 10C, 10D, 10E, 10F, 10G, 10H, 10I, and 10J show a patient with active MAS at sampling (red circles) and a patient with active sJIA without MAS at sampling ( Fig. 4 is a series of graphs showing the correlation of IFNγ and CXCL9 levels with ferritin levels, neutrophil and platelet counts, and with LDH and ALT levels, in black triangles). The Spearman correlation coefficient (Rs) and significance level (p) for each correlation are shown in Table 3.

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 of IFNγ levels with CXCL9 and CXCL10 levels in patients with MAS at sampling. The Spearman correlation coefficient (Rs) and significance level (p) for each correlation are shown in Table 3. 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 of IFNγ levels with CXCL9 and CXCL10 levels in patients with MAS at sampling. The Spearman correlation coefficient (Rs) and significance level (p) for each correlation are shown in Table 3.

12 is a schematic representation of the screening, treatment, and follow-up portions of the study presented in Example 7. FIG.

Figures 13A and 13B are graphs showing the effect of NI-0501 administration on body temperature in two patients with temperatures >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 counts in patients.

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

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

Figure 17 is a series of graphs and tables showing the effect of NI-0501 administration on glucocorticoid taper in patients.

FIG. 18 is a graph showing that administration of NI-0510 maintained IFNγ neutralization through HSCT. HLH responses to treatment with NI-0501 also persisted until transplantation.

19 is a schematic representation of the screening, treatment, and follow-up portion of the study presented in Example 8. FIG.

In the compositions and methods presented herein, a fully human IgG1 anti-interferon gamma (IFNγ) monoclonal antibody (mAb), referred to herein as NI-0501, that binds to and neutralizes IFNγ use. NI-0501 binds to soluble and receptor (IFNγR1)-bound forms of IFNγ. Because NI-0501 is a human IgG1, it retains the properties of this immunoglobulin isotype, including the ability to associate with Fcγ receptors and fix complement. IFNγ is one of the most potent and pleiotropic 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 last two decades, numerous studies have linked IFNγ to the pathogenesis and maintenance of inflammatory diseases (see, eg, Billiau A., "Interferon-gamma: biology and role in pathogenesis." Adv. Immunol. 1996; 62: 61-130; Schoenborn JR, Wilson CB., "Regulation of interferon-gamma during innate and adaptive immune responses.", Adv. Immunol., 2007; Zhang SY, Boisson-Dupuis S, Chapgier A, et al., "Inborn errors of interferon (IFN) - mediated immunity in
Humans: Insights into the reactive roles of IFN-alpha/beta, IFN-gamma, and IFN-lambda in host defense. , Immunol. Rev. 2008; 226:29-40). As part of the innate immune response, IFNγ is primarily associated with natural killer (NK) and natural killer T (NKT) when antigen-specific immunity is generated by CD4 Th1 and CD8 cytotoxic T lymphocyte (CTL) effector T cells. Produced by cells.

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

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

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

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

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).

HLH can also occur during the course of infections, rheumatic or neoplastic diseases, in which case it is referred to as secondary HLH. Secondary HLH has the same signs and symptoms as the primary form and can be equally severe. Current treatments for secondary HLH are aimed at addressing the cause of the underlying disease. This is certainly the case for HLH caused by infections such as leishmaniasis. Of note, the presence of certain infections, particularly viral infections such as those caused by CMV or EBV, very often triggers manifestation of the primary form of HLH. This finding is also supported by evidence that infection with lymphocytic choriomeningitis virus (LCMV) is required for the development of the disease in animal models of primary 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年；１０４巻：７３５～７４３頁；Ｐａｃｈｌｏｐｎｉｋ ＳＪ、Ｈｏ ＣＨ、Ｃｈｒｅｔｉｅｎ Ｆら、Ｎｅｕｔｒａｌｉｚａｔｉｏｎ ｏｆ ＩＦＮｇａｍｍａ ｄｅｆｅａｔｓ ｈａｅｍｏｐｈａｇｏｃｙｔｏｓｉｓ ｉｎ ＬＣＭＶ－ｉｎｆｅｃｔｅｄ ｐｅｒｆｏｒｉｎ－ ａｎｄ Ｒａｂ２７ａ－ｄｅｆｉｃｉｅｎｔ ｍｉｃｅ．、ＥＭＢＯ Ｍｏｌ． Ｍｅｄ．、２００９年；１巻：１１２～１２４頁；Ｋｏｇｌ Ｔ、Ｍｕｌｌｅｒ Ｊ、Ｊｅｓｓｅｎ Ｂら、Ｈｅｍｏｐｈａｇｏｃｙｔｉｃ ｌｙｍｐｈｏｈｉｓｔｉｏｃｙｔｏｓｉｓ ｉｎ ｓｙｎｔａｘｉｎ -11-deficient mice: T-cell extrusion limits fatal disease., Blood., 2013;121:604-613; and Sepulveda FE, Debeume F, Menasche G, et al.
HLH in both human and murine mutants with complete loss of cytotoxic effectors PRF1, RAB27A, and STX11, Blood. 2013; 121:595-603).

When HLH manifests itself during neoplastic disease, particularly hematologic malignancies, often the severity of the patient's condition requires immediate treatment of HLH before specifically addressing the underlying disease.

The presence of HLH signs and symptoms in patients with rheumatic diseases such as systemic juvenile idiopathic arthritis (sJIA) and systemic lupus erythematosus (SLE) is often described by rheumatologists as macrophage activation syndrome ( MAS) and may precede the appearance of the rheumatic disease itself. The majority of patients with MAS have impairments in NK and perforin function tests, and a significant number of patients exhibit polymorphic or heterozygous mutations in PRF1 and UNC13D. MAS is a very serious and life-threatening condition that usually resolves when appropriate treatment, most often consisting of corticosteroids and cyclosporine, is initiated. 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ら、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).

Although primary HLH is primarily understood as a childhood disease, HLH is a condition that can be seen in adults, and with increasing awareness, it may occur more frequently than previously understood. It is shown that there is In the majority of adult patients, the disease occurs between malignancies (mainly non-Hodgkin's lymphoma), infections, autoinflammatory or autoimmune diseases and iatrogenic immune deficiencies.

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

Management of patients with primary HLH currently includes the following steps (Henter et al., Blood Cancer, 2007): (i) corticosteroids and immunosuppressants (e.g., etoposide, CsA, alemtuzumab, antithymocyte globulin); (ii) maintenance treatment until transplantation; and (iii) all patients with identified genetic defects and, ultimately, no disease-associated mutations. Transplantation in cases of very severe HLH.

A major goal of induction therapy is to suppress the life-threatening inflammatory processes that characterize HLH, thereby permitting transplantation in patients in need of transplantation (Horne A, Janka G, Maarten ER et al. Haematopoietic stem Br. J. Haematol., 2005;129:622-630). Transplantation is the only curative treatment for HLH associated with highly penetrant genetic mutations (Henter et al., Blood, 2002).

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

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

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

All mutations in genes that characterize the primary form of HLH affect proteins involved in the same process, ultimately impairing cytotoxic activity. Perforin mutations were the first to be identified in HLH patients.

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

Neutralization of high circulating levels of IFNγ by administration of anti-IFNγ antibodies was demonstrated not only to reverse clinical and laboratory abnormalities, but also to dramatically improve survival. In contrast, ablation of any other cytokine had no effect on survival (Jordan et al., Blood, 2004; Pachlopnik et al., EMBO Mol. Med., 2009).

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

To examine the physiopathology of secondary HLH that occurs in the context of rheumatic disease, high levels of HLH, similar to those occurring in patients with sJIA, the rheumatic disease most frequently associated with secondary forms of HLH, were examined. An animal model was generated using IL-6 transgenic mice that express IL-6. When challenged with Toll-like receptor (TLR) ligands, these mice die with many of the hallmarks 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 & 6 Rheumatism, 2012;604:186-18). In these mice, administration of anti-IFNγ antibodies to neutralize IFNγ markedly improved survival and restored laboratory parameters (Prencipe G et al., manuscript in preparation).

The high circulating IFNγ levels in patients with primary HLH further reinforce 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), especially 53.5% above 1000 pg/mL. had a level. It has also been reported that IFNγ levels rise rapidly initially and can decline from >5000 pg/mL to normal in 48 hours after effective treatment of HLH.

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

Since hypercytokinemia and tissue infiltration by activated lymphocytes and histiocytes are involved in all HLH symptoms and are dependent on CD8+ T cell hyperactivity and high IFNγ levels, neutralization of IFNγ is a rational treatment. Configure method. Indeed, there are currently no agents specifically targeting CD8+ T cells available, and targeting individual cytokines downstream of IFNγ is not always feasible.

Thus, data from animal models of primary and secondary HLH and both patients with primary and secondary HLH confirm the pivotal role played by IFNγ in the pathogenesis of this disease. Neutralization of IFNγ provides a robust rationale for developing targeted therapies against HLH that should be effective with no or limited toxicity, based on data from observations made in .

The present disclosure is compositions and methods useful in identifying, or alternatively pinpointing, patient populations afflicted with a disorder, wherein the patient receives CXCL9 alone or one or more additional interferons Also provided are compositions and methods having elevated levels of combinations with gamma (IFNγ)-related biomarkers. In particular, the present disclosure provides compositions for detecting CXCL9 levels as a biomarker for IFNγ production in hemophagocytic lymphohistiocytosis (HLH), secondary HLH, and/or macrophage activation syndrome (MAS) and provide a way.

A body of evidence in animal models points to a central pathogenic role for IFNγ in primary hemophagocytic lymphohistiocytosis (HLH). High levels of IFNγ are also found in humans with HLH. High levels of IFNγ and three IFNγ-related chemokines, CXCL9, CXCL10 and CXCL11, were observed in patients with active MAS, a form of secondary HLH that occurs in the context of systemic juvenile idiopathic arthritis (sJIA) have been previously reported (e.g., Bracaglia C., Caiello
I, De Graaf K.; et al., Pediatric Rheumatology, 2014, 12 (Supplement No. 1): O3). Indirect evidence in mice suggests that IFNγ may be produced predominantly in peripheral tissues, with relatively low blood levels.

The term macrophage activation syndrome (MAS) refers to a severe and 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 this syndrome during the course of the disease. MAS can also occur in systemic lupus erythematosus, Kawasaki disease, and other autoimmune and autoinflammatory disorders, albeit less commonly. In sJIA, MAS generally occurs during disease activity, including at the onset of the disease. Infectious triggers can be identified in a high percentage of patients. Typical features of MAS include fever, splenomegaly, hemorrhage, and signs of liver, central nervous system and kidney involvement that can lead to multiple organ failure. Laboratory abnormalities include decreased leukocytes, platelets, and hemoglobin, hypertransaminasemia, marked increases in ferritin, and evidence of intravascular activation of the coagulation system (Ravelli, A. et al., Macrophage activation syndrome as part of systemic juvenile idiopathic arthritis: diagnosis, genetics, pathophysiology and treatment., Genes Immun., 13(4):289-98). MAS causes significant morbidity and mortality accounting for a relevant portion of deaths attributed to sJIA (Minoia, F., et al., Clinical features, treatments, and outcomes of macrophage activation syndrome complicating systematic juvenile id: ａ ｍｕｌｔｉｎａｔｉｏｎａｌ， ｍｕｌｔｉｃｅｎｔｅｒ ｓｔｕｄｙ ｏｆ ３６２ ｐａｔｉｅｎｔｓ．、Ａｒｔｈｒｉｔｉｓ Ｒｈｅｕｍａｔｏｌ、２０１４年、６６巻（１１号）：３１６０～９頁；Ｈａｓｈｋｅｓ， Ｐ．Ｊ．ら、Ｍｏｒｔａｌｉｔｙ ｏｕｔｃｏｍｅｓ ｉｎ ｐｅｄｉａｔｒｉｃ ｒｈｅｕｍａｔｏｌｏｇｙ ｉｎ ｔｈｅ ＵＳ．、Ａｒｔｈｒｉｔｉｓ Ｒｈｅｕｍ、２０１０ 62(2):599-608). A better understanding of disease pathogenesis, along with the resulting identification of new therapeutic targets and the potential development of targeted therapies, may lead to significant improvements in the management and outcome of MAS.

MAS shares most of the clinical features and laboratory abnormalities of hemophagocytic lymphohistiocytosis (HLH) and indeed is currently classified among secondary or reactive HLH (sec-HLH). (Jordan, MB et al., How I treat hemophagocytic lymphohistiocytosis. Blood, 2011, 118(15):4041-52). The primary form of HLH (p-HLH) is involved in granule exocytosis, including PRF1, UNC13D, STXBP2, STX11, RAB27A and XIAP, generally leading to defective cytotoxic activity of CD8+ lymphocytes and NK cells caused by mutations in genes that encode proteins that According to current classification, HLH is defined as secondary or reactive when there is no identifiable genetic cause and/or familial inheritance. sec-HLH may occur in the absence of demonstrable triggers or in the context of infection, malignancy or rheumatic disease, the latter commonly referred to as MAS. The genetic basis for the development of MAS is increasingly being elucidated, and several studies have shown heterozygous MAS, and sec-HLH in general, for low-penetrance variants or mutations in the same causative genes as p-HLH.性との関連性が示されている（Ｋａｕｆｍａｎ， Ｋ．Ｍ．ら、Ｗｈｏｌｅ－ｅｘｏｍｅ ｓｅｑｕｅｎｃｉｎｇ ｒｅｖｅａｌｓ ｏｖｅｒｌａｐ ｂｅｔｗｅｅｎ ｍａｃｒｏｐｈａｇｅ ａｃｔｉｖａｔｉｏｎ ｓｙｎｄｒｏｍｅ ｉｎ ｓｙｓｔｅｍｉｃ ｊｕｖｅｎｉｌｅ ｉｄｉｏｐａｔｈｉｃ ａｒｔｈｒｉｔｉｓ ａｎｄ ｆａｍｉｌｉａｌ ｈｅｍｏｐｈａｇｏｃｙｔｉｃ ｌｙｍｐｈｏｈｉｓｔｉｏｃｙｔｏｓｉｓ．、Ａｒｔｈｒｉｔｉｓ Ｒｈｅｕｍａｔｏｌ、２０１４年、６６巻（１２号）：３４８６～９５頁；Ｖａｓｔｅｒｔ， Ｓ．Ｊ．ら、Ｍｕｔａｔｉｏｎｓ ｉｎ ｔｈｅ ｐｅｒｆｏｒｉｎ ｇｅｎｅ ｃａｎ ｂｅ ｌｉｎｋｅｄ ｔｏ ｍａｃｒｏｐｈａｇｅ ａｃｔｉｖａｔｉｏｎ ｓｙｎｄｒｏｍｅ ｉｎ ｐａｔｉｅｎｔｓ ｗｉｔｈ ｓｙｓｔｅｍｉｃ ｏｎｓｅｔ ｊｕｖｅｎｉｌｅ ｉｄｉｏｐａｔｈｉｃ ａｒｔｈｒｉｔｉｓ．、Ｒｈｅｕｍａｔｏｌｏｇｙ（Ｏｘｆｏｒｄ）、２０１０年、４９巻（３号）：４４１～９頁．；Ｚｈａｎｇ， Ｋ．ら、Ｍａｃｒｏｐｈａｇｅ ａｃｔｉｖａｔｉｏｎ ｓｙｎｄｒｏｍｅ ｉｎ ｐａｔｉｅｎｔｓ ｗｉｔｈ ｓｙｓｔｅｍｉｃ ｊｕｖｅｎｉｌｅ ｉｄｉｏｐａｔｈｉｃ ａｒｔｈｒｉｔｉｓ ｉｓ ａｓｓｏｃｉａｔｅｄ ｗｉｔｈ ＭＵＮＣ １３－４ ｐｏｌｙｍｏｒｐｈｉｓｍｓ．、Ａｒｔｈｒｉｔｉｓ Ｒｈｅｕｍ、２００８年、５８巻（９号）：２８９２～ and Zhang, M. et al., Genetic defects in cytolysis in macrophage activation syndrome Curr Rheumatol Rep 2014, 16(9):439; family hemophagocytic lymphocytosis (FHL) related genes and abnormalities of cytotoxicity function tests in patients with macrophage activation syndrome (MAS) occurring in systemic juvenile idiopathic. , Pediatric Rheumatology, 2014, 12 (Supplement 1):53). These genetic background similarities between p-HLH and MAS further support a shared pathogenic mechanism.

Studies in patients with p-HLH and studies in mouse models of p-HLH show defective silencing and abnormalities of the immune response due to defective cytotoxic activity and abnormal antigen presenting cell (APC)-CD8+ T cell crosstalk. It supports the hypothesis that a significant T-cell activation is induced. This leads to uncontrolled immune activation and production of proinflammatory cytokines by T lymphocytes and macrophages, leading to organ damage. Studies in animal models of p-HLH performed in perforin-deficient and Rab27-deficient mice demonstrate a pivotal role for interferon-gamma (IFNγ) produced by activated CD8+ T cells. In perforin-deficient mice, neutralization of IFNγ leads to reversal of biochemical and hematologic abnormalities along with survival of another lethal syndrome (Jordan, MB et al., Animal model of hemophagocytic lymphohistiocytosis). ＨＬＨ）： ＣＤ８＋Ｔ ｃｅｌｌｓ ａｎｄ ｉｎｔｅｒｆｅｒｏｎ ｇａｍｍａ ａｒｅ ｅｓｓｅｎｔｉａｌ ｆｏｒ ｔｈｅ ｄｉｓｏｒｄｅｒ．、Ｂｌｏｏｄ、２００４年、１０４巻（３号）：７３５～４３頁；Ｐａｃｈｌｏｐｎｉｋ Ｓｃｈｍｉｄ， Ｊ．ら、Ｎｅｕｔｒａｌｉｚａｔｉｏｎ ｏｆ ＩＦＮｇａｍｍａ ｄｅｆｅａｔｓ ｈａｅｍｏｐｈａｇｏｃｙｔｏｓｉｓ ｉｎ ＬＣＭＶ－ｉｎｆｅｃｔｅｄ ｐｅｒｆｏｒｉｎ－ and Rab27a-deficient mice., EMBO Mol Med, 2009, 1(2):112-24). In Rab27-deficient mice, whose disease does not lead to death, IFNγ neutralization causes marked improvement in peripheral organ involvement, including the central nervous system (Pachlopnik, 2009). High circulating IFNγ levels have also been found in patients with HLH diagnosed according to the HLH 2004 diagnostic guidelines (My, LT et al., Comprehensive analyzes and characterization of haemophagocytic lymphocytosis in Vietnam children, 2010, Berlin, Germany). 148(2): 301-10; Takada, H. et al., Increased serum levels of
interferon-gamma-inducible protein 10 and monokine induced by gamma interferon in patients with haemophagocytic lymphocytosis. , 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 haemophagocytic syndrome. Br J Haematol, 2008, 143(1):84-91; Xu, X.; J. et al., Diagnostic accuracy of a specific cytokine pattern in hemophagocytic lymphohistiocytosis in children. , J Pediatr, 2012, 160(6):984-90e1), and thus are not necessarily based on the presence of a gene mutation. It should be noted that these trials included a variable but significant proportion of patients without a demonstrable genetic cause (Ibid).

Serum levels of IFNγ and three IFNγ-related chemokines with themselves and disease activity in patients with active MAS to search for biomarkers of in vivo production of IFNγ. The study presented herein was designed to assess the correlation of . Specifically, circulating levels of IFNγ, CXCL9, CXCL10, CXCL11 and IL-6 were measured in patients with sJIA, approximately 37% (20 of 54) of patients had MAS at sampling. Relationships between circulating levels and disease activity parameters were also assessed, as were correlations between levels of IFNγ and levels of CXCL9, CXCL10 and CXCL11. In some embodiments, the biomarker is total IFNγ levels, which are useful as pharmacodynamic biomarkers.

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

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

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

The present disclosure uses a neutralizing anti-IFNγ antibody or antigen-binding fragment thereof to treat inflammatory conditions associated with graft failure, graft rejection, and/or solid organ transplant failure, acute bone marrow graft rejection, and the like. Kind Code: A1 Compositions and methods for treating, preventing and/or delaying the onset or progression of, or ameliorating symptoms associated with the disorder are provided. The present disclosure uses a neutralizing anti-IFNγ antibody or antigen-binding fragment thereof to treat graft-versus-host disease (GvHD) in a subject who has undergone or has undergone a transplant or series of transplants containing biological material. The present invention provides compositions and methods for treating, inhibiting, delaying the progression of, or alternatively ameliorating the symptoms of ). The present disclosure provides compositions and methods that use neutralizing anti-IFNγ antibodies or antigen-binding fragments thereof to extend the survival of implanted biological material. The present disclosure uses neutralizing anti-IFNγ antibodies or antigen-binding fragments thereof to treat conditions associated with paraneoplastic cerebellar degeneration, hemorrhagic fever, sarcoidosis, adult-onset Still's disease or CART-T cell therapy Compositions and methods for preventing, preventing, and/or delaying the onset or progression of, or ameliorating the same are provided.

The compositions and methods provided herein can be used, for example, in cells, tissue(s), bone marrow, and/or organs including, by way of non-limiting example, heart, kidney, pancreas, liver, and/or intestine. Useful in the implantation of any biological material, including the(s). In some embodiments, the implanted biomaterial is an allogeneic biomaterial. In some embodiments, the implanted biological material is bone marrow. In some embodiments, the implanted biomaterial is a population of hematopoietic stem cells. In some embodiments, the implanted biological material is or is derived from one or more hepatocytes.

The compositions and methods provided herein treat, prevent, and treat symptoms associated with graft failure, graft rejection, and/or inflammatory disorders associated with graft rejection in subjects in need thereof. and/or delay the onset or progression of or alleviate it. In some embodiments, graft rejection, also referred to herein as graft failure, is acute. In some embodiments, graft rejection is hyperacute.

表１Ｂ．ＩＦＮγに結合し、それを中和する抗体クローン由来のＶＬ ＣＤＲ配列

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

Table 1B. VL CDR sequences from antibody clones that bind and neutralize IFNγ

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

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

Suitable anti-IFNγ antibodies include those described in US Pat. 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_C9 (“C9”), among others. 2R3P2_C10 ("C10"), ARC1.2R3P2_D3 ("D3"), ARC1.2R3P2_D6 ("D6"), 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.

In some embodiments, the IFNγ antibody is formatted in the IgG isotype. In some embodiments, the IFNγ antibody is formatted in the IgG1 isotype.

In some embodiments, the IFNγ antibodies of the invention specifically bind to human and/or cynomolgus monkey IFNγ, wherein the antibodies are NI-0501 antibody, A6 antibody, B4 antibody, B9 antibody, C9 antibody, C10 antibody, Binds to the same epitope as the D3, D6, D8, E1, F8, F9, G7, G9 and/or G10 antibodies.
method of treatment

The compositions and methods are useful in treating any of a variety of disorders associated with interferon-gamma (IFNγ) expression and/or activity, including aberrant IFNγ expression and/or activity. The compositions and methods of the present disclosure are useful in treating hemophagocytic lymphohistiocytosis (HLH). HLH is associated with clinical signs and symptoms of extreme inflammation (fever, splenomegaly, cytopenia, coagulopathy) that lead to the development of aberrant immune-mediated pathologies 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 lymphocytosis., Blood, 1991). 78:2918-2922). HLH includes primary (genetic/familial) HLH and secondary HLH.

Primary HLH is a heterogeneous autosomal recessive genetic disorder that occurs mostly in infancy and early childhood, with an estimated prevalence of 1 in 50,000 live births in Europe (Janka GE : Familial hemophagocytic lymphohistiocytosis., Eur. J. Pediatr., 1983, 140:221-230). The disease is invariably fatal and, without treatment, median survival is less than 2 months after onset of symptoms (Filipovich AH: Hemophagocytic lymphohistiocytosis (HLH) and related disorders. Hematology Am Soc Hematol Educ Prog 9 Year: pp. 127-131).

Genetic defects in primary HHL all encode proteins for perforin synthesis, cytolytic granule maturation, granule exocytosis and release granule exocytosis or NK required to eliminate activated macrophages. Affecting genes involved in the cytotoxic pathway of cells and/or cytotoxic lymphocytes (Filipovich, A., K. McClain, and A. Grom., 2010, Histiocytic disorders: recent insights into pathophysiology and practical guidelines , Biol., Blood Marrow Transplant., 16 (1 Supplement): S82-S89). In approximately 20-40% of patients with primary HLH, the cytotoxic dysfunction that characterizes HLH syndrome is a cytolytic protein of cytotoxic granules that is a key regulator of T-cell and natural killer cell-mediated cytolysis. It results from mutations in the gene encoding perforin (PRF1). 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. In addition, some immunodeficiency syndromes such as Glyceri syndrome type 2 (GS-2) and Chediak-Higashi syndrome (CHS) are frequently present with HLH (Janka GE, Lehmberg K: Hemophagocytic lymphohistiocytosis: pathogenesis and treatment ., Hematology Am Soc Hematol Educ
Program, 2013, 2013:605-611).

Secondary forms of HLH can occur during the course of infections, autoimmune/rheumatic diseases, or in association with malignancies. Secondary forms display the same signs and symptoms as primary HLH and can be equally severe.

The compositions and methods of the present disclosure are useful in treating secondary HLH. The compositions and methods of this disclosure are useful in treating macrophage activation syndrome (MAS).

MAS is a severe, potentially life-threatening complication of rheumatic disease and is caused by excessive activation and expansion of T lymphocytes and macrophages. Uncontrolled expansion of these immune cells leads to marked hypercytokinemia and a hyperinflammatory state with fever, cytopenia, hepatosplenomegaly, liver dysfunction, coagulopathy and hyperferritinemia, May 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. 2015:45-661). ). MAS is classified among the secondary or acquired forms of HLH because of its strong clinical and pathological similarities to HLH. Indeed, it was recently demonstrated that the majority of patients with MAS have impairments in 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, 16:439).

MAS occurs most frequently in patients with sJIA, and more rarely in patients with systemic lupus erythematosus (SLE), albeit less commonly, in patients with vasculitis, especially Kawasaki disease. It is Overt MAS develops in approximately 7-17% of patients with SJIA (Sawhney S, Woo P, Murray KJ: Macrophage activation syndrome: a potentially fatal complication of rheumatic disorders., 2000, Arch. Ild. Dis. 85:421-426; Moradinejad MH, Ziaee V: The incidence
of macrophage activation syndrome in children with rheumatic disorders. , Minerva
Pediatr. 2011, 63:459-466), some evidence suggests that asymptomatic MAS may be seen in as many as one-third of patients with active systemic disease (Behrens et al. EM, Beukelman T, Paessler M, Cron RQ: Occult macrophage activation syndrome in patients with systematic juvenile idiopathic arthritis., J. Rheumatol., 2007, 34:1383-1383).

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

Different sets of criteria have been proposed for the diagnosis of MAS in patients with sJIA. HLH-2004 diagnostic guidelines developed primarily for the primary (hereditary) form of HLH (Henter J, Horne A, Arico M, Egeler RM, et al.: HLH-2004: Diagnostic and therapeutic guidelines for hemophagocytic lympho- dialysis . Blood Cancer, 2007, 48:124-131) are sometimes recommended. However, they present some limitations and do not apply to patients with sJIA. For example, these patients often have elevated white blood cell and platelet counts as part of the sJIA inflammatory response, as well as elevated serum levels of fibrinogen, thus reducing cytopenias below the threshold required by HLH-2004. Criteria such as hypertension and hypofibrinogenemia 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. 2005, Med. 66:145-159). ＭＡＳを有する患者の有意な割合で、診察時には血球貪食が存在しない可能性がある（Ｍｉｎｏｉａ Ｆ、Ｄａｖｉ Ｓ、Ｈｏｒｎｅ Ａ、Ｄｅｍｉｒｋａｙａ Ｅら：Ｃｌｉｎｉｃａｌ ｆｅａｔｕｒｅｓ， ｔｒｅａｔｍｅｎｔ， ａｎｄ ｏｕｔｃｏｍｅ ｏｆ ｍａｃｒｏｐｈａｇｅ ａｃｔｉｖａｔｉｏｎ ｓｙｎｄｒｏｍｅ ｃｏｍｐｌｉｃａｔｉｎｇ ｓｙｓｔｅｍｉｃ ｊｕｖｅｎｉｌｅ ｉｄｉｏｐａｔｈｉｃ arthritis: a multinational, multicenter study of 362 patients., Arthritis & rheumatology (Hoboken, NJ), 2014, 66:3160-3169). Furthermore, hemophagocytosis, NK cell activity and sCD25 are not routinely assessed for MAS.

An alternative approach is based on the application of Preliminary Diagnosis Guidelines (PDG) for MAS comorbid with sJIA, generated by an analysis comparing a cohort of patients with MAS to a group of patients with relapse of sJIA1.

Recently, HLH-2004 diagnostic and preliminary diagnostic guidelines for sJIA-associated MAS were compared in a large patient population for their ability to distinguish between sJIA/MAS and sJIA (in the absence of MAS) and systemic infection (Davi S. 、Ｍｉｎｏｉａ Ｆ、Ｐｉｓｔｏｒｉｏ Ａ、Ｈｏｒｎｅ Ａら：Ｐｅｒｆｏｒｍａｎｃｅ ｏｆ ｃｕｒｒｅｎｔ ｇｕｉｄｅｌｉｎｅｓ ｆｏｒ ｄｉａｇｎｏｓｉｓ ｏｆ ｍａｃｒｏｐｈａｇｅ ａｃｔｉｖａｔｉｏｎ ｓｙｎｄｒｏｍｅ ｃｏｍｐｌｉｃａｔｉｎｇ ｓｙｓｔｅｍｉｃ ｊｕｖｅｎｉｌｅ ｉｄｉｏｐａｔｈｉｃ ａｒｔｈｒｉｔｉｓ．、Ａｒｔｈｒｉｔｉｓ ＆ ｒｈｅｕｍａｔｏｌｏｇｙ（Ｈｏｂｏｋｅｎ、Ｎ．Ｊ．）、２０１４年、６６巻：２８７１～２８８０頁). Despite some limitations due to its retrospective nature, this test provides the best balance of sensitivity and specificity and best agreement with diagnoses made by treating physicians according to preliminary MAS guidelines. It seems to indicate that The sensitivity of the HLH-2004 reference set was <30%. Nonetheless, the proportion of patients meeting each single PDG criterion is highly variable, and several clinical features (e.g., CNS dysfunction and hemorrhage) may manifest later in MAS. 、それにより、初期ＭＡＳではそれらの感度が低くなることも報告されている（Ｌｅｈｍｂｅｒｇ Ｋ、Ｐｉｎｋ Ｉ、Ｅｕｌｅｎｂｕｒｇ Ｃ、Ｂｅｕｔｅｌ Ｋら：Ｄｉｆｆｅｒｅｎｔｉａｔｉｎｇ ｍａｃｒｏｐｈａｇｅ ａｃｔｉｖａｔｉｏｎ ｓｙｎｄｒｏｍｅ ｉｎ ｓｙｓｔｅｍｉｃ ｊｕｖｅｎｉｌｅ ｉｄｉｏｐａｔｈｉｃ ａｒｔｈｒｉｔｉｓ ｆｒｏｍ ｏｔｈｅｒ ｆｏｒｍｓ ｏｆ ｈｅｍｏｐｈａｇｏｃｙｔｉｃ ｌｙｍｐｈｏｈｉｓｔｉｏｃｙｔｏｓｉｓ．、 The Journal of
pediatrics, 2013, 162:1245-1251).

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

9 variables (3 clinical variables [i.e. known underlying immunosuppression, hyperthermia, organ hypertrophy], 5 biological variables [i.e. triglycerides, ferritin, serum glutamate oxaloacetate transaminase, fibrinogen levels, and cytopenia], and one cytological variable [i.e., hemophagocytic characteristics on bone marrow aspirates]) were retained in HScore, and the probability of having hemophagocytic syndrome increased from <1% with HScore ≤ 90 to HScore >250 and >99%.

Until a definitive consensus on validated diagnostic criteria for MAS is achieved, expert clinical diagnosis remains critical to the challenge of distinguishing MAS from conditions with overlapping characteristics such as relapses of SJIA or sepsis-like syndromes. is.

There are currently no drugs approved for the treatment of MAS. High-dose glucocorticoids are usually the treatment of choice for MAS. In patients who fail to respond to glucocorticoids, cyclosporine A (CsA) has been 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 informatics disorders. A retrospective study of
24 patients. , Rheumatology (Oxford, England), 2001, 40:1285-1292).

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

The utility of biologics that inhibit the IL-1, IL-6R or TNFα pathways in treating MAS remains unclear. Biologics that inhibit these pathways have been reported to be effective in isolated cases, but only a few patients develop MAS in the setting of these treatments (Stern A, Riley R, Buckley L: Worsening of macrophage activation). ｓｙｎｄｒｏｍｅ ｉｎ ａ ｐａｔｉｅｎｔ ｗｉｔｈ ａｄｕｌｔ ｏｎｓｅｔ Ｓｔｉｌｌ'ｓ ｄｉｓｅａｓｅ ａｆｔｅｒ ｉｎｉｔｉａｔｉｏｎ ｏｆ ｅｔａｎｅｒｃｅｐｔ ｔｈｅｒａｐｙ．、Ｊ Ｃｌｉｎ Ｒｈｅｕｍａｔｏｌ、２００１年、７巻：２５２～２５６頁；Ｒａｍａｎａｎ ＡＶ、Ｓｃｈｎｅｉｄｅｒ Ｒ：Ｍａｃｒｏｐｈａｇｅ ａｃｔｉｖａｔｉｏｎ ｓｙｎｄｒｏｍｅ ｆｏｌｌｏｗｉｎｇ ｉｎｉｔｉａｔｉｏｎ ｏｆ ｅｔａｎｅｒｃｅｐｔ
in a child with systematic onset juvenile
rheumatoid arthritis. , J. Rheumatol. 2003, 30:401-403; De Benedetti F, Brunner HI, Ruperto N, Kenwright A, et al.: Randomized trial of tocilizumab in systematic 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 systematic juvenile idiopathic arthritis. , N. Engl. J. Med. 2012, 367:2396-2406), as well as patients who did not respond to these treatments, whereby inhibition of IL-1, IL-6R or TNFα completely protected against MAS development. have been shown to provide no effective treatment for end-stage syndrome.

A large retrospective multicenter study investigated the clinical, laboratory, and histopathologic characteristics of MAS/sJIA and current treatments and outcomes in a total of 362 patients (Minoia F, Davi S, Horne A 、Ｄｅｍｉｒｋａｙａ Ｅら：Ｃｌｉｎｉｃａｌ ｆｅａｔｕｒｅｓ， ｔｒｅａｔｍｅｎｔ， ａｎｄ ｏｕｔｃｏｍｅ ｏｆ ｍａｃｒｏｐｈａｇｅ ａｃｔｉｖａｔｉｏｎ ｓｙｎｄｒｏｍｅ ｃｏｍｐｌｉｃａｔｉｎｇ ｓｙｓｔｅｍｉｃ ｊｕｖｅｎｉｌｅ ｉｄｉｏｐａｔｈｉｃ ａｒｔｈｒｉｔｉｓ： ａ ｍｕｌｔｉｎａｔｉｏｎａｌ， ｍｕｌｔｉｃｅｎｔｅｒ ｓｔｕｄｙ ｏｆ ３６２ ｐａｔｉｅｎｔｓ．、Ａｒｔｈｒｉｔｉｓ ＆ ｒｈｅｕｍａｔｏｌｏｇｙ（Ｈｏｂｏｋｅｎ、Ｎ．Ｊ．）、２０１４年、６６巻： 3160-3169). At disease onset, MAS occurred in the setting of active sJIA in approximately half of the patients or during sJIA flares in 30%. Infectious triggers were identified in one-third of patients. Among the 24 patients for whom the type of infection was reported, EBV was the most common causative factor (25%). MAS appeared to be associated with treatment side effects in 11 patients (3.8%), 8 of whom had IL-6 (N=4), IL-1 (N=3) or TNFα (N=1 ) with pathway-targeting biological agents. Glucocorticoids were given to nearly all patients. Cyclosporine, biologics and etoposide were given to 61%, 15% and 12% of patients, respectively.

Therefore, identification of effective therapeutic regimens for MAS is an area of high unmet medical need. More than 50% of patients with sJIA and MAS either do not respond to systemic glucocorticoids alone or may require long-term treatment at high doses, with significant associated morbidity. Good evidence-based data on the efficacy of additional treatments such as CsA or etoposide are not available if patients fail to respond to glucocorticoids. The MAS process rapidly becomes irreversible and can lead to fatal outcomes. Current data suggest that the mortality rate for sJIA-associated MAS is 8%, with approximately one-third of patients requiring admission to the ICU. Recent findings on the central role of IFNγ in disease pathogenesis suggest that IFNγ blockade potentially represents a novel therapeutic target.

Compositions and methods, including the NI-0501 compositions of the present disclosure, are advantageous over current treatments for primary and secondary HLH.

MAS and HLH are characterized by sustained immune cell activation and a cytokine storm of proinflammatory cytokines with concomitant overproduction of IFNγ, TNFα, IL-1 and IL-6 (Henter JI, Elinder G. Soder
O, Hansson M, et al.: Hypercytokinemia in family hemophagocytic lymphohistiocytosis. , Blood 1991, 78:2918-2922; Imashuku S, Hibi S, Fujiwara F, Todo S: Hyper-interleukin (IL)-6-naemia in haemophagocytic lymphocytosis. , 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年、１６０巻：９８４～９０頁ｅ１；Ｐｕｔ Ｋ、Ａｖａｕ Ａ、Ｂｒｉｓｓｅ Ｅ、Ｍｉｔｅｒａ Ｔら：Ｃｙｔｏｋｉｎｅｓ ｉｎ ｓｙｓｔｅｍｉｃ ｊｕｖｅｎｉｌｅ ｉｄｉｏｐａｔｈｉｃ ａｒｔｈｒｉｔｉｓ ａｎｄ ｈａｅｍｏｐｈａｇｏｃｙｔｉｃ ｌｙｍｐｈｏｈｉｓｔｉｏｃｙｔｏｓｉｓ：ｔｉｐｐｉｎｇ ｔｈｅ ｂａｌａｎｃｅ ｂｅｔｗｅｅｎ ｉｎｔｅｒｌｅｕｋｉｎ－１８ ａｎｄ ｉｎｔｅｒｆｅｒｏｎ－γ． , Rheumatology (Oxford), 2015).ここ数年の間に、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年、１０４ Volume: 735-743; Pachlopnik
Schmid J, Ho C, Chretien F, Lefebvre JM et al.: Neutralization of IFNgamma defeats haemophagocytosis in LCMV-infected perforin- and Rab27a-deficient mice. EMBO Mol Med, 2009, 1:112-124; Zoller EE, Lykens JE, Terrell CE, Aliberti J, et al.: Hemophagocytosis causes a consumptive anemia of inflammation. , J. Exp. Med. 2011, 208: 1203-1214) and MAS (Behrens EM, Canna SW, Slade K, Rao S et al.: Repeated TLR9 stimulation results in macrophage activation syndrome, in J. Syndrome-like in. Disease. 2011, 121:2264-2277), evidence is accumulating supporting a central role for IFNγ in both development.

With respect to primary HLH, perforin knockout mice are considered a relevant model as they develop all of the diagnostic and many of the clinical and laboratory unique features of human disease upon infection with LCMV. be done. HLH-like diseases that develop in mice are dependent on CD8+ T cells and IFNγ produced in response to antigenic stimulation (Imashuku S, Hibi S, Fujiwara F, Todo S: Hyper-interleukin (IL)-6-naemia in haemophagocytic Lymphohistiocytosis., Br. J. Haematol., 1996, 93:803-807). It was demonstrated that neutralization of high circulating levels of IFNγ by administration of anti-IFNγ antibodies not only reversed clinical and laboratory abnormalities, but also dramatically improved survival. In contrast, removal of many other cytokines had no effect on 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 lymphocytosis in children. , J. Pediatr. 2012, 160:984-90 e1). The high circulating IFNγ levels found in these patients further strengthen the importance of IFNγ in HLH (Henter JI, Elinder G, Soder O, Hansson M, et al.: Hypercytokinemia in familial hemophagocytic lymphocytosis. Blood, 1991). ７８巻：２９１８～２９２２頁；Ｘｕ Ｘ、Ｔａｎｇ Ｙ、Ｓｏｎｇ Ｈ、Ｙａｎｇ Ｓら：Ｄｉａｇｎｏｓｔｉｃ ａｃｃｕｒａｃｙ ｏｆ ａ ｓｐｅｃｉｆｉｃ ｃｙｔｏｋｉｎｅ ｐａｔｔｅｒｎ ｉｎ ｈｅｍｏｐｈａｇｏｃｙｔｉｃ ｌｙｍｐｈｏｈｉｓｔｉｏｃｙｔｏｓｉｓ ｉｎ ｃｈｉｌｄｒｅｎ．、Ｊ． Ｐｅｄｉａｔｒ．、２０１２年、１６０巻：９８４～９０頁e1). In a series of 71 patients monitored from HLH diagnosis to treatment and follow-up, IFNγ levels were above the upper limit of normal (17.3 pg/mL) in all patients, notably 53.5% above 1000 pg/mL. had a higher level. It has also been reported that IFNγ levels rise rapidly initially and can decline from >5000 pg/mL to normal in 48 hours after effective treatment of HLH.

To elucidate the potential pathogenic role of IFNγ, two animal models of secondary HLH were investigated in the context of the NI-0501 development program. First, in a mouse model that mimics infection-driven HLH, repeated administration of CpG resulted in clinical (e.g., weight loss, splenomegaly) and laboratory characteristics (e.g., weight loss, splenomegaly) of HLH33 through activation of TLR9. hypercytokinemia leading to cytopenia, hyperferritinemia) was induced. Neutralization of IFNγ by administration of anti-IFNγ antibodies reversed the clinical and laboratory features of the disease. Neutralization of IFNγ was also shown to be complete in relevant target tissues such as liver and spleen. Interestingly, administration of anti-IFNγ antibodies revealed that the amount of IFNγ was 500-2,000-fold higher than the amount of IFNγ measured in the blood, likely more reflective of IFNγ production in tissues. Became. Two IFNγ-inducible chemokines (CXCL9 and CXCL10) were upregulated in both blood and liver after TLR9 stimulation, and a significant correlation was observed between serum levels of IFNγ and serum concentrations of CXCL9 and CXCL10. . Neutralization of IFNγ induced a significant reduction in serum CXCL9 and CXCL10 and 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).

Second, because animal models of IL-6 transgenic mice expressing high levels of IL-6 mimic the condition of patients with sJIA, the rheumatic disease most frequently associated with secondary forms of HLH, I tested this. Upon induction with Toll-like receptor (TLR) ligands, increased lethality, increased inflammatory cytokine production and hyperactivation of inflammatory signaling pathways were observed. In addition, these mice exhibited depressed platelet and neutrophil counts, elevated sCD25, ferritin and LDH levels, resembling many of the features commonly present in patients with MAS (Strippoli R, Carvello
F, Scianaro R, De Pasquale L, et al.: Amplification of the response to Toll-like receptor
Ligands by prolonged exposure to interleukin-6 in mice: Implication for the pathogenesis of macrophage activation syndrome. , Arthritis Rheum. 2012, 64:1680-1688). In these mice, administration of anti-IFNγ antibodies to neutralize IFNγ markedly improved survival and restored laboratory parameters (Prencipe G et al., manuscript in preparation).

Patients with secondary forms of HLH, either secondary to infection or of unknown origin (pHLH must have normal cytotoxic activity, absence of known gene mutations that cause pHLH, and no family history). Similar evidence was recently gathered in observational studies conducted in patients with MAS occurring in the setting of sJIA) or in the setting of sJIA.

Serum samples were analyzed during active end-stage disease and during disease remission in 14 patients with secondary HLH (7 of whom had an identifiable underlying infection). Levels of IFNγ, CXCL9 and CXCL10 were significantly higher in active phase 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 levels of CXCL9 (p=0.0018) and to a lesser extent with levels of CXCL10 (p=0.014). Levels of IFNγ and chemokines (especially CXCL9) correlated significantly with parameters of disease severity such as neutrophil and platelet counts, ferritin and ALT, thereby suggesting the pathogenic role of IFNγ in secondary HLH and chemokines (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 targets in secondary HLH).

Similar findings were shown in patients with MAS that occur in patients with sJIA. Serum concentrations of IFNγ, IFNγ-inducible chemokines (CXCL9, CXCL10, CXCL11) and IL-6 were measured in 54 patients with sJIA, 20 of whom had MAS. Levels of IL-6 were comparable in patients with end-stage MAS and those with active sJIA but no MAS at sampling. In contrast, circulating IFNγ and chemokine levels were significantly higher in MAS, particularly for CXCL9, with median levels approximately 15-fold higher than in patients with active sJIA without MAS (13392 vs. 837 pg/mL; p=0.005). Of note, only in patients with MAS did CXCL9 levels correlate with ferritin (p=0.041), neutrophil (p=0.010) and platelet (p=0.022) counts, ALT (p=0.022) = 0.044) and LDH (p = 0.013). Levels of IFNγ also correlated with laboratory parameters of disease severity, with the exception of LDH, for which statistical significance was not achieved (Bracaglia et al., manuscript in preparation).

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

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

The rationale for identifying this patient population as benefiting 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 relevant to MAS in sJIA showed that IFNγ neutralization markedly improved survival and reversed changes in laboratory parameters. Observational data in patients with MAS/sHLH then demonstrated the presence of high levels of IFNγ and, more importantly, extremely elevated levels of the IFNγ-inducible chemokines, CXCL9, CXCL10 and CXCL11. be done. Third, in patients with MAS/sHLH, IFNγ and CXCL9 concentrations are significantly correlated with disease parameters such as ferritin, platelet count and transaminases. Next, in previous studies in which all infusions administered were well tolerated, a favorable tolerability profile and no relevant safety concerns were observed in patients with pHLH. , this leads to observations made in healthy volunteers, that no infections caused by pathogens known to be beneficial for IFNγ neutralization have been reported, and that the number of infections that have occurred in some patients with pHLH has been reported. None were found to be related to treatment with NI-0501, but were found to be related to their immune status, disease duration and previous or concurrent treatments. Fifth, preliminary data from previous clinical trials have shown favorable effects on disease parameters, with visible effects occurring within the first day of treatment: typical clinical signs and symptoms of HLH. Improvement began rapidly after the first dose of NI-0501 (fever within hours, splenomegaly/hepatomegaly within days), and of the 18 evaluable patients at cutoff, NI-0501 was used. This procedure allowed 10 patients to be transferred to HSCT. Evidence from PK modeling and simulation approaches then demonstrates that a predictable pharmacokinetic profile of NI-0501 and neutralization of IFNγ is achieved and maintained. Finally, should NI-0501 fail to adequately control disease, conventional therapy (eg, CsA) can be started immediately without the need for a washout period.

In conclusion, based on preclinical and clinical evidence, there is a strong rationale for neutralizing IFNγ in MAS/sHLH secondary to rheumatic disease, and preliminary data in pHLH patients suggest that A favorable benefit-risk profile of NI-0501 with significant improvement for normalizing HLH features is demonstrated.

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

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

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

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

A therapeutically effective amount of an antibody of the invention generally relates to the amount necessary to achieve a therapeutic goal. As noted above, this may be the binding interaction of an antibody with its target antigen that interferes with target function in certain cases. The amount that needs to be administered will further depend on the binding affinity of the antibody for its specific antigen, as well as the rate at which the administered antibody is depleted from the free volume of other subjects to whom it is administered. Dependent. A general range for a therapeutically effective dosage of an antibody or antibody fragment of the invention can be, as a non-limiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight. Preferred doses include 1, 3, 6, 10 mg/kg body weight. Typical dosing frequencies can range, for example, from once daily to twice weekly. Treatment may last 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks or longer.

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 preparing 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 (Alfonso R. Gennaro et al., eds.) 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; Dekker, New York.

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

In one embodiment, an active compound, eg, an anti-IFNγ antagonist, is administered in combination therapy, ie, combined with one or more additional agents useful for treating a medical condition or disorder. The term "in combination" in this context means that the agents are given substantially simultaneously, simultaneously or sequentially. When given sequentially, it is preferred 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.

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

In some embodiments, the additional agent is an immunosuppressive agent. In some embodiments, the immunosuppressive agent is cyclosporin A (CsA). In some embodiments, the subject has received CsA prior to administration of NI-0501. In some embodiments, the additional agent comprises at least etoposide. In some embodiments, the subject has received etoposide prior to administration of NI-0501.

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

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

In some embodiments, the one or more additional agents are analgesic treatments, blood product transfusions, electrolyte and glucose infusions, antibiotics, antifungal and antiviral treatments and/or general supportive care. be.

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

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

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

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

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

A second dose is administered over one or more treatment periods. A second dose is administered over the first treatment period. A second dose is administered every three days after the first dose during the first treatment period. The first treatment period lasts about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks or longer. Preferably, the first treatment period is two weeks. Optionally, a second dose is administered over a second treatment period after completion of the first treatment period. A second dose is administered twice weekly during the second treatment period. The second treatment period is 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- Lasts 10 weeks, 4-10 weeks, 5-10 weeks, 6-10 weeks. The second treatment period is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks or longer.

In some embodiments, the second dose is increased or decreased during the course of the first or second treatment period. Increased or decreased doses are referred to as tertiary doses. A third dose can be 1.0, 3.0 or 6.0 mg per kg body weight. A third dose is administered over the remaining first and second treatment periods. Alternatively, a 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.

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

The first dose is 6.0 mg/kg body weight. The second dose is 3.0 mg/kg body weight. 10.0 mg per kg body weight.

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

A second dose is administered over one or more treatment periods. A second dose is administered over the first treatment period. A second dose is administered every three days after the first dose during the first treatment period. The first treatment period lasts about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks or longer. Preferably, the first treatment period is two weeks. Optionally, a second dose is administered over a second treatment period after completion of the first treatment period. A second dose is administered twice weekly during the second treatment period. The second treatment period is 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- Lasts 10 weeks, 4-10 weeks, 5-10 weeks, 6-10 weeks. The second treatment period is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks or longer.

In some embodiments, the second dose is increased or decreased during the course of the first or second treatment period. Increased or decreased doses are referred to as tertiary doses. Preferably, the third dose is 6.0 mg/kg body weight. A third dose is administered over the remaining first and second treatment periods. Alternatively, a 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.

A multiple varying dose regimen for treating secondary hemophagocytic lymphohistiocytosis (HLH) in adult humans includes an induction dose or first dose and a treatment dose or second dose of an anti-IFN-g monoclonal antibody. Including dose.

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

A second dose is administered over one or more treatment periods. A second dose is administered over the first treatment period. A second dose is administered every three days after the first dose during the first treatment period. The first treatment period lasts about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks or longer. Preferably, the first treatment period is two weeks. Optionally, a second dose is administered over a second treatment period after completion of the first treatment period. A second dose is administered twice weekly during the second treatment period. The second treatment period is 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- Lasts 10 weeks, 4-10 weeks, 5-10 weeks, 6-10 weeks. The second treatment period is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks or longer.

In some embodiments, the second dose is increased or decreased during the course of the first or second treatment period. Increased or decreased doses are referred to as tertiary doses. A third dose can be 1.0, 3.0 or 6.0 mg per kg body weight. A third dose is administered over the remaining first and second treatment periods. Alternatively, a 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.

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

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

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

A second dose is administered over one or more treatment periods. A second dose is administered over the first treatment period. A second dose is administered every three days after the first dose during the first treatment period. The first treatment period lasts about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks or longer. Preferably, the first treatment period is two weeks. Optionally, a second dose is administered over a second treatment period after completion of the first treatment period. A second dose is administered twice weekly during the second treatment period. The second treatment period is 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- Lasts 10 weeks, 4-10 weeks, 5-10 weeks, 6-10 weeks. The second treatment period is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks or longer.

In some embodiments, the second dose is increased or decreased during the course of the first or second treatment period. Increased or decreased doses are referred to as tertiary doses. A third dose can be 1.0, 3.0, 6.0 or 10.0 mg per kg body weight. A third dose is administered over the remaining first and second treatment periods. Alternatively, a 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.

In many variable dosing regimens according to the invention, the first and second time periods for the second dose can be adjusted from time to time, eg, based on the health of the subject. For example, time adjustments between doses can be adjusted ±1, 2, 3 or 4 days. ±2 days is preferred.

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

Doses are administered as a single injection. Antibodies are administered as monotherapy or combination therapy for the disease or condition being treated. For example, the subject is administered a second agent. A second agent is, for example, an anti-inflammatory agent and/or an immunosuppressive agent.

Optionally, the subject has been administered dexamethasone immediately prior to administration of the antibody. Dexamethasone is administered at a dose of at least 10 mg/ ^m2 . Alternatively, dexamethasone is administered at a dose of at least 5 mg/ ^m2 .

In some embodiments, the subject has not previously received treatment for HLH.
Pharmaceutical composition

The antibodies or soluble chimeric polypeptides of the invention (also referred to herein as "active compounds"), and derivatives, fragments, analogs and homologs thereof, 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 and all solvents, dispersion media, coatings, antibacterial and antifungal agents, tonicity and absorption agents compatible with pharmaceutical administration. Including retardants and the like. Suitable carriers include Remington's, a standard reference text in the field.
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, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as liposomes and fixed oils can also be used. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the composition is contemplated. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (ie, topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal or subcutaneous application may contain the following components: water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic 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, citric acid or phosphate. , and agents to adjust tonicity, such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. A parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

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

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. The composition must be stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols such as glycerol, propylene glycol, and liquid polyethylene glycols, and suitable mixtures thereof. Adequate 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 the action of microorganisms can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Inclusion of agents that delay absorption, such as aluminum monostearate and gelatin, in the composition can result in prolonged absorption of the injectable composition.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. can do. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying, in which a powder of the active ingredient plus any additional desired ingredient is obtained from a previously sterile-filtered solution thereof.

Preferred injectable pharmaceutical compositions include excipients such as g L-histidine, 3L-histidine monohydrochloride monohydrate, sodium chloride (NaCl), polysorbate 80 and the like.

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

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

In some aspects, the pharmaceutical composition is packaged in unit doses. Unit doses are packaged in containers. The container is a glass or plastic container. The container is a syringe, vial, infusion bottle, ampoule or carpoule. The container can hold a volume of 1-25 ml. For example, a container can hold a volume of 2, 5, 10 or 20 ml.

A unit dose of a fully human anti-interferon gamma (IFNγ) monoclonal antibody (eg, NI-501) is 5-25 mg/ml. Preferably the unit dose is 5 mg/ml or 25 mg/ml. Antibodies were prepared in a solution (e.g., water for injection ) inside.

In some embodiments, the invention provides a fully human anti-interferon gamma (IFNγ) monoclonal antibody at a concentration of 5 mg/ml or 25 mg/ml of antibody and a pH of the solution between 5.8 and 6.2. Provide a unit dose container with 20 ml of solution. The antibody is solubilized in solution, so the solution is clear, colorless and free of precipitate. Antibodies were prepared in a solution (e.g., water for injection ).

In other embodiments, the invention provides 10 ml or 20 ml of a fully human anti-interferon gamma (IFNγ) monoclonal antibody solution having a concentration of 25 mg/ml antibody and a pH of the solution between 5.8 and 6.2. a unit dose container comprising: The antibody is solubilized in solution, so the solution is clear, colorless and free of precipitate. Antibodies were prepared in a solution (e.g., water for injection ).

In another embodiment, the invention provides 2 ml or 10 ml of a fully human anti-interferon gamma (IFNγ) monoclonal antibody solution at a concentration of 5 mg/ml of antibody and a pH of the solution between 5.8 and 6.2. a unit dose container comprising: The antibody is solubilized in solution, so the solution is clear, colorless and free of precipitate. Antibodies were prepared in a solution (e.g., water for injection ).

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is orally applied and swirled in the mouth, expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, lozenges and the like may contain any of the following ingredients, or compounds of similar nature: binders such as microcrystalline cellulose, gum tragacanth 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; flavoring agents.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, eg a gas such as carbon dioxide, or a nebulizer.

Systemic administration can be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

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

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

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages for the subjects to be treated, each unit containing the desired pharmaceutical carrier in association with the required pharmaceutical carrier. It contains a predetermined quantity of active compound calculated to produce a therapeutic effect. The unit dosage form specifications of the present invention are defined by the unique properties of the active compounds and the particular therapeutic effects to be achieved, as well as limitations inherent in the art of formulating such active compounds to treat individuals. , which directly depends on it.

Pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
Detection of CXCL9 and other biomarkers

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

The term "biological sample" shall include tissues, cells and biological fluids isolated from a subject as well as tissues, cells and fluids present within a subject. Thus, use of the term "biological sample" includes blood and any fraction or component of blood, including serum, plasma, or lymph. Body fluids include, but are not limited to, blood, plasma, serum, synovial fluid, urine, sputum, spinal fluid, cerebrospinal fluid, pleural fluid, respiratory fluid, intestinal fluid, and genitourinary fluid, saliva, organ systems It can be a fluid isolated from anywhere within the subject, preferably from a peripheral location, including internal fluid, ascites, tumor cyst fluid, amniotic fluid, and combinations thereof. A biological sample also includes all experimentally isolated fractions of the aforementioned fluids. Biological samples also include solutions or mixtures containing homogenized solid materials, such as stool, tissue, and biopsy samples. The detection methods of the invention can be used to detect analyte mRNA, protein, or genomic DNA in biological samples in vitro as well as in vivo. For example, in vitro techniques for detection of analyte mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of analyte proteins include enzyme-linked immunosorbent assays (ELISA), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of analyte genomic DNA include Southern hybridizations. Procedures for immunoassay are described, for example, in "ELISA: Theory and Practice: Methods in Molecular Biology", Vol. 42, J. Am. R. Crowther (ed.) Human Press, Totowa, NJ, 1995; Diamandis and T. Christopoulus, Academic Press, Inc.; , San Diego, Calif., 1996; and "Practice and Theory of Enzyme Immunoassays," P.S. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Additionally, in vivo techniques for detecting an analyte protein include introducing into a subject a labeled anti-analyte protein antibody. For example, antibodies can be labeled with radioactive markers whose presence and location in a subject can be detected by standard imaging techniques.
Definition:

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

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

The term "multiple varying 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 therapy” describes a treatment schedule based on administering various amounts of an anti-IFN-gamma antibody at various times throughout the course of treatment. In one embodiment, the invention describes a multiple variable dose treatment method comprising an induction phase and a treatment phase, wherein the anti-FN-gamma antibody is administered at a higher or lower dose during the induction phase than during the treatment phase. . In another embodiment, the invention comprises an induction phase, a first treatment phase, and a second treatment phase, wherein the anti-IFN-gamma antibody is administered at a higher or lower dose during the induction phase than during the treatment phase. , describe a method of treatment with multiple varying doses. The anti-IFN-gamma antibody can be administered at a higher or lower dose during the first treatment period than during the second treatment period. Preferably, the dose is the same for the first and second treatment phases.

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

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

The terms "loading dose" or "loading dose" are used interchangeably herein and refer to the first dose of anti-IFN-gamma antibody, which is greater or lesser than the maintenance or treatment dose. . The induction dose can be a single dose or a set of doses. An induction dose is often used to bring the body's drug to a steady state amount and can be used to rapidly achieve maintenance drug levels. Following the induction dose, a smaller or larger dose of anti-IFN-gamma antibody, ie, a treatment dose, is administered. An induction dose is administered during the induction phase of treatment.

The terms "treatment phase" or "maintenance phase" as used herein refer to a period of treatment comprising administering an anti-IFN-gamma antibody to a subject to maintain the desired therapeutic effect. Point. The treatment phase follows the induction phase and thus begins once the threshold level is achieved. There can be more than one treatment phase, which is a treatment phase that is shortened or lengthened to maintain a certain threshold or achieve a desired clinical response. Preferably, in the treatment phase, the substance is administered every 3 days after the induction dose (eg the initial dose). Alternatively, the substance is administered once or twice weekly.

The term "treatment dose" or "maintenance dose" is the amount of anti-IFN-gamma antibody taken by a subject to maintain or perpetuate the desired therapeutic effect. The treatment dose is administered after the induction dose. A treatment dose can be a single dose or can be a set of doses. Treatment doses are administered during the treatment phase of therapy. The treatment doses may be smaller or larger than the induction dose and equivalent to each other when administered sequentially. Where there is more than one treatment phase, there can also be more than one treatment dose.

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

The term "dosing," as used herein, refers to administering substances (eg, anti-IFN-gamma antibodies) to achieve a therapeutic purpose (eg, treatment of IFN-gamma-related disorders). It means administering.

The terms "biweekly dosing regimen," "biweekly dosing," and "biweekly administration," as used herein, are used for therapeutic purposes (e.g., , treatment of IFN-gamma-related disorders) refers to the time course of administering a substance (eg, an anti-IFN-gamma antibody) to a subject. A biweekly dosing regimen does not include a once weekly dosing regimen.

The term "combination" as in the phrase "a combination of a first agent and a second agent" includes co-administration of a first agent and a second agent, e.g. It may be dissolved or admixed in an acceptable carrier, or may be a first agent followed by a second agent, or a second agent followed by a second agent. It may be one that administers one drug. Accordingly, the present invention includes methods of combination therapeutic treatment and combination pharmaceutical compositions.

The term "concurrently" as in the phrase "concurrent therapeutic treatment" includes administering an agent in the presence of a second agent. Co-therapeutic treatment methods include methods of co-administering the first, second, third, or additional agents. Co-therapeutic treatment methods also include methods of administering a first or additional agent in the presence of a second or additional agent, wherein the second or additional agent is, for example, It may have been previously administered. Concurrent therapeutic treatment methods may be performed stepwise by different practitioners. For example, one practitioner can administer a first agent to a subject and a second practitioner can administer a second agent to a subject, the administering steps simultaneously or substantially simultaneously Alternatively, it can be performed at a separate time so long as the first drug (and additional drug) is administered in the presence of the second drug (and additional drug). The actor and subject may be the same entity (eg, human).

The term "combination therapy," as used herein, refers to the administration of two or more therapeutic agents, such as an anti-IFN-gamma antibody and another drug, such as a DMARD or NSAID. Point. The other drug(s) can be administered concurrently, before, or after administration of the anti-IFN-gamma antibody.
embodiment

The compositions and methods presented herein, NI-0501, are formulated as a sterile concentrate for injection (per 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 0.05 mg polysorbate 80, 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 0.05 mg polysorbate 80, pH 6.0.

In the compositions and methods provided herein, NI-0501 is used to treat, prevent, and/or delay the onset or progression of, or ameliorate symptoms associated with HLH. administered to a subject in need thereof. In some embodiments, NI-0501 is administered to a subject in need thereof at an initial dose of 1 mg/kg over 1 hour by IV infusion. In certain patient populations, e.g., underweight and/or very young patient populations, the IV infusion is longer than 1 hour, e.g., at least 90 minutes, at least 2 hours, or at least 3 hours or longer. can continue.

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

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

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

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, wherein the dose of the second additional IV infusion is the dose of the first additional IV infusion. Higher than additional IV infusions. In some embodiments, the first additional IV infusion and the second additional IV infusion are administered at different doses, wherein the dose of the second additional IV infusion is the dose of the first additional IV infusion. Higher than the additional IV infusion, and both the dose 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 are 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 first IV infusion. In some embodiments, the first additional IV infusion is 3 days after the first IV infusion, 6 days after the first IV infusion, 9 days after the first IV infusion, 12 days after the first IV infusion, and 12 days after the first IV infusion. Administer at a time selected from the group consisting of 15 days after injection. In some embodiments, the first additional IV infusion is 3 days after the first IV infusion, 6 days after the first IV infusion, 9 days after the first IV infusion, 12 days after the first IV infusion, and the first 15 days after injection of In some embodiments, a second additional IV infusion is given 3 weeks after the first IV infusion, 4 weeks after the first IV infusion, 5 weeks after the first IV infusion, and 6 weeks after the first IV infusion. 7 weeks after the first injection, and 8 weeks after the first injection. In some embodiments, a second additional IV infusion is given 3 weeks after the first IV infusion, 4 weeks after the first IV infusion, 5 weeks after the first IV infusion, and 6 weeks after the first IV infusion. 7 weeks after the first injection, and 8 weeks after the first injection. In some embodiments, the first additional IV infusion is 3 days after the first IV infusion, 6 days after the first IV infusion, 9 days after the first IV infusion, 12 days after the first IV infusion, and the first followed by a second additional IV infusion 3 weeks after the first IV infusion, 4 weeks after the first IV infusion, 5 weeks after the first IV infusion, and 15 days after the first IV infusion. After 6 weeks, 7 weeks after the first injection, and 8 weeks after the first injection.

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

In some embodiments, after the initial dose, infusions are performed every 3 days until 15 days after the initial dose. In some embodiments, the first dose is followed by infusions every 3 days until 15 days after the first dose, and then twice weekly infusions starting at least 15 days after the first dose. In some embodiments, the infusion dose is increased to 3 mg/kg any time after the initial dose. In some embodiments, the dose of NI-0501 is increased to 6 mg/kg for up to 4 infusions after a minimum of 2 infusions at 3 mg/kg.

In the compositions and methods provided herein, NI-0501 is used to treat, prevent, and/or delay the onset or progression of, or ameliorate symptoms associated with HLH. to a subject in need thereof. In some embodiments, NI-0501 is administered to a subject in need thereof at an initial dose of 1 mg/kg over 1 hour by IV infusion. In some embodiments, after the initial dose, infusions are performed every 3 days until 15 days after the initial dose. In some embodiments, the first dose is followed by infusions every 3 days until 15 days after the first dose, and then twice weekly infusions starting at least 15 days after the first dose. In some embodiments, the infusion dose is increased to 3 mg/kg any time after the initial dose. In some embodiments, the dose of NI-0501 is increased to 6 mg/kg for up to 4 infusions after a minimum of 2 infusions at 3 mg/kg.

In the compositions and methods provided herein, NI-0501 is used to treat, prevent, and/or delay the onset or progression of, or ameliorate symptoms associated with HLH. administered to a subject in need thereof. In some embodiments, NI-0501 is administered to a subject in need thereof at a dose of greater than 6 mg/kg by IV infusion. In some embodiments, NI-0501 is administered to a subject in need thereof at a second dose of greater than 6 mg/kg by IV infusion after the first dose. In some embodiments, the second dose is at least 10 mg/kg. In some embodiments, the second dose is 10 mg/kg. In some embodiments, the second dose is 10 mg/kg, repeated daily. In some embodiments, the second dose is 10 mg/kg, repeated daily for one week. In some embodiments, the second dose is 10 mg/kg, repeated daily for two weeks. In some embodiments, the second dose is 10 mg/kg, repeated daily for more than 2 weeks.

In some embodiments, NI-0501 is used to treat, prevent, and/or delay the onset or progression of, or ameliorate symptoms associated with secondary HLH. administered to a subject who In some embodiments, NI-0501 is used to treat, prevent, and/or delay the onset or progression of, or ameliorate symptoms associated with secondary HLH in the context of sJIA. , to a subject in need thereof. In some embodiments, NI-0501 is administered to a subject in need thereof as a starting 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 NI-0501 doses of 3 mg/kg every 3 days for at least 4 weeks (ie, until SD27).

In some embodiments, once the desired clinical outcome is achieved, treatment with NI-0501 is reduced, stopped, or alternatively shortened. In some embodiments, treatment with NI-0501 is curtailed once complete clinical response, ie, MAS remission, is demonstrated.

In some embodiments, after 4 weeks, treatment with NI-0501 is continued as needed for up to an additional 4 weeks (ie, until SD56) as maintenance until MAS remission is achieved. In some embodiments, after 4 weeks, treatment with NI-0501 is continued as maintenance as needed until MAS remission is achieved, up to an additional 4 weeks (i.e., until SD56) and the dose is reduced to 1 mg/kg. There is a possibility of decreasing and extending the interval between injections to once weekly dosing.

In the compositions and methods provided herein, NI-0501 is used to treat, prevent, and/or delay the onset or progression of, or ameliorate symptoms associated with HLH. to a subject in need thereof, wherein the subject is receiving dexamethasone as a background. In some embodiments, the subject is a treatment naive patient (ie, 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 doses ranging from 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 of less than 5 mg/m ² .

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

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

In some embodiments, the additional agent is an immunosuppressive agent. In some embodiments, the immunosuppressive agent is cyclosporin A (CsA). In some embodiments, the subject has received CsA prior to administration of NI-0501. In some embodiments, the additional agent comprises at least etoposide. In some embodiments, the subject has received etoposide prior to administration of NI-0501.

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

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

In some embodiments, the one or more additional agents are analgesic treatments, blood product transfusions, electrolyte and glucose infusions, antibiotics, antifungal and antiviral treatments and/or general supportive care. be.

The compositions and methods provided herein treat, prevent, and/or prevent the onset or progression of symptoms associated with graft failure, graft rejection, and/or inflammatory disorders associated with graft rejection. is useful in delaying or alleviating The compositions and methods provided herein treat symptoms of graft-versus-host disease (GvHD) in a subject who has undergone or has undergone a transplant or series of transplants containing biological material. It is useful in inhibiting, slowing its progression, or alternatively reversing it. The compositions and methods presented herein are useful in prolonging the survival of implanted biological material.

The compositions and methods provided herein include, for example, cells, tissue(s), bone marrow, and/or heart, kidney, pancreas, liver, and/or intestine as non-limiting examples. It is useful in the transplantation of any biological material containing organ(s) or at least part of an organ. In some embodiments, the implanted biomaterial is an allogeneic biomaterial. In some embodiments, the implanted biological material is bone marrow. In some embodiments, the implanted biomaterial is a population of hematopoietic stem cells. In some embodiments, the implanted biological material is or is derived from one or more hepatocytes.

The compositions and methods provided herein treat, prevent, and treat symptoms associated with graft failure, graft rejection, and/or inflammatory disorders associated with graft rejection in subjects in need thereof. and/or delay the onset or progression of or alleviate it. In some embodiments, graft rejection, also referred to herein as graft failure, is acute. In some embodiments, graft rejection is hyperacute.

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

In some embodiments, graft rejection is chronic. In some embodiments, NI-0501 is administered to a subject in need thereof at an initial dose of 1 mg/kg over 1 hour by IV infusion. In certain patient populations, e.g., underweight and/or very young patient populations, the IV infusion is longer than 1 hour, e.g., at least 90 minutes, at least 2 hours, or at least 3 hours or longer. can continue.

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

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

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

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, wherein the dose of the second additional IV infusion is the dose of the first additional IV infusion. Higher than additional IV infusions. In some embodiments, the first additional IV infusion and the second additional IV infusion are administered at different doses, wherein the dose of the second additional IV infusion is the dose of the first additional IV infusion. Higher than the additional IV infusion, and both the dose 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 are 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 first IV infusion. In some embodiments, the first additional IV infusion is 3 days after the first IV infusion, 6 days after the first IV infusion, 9 days after the first IV infusion, 12 days after the first IV infusion, and 12 days after the first IV infusion. Administer at a time selected from the group consisting of 15 days after injection. In some embodiments, the first additional IV infusion is 3 days after the first IV infusion, 6 days after the first IV infusion, 9 days after the first IV infusion, 12 days after the first IV infusion, and the first 15 days after injection of In some embodiments, a second additional IV infusion is given 3 weeks after the first IV infusion, 4 weeks after the first IV infusion, 5 weeks after the first IV infusion, and 6 weeks after the first IV infusion. 7 weeks after the first injection, and 8 weeks after the first injection. In some embodiments, a second additional IV infusion is given 3 weeks after the first IV infusion, 4 weeks after the first IV infusion, 5 weeks after the first IV infusion, and 6 weeks after the first IV infusion. 7 weeks after the first injection, and 8 weeks after the first injection. In some embodiments, the first additional IV infusion is 3 days after the first IV infusion, 6 days after the first IV infusion, 9 days after the first IV infusion, 12 days after the first IV infusion, and the first followed by a second additional IV infusion 3 weeks after the first IV infusion, 4 weeks after the first IV infusion, 5 weeks after the first IV infusion, and 15 days after the first IV infusion. After 6 weeks, 7 weeks after the first injection, and 8 weeks after the first injection.

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

In some embodiments, after the initial dose, infusions are performed every 3 days until 15 days after the initial dose. In some embodiments, the first dose is followed by infusions every 3 days until 15 days after the first dose, and then twice weekly infusions starting at least 15 days after the first dose. In some embodiments, the infusion dose is increased to 3 mg/kg any time after the initial dose. In some embodiments, the dose of NI-0501 is increased to 6 mg/kg for up to 4 infusions after a minimum of 2 infusions at 3 mg/kg.

The present disclosure provides a patient population afflicted with a disorder, wherein the patient has elevated levels of CXCL9 alone or in combination with one or more additional interferon gamma (IFNγ)-related biomarkers. Also provided are compositions and methods useful in identifying, or alternatively precisely distinguishing, populations. In particular, the present 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). offer. In particular, the present disclosure provides compositions for detecting CXCL9 levels as a biomarker for IFNγ production in patients with or suspected of having secondary hemophagocytic lymphohistiocytosis (HLH). Provide an object and method. In some embodiments, 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 compositions and methods are used as biomarkers for IFNγ production in patients suffering from or suspected of suffering from MAS in the context of an autoimmune disease or inflammatory disorder. Detect CXCL9 levels. In some embodiments, compositions and methods are used to bio-analyze IFNγ production in patients suffering from or suspected of suffering from MAS in the context of a systemic autoimmune disease or inflammatory disorder. CXCL9 levels are detected as a marker. In some embodiments, compositions and methods are used to assess IFNγ production in patients suffering from or suspected of suffering from MAS in the setting of systemic juvenile idiopathic arthritis (sJIA). CXCL9 levels are detected as a biomarker. In some embodiments, compositions and methods are used to use CXCL9 as a biomarker for IFNγ production in patients suffering from or suspected of suffering from MAS in the setting of systemic lupus erythematosus (SLE). Detect levels.

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

In some patients suffering from or suspected of suffering from the disorder, fluids and other biological samples show levels of CXCL9 alone or in combination with other IFNγ-related biomarkers such as CXCL10 and/or CXCL11. contains an increase in

CXCL9 and these other biomarkers are indicators of IFNγ production in vivo. Thus, anti-IFNγ antagonists such as neutralizing anti-IFNγ antibodies or other polypeptide-based therapeutics, peptide-based therapeutics, small molecules that interfere with, inhibit, reduce or alternatively antagonize IFNγ signaling. IFNγ activity is blocked or alternatively inhibited by the use of inhibitors, nucleic acid-based therapeutics and derivatives thereof. Accordingly, compositions and methods provide anti-IFNγ antagonists, such as neutralizing anti-IFNγ antibodies or other polypeptide-based therapeutics, peptide-based, to patients exhibiting elevated levels of expression of CXCL9 and/or other biomarkers. Administration of therapeutic agents, small molecule inhibitors, nucleic acid-based therapeutic agents and derivatives thereof, resulting in aberrant IFNγ expression and/or activity, e.g., elevated, aberrant pro-inflammatory cytokine production, and/or combinations thereof , in treating, slowing the progression of, or alternatively ameliorating the symptoms of a disorder driven by, associated with, or alternatively affected by. Patients likely to be good candidates for treatment with an anti-IFNγ antagonist, e.g., a neutralizing anti-IFNγ antibody such as those described herein, may be treated with CXCL9 alone or with one or more IFNγ-related ligands or other Identified by detecting the level of a combination of 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 neutralizing anti-IFNγ antibodies or other polypeptides described herein. Treatment can be with anti-IFNγ antagonists, including therapeutic agents, peptide-based therapeutic agents, small molecule inhibitors, nucleic acid-based therapeutic agents and any of their derivatives.

Patients with elevated levels of CXCL9 alone or in combination with one or more additional IFNγ-related biomarkers are treated with one or more anti-IFNγ antagonists, such as the neutralizing anti-IFNγ antibodies described herein identified as suitable candidates for treatment with As used herein, the phrase "elevated levels of expression" refers to a sample from a patient not suffering from or suspected of suffering from primary or secondary HLH or an HLH-related disorder, or 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 another control sample. In some embodiments, the increased level of expression of CXCL9 and/or other biomarkers is a significant level increase.

Detected levels of CXCL9 alone or in combination with one or more other IFNγ-related biomarkers are useful to accurately identify or alternatively stratify patient populations. In some embodiments, the levels detected are used to determine the dosage of anti-IFNγ antagonist to be administered to a given patient. In some embodiments, detected levels are used to categorize or alternatively stratify patient populations. For example, a patient can be classified as having "severe" or high-grade MAS, or conversely, as having less severe or low-grade MAS, based on detected levels of CXCL9.

The sample is eg blood or a blood component eg 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.

In addition to detecting levels of IFNγ and/or other IFNγ-associated biomarkers, several additional techniques improve the sensitivity and specificity of biomarkers to identify or alternatively accurately discriminate patient populations. Patients suitable for treatment with an anti-IFNγ antagonist can also be identified by evaluating any biological and clinical parameter. Alternatively, these additional biological and clinical parameters can be used alone as a means of identifying patients who are suitable candidates for treatment with an anti-IFNγ antagonist or other suitable therapy. These biological and clinical parameters include, as non-limiting examples, any of the following: ferritin levels, neutrophil counts, platelet counts, alanine aminotransferase levels, and/or lactate dehydrogenase levels.

Disorders for which the compositions and methods of the present invention are useful include any disorder in which there is aberrant, e.g., elevated, IFNγ expression and/or activity, particularly HLH, including secondary HLH, MAS, and/or sJIA. be done.

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

Once a patient is identified as having elevated levels 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, an anti-IFNγ antagonist is a neutralizing anti-IFNγ antibody or an immunologically active (eg, antigen-binding) fragment thereof. Suitable neutralizing anti-IFNγ antibodies include any of the anti-IFNγ antibodies described herein.

In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof has at least 90%, 92%, 95%, 96%, 97%, 98%, a variable heavy chain complementarity determining region 1 (VH CDR1) comprising an amino acid sequence that is 99% or greater identical; a variable heavy chain complementarity determining region 2 (VH CDR2) comprising an amino acid sequence that is 97%, 98%, 99% or greater identical; and at least 90%, 92% with the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO: 3) , a variable heavy chain complementarity determining region 3 (VH CDR3) comprising an amino acid sequence that is 95%, 96%, 97%, 98%, 99% or greater identical; variable light chain complementarity determining region 1 (VL CDR1) comprising amino acid sequences that are at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or greater identical; EDNQRPS (SEQ ID NO: 5) a variable light chain complementarity determining region 2 (VL CDR2 ); and a variable light chain complement comprising an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or greater identical to the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6). Contains sex determining region 3 (VL CDR3).

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

In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof is a heavy chain comprising a combination of VH CDR1, VH CDR2, and VH CDR3 sequences, wherein the combination is one row of Table 1A. The three heavy chain CDR sequences (VH CDR1, VH
CDR2, VH CDR3) combination, heavy chain.

In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof is a light chain comprising a combination of VL CDR1, VL CDR2, and VL CDR3 sequences, wherein the combination is The three light chain CDR sequences (VL CDR1, VL
CDR2, VL CDR3) combination light chain.

In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof is a heavy chain comprising a combination of VH CDR1, VH CDR2, and VH CDR3 sequences, wherein the combination is The three heavy chain CDR sequences (VH CDR1, VH
CDR2, VH CDR3), a heavy chain, and a light chain comprising a combination of VL CDR1, VL CDR2, and VL CDR3 sequences, the combination being shown in one row of Table 1B. Includes light chains that are combinations of three light chain CDR sequences (VL CDR1, VL CDR2, VL CDR3).

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

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

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

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

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

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

In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof has at least 90%, 92%, 95%, 96%, 97%, It includes amino acid sequences that are 98%, 99% or greater identical.

In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof has at least 90%, 92%, 95%, 96%, 97%, It includes amino acid sequences that are 98%, 99% or greater identical.

In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof has at least 90%, 92%, 95%, 96%, 97%, amino acid sequences that are 98%, 99% or greater identical and at least 90%, 92%, 95%, 96%, 97%, 98%, 99% with the light chain amino acid sequence that is the amino acid sequence of SEQ ID NO:46 % or greater amino acid sequences are included.

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

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

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

In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises Amino acid sequences that are 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 102.

In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises Amino acid sequences that are 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 104.

In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or greater identical to a heavy chain variable amino acid sequence selected from the group consisting of 102 and SEQ ID NO:52 , 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, and 104 and at least 90%, 92%, 95%, It includes amino acid sequences that are 96%, 97%, 98%, 99% or more identical. In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises A heavy chain variable amino acid sequence selected from the group consisting of 102.

In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises A light chain variable amino acid sequence selected from the group consisting of 104.

In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises a heavy chain variable amino acid sequence selected from the group consisting of 102 and selected from the group consisting of SEQ ID NOS: 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, and 104 comprising a light chain variable amino acid sequence defined as

In some embodiments, an anti-IFNγ antibody or immunologically active fragment thereof is administered in a therapeutically effective amount. A therapeutically effective amount of an antibody of the invention generally relates to the amount necessary to achieve a therapeutic goal. This therapeutic purpose may, in certain cases, be the binding interaction of an antibody with its target antigen that interferes with the target's function. A general range for a therapeutically effective dosage of an antibody or antibody fragment of the invention can be, as a non-limiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight. Typical dosing frequencies can range, for example, from twice daily to once weekly.

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

In some embodiments, an anti-IFNγ antibody or immunologically active fragment thereof is administered as an initial loading dose, followed by one or more maintenance doses. In some embodiments, the one or more maintenance doses are doses substantially similar to the initial loading dose. In some embodiments, one or more maintenance doses are lower doses than the initial loading dose. In some embodiments, one or more maintenance doses are higher doses than the initial loading dose.

In some embodiments, the one or more maintenance doses comprises at least two or more doses, each maintenance dose being the same dose. In some embodiments, the two or more maintenance doses are substantially similar to the initial loading dose. In some embodiments, the two or more maintenance doses are greater than the initial loading dose. In some embodiments, the two or more maintenance doses are less than the initial loading dose.

In some embodiments, the one or more maintenance doses comprises at least two or more doses, and each maintenance dose is not the same dose. In some embodiments, two or more maintenance doses are administered in ascending doses. In some embodiments, two or more maintenance doses are administered in tapering doses.

In some embodiments, the one or more maintenance doses comprise at least two or more doses, each maintenance dose being administered at periodic time intervals. In some embodiments, two or more doses are administered at increasing time intervals. In some embodiments, two or more doses are administered with tapering time intervals.

In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof ranges from about 0.5 mg/kg to about 2 mg/kg, such as from about 0.5 mg/kg to about 1.5 mg/kg. , and/or at an initial loading dose ranging from about 0.5 mg/kg to about 1.0 mg/kg, followed by at least one, such as two or more, three or more 4 or more, or 5 or more maintenance doses. In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof is administered at an initial loading dose of about 1.0 mg/kg, followed by at least one, such as two or more Many, three or more, four or more, or five or more maintenance doses are administered.

A pharmaceutical composition according to the invention can comprise an anti-IFNγ antibody of the invention and a carrier. These pharmaceutical compositions may be included in kits, such as diagnostic kits.

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

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

(Example 1)
CXCL9 Levels as a Biomarker for IFNγ Production in Macrophage Activation Syndrome (MAS) To search for potential biomarkers of IFNγ production in vivo, the study presented here was designed to We evaluated the correlation of serum levels of IFNγ and three IFNγ-related chemokines with themselves and with laboratory parameters of disease activity in patients with active MAS.

Circulating levels of IFNγ, CXCL9, CXCL10, CXCL11 and IL-6 were measured using Luminex multiplex assays in patients with sJIA (n=54), 20 of whom had MAS at sampling. Associations between these circulating levels and disease activity parameters were evaluated, along with correlations between levels of IFNγ and levels of CXCL9, CXCL10 and CXCL11.

Active MAS had significantly elevated levels of IFNγ and three IFNγ-related chemokines (CXCL9, CXCL10 and CXCL11) compared to active sJIA without MAS at 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 to a lesser extent with CXCL10 and CXCL11; No correlation with IL-6 levels was seen. 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 correlated significantly with levels of CXCL9 (r=0.69; r ² =0.47; p=0.001) and to a lesser extent with levels of CXCL10 (r =0.53; r ² =0.28; p=0.015) and did not correlate with levels of CXCL11 (r=−0.04; p=0.886).
Table 7. Correlation of laboratory parameters of disease activity with IFNγ, CXCL9, CXCL10, CXCL11, and IL-6 in patients with MAS and patients with active sJIA.

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

The studies presented here are from an ongoing Phase 2 pilot study in primary HLH patients who received NI-0501 antibody and from patients who received NI-0501 antibody for humane use. belongs to.

As shown in FIG. 1, serum levels of CXCL9 and IFNγ in samples from 6 primary HLH patients and 3 humanitarian use patients were measured using Luminex and Meso Scale Discovery (MSD) techniques, respectively. measured by Correlation between CXCL9 levels and total IFNγ levels was performed. Statistics were performed and p-values obtained using Spearman's test.

As shown in FIG. 2, predose serum levels of CXCL9 and IFNγ in samples obtained from 6 primary HLH patients and 3 humanitarian use patients were measured by Luminex and MSD techniques, respectively. Correlation between CXCL9 levels and total IFNγ levels was performed. Statistics were performed and p-values obtained using Spearman's test.
(Example 3)
Correlation between CXCL9 and IFNγ levels in patients with secondary hemophagocytic lymphohistiocytosis (HLH)

The studies presented herein are from observational studies in secondary HLH patients who received NI-0501 antibody and from patients who received humane use of NI-0501 antibody. It is a thing.

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

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

Patients with MAS secondary to sJIA and sampling as shown in FIGS. Serum levels of CXCL9 and IFNγ were measured by multiplex assays using Luminex technology from patients with occasional active sJIA. Correlation of IFNγ or CXCL9 levels with ferritin, platelet count, neutrophil count or ALT (alanine aminotransferase) was performed. Statistics were performed and p-values obtained using Spearman's test.
(Example 4)
Correlation between CXCL9 and IFNγ levels in patients with severe hemophagocytic lymphohistiocytosis (HLH)

The studies presented here are from patients who received compassionate use of the NI-0501 antibody. This patient presented with symptoms of NLRC4-related disease and severe hemophagocytic lymphohistiocytosis (HLH). It was recently reported that mutations in the NLRC4 gene cause recurrent macrophage activation syndrome and increased production of IL-18, which is known to induce IFNγ.

Patients in this study had the following characteristics: onset at 20 days of age, fever, rash, marked hepatosplenomegaly, pancytopenia, hypofibrinogenemia, hypertriglyceridemia, marked increases in ferritin and sCD25. Accompanied by Multiple organ failure followed, requiring admission to the ICU. HLH diagnosis was based on 6 of the 8 HLH-2004 criteria. Genes that cause primary HLH (PRF1, UNC13D, STXBP2, STX11, RAB27A, XIAP) and functional tests (perforin expression, degranulation and cytotoxicity) were negative. High dose i. with progressive improvement in performance status and laboratory abnormalities. v. glucocorticoids and i. v. Cyclosporin A. Infection-induced (Candida Albicans and Klebsiella Pneumoniae sepsis) induced HLH reactivation, rapid deterioration of performance status, and new ICU admissions. Treatment with etoposide and/or ATG was not considered due to the presence of active infection in already immunocompromised subjects.

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

Dexamethasone (13.6 mg/m ² ) and i. v. In the background of cyclosporin A, treatment was initiated with NI-0501 ethical use. NI-0501 was dosed every 3 days and then every 7 days according to pharmacokinetics. No infusion reactions were observed. NI0501 was well tolerated. The clinical features of HLH and laboratory abnormalities progressively improved. Active ongoing infections were rapidly cleared. After 5 months of treatment the patient remained in excellent condition. Patients continued to receive oral cyclosporin A (6 mg/kg) and prednisone (0.3 mg/kg, equivalent to dexamethasone at 0.9 mg/ ^m2 ). All HLH parameters normalized.

Subject still exhibited episodes of unexplained inflammation. Analysis of NLRC4 revealed a novel missense mutation (T337N). An elevation of serum IL-18 was demonstrated, confirming the relevance of the NLRC4 mutation. High production of IFNγ was demonstrated by the high levels of IFNγ complexed with NI-0501. IFNγ was fully neutralized, as indicated by undetectable levels of IFNγ-inducible chemokines (Figure 5 and Table 8). Circulating levels of IL-18 were persistently elevated.

Thus, this study demonstrated that blocking IFNγ with NI-0501 in patients with severe refractory HLH (due to NLRC4 mutations) was well tolerated and was not associated with safety concerns. It has been demonstrated to allow control of all HLH hallmarks without delay, permit rapid glucocorticoid taper, and be accompanied by resolution of ongoing active infection.
(Example 5)
Targeted approach for treating hemophagocytic lymphohistiocytosis (HLH) with NI-0501

The study presented here is from a pilot Phase 2 study in children with primary HLH. Primary HLH (pHLH) is a rare immunomodulatory disorder that, if untreated, is invariably fatal. pHLH is driven by pathological immune activation leading to the development of fever, splenomegaly, cytopenia and coagulopathy, which can lead to multiple organ failure and death. Based on data from mouse models of primary and secondary HLH (sHLH) treated with anti-IFNγ antibodies, and observational studies in patients with HLH, high production of IFNγ reduces disease development. It is considered to be a very important driving factor. Immunochemotherapy, primarily etoposide-based regimens, are currently the only pharmacological approach to control HLH and lead patients to curative allogeneic hematopoietic stem cell transplantation (allo-HSCT). Despite recent attempts to further intensify treatment regimens, mortality and morbidity rates remain high, due in part to drug-related toxicities.

As noted above, NI-0501 is a fully human, high-affinity, anti-IFNγ mAb that binds to and neutralizes human IFNγ, providing a novel targeting approach for regulating HLH.

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

Study Population: A total of 13 patients were enrolled: 8F/5M, median age 1.0 years (range 2.5 months to 13 years). 12 patients who reactivated, had an inadequate response, or were intolerant to therapy after receiving conventional therapy received NI-0501 as second-line treatment . One patient was treated with NI-0501 as first line. Nine patients had known HLH gene defects (3 with FHL2, 2 with FHL3, 2 with GS-2, 1 with XLP1, one had XLP2). The majority of patients were end-stage on the HLH spectrum, had impaired performance status, and had significant toxicity from prior HLH treatment. 12 of 13 patients had elevated ferritin, 8 had elevated sCD25, 10 had cytopenia, 8 had splenomegaly, 9 had Hypofibrinogenemia and hypertriglyceridemia were present. Liver dysfunction and CNS complications were present in 7 and 3 patients, respectively.

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

Assessment of biomarkers, particularly CXCL9, a chemokine known to be subtly induced by IFNγ, not only allowed the demonstration of complete IFNγ neutralization, but also correlated with IFNγ production, to diagnose HLH. (Figs. 7A and 7B).

NI-0501 was well tolerated and no safety concerns were identified. No infections were reported for which IFNγ neutralization was known to be beneficial, nor did infections occur in patients who had not received prior chemotherapy. At least one SAE was reported in seven patients, all assessed by the DMC as not related to NI-0501 administration. No unexpected events were observed due to "off-target" effects of NI-0501 (eg, myelotoxicity, hemodynamic effects).

Conclusion: Targeted neutralization of IFNγ by NI-0501 provides an innovative and potentially less toxic approach to manage HLH. The results of this study demonstrate that NI-0501 is a safe and effective treatment option in patients with primary HLH who have responded poorly to or been intolerant to conventional therapy. Moreover, 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 it is anticipated that similar significant clinical benefit may be realized.
(Example 6)
Elevated circulating levels of interferon-γ and interferon-induced chemokines characterize patients with macrophage activation syndrome associated with systemic JIA

Interferon gamma (IFNγ) is a central mediator in mouse models of primary hemophagocytic lymphohistiocytosis (HLH). Given the similarities between primary HLH and secondary HLH (sec-HLH), including macrophage activation syndrome (MAS), IFNγ levels and Its biological activity was analyzed.

In the study presented here, using the Luminex multiplex assay, patients with sec-HLH (n=11) and patients with sJIA, 20 of whom had MAS at sampling (n = 54) serum levels of IL-1β, IL-6, IFNγ, and the IFN-induced and/or IFN-associated chemokines, CXCL9, CXCL10, and CXCL11, were assessed. Expression of IFNγ-inducible chemokines (CXCL9 and CXCL10 mRNA levels in liver and spleen) and their correlation with serum ferritin levels were examined in IL-6 transgenic mice in which MAS hallmarks were induced by TLR4 stimulation with LPS. evaluated in the model.

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

Thus, the studies presented below suggest that IFNγ plays a central role in MAS, due to high levels of IFNγ and IFN-induced chemokines and their correlation, particularly CXCL9, with the severity of laboratory abnormalities in MAS. Demonstrate that Elevated circulating levels of interferon-γ and interferon-induced chemokines characterize patients with macrophage activation syndrome associated with systemic JIA.

Materials and Methods: Patients and Samples. ３カ所のＰａｅｄｉａｔｒｉｃ Ｒｈｅｕｍａｔｏｌｏｇｙ Ｃｅｎｔｒｅｓ：ｔｈｅ Ｏｓｐｅｄａｌｅ Ｐｅｄｉａｔｒｉｃｏ Ｂａｍｂｉｎｏ Ｇｅｓｕ ｉｎ Ｒｏｍｅ、ｔｈｅ Ｉｓｔｉｔｕｔｏ Ｇｉａｎｎｉｎａ Ｇａｓｌｉｎｉ ｉｎ Ｇｅｎｏａ ａｎｄ ｔｈｅ Ｃｉｎｃｉｎｎａｔｉ Ｃｈｉｌｄｒｅｎ'ｓ Ｈｏｓｐｉｔａｌ Ｍｅｄｉｃａｌ Ｃｅｎｔｒｅにおいて、ＭＡＳを伴うまたは伴わないｓＪＩＡを有する患者から末梢血試料を採取した。 Fifty-four patients with sJIA who met the ILAR classification criteria for systemic arthritis (age at onset 7.9 years, interquartile range 4.6-13.6 years; 48% female) were studied (Petty, R.; E. et al., International League of Associations for Rheumatology classification of juvenile idiopathic arthritis: Rev. 2nd edition, Edmonton, 2001, J Rheumatol, 2004, 31(0-2):39. Twenty of the SJIA patients were sampled during an episode of active end-stage MAS diagnosed by the treating physician 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 systematic juvenile idiopathic arthritis. Pediatric Rheumatology, 2014, vol. Samples of 28 patients with active SJIA without evidence of MAS were available. From 35 sJIA patients (both with and without history of MAS), 35 samples were available during clinically inactive disease defined according to Wallace criteria (Wallace, C. A. et al., Preliminary criteria for clinical remission for select
Categories of juvenile idiopathic arthritis. J Rheumatol, 2004, 31(11):2290-4).

Since IFNγ has been shown to be elevated in patients with sec-HLH (rheumatic diseases excluded), 11 patients with sec-HLH found in Ospedale Pediatrico Bambino Gesu (age 8.6 at onset) age, interquartile range 4.1-12.9 years; 36% female) was also collected and used as a positive control. All sec-HLH patients fulfilled the 2004-HLH diagnostic guidelines (Henter, JI, et al., HLH-2004: Diagnostic and therapeutic guidelines for hemophagocytic lymphocytosis. Pediatr Blood Cancer vol. 31), 6 patients met 5 criteria and 5 patients met 4 criteria. It should be noted that levels of sCD25 in U/ml were not available as this study is not routinely performed at the institution where these patients were recruited. Diagnosis of primary HLH is based on no family history, absence of pathogenic mutations in genes known to cause HLH, and presence of normal functional tests (NK activity, perforin expression and CD107 degranulation). excluded on that basis. All 11 patients with sec-HLH each contributed one sample obtained during active disease.

Clinical and laboratory characteristics of all patients regarding diagnosis and at sampling were collected in a centralized web database by each center's principal investigator. Of the 20 MAS patients sampled during active disease, 6 were not receiving any treatment at the time of sampling and the remaining 14 patients received glucocorticoid pulse, cyclosporine A, anakinra or cyclophosphamide. had already received one of the MAS-specific treatments, including Of the 11 patients with active disease sec-HLH, 6 had not yet received a 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 consent was collected for all participants.

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

Animal experimentation. The generation and phenotype of IL-6 transgenic mice and the characteristics of the 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: an implication for the pathogenesis of macrophage activation syndrome., Arthritis Rheum, 2012, 64(5):1680-8). Mice were maintained under specific pathogen-free conditions and handled according to national police. The study 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) of 5 μg/g body weight. Mice were sacrificed after 30 hours. 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) with mouse Cxcl9 and Cxcl10 gene expression assays (Applied Biosystems). Gene expression data were normalized using mouse Hprt (Applied Biosystems). Data are expressed as arbitrary units (AU) determined using the 2 ^-Δct method. Serum ferritin concentrations were determined using a commercial ELISA kit (ALPCO Diagnostics) according to the manufacturer's instructions.

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

Results: Elevated levels of IFNγ and IFNγ-induced chemokines in patients with MAS. Comparing patients with active sJIA without MAS at sampling to patients sampled during clinically inactive disease, as expected (de Benedetti, F. et al., Correlation of serum interleukin-6 levels with joint involvement and thrombocytosis in systematic juvenile rheumatoid arthritis., Arthritis Rheum, 1991, 34(9):1158-63), IL-6 levels are clinically inactive in patients with active sJIA. It was found to be significantly higher (p<0.01) compared to patients with disease. As reported in several previous studies of active SJIA, serum IL-1β levels were below the limit of detection in the majority of patients, regardless of disease activity status. It is noteworthy that levels of IFNγ and three IFNγ-inducible chemokines did not differ between patients with clinically active sJIA and patients with clinically inactive disease.

Comparing patients with MAS at sampling to those with active sJIA without MAS at sampling, levels of IL-1β and IL-6 were comparable, thereby playing a central role in active sJIA It is suggested that the levels of these two known cytokines are not elevated during end-stage MAS. It should be noted that circulating IL-1β levels were below the limit of quantification (ie, 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 those with active sJIA without MAS at sampling. Levels of three IFNγ-related chemokines, CXCL9, CXCL10 and CXCL11, were also significantly higher in patients with active MAS compared to those with active sJIA without MAS at sampling. This difference was particularly evident for CXCL9, whose median levels were approximately 15-fold higher in patients with MAS compared to those with active sJIA without MAS.

Levels of IFNγ as well as three IFNγ-related chemokines were significantly elevated in patients with sec-HLH. Levels of IFNγ and IFNγ-related chemokines were nearly indistinguishable from levels in patients with MAS, and differences were not statistically significant. Coincidentally, levels of IFNγ and three IFNγ-inducible chemokines in patients with active MAS and in patients with active sec-HLH were comparable to treatment-naïve and previously treated patients. there were.

Levels of IFNγ and CXCL9, CXCL10 and CXCL11 are associated with the presence of MAS in individual patients. Figures 8A-8D show levels of IFNγ and CXCL9, CXCL10 and CXCL11 in individual patients for whom matched samples were available during active MAS and during 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 the samples obtained by the MAS-paired sample analysis. Moreover, in some patients, samples were available both before and after the MAS episode, demonstrating that IFNγ and IFNγ-induced chemokine levels returned to normal once MAS clinical symptoms resolved. For example, one patient in this study experienced three episodes of MAS and serum samples were obtained during these episodes as well as during the disease phase without MAS at the time of sampling. Elevated levels of IFNγ and three IFNγ-related chemokines were found only during MAS episodes in this patient, further confirming the relationship between increased production of IFNγ and three IFNγ-induced chemokines and active MAS. (FIGS. 9A-9B).

Levels of IFNγ and IFNγ-related chemokines correlate with laboratory abnormalities in MAS. Correlations between levels of IFNγ and three IFNγ-inducible chemokines and laboratory parameters of MAS at sampling were then investigated. In patients with active sJIA without MAS, levels of IFNγ and three IFNγ-inducible chemokines did not correlate with laboratory parameters of MAS with one exception: levels of CXCL9, CXCL10 and CXCL11 Weakly correlated with ALT levels with r2 ranging from 0.17 to 0.25 (Table ² ). The significance of this association is unclear, but it should be noted that ALT levels were within the normal range in all patients with active sJIA without MAS. No significant correlations between laboratory characteristics of MAS and IL-1 and IL-6 were found in patients with MAS at sampling. In contrast, in patients with MAS at the time of sampling, levels of IFNγ and IFNγ-induced chemokines were all generally abnormal in patients with MAS, levels of ferritin, neutrophil and platelet counts, and LDH and ALT. associated with elevation (Table 2). Correlations with laboratory abnormalities were particularly evident for IFNγ and CXCL9, with the sole exception that the correlation between IFNγ and LDH did not reach statistical significance (Table 2 and FIGS. 10A-10J). Again, as noted above, these correlations were absent in patients with active s-JIA without MAS at sampling. One patient in this group had significantly elevated 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 provides further support for the hypothesis that there is a strong association between IFNγ and CXCL9 levels and disease severity. Taken together, these results indicate that increased production of IFNγ and IFNγ-related chemokines is a hallmark of active MAS that strongly correlates with the severity of laboratory abnormalities in MAS.

Correlation of IFNγ and IFNγ-induced chemokine levels in patients with MAS. To further characterize the relationship between IFNγ and the three IFNγ-inducible chemokines in patients with MAS, correlations between IFNγ levels and levels of each individual chemokine were assessed. In particular, CXCL9 appears to be primarily and specifically induced by IFNγ, while CXCL10 and CXCL11 are also induced by type I interferons. Consistent with this, in patients with active MAS, circulating levels of IFNγ were significantly correlated with CXCL9 (r=0.693; r ² =0.48; p=0.001), whereas CXCL10 levels was weaker (r=0.535; r ² =0.29; p=0.015) (FIGS. 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).

IFNγ-induced chemokines correlate with disease activity in mouse models of MAS. To further investigate the relevance of IFNγ-induced chemokine production to MAS, the expression of these chemokines in target tissues (liver and spleen) was investigated in a mouse model of MAS. In this model, against a background of high levels of IL-6 in IL-6 transgenic mice, the TLR4 agonist lipopolysaccharide (LPS) is used to induce clinical and laboratory features of MAS by mimicking acute infection ( Strippoli et al., Arthritis Rheum, 2012). This approach 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. High mRNA levels of CXCL9 and CXCL10 were present in the liver and spleen of IL-6 transgenic mice after induction with LPS. In particular, serum levels of ferritin were significantly correlated with levels of CXCL9 expression in the spleen and liver and CXCL10 expression in the liver, thereby indicating upstream events associated with IFNγ in target tissues (i.e., liver and liver). CXCL9 and CXCL10 production in the spleen) and typical downstream laboratory abnormalities such as high ferritin levels are shown. Taken together, data in patients with MAS and in MAS mouse models demonstrate a clear relationship between increased production of IFNγ and increased expression of CXCL9 and, to a lesser extent, CXCL10, and laboratory abnormalities of MAS.

Studies in both patients and animal models of p-HLH have demonstrated a central role for IFNγ in disease pathogenesis. However, the role of IFNγ in sec-HLH, including MAS in the context of sJIA, remains unclear. This study conclusively demonstrates that high levels of IFNγ and IFNγ-inducible chemokines were present in patients with MAS occurring in sJIA. In addition, levels of IFNγ, CXCL9 and CXCL10 strongly correlated with laboratory parameters of MAS severity. In this study, serum levels of IFNγ and three IFNγ-related chemokines were found to be similar in patients with active sJIA and those with clinically inactive disease. This result contradicts the pathogenic role of IFNγ in sJIA and indeed agrees with some findings by other authors. Three gene expression studies have failed to find a pronounced IFNγ-inducible signature in peripheral blood mononuclear cells (PBMCs) of patients with active sJIA without MAS at sampling (Fall, N. et al. Gene expression profiling of peripheral blood from patients with untreated new-onset systematic juvenile idiopathic arthritis reveals molecular heterogeneity that may predict
macrophage activation syndrome. , Arthritis Rheum, 2007, 56(11):3793-804; Ogilvie, E.; M. et al., Specific gene expression profiles in systematic juvenile idiopathic arthritis. , Arthritis Rheum, 2007, 56(6):1954-65; Pascual, V.; et al., Role of interleukin-1 (IL-1) in the pathogenesis of systematic onset juvenile idiopathic arthritis and clinical response to IL-1 blockade. J Exp
Med, 2005, 201(9):1479-86). After ex vivo stimulation of PBMCs, the number of IFNγ-producing cells in patients with active sJIA was similar to controls (Lasiglie, D. et al., Role of IL-1 beta in the development of human T. H) 17
cells: lesson from NLPR3 mutated patients. , PLoS One, 2011, 6(5):e20014). Consistently, neither patients with active nor inactive SJIA show elevated serum or synovial fluid IFNγ levels (de Jager, W. et al., Blood and synovial fluid cytokine signatures in patients with juvenile idiopathic arthritis: a cross-sectional study., Ann Rheum Dis, 2007, 66(5):589-98). Although CXCL9 and CXCL10 are largely undetectable in synovial tissue from patients with sJIA, high levels of these chemokines can be found in synovial tissue from patients with oligoarticular or polyarticular JIA, indicating that , supporting that IFNγ has no role in sJIA joint inflammation (Sikora, KA et al., The limited role of interferon-gamma in systemic juvenile idiopathic
arthritis cannot be explained by cellular hyperresponsiveness. , Arthritis Rheum, 2012, 64(11):3799-808). Recent data in mice have shown that immunostimulation of IFNγ knockout mice with Freund's complete adjuvant results in a systemic inflammatory syndrome with features of sJIA, thereby limiting the 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, 66(5):1340-51).

In striking 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 graded samples obtained both during active MAS and during active sJIA without MAS. Fortuitously, this study did not find any significant elevation of IL-6 or IL-1β levels in patients sampled during MAS, nor any association with laboratory parameters of MAS, thereby indicating that these are critically involved in the pathogenesis of sJIA (De Benedetti, F. et al., Randomized trial of tocilizumab in systemic juvenile idiopathic arthritis., N Engl J Med, 2012, 367(25) : 2385-95;Ruperto, N. et al., Two randomized trials of canakinumab
in systematic juvenile idiopathic arthritis. , N Engl J Med, 2012, 367(25):2396-406), suggesting that it may not be critical in maintaining MAS. This finding of elevated levels of IFNγ and IFNγ-related chemokines is consistent with several previous findings. Shimizu et al. reported that levels of neopterin, a catabolite of guanosine triphosphate synthesized by human macrophages upon stimulation with IFNγ, were significantly higher in patients with MAS during sJIA than in patients with active sJIA without MAS. (Shimizu, M. et al., Distinct cytokine profiles of systematic-onset juvenile idiopathic arthritis-associated macrophage activation syndrome with partial
Emphasis on the role of interleukin-18 in its pathogenesis. , Rheumatology (Oxford), 2010, 49(9):1645-53). More recently, Put et al. reported elevated levels of IFNγ and CXCL10 in 5 patients with primary and secondary HLH, 3 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 5, Rheum 0. Consistent with these results, five patients with active sJIA without MAS at sampling had significantly lower levels of IFNγ and CXCL10 (Put et al., Rheumatology, 2015).

Interestingly, this study found that not only were the levels of IFNγ and IFNγ-related chemokines significantly elevated, but that their levels, particularly that of CXCL9, were closely correlated with the laboratory characteristics of MAS. shows an association with disease severity. This study found significantly higher levels of IFNγ and CXCL9 and CXCL10 in one patient with critical illness with multiorgan failure and central nervous system complications with generalized seizures requiring prolonged intensive care unit admission. , which further supports the association with disease severity.

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

These results in MAS and sec-HLH, combined with the findings available in the literature in patients with p-HLH, suggest that elevation of IFNγ and IFNγ-related chemokines, particularly CXCL9, may be the hallmark of HLH, regardless of underlying cause. support the hypothesis that the 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., Activating NLRC4 inflammasome mutation causes). autoinflammation with recurrent macrophage activation syndrome., Nat Genet, 2014, 46(10):1140-6), which allows IFNγ excess even in the context of HLH induced only by inflammasome dysregulation. It is suggested that production may occur.

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

To determine the safety and tolerability profile of multiple intravenous (IV) doses of an anti-IFNγ antibody, referred to herein as NI-0501; the efficacy and benefit/risk profile of NI-0501 in HLH patients. to describe the pharmacokinetic (PK) profile of NI-0501 in HLH patients; to define a reasonable NI-0501 therapeutic dose regimen for HLH; and to assess the immunogenicity of NI-0501 designed the study presented here.

Preclinical Studies: Previous studies have shown that NI-0501 has similar binding affinity and blocking activity against IFNγ from non-human primate species, including rhesus and cynomolgus monkeys, but dogs, cats, pigs, and rabbits. , demonstrated that it does not against IFNγ from rats or mice. Toxicity and safety studies in cynomolgus monkeys showed no off-target toxicities due to administration of NI-0501, weekly dosing of NI-0501 was well tolerated and required antibiotic prophylaxis and no abnormal histopathological or behavioral findings were observed during these previous studies.

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

A Phase I Clinical Trial: A Phase 1, Randomized, Double-Blind Study in 20 Healthy Adult Volunteers Investigating the Safety, Tolerability and Pharmacokinetic Profile of a Single Intravenous (IV) Administration of NI-0501 Placebo-controlled single escalating dose study. During this study, 6 subjects received placebo and 3, 3, 4, and 4 subjects (14 subjects in total) received 0.01, 0.1, 1, respectively. , and a dose of 3 mg/kg of NI-0501.

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

A total of 41 adverse events (AEs) were observed after initiation of drug infusion in 14 of 20 subjects (70%), 10 of which were reported by 4 subjects receiving placebo. Thirty-six (87.8%) AEs were of mild intensity and five (12.2%) were of moderate intensity. No severe or life-threatening AEs were reported. Twenty-three AEs (56.1%) in 10 of 14 subjects who experienced an AE were reported to be drug-related (at least plausible). The majority of AEs were single and no trend related to escalating NI-0501 doses was observed. All NI-0501 infusions were successful.

In summary, infusion of NI-0501 was well tolerated and the effects observed during 8-week post-drug infusion monitoring did not include any serious or unexpected off-target safety or immunogenicity. No concerns were expressed.

Phase 2/3 Clinical Trial Materials and Methods: These studies are conducted in patients with primary HLH. Trials are divided into three parts: screening, treatment, and follow-up. An outline is shown in FIG.

In these studies, suitable patients may be naive to HLH treatment (also referred to herein as "first-line patients") or may have already received conventional HLH therapy, e.g. , patients who have failed to respond adequately or have shown signs of intolerance by the treating physician (also referred to herein as "second-line patients"). Patients receiving NI-0501 after failure or intolerance to conventional HLH therapy are central to trials to demonstrate the efficacy of NI-0501 as a second-line treatment for primary HLH. cohort. Treatment-naive patients will be enrolled for the collection of efficacy and safety data in the first-line setting.

The following patients are excluded from this study: patients who had a diagnosis of HLH secondary as a result of documented rheumatic or neoplastic disease; previously treated with anti-thymocyte globulin (ATG), anti-CD52 therapy, etc.) or treated within 5 times their defined half-life with any other biologic active mycobacterial, Histoplasma Capsulatum, Shigella, Salmonella, Campylobacter and Leishmania infections; previous history of tuberculosis or latent tuberculosis patients with positive serologic tests for HIV antibodies, hepatitis B surface antigen, or hepatitis C antibodies; patients with malignancy; severe cardiovascular, pulmonary, hepatic, or renal function Patients with another compelling complication or dysplasia; Patients with a history of hypersensitivity or allergy to any component of the study regimen; Live or attenuated live (including BCG) within the previous 12 weeks after screening vaccinated patients; and/or pregnant or lactating female patients.

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

In these studies, NI-0501 is administered at an initial dose of 1 mg/kg over 1 hour by IV infusion. This dose is expected to inhibit at least 99% of the effects of IFNγ over 3 days in patients with baseline IFNγ concentrations less than or equal to 3400 pg/mL. Infusions are given every 3 days until Study Day 15 (SD15) (Infusion #6) and then twice weekly. The NI-0501 dose can be escalated to 3 mg/kg at any time during the study according to predetermined criteria guided by clinical and laboratory response in each patient (as described in Table 5 below). ). After a minimum of two infusions at 3 mg/kg, at re-evaluation, the same clinical and laboratory criteria that made the patient eligible to receive 3 mg/kg NI-0501 were found to still apply. If so, the dose of NI-0501 can be increased to 6 mg/kg over up to 4 infusions with regular monitoring of clinical and laboratory HLH parameters. Based on the evolution of these parameters, the dose of NI-0501 could be i) reduced back to 3 mg/kg or ii) due to evidence of PK and PD resulting in excessively high IFNγ production and therefore rapid NI-0501. Additional IV infusions can be given while still at 6 mg/kg (or increased above 6 mg/kg) if elimination is indicated. Dose escalation may occur at any time during the study provided the clinical and laboratory criteria described herein are met.

In these studies, NI-0501 is administered for 8 weeks and the treatment period is divided into two separate periods: treatment period 1 and 2, as shown in FIG.

After 8 weeks of administration of NI-0501, transplant conditioning can begin in preparation for hematopoietic stem cell transplantation (HSCT). If patient condition and donor availability permit transplantation, the expected duration of treatment can be shortened, but not less than 4 weeks. Established favorable benefit/risk for the patient if no suitable donor has been identified by week 8 or if the transplant schedule needs to be delayed for reasons unrelated to NI-0501 administration Treatment with NI-0501 can be continued in the setting of a long-term follow-up study if .

In these studies, NI-0501 is administered against a background of dexamethasone that can be tapered depending on the patient's condition. Treatment-naive patients receive NI-0501 in the background of 10 mg/m ² dexamethasone. Patients receiving NI-0501 as second-line HLH treatment should receive dexamethasone at a dose of at least 5 mg/ ^m2 or, if higher, at the same dose given prior to screening . Patients must be receiving dexamethasone from SD-1.

Dexamethasone may be tapered according to the patient's condition and as determined by the treating physician. A tapering scheme can be selected by the treating physician, provided that the dexamethasone dose at each step does not exceed halving and the frequency of change does not exceed weekly.

If disease worsens after tapering off dexamethasone, the dose of dexamethasone can be increased and maintained until an adequate response is achieved by the treating physician.

As recommended in the HLH treatment guidelines, patients receive prophylactic treatment against Pneumocystis jiroveci, fungal and herpes zoster virus infections from the day before starting treatment with NI-0501 until the end of the study. Patients will receive prophylactic treatment from the day before starting treatment with NI-0501 (ie, SD-1) until the end of the study. For example, for the prevention of Pneumocystis jiroveci, the patient receives, for example, 750 mg/m ² of sulfamethoxa per day given orally in equally divided doses twice daily for three consecutive days per week. Zol can be received with 150 mg/m ² of trimethoprim per day. For the prevention of fungal infections, patients may receive, for example, fluconazole 12 mg/kg daily at a daily dose of up to 400 mg. For HZ virus prophylaxis, patients may receive, for example, acyclovir 200 mg four times daily for children over two years of age and 100 mg four times daily for children under two years of age. These treatments are delivered orally whenever possible, otherwise intravenously.

Patients may also receive any of a variety of combination therapies such as, for example, cyclosporine A, intrathecal methotrexate and glucocorticosteroids, and others. If cyclosporine A (CsA) has already been administered to the patient prior to screening, it can be continued. CsA can be withdrawn at any time. CsA is not newly introduced during the course of the study once NI-0501 administration is initiated.

If patients are receiving intrathecal methotrexate and glucocorticoids at the start of treatment with NI-0501, continue this treatment as needed. If the appearance of CNS symptoms occurs prior to initiation of treatment with NI-0501, treatment with intrathecal methotrexate and glucocorticoids should be initiated prior to the first dose of NI-0501.

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

Analgesic treatments, transfusions of blood products, electrolyte and glucose infusions, antibiotics, antifungal and antiviral treatments and general supportive care are permitted. Additional HLH treatment may be permissible if HLH improvement is unsustainable or limited when maximum NI-0501 dose levels are achieved. As used herein, non-sustained HLH improvement refers to the patient's inability to maintain at least a 50% improvement from baseline for the three HLH parameters (see Table 6 below). Lack of HLH improvement must be demonstrated by at least two consecutive measurements. As used herein, limited HLH improvement refers to less than 50% change from baseline in at least three HLH clinical and laboratory criteria. Etoposide should be administered as an additional HLH treatment unless prior medical history leads to lack of response or clear evidence of intolerance to the drug.

The following treatments should not be used concurrently with NI-0501 administration: Etoposide, T-cell depleting agents, or any other biologics are generally not tolerated except for: G-CSF, long-term rituximab, for documented B-cell EBV infection; and additional HLH treatment, unsustained or limited HLH improvement at maximal NI-0501 dose levels (see here defined). Etoposide should be administered unless previous medical history leads to lack of response or clear evidence of intolerance to the drug. Vaccination with live or attenuated (including BCG) vaccines must be avoided throughout the study, including the 4-week follow-up period. If NI-0501 concentrations remain at therapeutic levels after study completion, the period without vaccination should be extended until measurable concentrations of NI-0501 are no longer detectable.

The development of clinical signs that characterize the disease (fever, splenomegaly, CNS symptoms) and laboratory parameters (CBC, fibrinogen, ferritin, sCD25 levels) are used to assess onset of response and time to response. The primary efficacy endpoint includes overall response rate, ie, achievement of either complete or partial response or HLH improvement at end of treatment (EoT), as defined in Table 6 below. Secondary efficacy endpoints were time to response at any time point during the study; maintenance (including data collected in any long-term follow-up study); number of patients able to reduce glucocorticoids by 50% of baseline dose or greater; HSCT if deemed necessary survival at week 8 (or EoT) and study termination; serum concentrations of NI-0501 to determine the NI-0501 pharmacokinetic (PK) profile; circulating total IFNγ and Determination of pharmacodynamic (PD) effects, including levels of sum markers, ie CXCL9 and CXCL10; and determination of other biomarkers, eg, sCD25, IL-10.

Safety parameters collected and evaluated include incidence, severity, causality and outcome of adverse events (AEs) (serious and non-serious), with particular attention to infection; red blood cells (hemoglobin); development of laboratory parameters such as complete blood cell counts (CBC), liver tests, renal function tests and coagulation with a focus on spheres and platelets; number of patients withdrawn for safety reasons; and immunogenicity (ADA). Include other parameters such as the level of circulating antibody to NI-0501 (if any) to determine.

The primary endpoint (overall response rate) will be assessed using an exact one-tailed binomial test at the 0.025 level. Time to response, durability of response and survival time are shown using Kaplan-Meier curves and median values are calculated when available. A 95% confidence interval is calculated for the median for each of these endpoints. Additional endpoints based on binary outcomes, including the number of patients with a 50% or greater reduction in glucocorticoids, and the number of patients who were able to proceed to HSCT, were converted to proportions and associated Calculate the 95% confidence interval for Statistical significance by p-value is obtained only for the primary endpoint. All other endpoints are considered supportive of the primary endpoint and, as a result, no formal hierarchy of endpoints is presented.

Administration of NI-0501 in patients leads to rapid normalization of fever within hours after the first infusion of NI-0501. Figures 13A and 13B show the effect of NI-0501 infusion on body temperature in two patients with temperatures >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 counts in patients. Figure 15 is a series of graphs and tables showing the effect of NI-0501 administration on platelet counts in patients. Figure 16 is a series of graphs and tables showing the effect of NI-0501 administration on ferritin serum levels in patients. Figure 17 is a series of graphs and tables showing the effect of NI-0501 administration on glucocorticoid taper in patients. FIG. 18 is a graph showing that administration of NI-0510 maintained IFNγ neutralization through HSCT. HLH responses to treatment with NI-0501 also persisted until transplantation. Patients were also evaluated for any CNS complications after administration of NI-0501. A summary of baseline CNS complications and status to end of treatment (EOT) is shown in Table 11 below.

Of 10 patients who underwent hematopoietic stem cell transplantation (HSCT), all patients survived; 1 patient required a CD34 stem cell boost due to mixed chimerism at D+145 after HSCT. Secondary transplant failure occurred in one patient, followed by reactivation of HLH. This patient died of acute respiratory failure and bacterial infection on D+68 after HSCT. Another patient died at D+47 after HSCT (septic shock in the setting of severe GvHD). Mild GvHD was reported in 3 other patients and has resolved/is resolving.

Serum concentrations of NI-0501 at HSCT, as reflected by levels of CXCL9 (a chemokine subtly induced by IFNγ) below the limit of quantification in 8 of 10 transplanted patients neutralization was measured. Thus, these data indicate that NI-0501 can eliminate the short-term or long-term toxicity reported for etoposide-based regimens. This translates to a reduced risk of allo-HSCT-related complications.

These data demonstrate that treatment with NI-0501 can ameliorate and/or resolve associated clinical and laboratory abnormalities of HLH, including CNS signs and symptoms. The response to NI-0501 is independent of the presence and type of causative mutation and/or the presence and type of infectious trigger. NI-0501 was well tolerated. No safety concerns have been raised to date (eg no myelotoxicity, no extensive immunosuppression). No infections caused by pathogens known to be facilitated by IFNγ neutralization have been observed. Neutralization of IFNγ by NI-0501 could provide an innovative targeting approach for managing HLH.
(Example 8)
Short-term intravenous administration of NI-0501, an anti-interferon gamma (anti-IFNγ) monoclonal antibody, in patients with systemic juvenile idiopathic arthritis (sJIA) developing macrophage activation syndrome/secondary HLH (MAS/sHLH) Safety, Tolerability, Pharmacokinetics and Efficacy of Internal Administration

The study presented here was designed to demonstrate the efficacy and safety of NI-0501 for the treatment of MAS/sHLH in patients with sJIA and consisted of two parts (i) the PK profile of NI-0501; and (ii) a pivotal study to demonstrate the efficacy and safety of NI-0501; (study will continue once NI-0501 dosing regimen and positive benefit/risk profile are confirmed). An overview of this study design is shown in FIG.

The primary objectives of the pilot study were to (i) define a valid NI-0501 therapeutic dose regimen for sJIA patients with MAS/sHLH; (ii) benefit/risk profile of NI-0501 in sJIA patients with MAS/sHLH. and (iii) to describe the pharmacokinetic (PK) profile of NI-0501 in sJIA patients with MAS/sHLH. The primary objectives of the pivotal study were (i) to determine the efficacy of NI-0501 in sJIA patients with MAS/sHLH; (ii) short-term intravenous (i.e. v.) to evaluate the safety and tolerability profile of administration; (iii) to confirm the positive benefit/risk profile of NI-0501 in sJIA patients with MAS/sHLH; To conduct an exploratory evaluation of CXCL10 as a MAS/sHLH diagnostic biomarker and as a predictor of response to treatment with NI-0501; and (v) to assess the immunogenicity of NI-0501 in sJIA patients with MAS/sHLH It is to evaluate.

The study population includes sJIA patients with MAS/sHLH who have shown an inadequate response to high-dose glucocorticoid treatment. Inclusion criteria include: (i) gender: male and female; (ii) age: <16 years at sJIA diagnosis; (iii) in the presence of at least two of the following laboratory and clinical criteria: Diagnosis of active MAS/sHLH confirmed by treating rheumatologist: (a) Laboratory criteria: platelet count ^≤262 x 109/L, WBC count ^≤4.0 x 109/L, AST level >59 U/L. (b) clinical criteria: hepatomegaly, hemorrhagic manifestations, and/or CNS dysfunction; (iv) for at least 3 days according to local standard of care high dose i. v. Patients who show an inadequate response to glucocorticoid treatment (including but not limited to pulses of 30 mg/kg mPDN for 3 consecutive days); (v) high dose i. v. Glucocorticoids should not be lowered below 2 mg/Kg mPDN per day which is equivalent to two separate daily doses of up to 60 mg per day. If patient condition and/or laboratory parameters worsen rapidly, high dose i. v. (vi) consent of patient (or consent of legally recognized representative(s)); and (vii) if patient is past puberty Acceptance of contraceptive measures.

Exclusion criteria were (i) diagnosis of suspected or confirmed primary HLH or HLH consequent to neoplastic disease; (ii) anakinra, tocilizumab, canakinumab, TNF inhibitors, rituximab or any other biologic (iii) active mycobacterial (typical and atypical), Histoplasma Capsulatum, Shigella, Salmonella, Campylobacter and Leishmania infections; (iv) evidence of latent tuberculosis; (v) positive serology for HIV antibodies; (vi) presence of malignancy; Patients with another comorbidity or dysplasia severely affecting cardiovascular, pulmonary, CNS, hepatic or renal function that may significantly affect sex assessment; (viii) study regimen (ix) having received a BCG vaccine within 12 weeks prior to screening; (x) having received any other live or attenuated vaccine within 6 weeks prior to screening and/or (xi) including pregnant or lactating female patients.

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

In Part 2, once the proposed dosing regimen is validated and positive benefit/risks of NI-0501 are demonstrated, the study will continue. If necessary, minor modifications of the dosing regimen can be applied based on the evidence obtained in Part 1.

Background therapy and concomitant medications: background at least 2 mg/kg methylprednisolone (mPDN) equivalent to a maximum of 60 mg per day (in patients 30 kg or greater) which can be tapered during treatment depending on patient condition Administer NI-0501 as Patients receive prophylactic treatment for herpes zoster infection, preferably starting the day before (in any event, prior to beginning treatment with NI-0501) until serum NI-0501 levels are no longer detectable. Cyclosporin A (CsA) can be continued if started at least 3 days before starting treatment with NI-0501. CsA dose adjustments are possible to maintain therapeutic levels. CsA may be withdrawn at any time during the study at the discretion of the attending physician. No de novo CsA can be introduced once NI-0501 administration is initiated. If the 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 (including BCG) vaccines must be avoided throughout the study, in any case until serum NI-0501 levels are undetectable. Analgesic treatments, blood product transfusions, electrolyte and glucose infusions, antibiotics, antifungal and antiviral treatments, and general supportive care are permitted.

Sample Size: Part 1 will enroll at least 5 evaluable patients. Part 2 will enroll at least 10 evaluable patients to achieve a total of 15 evaluable patients as the study continues. 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, at least 50% of patients respond inadequately to systemic glucocorticoids alone, i.e., 50% of patients receiving glucocorticoids achieve MAS remission by 8 weeks after initiation of treatment. This test has a power of 70% for improvements from 50% to 77% using a one-sided significance level of 5%.

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

Study Endpoints: Part 1 (pilot) of the study will assess the following to validate the dosing regimen in this patient population: (i) NI-0501 benefit/risk profile; (ii) NI-0501 PK profile. (iii) levels of chemokines known to be induced by IFNγ (e.g., CXCL9, CXCL10, CXCL11); (iv) cytopenia at 2, 4, 6 and 8 weeks after initiation of NI-0501; , development of the distinct features of MAS, liver dysfunction and coagulopathy; and (v) dose and duration of treatment with NI-0501. In Part 2 of the study (pivotal), the efficacy study endpoints are as follows: (a): Primary efficacy endpoint: MAS remission achieved by 8 weeks after initiation of treatment with NI-0501 and (b) secondary efficacy endpoints: time to MAS remission; time to first response as assessed by the principal investigator; number of patients able to be tapered to the same (or lower) dose that they were receiving prior to onset; time to achieve glucocorticoid taper; survival at the end of the study; Number of patients who discontinued the trial due to absence. In Part 2 of the study (pivotal), the safety study endpoints are: (a) incidence, severity, causality and outcome of AEs (serious and non-serious); attention; development of laboratory parameters, especially CBC (focusing on hemoglobin, neutrophils and platelets), LFT, and coagulation parameters; number of patients discontinued for safety reasons; and immunogenicity (ADA) Levels of circulating antibodies to NI-0501 to determine (if any).

Pharmacokinetics and pharmacodynamics are assessed by the following PK profile of NI-0501; levels of circulating free IFNγ before dosing, and total IFNγ after initiation of NI-0501 (free IFNγ + NI-0501 bound levels of chemokines known to be induced by IFNγ (e.g., CXCL9, CXCL10, CXCL11); chemokine levels (CXCL9, CXCL10) and free NI-0501, free IFNγ (predose) and total IFNγ chemokine and total IFNγ levels with laboratory parameters of MAS severity such as ferritin, platelet count, LFT (exploratory analysis); and other potential disease biomarkers such as sCD25 , IL-10, IL-6, IL-18, TNFα, neopterin) levels.
Other embodiment

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to be illustrative, not limiting, of the scope of the invention, which is defined by the scope of the appended claims. be done. 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 a human subject in need of treatment of primary hemophagocytic lymphohistiocytosis (HLH), comprising: on the body,
a) a first dose of 1.0 or 3.0 mg/kg body weight of said subject of an antibody that binds interferon gamma (IFNγ) administered to said subject within 12 hours; and b) 12 hours. intravenously administering a second dose of said antibody of 3.0, 6.0 or 10.0 mg per kg of body weight of said subject within a period of time;
The antibody comprises a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO: 1); a variable heavy chain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence of ); and a variable heavy chain complementarity determining region 3 (VH CDR3) comprising the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO:3); and a variable light chain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO:4). a variable light chain complementarity determining region 2 (VL CDR2) region comprising the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and a variable light chain complementarity determining region 3 (VL CDR3) region comprising the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6). A method involving a region.
(Item 2)
The method of item 1, wherein the antibody comprises a heavy chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO:47 and a light chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO:48.
(Item 3)
The method of item 1, wherein the dose of antibody is administered within 6 hours.
(Item 4)
4. The method of item 3, wherein the dose of antibody is administered within 1 hour.
(Item 5)
2. The method of item 1, wherein said second dose is administered for a first treatment period every three days after said first dose.
(Item 6)
said second dose is administered over a second treatment period following completion of said first treatment period of 1, 2, 3, 4, 5, 6, 7, 8 weeks after said first dose; The method of item 5.
(Item 7)
7. The method of item 6, wherein said second treatment period is twice a week.
(Item 8)
The method of item 1, further comprising administering to said subject a third dose of 1.0 mg/kg body weight of said subject within 12 hours of said antibody.
(Item 9)
The method of item 1, wherein the dose of antibody is administered as a single injection.
(Item 10)
The method of item 1, wherein said antibody is administered as monotherapy or combination therapy.
(Item 11)
The method of item 1, wherein the subject is an adult subject or a pediatric subject.
(Item 12)
A multiple variable dose method for treating secondary hemophagocytic lymphohistiocytosis (HLH) in a pediatric human subject in need of treatment of secondary hemophagocytic lymphohistiocytosis (HLH). to the subject,
a) a first dose of 6.0 mg/kg body weight of said subject of an antibody that binds interferon gamma (IFNγ) administered to said subject within 12 hours; and b) within 12 hours of said intravenously administering a second dose of said antibody of 3.0 mg per kg body weight of the subject;
The antibody comprises a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO: 1); a variable heavy chain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence of ); and a variable heavy chain complementarity determining region 3 (VH CDR3) comprising the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO:3); and a variable light chain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO:4). a variable light chain complementarity determining region 2 (VL CDR2) region comprising the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and a variable light chain complementarity determining region 3 (VL CDR3) region comprising the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6). A method involving a region.
(Item 13)
13. The method of item 12, wherein the dose of antibody is administered within 6 hours.
(Item 14)
14. The method of item 13, wherein the dose of antibody is administered within 1 hour.
(Item 15)
13. The method of item 12, wherein said second dose is administered for a first treatment period every three days after said first dose.
(Item 16)
16. The method of item 15, wherein said second dose is administered over a second treatment period after completion of said first treatment period.
(Item 17)
17. The method of item 16, wherein said second treatment period is twice a week.
(Item 18)
13. The method of item 12, further comprising administering an additional dose of said antibody of 6.0 mg/kg body weight of said subject within 12 hours after said second dose.
(Item 19)
13. The method of item 12, wherein the dose of antibody is administered as a single injection.
(Item 20)
13. The method of item 12, wherein said antibody is administered as monotherapy or combination therapy.
(Item 21)
A multiple variable dose method for treating secondary hemophagocytic lymphohistiocytosis (HLH) in a human adult subject in need of treatment of secondary hemophagocytic lymphohistiocytosis (HLH). to the subject,
a) a first dose of 3 mg or 6.0 mg/kg body weight of said subject that binds to interferon gamma (IFNγ) administered to said subject within 12 hours; and b) within 12 hours. intravenously administering a second dose of said antibody of 10.0 mg or less per kg body weight of said subject;
The antibody comprises a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO: 1); a variable heavy chain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence of ); and a variable heavy chain complementarity determining region 3 (VH CDR3) comprising the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO:3); and a variable light chain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO:4). a variable light chain complementarity determining region 2 (VL CDR2) region comprising the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and a variable light chain complementarity determining region 3 (VL CDR3) region comprising the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6). A method involving a region.
(Item 22)
22. The method of item 21, wherein the dose of antibody is administered within 6 hours.
(Item 23)
23. The method of item 22, wherein the dose of antibody is administered within 1 hour.
(Item 24)
23. The method of item 22, wherein said second dose is administered for a first treatment period every 3 days after said first dose.
(Item 25)
25. The method of item 24, wherein said second dose is administered over a second treatment period after completion of said first treatment period.
(Item 26)
26. The method of item 25, wherein said second treatment period is twice a week.
(Item 27)
22. The method of item 21, further comprising administering an additional dose of said antibody of 1.0, 3.0 or 6.0 mg/kg body weight of said subject within 12 hours after said second dose. the method of.
(Item 28)
22. The method of item 21, wherein the dose of antibody is administered as a single injection.
(Item 29)
22. The method of item 21, wherein said antibody is administered as monotherapy or combination therapy.
(Item 30)
A method of treating a condition in a human subject in need thereof by multiple variable doses comprising:
a) a first dose of an antibody that binds interferon gamma (IFNγ) administered to said subject within 12 hours; and b) a second dose of said subject's body weight within 12 hours. administering the antibody intravenously;
The antibody comprises a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO: 1); a variable heavy chain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence of ); and a variable heavy chain complementarity determining region 3 (VH CDR3) comprising the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO:3); and a variable light chain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO:4). a variable light chain complementarity determining region 2 (VL CDR2) region comprising the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and a variable light chain complementarity determining region 3 (VL CDR3) region comprising the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6). A method involving a region.
(Item 31)
31. The method of item 30, wherein said antibody comprises a heavy chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO:47 and a light chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO:48.
(Item 32)
31. The method of item 30, wherein the dose of antibody is administered within 6 hours.
(Item 33)
33. The method of item 32, wherein the dose of antibody is administered within 1 hour.
(Item 34)
31. The method of item 30, wherein said second dose is administered for a first treatment period every three days after said first dose.
(Item 35)
35. The method of item 34, wherein said second dose is administered over a second treatment period after completion of said first treatment period.
(Item 36)
36. The method of item 35, wherein said second treatment period is twice a week.
(Item 37)
31. The method of item 30, wherein said first dose is 1.0-10 mg/kg body weight of said subject.
(Item 38)
31. The method of item 30, wherein said dose is 1.0-10 mg/kg body weight of said subject and said second dose is lower or higher than said first dose.
(Item 39)
31. The method of item 30, further comprising administering a third dose of said antibody of 1.0-10 mg/kg body weight of said subject administered to said subject within 12 hours.
(Item 40)
31. The method of item 30, wherein the dose of antibody is administered as a single injection.
(Item 41)
31. The method of item 30, wherein said antibody is administered as monotherapy or combination therapy.
(Item 42)
31. The method of item 30, wherein said subject is an adult subject or a pediatric subject.
(Item 43)
31. The method of item 30, wherein the condition is graft rejection.
(Item 44)
44. The method of item 43, wherein said graft rejection is solid organ transplant injury, acute bone marrow graft rejection.
(Item 45)
31. The method of item 30, wherein the condition is graft-versus-host disease, paraneoplastic cerebellar degeneration, hemorrhagic fever, sarcoidosis, adult-onset Still's disease.
(Item 46)
31. The method of item 30, administered to the subject after receiving CAR T cell therapy.
(Item 47)
The method of any one of the preceding items, wherein said subject has been administered dexamethasone immediately prior to dosing of said antibody.
(Item 48)
31. The method of item 30, wherein said dexamethasone is administered at a dose of at least 10 mg/ ^m2 .
(Item 49)
31. The method of item 30, wherein said dexamethasone is administered at a dose of at least 5 mg/ ^m2 .
(Item 50)
The method of any one of the preceding items, wherein the subject has not previously received treatment for HLH.
(Item 51)
13. The method of any one of the preceding items, further comprising administering at least a second agent to said subject.
(Item 52)
524. The method of item 523, wherein said second agent is a therapeutic agent, an anti-inflammatory agent, and/or an immunosuppressive agent.
(Item 53)
Per mL:
a) 5 mg or 25 mg fully human anti-interferon gamma (IFNγ) monoclonal antibody; and b) 1.55 mg L-histidine, 3.14 mg L-histidine monohydrochloride monohydrate, 7.31 mg sodium chloride (NaCl), and An injectable pharmaceutical formulation containing 0.05 mg polysorbate 80, pH between 5.8 and 6.2.
(Item 54)
The antibody comprises a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO: 1); a variable heavy chain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence of ); and a variable heavy chain complementarity determining region 3 (VH CDR3) comprising the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO:3); and a variable light chain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO:4). a variable light chain complementarity determining region 2 (VL CDR2) region comprising the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and a variable light chain complementarity determining region 3 (VL CDR3) region comprising the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6). 54. The formulation of item 53, comprising a region.
(Item 55)
55. The formulation of item 54, wherein said antibody comprises a heavy chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO:47 and a light chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO:48.
(Item 56)
A unit dose vial containing 20 ml of a fully human anti-interferon gamma (IFNγ) monoclonal antibody solution suitable for injection, wherein the concentration of antibody is 5 mg/ml or 25 mg/ml, and the pH of said solution is 5.8 to 6.5 mg/ml. 2 unit dose vials.
(Item 57)
57. The unit dose vial of item 56, wherein said antibody is solubilized in said solution so that said solution is clear, colorless and free of precipitate.
(Item 58)
The antibody comprises a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO: 1); a variable heavy chain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence of ); and a variable heavy chain complementarity determining region 3 (VH CDR3) comprising the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO:3); and a variable light chain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO:4). a variable light chain complementarity determining region 2 (VL CDR2) region comprising the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and a variable light chain complementarity determining region 3 (VL CDR3) region comprising the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6). 57. The unit dose vial of item 56, comprising a region.
(Item 59)
59. The unit dose vial of item 58, wherein said antibody comprises a heavy chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO:47 and a light chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO:48.
(Item 60)
A unit dose vial containing 10 ml or 20 ml of a fully human anti-interferon gamma (IFNγ) monoclonal antibody solution suitable for injection, wherein the concentration of antibody is 25 mg/ml and the pH of said solution is 5.8 to 6.2. Between, unit dose vials.
(Item 61)
61. The unit dose vial of item 60, wherein said antibody is solubilized in said solution so that said solution is clear, colorless and free of precipitate.
(Item 62)
The antibody comprises a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO: 1); a variable heavy chain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence of ); and a variable heavy chain complementarity determining region 3 (VH CDR3) comprising the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO:3); and a variable light chain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO:4). a variable light chain complementarity determining region 2 (VL CDR2) region comprising the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and a variable light chain complementarity determining region 3 (VL CDR3) region comprising the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6). 61. The unit dose vial of item 60, comprising a region.
(Item 63)
63. The unit dose vial of item 62, wherein said antibody comprises a heavy chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO:47 and a light chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO:48.
(Item 64)
A unit dose vial containing 2 ml or 20 ml of a fully human anti-interferon gamma (IFNγ) monoclonal antibody solution suitable for injection, wherein the concentration of antibody is 5 mg/ml and the pH of said solution is 5.8 to 6.2. Between, unit dose vials.
(Item 65)
65. The unit dose vial of item 64, wherein said antibody is solubilized in said solution so that said solution is clear, colorless and free of precipitate.
(Item 66)
The antibody comprises a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO: 1); a variable heavy chain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence of ); and a variable heavy chain complementarity determining region 3 (VH CDR3) comprising the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO:3); and a variable light chain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO:4). a variable light chain complementarity determining region 2 (VL CDR2) region comprising the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and a variable light chain complementarity determining region 3 (VL CDR3) region comprising the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6). 65. The unit dose vial of item 64, comprising a region.
(Item 67)
67. The unit dose vial of item 66, wherein said antibody comprises a heavy chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO:47 and a light chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO:48.

Claims (20)

A composition for the treatment of symptoms of macrophage activation syndrome (MAS) or secondary hemophagocytic lymphohistiocytosis (HLH) in a subject, said composition comprising an antibody that binds interferon gamma (IFNγ) wherein the antibody comprises
i) a first dose of 1.0 mg to 10 mg per kg body weight of said subject; and
ii) a second dose of 1.0 mg to 10 mg per kg body weight of said subject
administered at
The antibody comprises a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO: 1); a variable heavy chain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence of ); and a variable heavy chain complementarity determining region 3 (VH CDR3) comprising the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO:3); and a variable light chain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO:4). a variable light chain complementarity determining region 2 (VL CDR2) region comprising the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and a variable light chain complementarity determining region 3 (VL CDR3) region comprising the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6). A method involving a region. 2. The antibody of claim 1, wherein the antibody is administered at i) a first dose of 6.0 mg/kg body weight of the subject and ii) a second dose of 3.0 mg/kg body weight of the subject. composition. 2. The composition of claim 1, wherein said antibody comprises a heavy chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO:47 and a light chain variable amino acid sequence that is the amino acid sequence of SEQ ID NO:48. 2. The composition of claim 1, wherein the first dose of antibody is administered over an hour or longer. 2. The claim 1, wherein the second dose is administered over a first treatment period, and wherein the second dose is administered every three days after the first dose during the first treatment period. composition. 6. The composition of Claim 5, wherein said first treatment period is about two weeks. 6. The composition of claim 5, wherein said second dose is administered over a second treatment period after completion of said first treatment period. 8. The composition of claim 7, wherein said second dose is administered twice weekly during said second treatment period. 8. The composition of Claim 7, wherein said second treatment period is about two weeks. 2. The composition of claim 1, wherein said first dose and said second dose are administered intravenously. 2. The composition of claim 1, wherein said antibody is in the pharmaceutical composition at a concentration of 5 mg/mL. 2. The composition of claim 1, wherein said antibody is in the pharmaceutical composition at a concentration of 25 mg/mL. The antibody is administered as a pharmaceutical formulation, the pharmaceutical formulation comprising L-histidine, L-histidine monohydrochloride monohydrate, sodium chloride (NaCl), and polysorbate 80, the pH of the formulation being from pH 5.8. 2. The composition of claim 1, wherein the pH is between 6.2. 2. The composition of claim 1, wherein the subject has systemic juvenile idiopathic arthritis (sJIA). 15. The composition of claim 14, wherein said subject has shown an inadequate response to high-dose glucocorticoid treatment. 2. The composition of claim 1, wherein said subject has an immune-related disorder, such as an autoimmune disease or an inflammatory disorder. 2. The composition of claim 1, wherein the subject has adult-onset Still's disease (AOSD). 2. The composition of claim 1, wherein said subject has systemic lupus erythematosus (SLE). 2. The composition of claim 1, wherein said subject is an adult subject. 2. The composition of claim 1, wherein said subject is a pediatric subject.

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