EA042261B1 - METHODS, COMPOSITIONS AND DOSAGE REGIMENS FOR THE TREATMENT OR PREVENTION OF INTERFERON-GAMMA RELATED DISEASES - Google Patents

Description

Related Applications

The present application claims the priority of U.S. Provisional Application No.

62/411783, filed October 24, 2016, the contents of which are incorporated herein by reference in their entirety.

Field of technology to which the invention relates

The invention relates primarily 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 (IFNy, IFN-gamma), such as, for example, hemophagocytic lymphohistiocytosis (HLH) , hemorrhagic fever, sequelae of CART cell therapy, graft failure, graft rejection, graft-versus-host disease (GvHD), and/or inflammatory disease associated with graft rejection. The invention also relates to methods and compositions for extending the viability of transplanted biological material.

Background of the invention

Human interferon-gamma (IFNy, IFN-gamma) is a lymphokine produced by activated T lymphocytes and natural killer cells. It exhibits antiproliferative and immunomodulatory activities, binds to IFNy-R, a heterodimeric receptor on most primary cells of the immune system, and triggers a cascade of events leading to inflammation. It is known that the immunomodulatory activity of IFNy has a beneficial effect in many clinical conditions. However, as is known, there are many clinical conditions in which the activity of IFNy leads to detrimental effects. For example, autoimmune diseases are associated with high levels of IFNy in the blood and diseased tissues in patients with autoimmune diseases. IFNy activity has also been shown to be associated with disease states such as cachexia and septic shock.

IFNy has been implicated in a number of diseases; and drugs based on anti-IFNy agents have been developed.

The essence of the invention

Various aspects of the invention relate to a treatment regimen using multiple varying doses for the treatment of diseases, disorders or conditions associated with elevated levels of IFNy.

In one aspect, the invention relates to a method of treating primary hemophagocytic lymphohistiocytosis (HLH) in a human by intravenously administering a first dose and a second dose of an antibody that binds interferon gamma (IFNy) to an individual. The individual is an adult individual or a child individual. The first dose is 1.0 or 3.0 mg/kg of the individual's body weight and the second dose is 3.0, 6.0 or 10.0 mg/kg of the individual's body weight. Optionally, a third dose of 1.0 mg/kg of the individual's body weight is administered.

In another aspect, the invention relates to a method of treating secondary hemophagocytic lymphohistiocytosis (HLH) in a child individual by intravenously administering to the individual a first dose and a second dose of an antibody that binds interferon gamma (IFNy). The first dose is 6.0 mg/kg of the individual's body weight and the second dose is 3.0 mg/kg of the individual's body weight. Optionally, a third dose of 6.0 mg/kg of the individual's body weight is administered.

In a further aspect, the invention relates to a method of treating secondary hemophagocytic lymphohistiocytosis (HLH) 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 (IFNy). The first dose is 3.0 mg/kg or 6.0 mg/kg of the individual's body weight and the second dose is not more than 10 mg/kg of the individual's body weight. For example, the second dose is 1.0, 3.0, 6.0 or 10.0 mg/kg. Optionally, a third dose of less than 10.0 mg/kg of the individual's body weight is administered. For example, the third dose is 1.0, 3.0, or 6.0 mg/kg of the individual's body weight.

In another aspect, the invention relates to a method of 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 (IFNy). The individual is an adult individual or a child individual. The condition is transplant rejection, such as solid organ transplant rejection or acute bone marrow transplant rejection. The condition is graft-versus-host disease, paraneoplastic cerebellar degeneration, hemorrhagic fever, sarcoidosis, or acquired Still's disease. Alternatively, the method is applied to an individual after receiving CART cell therapy. The first dose is from 1.0 to 10 mg/kg of the individual's body weight and the second dose is from 1.0 to 10 mg/kg of the individual's body weight. For example, the second dose is 1.0, 3.0, 6.0 or 10.0 mg/kg. Preferably, the second dose is higher or lower than the first dose. Optionally, a third dose of 1.0 to 10 mg/kg of the individual's body weight is administered. For example, the first, second, or third dose is 1.0, 3.0, 6.0, or 10.0 mg/kg of the individual's body weight.

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An antibody that binds interferon gamma (IFNy) contains the heavy chain variable region complementarity determining region 1 (VH CDR1) containing the amino acid sequence SYAMS (SEQ ID NO: 1); a heavy chain variable complementarity determining region 2 (VH CDR2) containing the amino acid sequence AISGSGGSTYYADSVKG (SEQ ID NO: 2) and a heavy chain variable complementarity determining region 3 (VH CDR3) containing the amino acid sequence DGSSGWYVPHWFDP (SEQ ID NO: 3); light chain variable region complementarity determining region 1 (VL CDR1) containing the amino acid sequence TRSSGSIASNYVQ (SEQ ID NO: 4); a light chain variable complementarity determining region 2 (VL CDR2) containing the amino acid sequence EDNQRPS (SEQ ID NO: 5); and a light chain variable complementarity determining region 3 (VL CDR3) containing the amino acid sequence QSYDGSNRWM (SEQ ID NO: 6). For example, an antibody contains a heavy chain variable region amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 47 and a light chain variable region amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 48.

The antibody dose is administered over 1, 6, or 12 hours.

The second dose is administered during the first treatment period every three days after the first dose. In addition, the second dose is administered during the second treatment period following the completion of the first treatment period. During the second treatment period, the dose is administered, for example, twice a week.

The antibody dose is administered as a single injection.

The antibody is administered as monotherapy or as part of combination therapy.

Optionally, the methods of the invention also include administering dexamethasone immediately prior to administration of the antibody dose. Dexamethasone is administered at a dose of at least 10 mg/m ² or at least 5 mg/m ² .

The individual has not previously received HLH treatment.

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

The scope of the invention also includes an injectable pharmaceutical composition containing in ml: 5 mg or 2 to 5 mg of a fully human monoclonal antibody against interferon gamma (IFNy); 1.55 mg L-histidine, 3.14 mg L-histidine monohydrochloride, monohydrate, 7.31 mg sodium chloride (NaCl) and 0.05 mg polysorbate 80, pH 5.8 to 6.2.

In a further aspect, the invention relates to a single-dose ampoule containing 20 ml of an injectable solution of a fully human monoclonal antibody against interferon gamma (IFNy), wherein the concentration of the antibody is 5 mg/ml or 25 mg/ml and the pH of the solution is between 5.8 up to 6.2. The antibody is solubilized in solution such that the solution is clear, colorless and free of precipitate.

In another aspect, the invention relates to a single-dose ampoule containing 10 ml or 20 ml of an injectable solution of a fully human anti-interferon gamma (IFNy) monoclonal antibody, wherein the concentration of the antibody is 25 mg/ml and the pH of the solution is from 5.8 to 6.2. The antibody is solubilized in solution such that the solution is clear, colorless and free of precipitate.

In another aspect, the invention relates to a single dose ampoule containing 2 ml or 10 ml of an injectable solution of a fully human anti-interferon gamma (IFNy) monoclonal antibody, the concentration of the antibody is 5 mg/ml and the pH of the solution is from 5.8 to 6 ,2. The antibody is solubilized in solution such that the solution is clear, colorless and free of precipitate.

An antibody that binds interferon gamma (IFNy) comprises: heavy chain variable region complementarity determining region 1 (VH CDR1) containing the amino acid sequence SYAMS (SEQ ID NO: 1); heavy chain variable region complementarity determining region 2 (VH CDR2) comprising the amino acid sequence AISGSGGSTYYADSVKG (SEQ ID NO: 2); and heavy chain variable region complementarity determining region 3 (VH CDR3) containing the amino acid sequence DGSSGWYVPHWFDP (SEQ ID NO: 3); light chain variable region complementarity determining region 1 (VL CDR1) containing the amino acid sequence TRSSGSIASNYVQ (SEQ ID NO: 4); light chain variable complementarity determining region 2 (VL CDR2) comprising the amino acid sequence EDNQRPS (SEQ ID NO: 5); and light chain variable region complementarity determining region 3 (VL CDR3) containing the amino acid sequence QSYDGSNRWM (SEQ ID NO: 6). For example, an antibody comprises a heavy chain variable region amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 47 and a light chain variable region amino acid sequence corresponding to

- 2 042261 amino acid sequence of SEQ ID NO: 48.

Any of the above aspects or embodiments may be combined with any other aspect or embodiment.

Unless otherwise indicated, all technical and scientific terms used in this document have the meaning given to them by experts in the field to which the present invention relates. While methods and materials similar or equivalent to those described herein may be used in the practice of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other literature referenced herein are expressly incorporated by reference in their entirety. In the event of a conflict, this specification, including definitions, shall prevail. In addition, the materials, methods, and examples described herein are illustrative only and should not be limiting.

Other features and advantages of the present invention will become apparent from, and are within the scope of, the following detailed description and claims.

Brief description of the drawings

In FIG. 1 is a graph showing the correlation between pre-dose serum CXCL9 levels and total IFNy levels 24 hours after NI-0501 antibody infusion in the current phase 2 pilot study in patients with primary HLH.

In FIG. 2 is a graph demonstrating the correlation between serum CXCL9 levels and pre-dose total IFNy levels 24 hours after NI-0501 antibody infusion in the current phase 2 pilot study in patients suffering from primary HLH.

In FIG. 3A and 3B are a series of graphs showing the correlation between serum CXCL9 levels and IFNy levels in patients suffering from macrophage activation syndrome (MAS) secondary to systemic juvenile idiopathic arthritis (sJIA) and in patients with active sJIA.

In FIG. 4A-1, 4A-2, 4B-1, 4B-2, 4C-1, 4C-2, 4D-1 and 4D-2 are a series of graphs showing the correlation between IFNy levels as well as serum CXCL9 levels and clinical scores. in patients with active sJIA and MAS secondary to sJIA.

In FIG. 5 is a graph demonstrating complete neutralization of IFNy as evidenced by undetectable levels of IFNy inducible chemokines.

In FIG. 6 is a graph showing the attenuation of HLH activity during NI0501 treatment (2 weeks and end of treatment): percentage of patients with platelet count >100x10 ⁹ /l, neutrophil count >1x10 ⁹ /l, fibrinogen level >1.5 g/l and a decrease in ferritin levels of at least 25%.

In FIG. 7A and 7B are a series of graphs showing the correlation between pre-dose CXCL9 levels and total IFNy levels 24 hours after NI-0501 infusion. In the inset shown in Fig. 7B shows an example of an individual IFNy and CXCL9 profile during treatment with NI-0501.

In FIG. 8A, 8B, 8C, and 8D are a series of graphs showing serum levels of IFNy as well as CXCL9, CXCL10, and CXCL11 in individual patients from whom paired samples were obtained during active MAS and during active sJIA without MAS at the time of sample collection (act .sJIA). Significance levels (p) were determined using the Wilcoxon rank test for paired samples.

In FIG. 9A and 9B are a series of graphs showing changes in white blood cell (WBC) and platelet (PLT) counts, as well as ferritin levels (FIG. 9A), and changes in serum levels of IFNy, CXCL9, CXCL10, and CXCL11 (FIG. 9B). in one patient who had 3 episodes of MAS during his sJIA.

In FIG. 10A, 10B, 10C, 10D, 10E, 10F, 10G, 10H, 10I, and 10J are a series of graphs showing the correlation of IFNy and CXCL9 levels with ferritin levels, neutrophil and platelet counts, and LDH and ALT levels in patients with active MAS. at time of sample collection (red circles) and in patients with active sJIA without MAS at time of sample collection (black triangles). Spearman's correlation coefficient (Rs) and significance level (p) for each correlation are given in Table. 3.

In FIG. 11A, 11B, 11C, 11D, 11E and 11F are a series of graphs showing the association of IFNy with CXCL9 and CXCL10 production in MAS. Panel A: Correlation of IFNy levels with CXCL9 and CXCL10 levels in MAS patients at the time of sample collection. Spearman's correlation coefficient (Rs) and significance level (p) for each correlation are given in Table. 3.

In FIG. 12 is a schematic representation of the screening, treatment, and follow-up steps in the studies described in Example 7.

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

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In FIG. 14 shows a series of graphs and a table, the number of neutrophils in patients.

In FIG. 15 shows a series of graphs and a table, the number of platelets in patients.

In FIG. 16 is a series of graphs and table, serum ferritin levels in patients.

In FIG. 17 is a series of graphs and a table showing the effect of NI-0501 administration on those showing the effect of NI-0501 administration on those showing the effect of NI-0501 administration on those showing the effect of NI-0501 administration on gradual glucocorticoid dose reduction in patients.

In FIG. 18 is a graph demonstrating that administration of NI-0510 provides neutralization of IFNy prior to HSCT. The HLH response to NI-0501 also persisted until transplantation.

In FIG. 19 is a schematic representation of the screening, treatment, and follow-up steps in the studies described in Example 8.

Detailed description of the invention

The compositions and methods provided herein utilize a fully human anti-interferon-gamma (IFNy) IgG1 monoclonal antibody (mAb), herein referred to as NI-0501, that binds and neutralizes IFNy. NI-0501 binds soluble and receptor-bound (IFNyR1) forms of IFNy. Since NI-0501 is a human IgG1, it retains the characteristics of this immunoglobulin isotype, including the ability to bind Fcy receptors and complement fixation. IFNy is one of the most potent and pleiotropic cytokines of the immune system. It is extremely important for innate and adaptive immunity against viral and intracellular bacterial infections. Once bound to its receptor, IFNy acts to elicit various physiological and cellular responses. Numerous studies over the past 20 years have demonstrated an association of IFNy with the pathogenesis and maintenance of inflammatory diseases (see, for example, Billiau A. Interferon-gamma: biology and role in pathogenesis, Adv. Immunol. 1996; 62:61-130; Schoenborn JR, Wilson CB, Regulation of interferon-gamma during innate and adaptive immune responses, Adv Immunol 2007, 96:41-101, and Zhang SY, Boisson-Dupuis S, Chapgier A et al, Inborn errors of interferon (IFN)-mediated immunity in humans: insights into the respective roles of IFNalpha/beta, IFN-gamma, and IFN-lambda in host defense, Immunol Rev. 2008;226:29-40 IFNy is predominantly produced by natural killer (NK) and T cells. natural killer β-cells (NKT) as part of the innate immune response, as well as effector CD4 Th1 cells and CD8 cytotoxic T-lymphocytes (CTL) during the development of antigen-specific immunity.

The compositions and methods provided herein are useful in the treatment of diseases, disorders and conditions associated with elevated levels of IFNy. Diseases, disorders, and conditions that can be treated or prevented using the compositions and methods of the invention include, for example, hemophagocytic lymphohistiocytosis (HLH), graft versus host disease, paraneoplastic cerebellar degeneration, hemorrhagic fever, sarcoidosis, acquired Still's disease, graft failure, graft rejection transplant and/or inflammatory disease associated with transplant rejection. Transplant rejection includes solid organ transplant rejection or acute bone marrow transplant rejection. In addition, the compositions and methods are also useful for treating or alleviating a side effect of CART cell therapy.

HLH is a syndrome characterized by a severe impairment or absence of cytotoxic function of NK and CD8+ T cells, with an abrupt activation of the immune system. HLH includes primary (genetic/familial) HLH and secondary HLH, both clinically characterized by immune system dysregulation leading to severe hypercytokinemia with detrimental effects on various tissues and organs (Henter JI, Elinder G, Soder O et al. Hypercytokinemia in familial hemophagocytic lymphohistiocytosis, Blood 1991; 78: 2918-2922). HLH develops in both adults and children.

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Table 9. HLH classification

Primary HLH Family HLH (HLH) Gene Protein Function FLH-1 unknown unknown FLH-2 PRF1 Perforin Pore-forming protein FLH-3 UNC13D Muncl3-4 Vesicle stimulation FLH-4 STX11 Syntax 11 Transport and fusion of vesicles FLH-5 STXBP2 (UNC18B) Muncl8-2 Transport and fusion of vesicles Syndromes of immunodeficiency Gene Protein Function CHS LYST Lyst Vesicle transport GS-2 RAB27A Rab27a Docking of vesicles XLP-1, XLP-2 SH2DIA, BIRC4 SAP, ΧΙΑΡ signal transduction and lymphocyte activation Secondary HLH infections Rheumatic diseases (macrophage activation syndrome) metabolic diseases Malignant neoplasms

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

The disruption of cytotoxic function that occurs in HLH leads to hypercytokinemia and hemophagocytosis. This, in turn, causes all the typical symptoms of HLH (Dhote R., Simon J., Raro T. et al. Reactive hemophagocytic syndrome in adult systemic disease: report of twenty-six cases and literature review.

Arthritis Rheum. 2003; 49:633-639; Risdall RJ, McKenna RW, Nesbit ME et al. Virus-associated hemophagocytic syndrome: a benign histiocytic proliferation distinct from malignant histiocytosis. Cancer 1979; 44:993-1002; and Risdall RJ, Brunning RD, Hernandez JI, Gordon DH. Bacteria-associated hemophagocytic syndrome. Cancer 1984; 54: 2968-2972). Typical symptoms of HLH include, for example, prolonged fever, splenomegaly, hepatomegaly, cytopenia, hyperferritinemia, hypertriglyceridemia, hypofibrinogenemia, hemophagocytosis, hypercytokinemia and/or lymphohistiocytic infiltration, bone marrow hypoplasia, meningeal infiltration.

Cytokines that are elevated in HLH patients include: IFNy, interleukin 6 (IL-6), IL-10, tumor necrosis factor (TNF)a, IL-8, macrophage colony-stimulating factor (MCSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF).

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

If HLH manifests itself in the course of a neoplastic disease, in particular a hematologic malignancy, often the severity of the patient's condition warrants urgent treatment of the HLH before treatment is initiated to address the underlying disease.

The presence of signs and symptoms of HLH in patients suffering from rheumatic disease such as systemic juvenile idiopathic arthritis (sJIA) and systemic lupus erythematosus (SLE),

- 5 042261 Rheumatologists often refer to macrophage activation syndrome (MAS), which may precede the onset of the rheumatic disease itself. In most patients with MAS, tests show impaired NK and perforin function, and a significant number of patients have polymorphisms or heterozygous mutations in PRF1 and UNC13D. Although this is an extremely serious and life-threatening condition, it usually resolves if adequate treatment is started, consisting in most cases of corticosteroids and cyclosporine. However, in about 15% of patients who develop MAS, the disease may be difficult to control and etoposide may be considered (Minoia F., Davi S., Home AC et al. Clinical Features, Treatment, and Outcome of Macrophage Activation Syndrome Complicating Systemic Juvenile Idiopathic Arthritis: A Multinational, Multicenter Study of 362 Patients Arthritis & Rheumatism 2014;66: 3160-3169).

Although primary HLH is thought to be predominantly a childhood disease, HLH is a condition that also occurs in adults, and emerging information indicates that it may occur more often than previously thought. Most adult patients develop the disease with malignancy (mainly in the case of non-Hodgkin's lymphomas), infections, autoinflammatory or autoimmune diseases, and iatrogenic immunodeficiencies.

There are currently no approved therapeutic agents for the treatment of HLH. However, experts in the field have developed guidelines for the care of patients with HLH (Henter JI, Home AC, Arico M., Egeler RM, Filipovich AH, Imashuku S. Ladisch S., McClain K., Webb D., Winiarski J, and Janka Diagnostic and Therapeutic Guidelines for Hemophagocytic Lymphohistiocytosis Blood Cancer 2007; 48: 124-13.1; Henter JI, Samuelsson-Horne A, Arico M et al. Treatment of hemophagocytic lymphohistiocytosis with HLH-94 immunochemotherapy and bone marrow transplantation. Blood 2002; 100: 2367-2373 and Jordan MB, Allen CE, Weitzman S, Filipovich AH, McClain KL How I treat hemophagocytic lymphohistiocytosis Blood 2011;118: 4041-4052).

Management of patients with primary HLH currently includes the following steps (Henter et al., Blood Cancer 2007): (i) 8 weeks of induction therapy with a combination of corticosteroids and immunosuppressive drugs (eg, etoposide, CsA, alemtuzumab, anti-inflammatory globulin). thymocytes); (ii) maintenance therapy until transplantation; and (iii) transplantation for all patients with identified genetic deficiency and, over time, in very severe cases of HLH without disease-associated mutations.

The main goal of induction therapy is to suppress the life-threatening inflammatory process that is characteristic of HLH, allowing transplantation for those patients who need it (Home A, Janka G, Maarten ER et al. Haematopoietic stem cell transplantation in haemophagocytic lymphohistiocytosis. Br. J. Haematol 2005; 129: 622-630). Transplantation is the only treatment for HLH associated with highly penetrating genetic mutations (Henter et al., Blood 2002).

Despite the application of such guidelines, the overall mortality rate in cases of primary HLH remains at 40-50% (Henter et al., Blood 2002; Trottestam H, Home 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 during the induction period, which are associated with severe short-term and long-term complications, makes an additional contribution to the already high mortality. The compositions and methods provided herein are designed as targeted therapies with guaranteed efficacy and less toxicity.

In recent years, an increasing amount of evidence has been obtained for the key role of IFNy in the development of HLH (Henter JI, Elinder G, Soder O et al. Hypercytokinemia in familial hemophagocytic lymphohistiocytosis. Blood 1991; 78: 2918-2922; Jordan MB, Hildeman D, Kappler J , Marrack P. An animal model of hemophagocytic lymphohistiocytosis (HLH): CD8+ T cells and interferon gamma are essential for the disorder Blood 2004;104: 735-743; Pachlopnik SJ, Ho CH, Chretien F et al. Neutralization of IFNgamma lesions 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 in Children J Pediatr 2011 and Risma K. , Jordan M.B. Hemophagocytic lymphohistiocytosis: updates and evolving concepts. Curr. Opin. Pediatr. 2012; 24:9-15).

All gene mutations characteristic of the primary forms of HLH affect proteins involved in the same process, ultimately leading to impaired cytotoxic activity. Perforin mutations were the first to be found in HLH patients.

Perforin knockout (KO) mice are considered a relevant model of human disease. Indeed, these mice develop all of the diagnostic and many of the clinical and laboratory characteristics of human disease upon infection with LCMV, and without treatment, these animals die. As a result, perforin-deficient KO mice have been used to study the pathophysiology of HLH. The HLH-like pathology that develops in these animals depends on CD8+ T

- 6 042261 cells and IFNy produced in response to antigen stimulation.

It was shown that when high circulating levels of IFNy were neutralized by the administration of anti-IFNy antibodies, not only did the abnormal clinical and laboratory parameters return to the previous state, but also dramatically increased survival rates. 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 were investigated in the context of the NI-0501 development program. In one model, repeated administration of CpG (causing TLR9 stimulation) was used to mimic chronic severe hyperstimulation in healthy mice (ie, with normal cytotoxic pathway genetics) as a model for HLH secondary to infection. Although these mice did not necessarily die, they developed typical clinical and laboratory signs of HLH. Upon neutralization of IFNy by administration of an anti-IFNy antibody, clinical and laboratory parameters returned to normal. Interestingly, it has been shown in this model that the administration of an anti-IFNy antibody leads to a complete neutralization of the effects of IFNy also in the respective target tissues, such as the liver and spleen (article in preparation).

To investigate the pathophysiology of secondary HLH occurring in the context of rheumatic diseases, an animal model was created using IL-6 transgenic mice in which IL-6 is highly expressed, similar to what occurs in patients with sJIA, a rheumatic disease, most commonly associated with secondary forms of HLH. When stimulated with Toll-like receptor (TLR) ligands, these mice die, exhibiting many of the signs of human disease (Strippolli R., Carvallo F., Scianaro R, et al. Amplification of the response to Tolllike receptor ligands by extended exposure to interleukin-6 in mice: Implication for the pathogenesis of macrophage activation syndrome Arthritis & Rheumatism 2012;64:1680-1688). In these mice, when IFNy is neutralized by administration of an anti-IFNy antibody, survival rates improve dramatically and laboratory values return to normal (Prencipe G. et al., forthcoming).

Additional evidence for the importance of IFNy in HLH is the high concentrations of circulating IFNy in patients with primary HLH (Henter et al., Blood 1991; Xu et al., J Pedatr 2011). In a group of 71 patients who were monitored from HLH diagnosis to treatment and follow-up, IFNy levels were above the upper limit of normal (17.3 pg/mL) in all patients and, in particular, 53.5% of patients had levels above 1000 pg/ml. It has also been reported that IFNy levels rise immediately and rapidly, and can fall from >5000 pg/mL to normal levels within 48 h with effective treatment of HLH.

More recently, in an observational study of patients with secondary forms of HLH, high levels of IFNy have been found both in patients with HLH due to infections and in patients with HLH occurring in the context of sJIA. Levels of CXCL9, CXCL10 and CXCL11, three chemokines known to be induced by IFNy, were also significantly elevated. Of note, levels of IFNy and the three IFNy-related chemokines were found to be significantly correlated with laboratory measures of disease severity, such as ferritin levels, platelet counts, and transaminase levels (Bracaglia et al., submitted for publication).

Since hypercytokinemia and organ infiltration with activated lymphocytes and histiocytes are responsible for all symptoms of HLH and depend on CD8+ T cell hyperactivity and high levels of IFNy, neutralization of IFNy is a rational therapeutic approach. Indeed, there are currently no agents specifically targeting CD8+ T cells, and targeting agents for individual IFNy-dependent cytokines may not be feasible.

Thus, based on data obtained using animal models of primary and secondary HLH, as well as on the basis of the observation of patients with both primary and secondary HLH, confirming the important role of IFNy in the pathogenesis of this disease, neutralization of IFNy seems to be a reliable basis for the development targeted therapy for HLH, which should be effective and be accompanied by no, or limited, toxicity.

The present invention also relates to compositions and methods that are useful for identifying or clarifying a population of patients suffering from a disease, where patients have an elevated level of CXCL9, alone or in combination with one or more additional interferon-y (IFNy) associated biomarkers. In particular, the invention provides compositions and methods for determining levels of CXCL9 as a biomarker for IFNy production in hemophagocytic lymphohistiocytosis (HLH), secondary HLH, and/or macrophage activation syndrome (MAS).

A wealth of evidence from animal models points to a key pathogenic role for IFNy in primary hemophagocytic lymphohistiocytosis (HLH). High levels of IFNy are also found in people suffering from HLH. It has previously been reported that high levels of IFNy and three IFNy-related chemokines, CXCL9, CXCL10 and CXCL11, were observed in patients with active MAS,

- 7 042261 a form of secondary HLH that develops in the context of systemic juvenile idiopathic arthritis (sJIA) (see, for example, Bracaglia C., Caiello I, De Graaf K., et al. Pediatric Rheumatology 2014, 12 (Suppl 1): 03) . The results obtained in mice indirectly indicate that IFNy is predominantly produced in peripheral tissues and blood concentrations may be relatively low.

The term macrophage activation syndrome (MAS) refers to a severe, potentially fatal complication of chronic inflammatory rheumatic diseases. It typically occurs in the context of systemic juvenile idiopathic arthritis (sJIA), with 10-20% of patients developing this syndrome during the course of the disease. It can also occur, although more rarely, in systemic lupus erythematosus, Kawasaki disease, and other autoimmune and autoinflammatory diseases. In sJIA, MAS typically occurs during the active stages of the disease, including the onset of the disease. An infectious trigger can be identified in a large proportion of patients. Typical signs of MAS include fever, splenomegaly, bleeding, and signs of liver, central nervous system, and kidney dysfunction, which can lead to multiple organ failure. Laboratory abnormalities include a decrease in the number of white blood cells, platelets and hemoglobin, hypertransaminasemia, a marked increase in ferritin levels, and evidence of intravascular activation of the blood 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): pp 289-98). MAS leads to serious complications and death, being the cause of a significant proportion of deaths due to sJIA (Minoia, F., et al., Clinical features, treatment, and outcome of macrophage activation syndrome complicating systemic juvenile idiopathic arthritis: a multinational, multicenter study of 362 patients Arthritis Rheumatol, 2014. 66(11): p. 3160-9 Hashkes, P. J., et al., Mortality outcomes in pediatric rheumatology in the US Arthritis Rheum, 2010. 62(2): p. 599-608 ). A better understanding of the pathogenesis of the disease, followed by the identification of new therapeutic targets, and the possible development of targeted therapies could lead to a significant improvement in disease control and outcome of MAS.

Most of the clinical signs and laboratory abnormalities in MAS are the same as in hemophagocytic lymphohistiocytosis (HLH) and, indeed, it is currently categorized as secondary or reactive HLH (sec-HLH) (Jordan, M.B., et al., How I treat hemophagocytic lymphohistiocytosis Blood, 2011. 118(15): pp. 4041-52. Primary forms of HLH (p-HLH) are caused by mutations in genes encoding proteins involved in granule exocytosis, including PRF1, UNC13D, STXBP2, STX11, RAB27A, and XIAP, which typically result in defective cytotoxic activity of CD8+ lymphocytes and NK cells. According to the current classification, in the absence of an identifiable genetic cause and/or family history, HLH is classified as secondary or reactive. Second-HLH can occur in the absence of an obvious trigger or in the context of an infection, malignancy, or rheumatic disease, in the latter case it is commonly referred to as MAS. The genetic basis for the development of MAS is gradually becoming better understood, with a number of studies pointing to the association of MAS, and sec-HLH in general, with heterozygosity for low-penetrance variants or mutations in the same genes as in the development of n-HLH (Kaufman K. M., et al., Whole-exome sequencing reveals overlap between macrophage activation syndrome in systemic juvenile idiopathic arthritis and familial hemophagocytic lymphohistiocytosis Arthritis Rheumatol, 2014, 66(12): p. 3486-95; Vastert, S. J., et al., Mutations in the perforin gene can be linked to macrophage activation syndrome in patients with systemic onset juvenile idiopathic arthritis Rheumatology (Oxford), 2010 49(3): p. 441-9 Zhang K., et al., Macrophage activation syndrome in patients with systemic juvenile idiopathic arthritis is associated with MUNC13-4 polymorphisms. Arthritis Rheum, 2008. 58(9): p. 2892-6; rr Rheumatol Rep, 2014. 16(9): p. 439; and Bracaglia C., Siem E., Da Ros M, et al. Mutations of familial hemophagocytic lymphohistiocytosis (FHL) related genes and abnormalities of cytotoxicity function tests in patients with macrophage activation syndrome (MAS) occurring in systemic juvenile idiopathic arthritis (sJIA). Pediatric Rheumatology 2014, 12 (Suppl 1): P53). This similarity in the genetic basis for p-HLH and MAS further supports a common pathogenic mechanism.

Results from studies of patients suffering from p-HLH, as well as mouse models of p-HLH, support the hypothesis that defective cytotoxic activity and abnormalities in the interaction of antigen presenting cells (AHK)-CD8+ T cells lead to defects in the attenuation of the immune response and abnormal activation of T cells. . This results in uncontrolled immune activation and production of pro-inflammatory cytokines by T-lymphocytes and macrophages, leading to organ damage. The results of studies in animal models of p-HLH using perforin and Rab27 deficient mice indicate an important role for interferon-gamma (IFNy) produced by activated CD8+ T cells. In perforin-deficient mice, neutralization of IFNy allows animals to survive this usually lethal syndrome, with a return to normal of abnormal biochemical and hematological parameters (Jordan, M.B., et al., An animal model of hemophagocytic lymphohistiocytosis (HLH): CD8+ T cells and interferon gamma are essential for the disorder.

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Blood, 2004. 104(3): p. 735-43; Pachlopnik Schmid, J., et al., Neutralization of IFNgamma lesions haemophagocytosis in LCMV-infected perforin- and Rab27a-deficient mice. EMBO Mol Med, 2009. 1(2): p. 112-24). In Rab27-deficient mice, in which the disease does not lead to death, neutralization of IFNy causes a marked improvement in the state of peripheral organs, including the central nervous system (Pachlopnik 2009). High levels of circulating IFNy have also been found in patients with HLH diagnosed according to the 2004 HLH diagnostic guidelines (My, L.T., et al., Comprehensive analyzes and characterization of haemophagocytic lymphohistiocytosis in Vietnamese children. Br J Haematol, 2010. 148( 2): p. 301-10, Takada H., et al., Increased serum levels of interferon-gamma-inducible protein 10 and monokine induced by gamma interferon in patients with haemophagocytic lymphohistiocytosis, Clin Exp Immunol, 2003. 133(3) : p. 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): pp. 84-91 Xu X.J., et al., Diagnostic accuracy of a specific cytokine pattern in hemophagocytic lymphohistiocytosis in children J Pediatr, 2012. 160(6): p. 984-90 e1) and therefore not necessarily based on the presence of a genetic mutation. It should be noted that these studies included a significant, albeit variable, proportion of patients with no apparent genetic cause for the disease (ibid.).

The studies described herein were designed to correlate serum levels of IFNy and the three IFNy-related chemokines with each other and with laboratory measures of disease activity in patients with active MAS in search of an in vivo biomarker of IFNy production. In particular, circulating levels of IFNy, CXCL9, CXCL10, CXCL11, and IL-6 were measured in patients with sJIA, with approximately 37% (20 of 54) of patients having MAS at the time of sample collection. Also evaluated the relationship of circulating levels with indicators of disease activity and, in addition, the correlation of IFNy levels with levels of CXCL9, CXCL10 and CXCL11. In some embodiments, the implementation of the biomarker is the total level of IFNy, which is useful as a pharmacodynamic biomarker.

As described herein, levels of IFNy and the 3 IFNy-related chemokines CXCL9, CXCL10 and CXCL11 were significantly elevated with active MAS compared with active sJIA without MAS at the time of sample collection. In active MAS, laboratory measures of disease severity such as ferritin, neutrophils, platelets, alanine aminotransferase, and lactate dehydrogenase levels correlated significantly with IFNy and CXCL9 levels, and to a lesser extent with CXCL10 and CXCL11 levels; no correlation was found with IL-b levels. In patients with active sJIA without MAS, there was no significant correlation between laboratory parameters and cytokine levels. In active MAS, IFNy levels correlated significantly with CXCL9 levels, to a lesser extent with CXCL10 levels, but not with CXCL11 levels.

High levels of IFNy and CXCL9 observed in patients with active MAS correlated significantly with laboratory measures of disease severity. In patients with active MAS, IFNy and CXCL9 levels were strongly correlated. Since CXCL9 has been shown to be induced only by IFNy and not by other interferons (see e.g. Groom J.R. and Luster A.D. Immunol Cell Biol 2011, Feb; 89(2):207-15), the results disclosed herein show that CXCL9 is a biomarker for IFNy production in MAS.

The studies described herein also showed that levels of IFNy and chemokine (C-X-C motif) ligand 9 (CXCL9), CXL10 and CXCL11, three chemokines known to be induced by IFNy, are elevated in patients with MAS, complication of sJIA, but not in patients with active sJIA without MAS. In addition, in these patients, IFNy, CXCL9, CXCL10, and CXCL11 levels correlated with laboratory measures of disease severity.

The present invention relates to compositions and methods that use a neutralizing anti-IFNy antibody, or an antigen-binding fragment thereof, to treat, prevent and/or delay the onset or progression of, or alleviate a symptom associated with graft failure, graft rejection, and/or inflammatory a disease associated with transplant rejection, such as solid organ transplant rejection or acute bone marrow transplant rejection. The invention relates to compositions and methods that use a neutralizing anti-IFNy antibody, or an antigen-binding fragment thereof, to treat, suppress, delay the progression of, or otherwise alleviate a symptom of a graft-versus-host disease (GvHD) reaction in an individual who has received or is receiving a graft containing a biological material, or a series of grafts containing biological material.

The invention relates to compositions and methods in which a neutralizing anti-IFNy antibody, or an antigen-binding fragment thereof, is used to prolong the viability of a transplanted biological material. The invention relates to compositions and methods in which a neutralizing anti-IFNy antibody, or antigen-binding fragment thereof, is used for the treatment, prevention and/or delay in onset or progression, or relief of a symptom associated with paraneoplastic cerebellar degeneration, hemorrhagic fever, sarcoidosis,

- 9 042261 Still's disease or CART cell therapy.

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

Using the compositions and methods provided herein, NI-0501 is administered to an individual in need thereof to treat, prevent and/or delay the onset or progression of, or alleviate a symptom associated with graft failure, graft rejection, and/or inflammatory disease associated with transplant rejection. In some embodiments, graft rejection, also referred to herein as graft failure, is acute. In some embodiments, graft rejection is hyperacute.

Neutralizing anti-IFNy antibodies of the invention comprise, for example, the heavy chain complementarity determining regions (CDRs) shown in Table 1 below. 1 A, light chain CDRs shown in Table. 1B, and combinations thereof. Amino acids constituting complementarity determining regions (CDRs) as defined according to Chothia et al. 1989, E.A. Rabat et al., 1991, underlined and italicized below (See Chothia C, et al., Nature 342: 877-883 (1989); Rabat, EA, et al., Sequences of Protein of immunological interest, fifth edition, US Department of Health and Human Services, US Government Printing Office (1991)).

Table 1 A. VH CDR sequences from antibody clones that bind and neutralize IFNy

clone name VH CDR1 VH CDR2 VH CDR3 NI-0501 SYAMS (SEQ ID NO:1) AI SGSGGSTYYADSVKG (SEQ ID NO:2) DGSSGWYVPHWFDP (SEQ ID NO:3) AC1.2R3P2_A6 SYAMS (SEQ ID NO:1) AI SGSGGSTYYADSVKG (SEQ ID NO:2) DGSSGWYVPHWFDP (SEQ ID NO:3) AC1R3P2_B4 SYAMS (SEQ ID NO:1) AI SGSGGSTYYADSVKG (SEQ ID NO:2) DHSSGWYVISGMDV (SEQ ID NO:9) AD14R4P1_B9 SYAMS (SEQ ID NO:1) AI SGSGGSTYYADSVKG (SEQ ID NO:2) DLTVGGPWYYFDY (SEQ ID NO:11) AD14R4P2_C9 SYAMS (SEQ ID NO:1) AI SGSGGSTYYADSVKG (SEQ ID NO:2) DGWNALGWLES(SEQ ID NO:14) AC1.4R4P2_C10 SYAMS (SEQ ID NO:1) AI SGSGGSTYYADSVKG (SEQ ID NO:2) DGSSGWYVPHWFDP (SEQ ID NO:3) AC1.2R3P7_D3 SNAMS (SEQ ID NO:20) TLTGSGGTAYYADSVEG (SEQ ID NO:21) GTELVGGGLDN (SEQ ID NO :22) AD1R2P2_D6 SYAMS (SEQ ID NO:1) AI SGSGGSTYYADSVKG (SEQ ID NO:2) DGWNALGWLES(SEQ ID NO:14) AC1.2R3P2_D8 SYAMS (SEQ ID NO:1) AI SGSGGSTYYADSVKG (SEQ ID NO:2) DGSSGWYVPHWFDP (SEQ ID NO:3) AD1.3R3P6_E1 SYAMS (SEQ ID NO:1) AI SGSGGSTYYADSVKG (SEQ ID NO:2) RSFDSGGSFEY (SEQ ID NO :28) AD1.3R3P5_F8 SYAMS (SEQ ID NO:1) AI SGSGGSTYYADSVKG (SEQ ID NO:2) VGSWYLEDFDI (SEQ ID NO :31) ADI.3R3P6_F9 SYAMS (SEQ ID NO:1) AI SGSGGSTYYADSVKG (SEQ ID NO:2) GGNYGDYFDYFDY (SEQ ID NO :34) AD14R4P2_G7 SYAMS (SEQ ID NO:1) AI SGSGGSTYYADSVKG (SEQ ID NO:2) DGWNALGWLES(SEQ ID NO:14) ADI.1R3P3_G9 SYAMS (SEQ ID NO:1) AI SGSGGSTYYADSVKG (SEQ ID NO:2) DFWVITSGNDY(SEQ ID NO :39) ADI.3R3P6_G10 SYAMS (SEQ ID NO:1) AI SGSGGSTYYADSVKG (SEQ ID NO:2) DGWNALGWLES(SEQ ID NO:14)

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

clone name VL CDR1 VL CDR2 VL CDR3 NI-0501 TRSSGSIASNYVQ (SEQ ID NO:4) EDNQRPS (SEQ ID NO:5) QSYDGSNRWM (SEQ ID NO:6) AC1,2R3P2_A6 TRSSGSIVSNYVQ (SEQ ID NO:7) EDNRPS (SEQ ID NO:8) QSYDGSNRWM (SEQ ID NO:6) AC1R3P2_B4 TRSSGSIASNYVQ (SEQ ID NO:4) EDNQRPS (SEQ ID NO:5) QSNDSDNW (SEQ ID NO:10) AD14R4F1_B9 TRSSGSIVSNYVQ (SEQ ID NO:7) DDDQRPS (SEQ 11 NO: 12) QSYDSSNW (SEQ ID NO:13) AD14R4P2_C9 TRSGGSIGSYYVQ (SEQ ID NO:15) DDKKRPS (SEQ ID NO:16) QSYDSNNLW (SEQ ID NO:17) AC1.4R4P2_C10 TRSSGTIASNYVQ (SEQ ID NO:18) EDNQRPS (SEQ ID NO:5) QSYDNSNHWV (SEQ ID NO:19) AC1.2R3P7_D3 TGSGGSIATNYVQ (SEQ ID NO:23) EDNQRPS (SEQ ID NO:5) QSYDSDNHHW (SEQ ID NO:24) AD1R2P2_D6 TGSSGSIASNYVQ (SEQ ID NO:25) EDNQRPS (SEQ ID NO:5) QSYDSSNQEW (SEQ ID NO:26) AC1.2R3P2_D8 TRSSGSIVSNYVQ (SEQ ID NO:7) EDNQRPS (SEQ ID NO:5) QSYDSNNFWV (SEQ ID NO:27) AD1.3R3P6_E1 TRSSGYIASSYVQ (SEQ ID NO:105) EDDRRPS (SEQ ID NO:29) QSYDDTTPWV (SEQ ID NO:30) AD1.3R3P5_F8 TRSSGSIASNYVH EDNRRPs QSSDTTYHGGW (SEQ ID NO:32) (SEQ ID NO:8) (SEQ ID NO:33) ADI.3R3P6_F9 TRSSGSIASNYVQ (SEQ ID NO:4) EDNQRPS (SEQ ID NO:5) QSYEGF (SEQ ID NO:35) AD14R4P2_G7 TGRNGNIASNYVQ (SEQ ID NO:36) EDTQRPS (SEQ ID NO:37) QSSDSNRVL (SEQ ID NO:38) ADI.1R3P3_G9 TRSSGSIASNYVQ (SEQ ID NO:4) EDNRPS (SEQ ID NO:8) QSFDSTNLW (SEQ ID NO:40) ADI .3R3P6_G10 AGSSGSIASNYVQ (SEQ ID NO:41) EDNQRPS (SEQ ID NO:5) QSYSYNNQW (SEQ ID NO:42)

Exemplary antibodies of the invention include, for example, the anti-IFNY antibodies described in PCT Publication No. WO 2006/109191, the entire contents of which are incorporated herein by reference.

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

Nucleotide sequence of the NI-0501 heavy chain:

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCC

TGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGG

GCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCC

GGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAG

GACACGGCCGTATATTACTGTGCGAAAGATGGTAGCAGTGGCTGGTACGTACCACACTGGTTCGACCC

CTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGG

CACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCC

GAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT

ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGA

CCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCT

TGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCT

CTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGG

ACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC

AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGTGGTCAGCGTCCTCACCGTCCTGCA

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CCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCG

AGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGG

GAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGC

CGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCG

ACGGCCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTC

TCATGCTCCGTGATGCATGAGGCTCTGCAAACCACTACACCGCAGAAGAGCCTCTCCCTGTCTCCGG

GTAAATAG (SEQ ID NO: 43)

Amino acid sequence of the NI-0501 heavy chain:

EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTY

YADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAKDGSSGWYVPHWFDPWGOGTLVTVSS

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS

LSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP

KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVL

HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY

PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT

QKSLSLSPGK (SEQ ID NO:44)

Nucleotide sequence of the NI-0501 light chain:

AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCAT

CTCCTGCACTCGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGTACCAACAGCGCCCG

GGCAGTTCCCCCACCACTGTCATCTATGAGGATAACCAGAGACCCTCTGGGGTCCCTGATCGGT

TCTCTGGCTCCATCGACAGCTCCTCCAATTCTGCCTCCCTCACCATCTCTGGGCTGAAGACTGA

GGACGAGGCTGACTACTACTGTCAGTCTTATGATGGCAGCAATCGTTGGATGTTCGGCGGAGGG

ACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCT

CTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGC

CGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCAACCC

TCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGA

AGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGC

CCCTACAGAATGTTCATAG (SEQ ID NO:45)

Amino acid sequence of the NI-0501 light chain:

NFMLTOPHSVSESPGKTVTISCTRSSGSIASNYVOWYOORPGSSPTTVIYEDNQRPSGV

PDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDGSNRWMFGGGTKLTVLGOPKAAPSVTLF

PPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTP

EQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO:46)

Amino acid sequence of the NI-0501 heavy chain variable region:

EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTY

YADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAKDGSSGWYVPHWFDPWGOGTLVTVSS (SEQ ID NO:47) Аминокислотная последовательность вариабельной области легкой цепи NI-0501: NFMLTOPHSVSESPGKTVTISCTRSSGSIASNYVOWYOORPGSSPTTVIYEDNQRPSGV PDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDGSNRWMFGGGTKLTVL (SEQ ID NO :4 8)

Suitable anti-IFNY antibodies include those described in US Pat. No. 7,700,098, the entire contents of which are incorporated herein by reference. Some exemplary antibodies include those named ARC1.2R3P2_A6 (A6), ARC1.2R3P2_B4 (B4), ARC1.2R3P2_B9 (B9), ARC1.2R3P2_C9 (C9), ARC1.2R3P2_C10 (C10), ARC1.2R3P2_D3 (D3) in said document. ) and ARC1.2R3P2_G10 (G10).

The sequences of these antibodies are shown below.

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Nucleotide sequence Ab UN:

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCA GGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACT CCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAA CAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGATGGTAGCAGTGGCTGGTAC GTACCACACTGGTTCGACCCCTGGGGCCGGGGCACCCTGGTCACCGTCTCGAGT (SEQ ID NO:4 9)

Amino acid sequence of Ab UN:

EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTY YADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAKDGSSGWYVPHWFDPWGRGTLVTVSS (SEQ ID NO:50)

Nucleotide sequence Ab VL:

AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCAT CTCCTGCACTCGCAGCAGTGGCAGCATTGTCAGCAACTATGTGCAGTGGTACCAACAGCGCCCG GGCAGTGCCCCCACCACTGTCATCTATGAGGATAACCGGAGACCCTCTGGGGTCCCTGATCGGT TCTCTGGCTCCATCGACAGCTCCTCCAATACTGCCTCCCTCACCATCTCTGGGCTGGAGGCTGA GGACGAGGCTGACTACTACTGTCAGTCTTATGATGGCAGCAATCGTTGGATGTTCGGCGGAGGG ACCAAGCTGACCGTCCTAGGT (SEQ ID NO:51)

Amino acid sequence of Ab VL:

NFMLTOPHSVSESPGKTVTISCTRSSGSIVSNYVQWYOORPGSAPTTVIYEDNRRPSGV

PDRFSGSIDSSSNTASLTISGLEAEDEADYYCOSYDGSNRWMFGGGTKLTVLG (SEQ ID NO:52)

Nucleotide sequence B4 UN:

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCA GGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACT CCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAA CAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGATCATAGCAGTGGCTGGTAC GTAATCTCCGGTATGGACGTCTGGGGCCGAGGGACAATGGTCACCGTCTCGAGT (SEQ ID NO:53)

Amino acid sequence B4 UN:

EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDHSSGWYVISGMDVWGRGTMVTVSS (SEQ ID NO:54)

Nucleotide sequence B4 VL:

AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCAT CTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGTACCAGCAGCGCCCG GGCAGTTCCCCCACCACTGTGATCTCTGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGT TCTCTGGCTCCGTCGACAGCTCCTCCAACTCTGCCTCCCTCACCATTTCTGGACTGAGGACTGA GGACGAGGCTGACTATTACTGTCAGTCTAATGATTCCGACAATGTGGTTTTCGGCGGAGGGACC AAGCTGACCGTCCTAGGT (SEQ ID NO:55)

Amino acid sequence B4 VL:

NFMLTOPHSVSESPGKTVTISCTRSSGSIASNYVOWYOORPGSSPTTVISEDNQRPSGV

PDRFSGSVDSSSNSASLTISGLRTEDEADYYCOSNDSDNWFGGGTKLTVLG (SEQ ID NO:56)

Nucleotide sequence B9 UN:

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCA GGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACT CCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATCCCAAGAACACGCTGTATCTGCAAATGAA CAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAGGACCTAACAGTGGGTGGTCCC TGGTACTACTTTGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGT (SEQ ID

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NO:57)

Amino acid sequence B9 UN:

EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTY YADSVKGRFTISRDNPKNTLYLOMNSLRAEDTAVYYCAKDLTVGGPWYYFDYWGOGTLVTVSS (SEQ ID NO:58)

Nucleotide sequence of B9 VL:

AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCAT CTCCTGCACCCGCAGCAGTGGCAGCATTGTCAGCAACTATGTGCAGTGGTACCAGCAGCGCCCG GGCAGTGCCCCCACCACTGTGATCTTTGACGATGACCAAAGACCCTCTGGGGTCCCTGGTCGGT TCTCTGGCTCCCTCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGGCTGCAGACTGA GGACGAGGCTGACTACTACTGTCAGTCTTATGATAGCAGCAATGTGGTATTCGGCGGGGGGACC AAGGTCACCGTCCTAGGT (SEQ ID NO:59)

Amino acid sequence of B9 VL:

NFMLTOPHSVSESPGKTVTISCTRSSGSIVSNYVQWYQQRPGSAPTTVIFDDDQRPSGV PGRFSGSLDSSSNSASLTISGLQTEDEADYYCQSYDSSNWFGGGTKVTVLG (SEQ ID NO:60)

Nucleotide sequence C9 UN:

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCA GGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACT CCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAA CAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAAGATGGATGGAACGCGCTGGGA TGGCTTGAATCCTGGGGCCGGGGCACCCTGGTCACCGTCTCGAGT (SEQ ID NO:61)

Amino acid sequence C9 UN:

EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTY

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDGWNALGWLESWGRGTLVTVSS (SEQ ID NO:62)

Nucleotide sequence of C9 VL:

AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAGGACGATAACCAT CTCCTGCACCCGCAGTGGTGGCAGCATTGGCAGCTACTATGTGCAGTGGTACCAGCAGCGCCCG GGCACTGCCCCCACCACTGTGATCTATGACGATAAAAAAAGACCCTCTGGGGTCCCTGATCGGT TCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGA GGACGAGGCTGACTACTATTGTCAGTCTTATGATAGCAACAATCTTGTGGTTTTCGGCGGAGGG ACCAAGGTCACCGTCCTAGGT (SEQ ID NO:63)

Amino acid sequence of C9 VL:

NFMLTOPHSVSESPGRTITISCTRSGGSIGSYYVQWYOORPGTAPTTVIYDDKKRPSGV

- 14 042261

PDRFSGSIDSSSNSASLTISGLKTEDEADYYCOSYDSNNLWFGGGTKVTVLG (SEQ ID NO:64)

Nucleotide sequence of CIO VH:

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCA GGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACT CCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAA CAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGATGGTAGCAGTGGCTGGTAC GTACCACACTGGTTCGACCCCTGGGGCAGGGGGACAATGGTCACCGTCTCGAGT (SEQ ID NO:65) Аминокислотная последовательность CIO VH: EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTY YADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAKDGSSGWYVPHWFDPWGRGTMVTVSS (SEQ ID NO:66) Нуклеотидная последовательность CIO VL: AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCAT CTCCTGCACCCGCAGCAGTGGCACCATTGCCAGCAACTATGTGCAGTGGTACCAGCAGCGCCCG GGCAGTTCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGT TCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGA GGACGAGGCTGACTACTACTGTCAGTCTTATGATAACAGCAATCATTGGGTGTTCGGCGGAGGG ACCAAGGTCACCGTCCTAGGT (SEQ ID NO:67) Аминокислотная последовательнос CIO VL: NFMLTQPHSVSESPGKTVTISCTRSSGTIASNYVQWYOORPGSSPTTVIYEDNQRPSGV

PDRFSGSIDSSSNSASLTISGLKTEDEADYYCOSYDNSNHWVFGGGTKVTVLG (SEQ ID NO:68)

Nucleotide sequence of D3 VH:

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCAGGGGGGTCCCTGAAACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCAATGCCATGAGTTGGGTCCGCCAGGCTCCA GGGAAGGGGCTGGAGTGGGTCTCAACTCTTACTGGTAGTGGTGGTACCGCATACTACGCAGACT CCGTGGAGGGCCGGTTCAGCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAA CAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAGGGCACGGAACTCGTGGGAGGA GGACTTGACAACTGGGGCCAAGGCACCCTGGTCACCGTCTCGAGT (SEQ ID NO:69) Аминокислотная последовательность D3 VH: EVOLLESGGGLVOPGGSLKLSCAASGFTFSSNAMSWVROAPGKGLEWVSTLTGSGGTAY YADSVEGRFSISRDNSKNTLYLOMNSLRAEDTAVYYCAKGTELVGGGLDNWGOGTLVTVSS (SEQ ID NO:70)

- 15 042261

Nucleotide sequence of D3 VL:

^TTTTATGCTGACTCAGCCCCACTCTCTGTCGGAGTCTCCGGGGAAGACGGTGACGAT CTCCTGCACCGGCAGCGGAGGCAGCATTGCCACCAACTATGTGCAGTGGTATCAGCAGCGCCCG GGCAGTGCCCCCACCACTGTGATCCATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGT TCTCTGGCTCCATCGACGGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGCAGCCTGA GGACGAGGCTGATTACTACTGTCAGTCTTATGATAGTGACAATCATCATGTGGTATTCGGCGGA GGGACCAAGCTGACCGTCCTAGGT (SEQ ID NO:71)

Amino acid sequence of D3 VL:

NFMLTOPHSLSESPGKTVTISCTGSGGSIATNYVOWYOORPGSAPTTVIHEDNQRPSGV

PDRFSGSIDGSSNSASLTISGLOPEDEADYYCOSYDSDNHHWFGGGTKLTVLG (SEQ ID

NO:72)

Nucleotide sequence of D6 VH:

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCA GGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACT CCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAA CAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAAGATGGATGGAACGCGCTGGGA TGGCTTGAATCCTGGGGCAAGGGGACAATGGTCACCGTCTCGAGT (SEQ ID NO:73)

Amino acid sequence of D6 VH:

EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTY

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDGWNALGWLESWGKGTMVTVSS (SEQ ID NO:74)

Nucleotide sequence of D6 VL:

AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCAT CTCCTGCACCGGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGTACCAGCAGCGCCCG GGCAGTGCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGT TCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGA GGACGAGGCTGACTACTACTGTCAGTCTTATGATAGCAGCAATCAAGAGGTGGTATTCGGCGGA GGGACCAAGCTGACCGTCCTAGGT (SEQ ID NO:75)

Amino acid sequence of D6 VL:

NFMLTOPHSVSESPGKTVTISCTGSSGSIASNYVOWYOORPGSAPTTVIYEDNQRPSGV

PDRFSGSIDSSSNSASLTISGLKTEDEADYYCOSYDSSNQEWFGGGTKLTVLG (SEQ ID NO :7 6)

Nucleotide sequence of D8 VH:

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCA

- 16 042261

GGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACT CCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAA CAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGATGGTAGCAGTGGCTGGTAC GTACCACACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO:77) Аминокислотная последовательность D8 VH: EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTY

YADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAKDGSSGWYVPHWFDPWGOGTLVTVSS (SEQ ID NO:78)

Nucleotide sequence of D8 VL:

AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTTACCAT CTCCTGCACCCGCAGCAGTGGCAGCATTGTCAGCAACTATGTACAGTGGTACCAGCAGCGCCCG GGCAGTTCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGT TCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGA GGACGAGGCTGACTACTACTGTCAGTCTTATGATAGCAACAATTTTTGGGTGTTCGGCGGAGGG ACCAAGCTGACCGTCCTAGGT (SEQ ID NO:79) Аминокислотная последовательность D8 VL: NFMLTOPHSVSESPGKTVTISCTRSSGSIVSNYVQWYOQRPGSSPTTVIYEDNQRPSGV

PDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSNNFWVFGGGTKLTVLG (SEQ ID NO :8 0)

Nucleotide sequence of El VH:

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCA GGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACT CCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAA CAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGTGAAAAGGTCCTTTGATAGTGGTGGG TCCTTTGAGTACTGGGGCCAGGGGACAATGGTCACCGTCTCGAGT (SEQ ID NO:81) Аминокислотная последовательность El VH: EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTY YADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCVKRS FDSGGS EEYWGOGTMVTVS S (SEQ ID NO:82) Нуклеотидная последовательность El VL: AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTCACCAT CTCCTGCACCCGCAGCAGTGGCTACATTGCCAGCTCCTATGTGCAGTGGTACCAGCAGCGCCCG GGCAGTTCCCCCACCACTGTAATCTTTGAGGATGACCGGAGACCCTCTGGGGTCCCTGATCGGT TCTCTGGCTCCATCGACGGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAGGACTGA

- 17 042261

GGACGAGGCTGACTACTACTGTCAGTCTTATGATGACACCACTCCCTGGGTGTTCGGCGGAGGG ACCAAGCTGACCGTCCTAGGT (SEQ ID NO:83)

El VL amino acid sequence:

NFMLTOPHSVSESPGKTVTISCTRSSGYIASSYVQWYOORPGSSPTTVIFEDDRRPSGV

PDRFSGSIDGSSNSASLTISGLRTEDEADYYCOSYDDTTPWVFGGGTKLTVLG (SEQ ID NO:84)

Nucleotide sequence of F8 UN:

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCA GGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACT CCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAA CAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGTCGGCAGCTGGTACCTGGAA GATTTTGATATCTGGGGCCGGGGGACAATGGTCACCGTCTCGAGT (SEQ ID NO:85)

Amino acid sequence of F8 UN:

EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGSWYLEDFDIWGRGTMVTVSS (SEQ ID NO:86)

Nucleotide sequence of F8 VL:

AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTTACCAT CTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAACTATGTTCACTGGTATCAGCAGCGCCCG GGCAGTTCACCCACCACTGTGATCTATGAGGATAACCGAAGACCCTCTGGGGTCCCTGCTCGGT TCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGGAGACTGA CGACGAGGCTGACTACTACTGTCAGTCTTCTGATACCACCTATCATGGAGGTGTGGTATTCGGC GGAGGGACCAAGCTGACCGTCCTAGGT (SEQ ID NO:87) Аминокислотная последовательность F8 VL: NFMLTOPHSVSESPGKTVTISCTRSSGSIASNYVHWYOQRPGSSPTTVIYEDNRRPSGV

PARFSGSIDSSNSASLTISGLETDDEADYYCOSSDTTYHGGWFGGGTKLTVLG (SEQ ID NO :8 8)

Nucleotide sequence of F9 UN:

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCA GGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACT CCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAA CAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAAGGCGGTAACTACGGTGATTAC TTCGACTACTTTGACTACTGGGGCAGAGGGACAATGGTCACCGTCTCGAGT (SEQ ID NO :8 9)

- 18 042261

Amino acid sequence F9 UN:

EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTY YADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAKGGNYGDYFDYFDYWGRGTMVTVSS (SEQ ID NO:90)

Nucleotide sequence of F9 VL:

AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCAT CTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAATTATGTGCAGTGGTACCAGCAGCGCCCG GGCAGTGCCCCCACCATTGTGATCTATGAAGATAACCAAAGACCCTCTGGGGTCCCTCATCGGT TCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGA GGACGAGGCTGACTACTACTGTCAGTCTTATGAGGGGTTCGGCGGAGGGACCAAGCTGACCGTC CTAGGT (SEQ ID NO:91)

Amino acid sequence of F9 VL:

NFMLTOPHSVSESPGKTVTISCTRSSGSIASNYVOWYOORPGSAPTIVIYEDNQRPSGV

PHRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYEGFGGGTKLTVLG (SEQ ID NO:92)

Nucleotide sequence of G7 VH:

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCA GGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACT CCGTGAAGGGCCGGTTCACTATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAA CAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAAGATGGATGGAACGCGCTGGGA TGGCTTGAATCCTGGGGCCAGGGGACAATGGTCACCGTCTCGAGT (SEQ ID NO:93)

Amino acid sequence of G7 VH:

EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTY

YADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAKDGWNALGWLESWGOGTMVTVSS (SEQ ID NO:94)

Nucleotide sequence of G7 VL:

AATTTTATGCTGACTCAGCCCCACGCTGTGTCGGAGTCTCCGGGGAAGACGGTGACCAT TTCCTGCACCGGCAGAAATGGCAACATTGCCAGCAACTATGTGCAGTGGTACCAGCAGCGCCCG GACAGTGCCCCCACCCTTATAATCTTTGAAGATACCCAAAGACCCTCTGGGGTCCCTACTCGGC TCTCAGGCTCCATCGACACCTCCTCCAATTCTGCCTCCCTCATCATCTCTTCATTGAGGACTGA GGACGAGGCTGATTACTACTGTCAATCTTCTGATTCCAACAGGGTGCTGTTCGGCGGAGGGACC AAGGTCACCGTCCTAGGT (SEQ ID NO:95)

Amino acid sequence of G7 VL:

NFMLTOPHAVSESPGKTVTISCTGRNGNIASNYVOWYOORPDSAPTLIIFEDTQRPSGV

PTRLSGSIDTSSNSASLIISSLRTEDEADYYCOSSDSNRVLFGGGTKVTVLG (SEQ ID

NO:96)

- 19 042261

Nucleotide sequence of G9 UN:

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT

CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCA

GGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACT

CCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAA

CAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAAGATTTTTGGGTTATTACGAGT GGGAATGACTACTGGGGGCGGGGGACCACGGTCACCGTCTCGAGT (SEQ ID NO:97) G9 CN amino acid sequence:

EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTY

YADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCAKDFWVITSGNDYWGRGTTVTVSS (SEQ ID NO:98)

Nucleotide sequence of G9 VL:

AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTGACCAT

CTCCTGCACCCGCAGCAGTGGCAGCATTGCTAGCAATTATGTGCAGTGGTACCAGCAGCGCCCG

GGCAGTTCCCCCACCACTGTGATCTTTGAAGATAACCGAAGACCCTCTGGGGTCCCTGATCGGT TTTCTGGCTCCATCGACACCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGA GGACGAGGCTGACTACTACTGTCAGTCTTTTGATAGCACCAATCTTGTGGTGTTCGGCGGAGGG ACCAAGCTGACCGTCCTAGGT (SEQ ID NO:99) Аминокислотная последовательность G9 VL: NFMLTOPHSVSESPGKTVTISCTRSSGSIASNYVQWYOQRPGSSPTTVIFEDNRRPSGV PDRFSGSIDTSSNSASLTISGLKTEDEADYYCQSFDSTNLWFGGGTKLTVLG (SEQ ID NO:100) Нуклеотидная последовательность G10 УН: GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACT CTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCA GGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACT CCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAA CAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAAGATGGATGGAACGCGCTGGGA TGGCTTGAATCCTGGGGGAAGGGGACCACGGTCACCGTCTCGAGT (SEQ ID NO:101) Аминокислотная последовательность G10 УН: EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWVSAISGSGGSTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDGWNALGWLESWGKGTТVTVSS (SEQ ID NO:1 02) Нуклеотидная последовательность G10 VL: AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCAT CTCCTGCGCCGGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGTACCAGCAGCGCCCG GGCAGTGCCCCCACCGCTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGAT TCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGA GGACGAGGCTGACTACTACTGTCAATCTTACTCTTACAACAATCAGGTCGTGTTCGGCGGAGGG ACCAAGGTCACCGTCCTAGGT (SEQ ID NO:103) Аминокислотная последовательность G10 VL: NFMLTOPHSVSESPGKTVTISCAGSSGSIASNYVOWYOORPGSAPTAVIYEDNQRPSGV PDRFSGSIDSSSNSASLTISGLKTEDEADYYCOSYSYNNQWFGGGTKVTVLG (SEQ ID NO:104)

In some embodiments, the anti-IFNY antibodies are of the IgG isotype. In some embodiments, the anti-IFNY antibodies are of the IgG1 isotype.

In some embodiments, the anti-IFNY antibodies of the invention specifically bind human and/or cynomolgus IFNy, wherein the antibody binds to the same epitope as NI-0501 antibody, Ab antibody, B4 antibody, B9 antibody, C9 antibody, C10 antibody. , D3 antibody, D6 antibody, D8 antibody, E1 antibody, F8 antibody, F9 antibody, G7 antibody, G9 antibody, and/or G10 antibody.

Methods of treatment

The compositions and methods are useful for treating any of a variety of diseases associated with interferon-gamma (IFNy) expression and/or activity, including aberrant IFNY expression and/or activity. The compositions and methods of the invention are useful in the treatment of hemophagocytic lymphohistiocytosis (HLH). HLH is a rare, serious, and life-threatening disorder of abnormal immune activation characterized by clinical signs and symptoms of extreme inflammation (fever, splenomegaly, cytopenias, coagulopathy) leading to immune-mediated pathologies that, through tissue damage, can eventually cause multiple organ failure and death (Henter JI, Elinder G, Soder O, Hansson M, et al.: Hypercytokinemia in familial hemophagocytic lymphohistiocytosis. Blood 1991, 78: 2918-2922). HLH includes primary (genetic/familial) HLH and secondary HLH.

Primary HLH is a heterogeneous autosomal recessive disorder that

- 20 042261 Infants and young children are mainly affected, with an estimated prevalence in Europe of 1/50,000 live births (Janka GE: Familial hemophagocytic lymphohistiocytosis. Eur. J. Pediatr.

1983, 140: 221-230). The disease is notoriously fatal with a median survival of less than 2 months after symptom onset if left untreated (Filipovich AH: Hemophagocytic lymphohistiocytosis (HLH) and related disorders. Hematology Am Soc Hematol Educ Program 2009: 127-131).

All genetic defects in primary HLH affect genes involved in the cytotoxic pathway of NK cells and/or cytotoxic lymphocytes required for the elimination of activated macrophages, encoding proteins of the perforin synthesis pathway, cytolytic granule maturation, granule exocytosis, and the function of releasing granule contents during exocytosis (Filipovich, A, K. McClain, and A. Grom 2010. Histiocytic disorders: recent insights into pathophysiology and practical guidelines Biol Blood Marrow Transplant 16(1 Suppl): S82-S89). In approximately 20-40% of patients with primary HLH, the cytotoxic dysfunction characteristic of the HLH syndrome results from mutations in the gene encoding perforin (PRF1), a cytolytic protein of cytotoxic granules that is a key regulator of T-cell and cell-mediated - natural killers of cytolysis. In about 10% of patients, the disease is caused by mutations in the UNC13D gene, which codes for a protein that is involved in the release of perforin into target cells. In addition, some immunodeficiency syndromes, such as Griscelli syndrome type 2 (GS-2) and Chediak-Higashi syndrome (CHS), are often accompanied by HLH (Janka GE, Lehmberg K: Hemophagocytic lymphohistiocytosis: pathogenesis and treatment. Hematology Am Soc Hematol Educ Program 2013, 2013: 605-611).

Secondary forms of HLH may occur during infection, autoimmune/rheumatic disease, or in association with malignancy. Secondary forms have the same signs and symptoms as primary HLH and can be equally severe.

The compositions and methods of the invention are useful in the treatment of secondary HLH. The compositions and methods of the invention are useful in the treatment of macrophage activation syndrome (MAS).

MAS is a severe, potentially life-threatening complication of rheumatic diseases that is caused by excessive activation and expansion of T-lymphocytes and macrophages. Uncontrolled expansion of these immune cells results in a marked hypercytokinemia and hyperinflammatory state that is associated with fever, cytopenias, hepatosplenomegaly, liver dysfunction, clotting abnormalities, and hyperferritinemia, and 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, 66: 145-159). Because of its strong clinical and pathological similarity to HLH, MAS is classified as a secondary or acquired form of HLH. Indeed, it has recently been found that in most MAS patients, tests show impaired NK and perforin function, and a significant number of MAS patients have polymorphisms or heterozygous mutations in PRF1 and UNC13D (Zhang M, Behrens EM, Atkinson TP, Shakoory B, et al.: Genetic defects in cytolysis in macrophage activation syndrome Curr Rheumatol Rep 2014, 16: 439).

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

Since MAS is a potentially fatal disease, timely diagnosis and immediate therapeutic intervention are essential for proper disease control. Reported mortality rates for MAS reach 20-30% and it remains the leading cause of death in pediatric rheumatology patients (Grom AA, Horne A, De Benedetti F: Macrophage activation syndrome in the era of biologic therapy. Nat Rev Rheumatol. 2016 Mar 24 doi: 10.1038/nrheum.2015.179).

Various sets of criteria have been proposed for diagnosing MAS in patients with sJIA. The 2004 HLH Diagnostic Guidelines are sometimes recommended for use (Henter J, Home A, Aric0 M, Egeler RM, et al.: HLH-2004: Diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer 2007, 48: 124-131 ), mainly developed for primary (genetic) forms of HLH. However, these criteria have some limitations and may not apply to patients with sJIA. For example, criteria such as cytopenias and hypofibrinogenemia below the HLH-2004 guidelines become evident only in the later stages of MAS, as these patients often have elevated white blood cell and platelet counts, as well as elevated serum fibrinogen levels as part of the inflammatory response in sJIA (Schulert GS, Grom AA: Pathogenesis of macrophage activation syndrome and potential for cytokine-directed therapies. Annu. Rev. Med. 2015, 66: 145-159). Hemophagocytosis may be absent in the clinical picture in a significant proportion of patients with MAS (Minoia F, Davi

S, Home A, Demirkaya E, et al.: Clinical features, treatment, and outcome of macrophage activation syndrome complicating systemic juvenile idiopathic arthritis: a multinational, multicenter study of 362 patients. Arthritis &

rheumatology (Hoboken, N.J.) 2014, 66: 3160-3169). In addition, hemophagocytosis, NK cell activity, and sCD25 are not usually assessed in the context of MAS.

An alternative approach is based on the application of a preliminary diagnostic guideline (PDG) for complications of MAS in sJIA, which was created by analyzing a group of patients with MAS in comparison with a group of patients with a sudden exacerbation of sJIA.

The HLH-2004 diagnostic guideline and the provisional diagnostic guideline for sLA-associated MAS were recently compared for their ability to distinguish sJIA/MAS from sJIA (in the absence of MAS) and systemic infection in a large patient population (Davi S, Minoia F, Pistorio A, Home A, et al.: Performance of current guidelines for diagnosis of macrophage activation syndrome complicating systemic juvenile idiopathic arthritis Arthritis & rheumatology (Hoboken, N.J.) 2014, 66: 28712880). Although with some limitations due to its retrospective nature, this study demonstrated that MAS provisional guidance achieves a better balance between sensitivity and specificity, and better agreement with the diagnosis made by the treating physician. The sensitivity of the HLH-2004 criteria set was <30%. However, it has also been reported that the proportion of patients meeting each PDG criterion is highly variable, and some clinical features (eg, CNS dysfunction and bleeding) may present in the late stage of MAS, reducing the sensitivity of these criteria in the early stages of MAS (Lehmberg K, Pink I, Eulenburg C, Beutel K, et al: Differentiating macrophage activation syndrome in systemic juvenile idiopathic arthritis from other forms of hemophagocytic lymphohistiocytosis The Journal of Pediatrics 2013, 162: 1245-1251.

A diagnostic scoring system (HScore) was recently developed and tested in a retrospective group of 312 patients, of which 162 were found 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, 66: 2613-2620).

Nine variables (3 clinical [i.e., known underlying immunosuppression, high fever, organomegaly], 5 biological [i.e., levels of triglycerides, ferritin, serum glutamatoxaloacetate transaminase, fibrinogen, and cytopenia] and 1 cytological [i.e., evidence of hemophagocytosis in bone marrow punctate ]) were stored in the HScore, and the probability of having a hemophagocytic syndrome ranges from <1% in the case of an HScore <90 to >99% in the case of an HScore >250.

Until a final consensus is reached on validated diagnostic criteria for MAS, the clinical diagnosis of a qualified clinician is still critical in distinguishing between MAS and conditions presenting with similar clinical features, such as acute exacerbation of sJIA or sepsis-like syndromes.

There are currently no approved drugs for the treatment of MAS. Generally, high-dose glucocorticoids are the first-line therapy for MAS. For patients not responding to glucocorticoids, the use of cyclosporine A (CsA) has been proposed as additional therapy (Stephan JL, Kone-Paut I, Galambrun C, Mouy R, Bader-Meunier B, Prieur AM: Reactive haemophagocytic syndrome in children with inflammatory disorders A retrospective study of 24 patients Rheumatology (Oxford, England) 2001, 40: 1285-1292).

The administration of etoposide, which is part of the treatment protocol developed for the treatment of pHLH according to HLH-94, is also being considered for patients not responding to high doses of glucocorticoids. However, the potential toxicity of the drug remains a serious concern. Another current first line therapy for HLH includes dexamethasone. However, therapeutic agents such as etoposide and/or dexamethasone are broad spectrum myelosuppressive and/or immunosuppressive agents. There is currently no standard second-line therapy for HLH, treatment with agents such as alemtuzumab/ATG results in profound immunosuppression, and survival rates with these treatments are considered very low.

The usefulness of biological compounds that inhibit the IL-1, IL-6R or TNFa pathways for the treatment of MAS is still unclear. Although biological compounds that inhibit these pathways have been reported to be effective in selected cases, there have also been reports of patients developing MAS under such treatment (Stern A, Riley R, Buckley L: Worsening of macrophage activation syndrome in a patient with adult onset Still's disease after initiation of etanercept therapy J Clin Rheumatol 2001, 7: 252256; Ramanan AV, Schneider R: Macrophage activation syndrome following initiation of etanercept in a child with systemic 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 systemic juvenile idiopathic arthritis. N. Engl. J. Med. 2012, 367: 2385-2395; Ruperto N, Brunner HI, Quartier P, Constantin T, et al.: Two randomized

- 22 042261 trials of canakinumab in systemic juvenile idiopathic arthritis. N. Engl. J. Med. 2012, 367: 2396-2406), as well as patients who do not respond to such therapy, this indicates that the inhibition of IL-1,

IL-6R or TNFa does not provide complete protection against the development of MAS and is not an effective treatment for the developed syndrome.

A large, retrospective, multicenter study examined clinical, laboratory, and histopathological characteristics, as well as current methods and outcomes of MAS/sJIA treatment, in a total of 362 patients (Minoia F, Davi S, Home A, Demirkaya E, et al.: Clinical features, treatment, and outcome of macrophage activation syndrome complicating systemic juvenile idiopathic arthritis: a multinational, multicenter study of 362 patients Arthritis & rheumatology (Hoboken, N.J.) 2014, 66: 3160-3169). In about half of the patients, MAS occurred in the context of active sJIA, or during an acute outbreak of sJIA in 30% of them at the onset of the disease. An infectious trigger was identified in a third of patients. Among 24 patients in whom the type of infection was determined, EBV was the most common pathogen (25%). In 11 patients (3.8%), MAS was considered to be associated with a treatment side effect: 8 of them were treated with a biologic agent targeting the IL-6 (N=4), IL-1 (N=3) or TNFa pathway. (N=1). Almost all patients received glucocorticoids. Cyclosporine, biologics, and etoposide received 61%, 15%, and 12% of patients, respectively.

Thus, there is a high unmet need to develop effective treatment regimens for MAS. More than 50% of patients with sJIA and MAS do not respond to systemic glucocorticoids alone or may require long-term high-dose treatment, which is associated with significant health risks. When patients do not respond to glucocorticoids, there are no reliable data on the effectiveness of additional therapeutic agents such as CsA or etoposide. The course of MAS can quickly become irreversible, leading to death. According to current data, the mortality rate from sJIA-associated MAS is 8%, with approximately one third of patients requiring hospitalization in the intensive care unit. Recent evidence for the critical role of IFNy in disease pathogenesis indicates that IFNy blockade is potentially a new type of targeted therapy.

Compositions, including NI-0501 compositions, and the methods of the invention offer advantages over current treatments for primary and secondary HLH.

MAS and HLH are characterized by sustained immune cell activation and an accompanying burst of pro-inflammatory cytokine production with overproduction of IFNy, TNFa, IL-1, and IL-6 (Henter JI, Elinder G, Soder O, Hansson M, et al.: Hypercytokinemia in familial hemophagocytic lymphohistiocytosis Blood 1991, 78: 2918-2922; Imashuku S, Hibi S, Fujiwara F, Todo S: Hyper-interleukin (IL)6-naemia in haemophagocytic lymphohistiocytosis. Br. J. Haematol 1996, 93: 803-807; Xu X, Tang Y, Song H, Yang S, et al.: Diagnostic accuracy of a specific cytokine pattern in hemophagocytic lymphohistiocytosis in children J. Pediatr 2012, 160: 984-90.e1 Put K, Avau A, Brisse E , Mitera T, et al.: Cytokines in systemic juvenile idiopathic arthritis and haemophagocytic lymphohistiocytosis: tipping the balance between interleukin-18 and interferon-y, Rheumatology (Oxford) 2015). In recent years, evidence has accumulated to support a crucial role of IFNy in the development of both forms of 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, 104: 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-like disease in mice J. Clin Invest 2011, 121: 2264-2277).

Perforin knockout mice are considered a relevant model for primary HLH because these mice develop all of the diagnostic and many of the clinical and laboratory characteristics of human disease after infection with LCMV. The HLH-like disease that develops in these animals depends on CD8+ T cells and IFNy produced in response to antigen challenge (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 shown that when high circulating levels of IFNy were neutralized by the administration of anti-IFNy antibodies, not only did the abnormal clinical and laboratory parameters return to the previous state, but also dramatically increased survival rates. In contrast, ablation of any other cytokine 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 lymphohistiocytosis in children J. Pediatr. 2012, 160:984-90.e1). Additional evidence for the importance of IFNy in HLH is the high concentrations of circulating IFNy in these patients (Henter JI, Elinder G, Soder O, Hansson M, et al.: Hypercytokinemia in familial hemophagocytic lymphohistiocytosis. Blood 1991, 78: 2918-2922; Xu X, Tang Y, Song H, Yang S, et al.: Diagnostic accuracy of a specific cytokine pattern in hemophagocytic lymphohistiocytosis in children J. Pediatr 2012, 160:

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984-90.e1). In a group of 71 patients who were monitored from HLH diagnosis to treatment and follow-up, IFNy levels were above the upper limit of normal (17.3 pg/mL) in all patients and, in particular, 53.5% of patients had levels above 1000 pg/ml.

It has also been reported that IFNy levels rise immediately and rapidly, and can fall from >5000 pg/mL to normal levels within 48 h with effective treatment of HLH.

Two animal models of secondary HLH were investigated in the context of the NI0501 development program to elucidate the potential pathogenic role of IFNy. First, in a mouse model that mimics infection-induced HLH, repeated administration of CpG via TLR9 activation initiated the development of hypercytokinemia, resulting in clinical (eg, weight loss, splenomegaly) and laboratory (eg, cytopenia, hyperferritinemia) signs of HLH. Upon neutralization of IFNy by administration of an anti-IFNy antibody, clinical and laboratory parameters returned to normal. It was shown that the neutralization of IFNy was also complete in the relevant target tissues such as the liver and spleen. Interestingly, the administration of an anti-IFNy antibody for the first time demonstrated an amount of IFNy 500-2000 times greater than that measured in blood, apparently more accurately reflecting tissue IFNy production. Expression of two IFNy-inducible chemokines (CXCL9 and CXCL10) was upregulated after TLR9 stimulation in both blood and liver, and a significant correlation was observed between serum IFNy levels and serum concentrations of CXCL9 and CXCL10. Neutralization of IFNy caused a significant decrease in serum levels of CXCL9 and CXCL10, as well as their mRNA levels in the liver (Buatois V, Chatel L, Cons L, Lory S, et al.: IFNy drives disease in the TLR9-mediated secondary HLH in mice: rationale for a new therapeutic target in secondary HLH, forthcoming).

Second, an animal model was studied in IL-6 transgenic mice in which IL-6 is highly expressed because it mimics the condition of patients with sJIA, the rheumatic disease most commonly associated with secondary forms of HLH. Ligand stimulation of Toll-like receptors (TLRs) in these animals showed increased mortality, increased production of inflammatory cytokines, and hyperactivation of inflammatory signaling pathways. In addition, these mice showed dramatic decreases in platelet and neutrophil counts, elevated levels of SCD25, ferritin, and LDH, which resembled many of the features typically seen 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 extended exposure to interleukin-6 in mice: implication for the pathogenesis of macrophage activation syndrome Arthritis Rheum 2012, 64: 1680-1688). In these mice, when IFNy is neutralized by administration of an anti-IFNy antibody, survival rates improve dramatically and laboratory values return to normal (Prencipe G et al., forthcoming).

Similar data were recently obtained in an observational study in patients with secondary forms of HLH arising either from infection or from an unknown cause (pHLH was excluded due to normal cytotoxic activity, the absence of mutations in known genes that cause pHLH, and the absence of a family history of the disease), or with a MAS existing in an sJIA context.

In 14 patients with secondary HLH (in 7 of whom an underlying infection could be identified), serum samples were analyzed during an acute outbreak of the disease and during remission of the disease. IFNy, CXCL9 and CXCL10 levels were markedly higher in the active phase compared with remission of the disease (IFNy: 34.7 vs. <3.5 pg/ml; CXCL9: 33598 vs. 745 pg/ml; CXCL10: 4420 vs. 132 pg/ml; median values). IFNy levels correlated significantly with CXCL9 levels (p=0.0018) and, to a lesser extent, with CXCL10 levels (p=0.014). IFNy and chemokine levels (particularly CXCL9) correlated significantly with measures of disease severity such as neutrophil and platelet counts, ferritin, and ALT, further supporting a pathogenic role for IFNy in secondary HLH and the potential use of chemokines as relevant disease biomarkers ( Buatois V, Chatel L, Cons L, Lory S, et al: IFNy drives disease in the TLR9mediated secondary HLH in mice: rationale for a new therapeutic target in secondary HLH).

Similar results were obtained in patients with MAS occurring in patients suffering from sJIA. Serum concentrations of IFNy, IFNy-inducible chemokines (CXCL9, CXCL10, CXCL11) and IL-6 were measured in 54 patients with sJIA, of whom 20 had MAS. IL-6 levels were comparable in patients with active MAS and patients with active sJIA but no MAS at the time of sample collection. In contrast, circulating levels of IFNy and chemokines were significantly higher in MAS, especially in the case of CXCL9, whose median levels were approximately 15-fold higher than in patients with active sJIA without MAS (13392 vs 837 pg/mL; p = 0.005). Notably, only in patients with MAS was a significant correlation found between CXCL9 levels and typically abnormal measures such as ferritin (p=0.041), neutrophils (p=0.010) and platelets (p=0.022), ALT (p =0.044) and LDH (p=0.013). IFNy levels also correlated with laboratory measures of disease severity, with the exception of LDH, for which statistical significance was not reached (Bracaglia et al., forthcoming).

Taken together, these results provide a sound basis for IFNy neutralization as a targeted therapy for secondary HLH and MAS, and for investigating this approach in the clinical setting.

Compositions, including NI-0501 compositions, and methods of the invention offer advantages over current treatments for sJIA. For example, compositions, including compositions of NI0501, and methods of the invention are useful for the treatment of MAS/sHLH in patients with sJIA, with the primary goal of achieving remission of MAS.

The rationale for identifying a patient population that would benefit from NI0501 treatment and for evaluating the efficacy of NI-0501 in MAS/sHLH relies on a number of factors. First, preclinical data from an animal model relevant to MAS in sJIA have shown that neutralization of IFNy leads to a marked improvement in survival rates and return to normal laboratory parameters. Further, observational data in patients with MAS/sHLH demonstrate high levels of IFNy and, more importantly, extremely elevated levels of the IFNy-induced chemokines CXCL9, CXCL10 and CXCL11. Third, in patients with MAS/sHLH, IFNy and CXCL9 concentrations are highly correlated with disease markers such as ferritin, platelet, and transaminase levels. Further, the favorable tolerability profile and lack of associated safety issues observed in patients with pHLH in previous studies in which all infused drugs were well tolerated, confirming the results of a study in healthy volunteers, as well as the lack of reports of infections caused by pathogens that, like it is known that neutralization of IFNy should contribute, in addition, all of the infections present in some patients with pHLH were found to be associated not with NI0501 treatment, but with the immune status of the patients, the duration of the disease and previous or concomitant treatment. Fifth, preliminary data from previous clinical trials demonstrate a beneficial effect on disease parameters, with a noticeable onset of effects within the first days of treatment: typical clinical signs and symptoms of HLH began to noticeably improve after the first administration of NI-0501 (fever for several hours, splenitis /hepatomegaly for several days); and of the 18 patients evaluated at the time data collection was stopped, treatment with NI-0501 enabled 10 patients to transition to HSCT. Further, data generated by the PK modeling and simulation approach demonstrate the predictable pharmacokinetic profile of NI-0501 and that IFNy neutralization is achieved and maintained. Finally, conventional therapy (eg, CsA) can be started immediately, without the need for a washout period, if NI-0501 is not sufficient to adequately control the disease.

In summary, based on preclinical and clinical data, there is a strong case for IFNy neutralization in MAS/sHLH secondary to rheumatic disease, and preliminary results in patients with pHLH indicate a favorable benefit/risk ratio with NI-0501. with significant changes and a return to the norm of indicators characteristic of HLH.

Thus, the use of NI-0501 represents an innovative and effective therapeutic approach to control this severe, life-threatening complication of rheumatic diseases, potentially limiting the side effects of long-term, high-dose glucocorticoid treatment.

Introduction of anti-IFNy antibodies

It should be understood that the therapeutic compounds of the present invention will be administered with appropriate carriers, excipients and other means that are included in the compositions to provide better transfer, delivery, tolerability, and the like. A description of a variety of suitable formulations can be found in reference books known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th edition, Mack Publishing Company, Easton, PA (1975)), in particular in chapter 87 by Blaug, Seymour in said collection. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids containing lipid (cationic or anionic) vesicles (such as Lipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in-water emulsions, and water-in-oil, carbovax emulsions (polyethylene glycols of various molecular weights), semi-solid gels and semi-solid mixtures containing carbovax. Any of the above mixtures may be suitable for treatment and therapy in accordance with the present invention, provided that the active ingredient in the composition is not inactivated by the components of the composition and the composition is physiologically compatible and tolerated by the route of administration used. See also Baldrick P. Pharmaceutical excipient development: the need for preclinical guidance, Regul. Toxicol Pharmacol. 32(2): 210-8 (2000), Wang W. 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 parenteral formulations PDA J Pharm Sci Technol. 52: 238-311 (1998) and references therein for additional information relating to formulations, excipients and carriers well known to pharmaceutical chemists.

The effectiveness of treatment is determined using well-known methods for diagnosing or treating a particular disease associated with the immune system. Relief of one or more symptoms associated with the immune system indicates that the introduction of antibodies leads to positive clinical results.

Antibodies of the invention, including polyclonal, monoclonal, humanized and fully human antibodies, can be used as therapeutic agents. Such agents will typically be used to treat or prevent a disease or condition associated with aberrant expression or activation of a particular target in an individual. An antibody formulation, preferably an antibody having high specificity and high affinity for its target antigen, is administered to the individual and will generally have an effect when it binds to the target. Administration of an antibody may result in the abolition or inhibition or interference of the signaling function of the target. Administration of an antibody may result in the abolition or inhibition, or interference, of the target's binding to an endogenous ligand to which the target naturally binds.

A therapeutically effective amount of an antibody of the invention generally means the amount necessary to achieve the therapeutic goal. As noted above, this may be a binding interaction between the antibody and its target antigen, which in some cases prevents the target from functioning. The amount to be administered will also depend on the binding affinity of the antibody to its specific antigen, and furthermore will depend on the rate at which the administered antibody is distributed from the void volume administered to the individual. Generally accepted ranges for a therapeutically effective dose 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. The generally accepted frequency of dosing may be in the range, for example, from once a day to twice a week. Treatment may be continued for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks.

For the treatment of various diseases and disorders, the antibodies or fragments thereof of the invention may be administered in the form of pharmaceutical compositions. The principles and considerations to be taken into account when creating such compositions, as well as guidance for selecting ingredients, can be found, for example, in Remington: The Science And Practice Of Pharmacy 19th edition (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

The composition may also contain more than one active compound, for example, anti-IFNy antagonist compounds needed to treat a particular condition, preferably compounds with complementary activities that will not adversely affect each other. Alternatively or additionally, the composition may contain an agent that enhances its function, such as, for example, a cytotoxic agent, a cytokine, a chemotherapeutic agent, or a growth inhibitory agent. Such molecules are preferably present in combination in amounts that are effective to achieve the intended purpose.

In one embodiment, the active compound, eg, an anti-IFNy antagonist compound, is administered as part of a combination therapy, that is, in combination with one or more additional agents useful in the treatment of conditions or diseases. As used herein, the term in combination means that the agents are administered at substantially the same time, either simultaneously or sequentially. When administered sequentially at the time the second compound is administered, the first of the two compounds is preferably still present at effective concentrations in the area being treated.

For example, combination therapy may include the use of one or more anti-IFNy neutralizing antibodies of the invention co-formulated and/or co-administered with one or more additional drugs, e.g., one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, inhibitors metabolism, enzyme inhibitors and/or cytotoxic or cytostatic agents, as described in more detail below. In such combination therapy, it is preferable that lower doses of administered therapeutic agents can be used, thus avoiding the possible toxicity or complications associated with different types of monotherapy.

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

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In some embodiments, the additional agent is intrathecally administered methotrexate and/or glucocorticoids. In some embodiments, the individual received intrathecal methotrexate and/or glucocorticoids prior to initiation of NI-0501 administration.

In some embodiments, the additional agent is an intravenous immunoglobulin (IV IG). In some embodiments, IV IG is administered as replacement therapy to an individual with established immunoglobulin deficiency. In some embodiments, when the individual has an established immunoglobulin deficiency, IV IG 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 analgesics, blood products for transfusion, electrolytes and infusion glucose, antibiotics, antifungal and antiviral agents, and/or general maintenance drugs.

When antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein and/or the smallest inhibitory fragment that interferes with or antagonizes IFNy signaling is preferred. For example, based on the sequences of the variable region of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or obtained by recombinant DNA methods. (See, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). The composition may also contain more than one active compound needed to treat a particular condition, preferably compounds with complementary activities that will not adversely affect each other. Alternatively or additionally, the composition may contain an agent that enhances its function, such as, for example, a cytotoxic agent, a cytokine, a chemotherapeutic agent, or a growth inhibitory agent. Such molecules are preferably present in combination in amounts that are effective to achieve the intended purpose.

Dosing regimens

The invention also relates to a dosage regimen for treating, preventing and/or delaying the onset or progression of, or alleviating a symptom associated with elevated levels of IFNy. Dosing regimens are multiple varying dose regimens. The dose of an antibody that binds interferon gamma (IFNy) is in the range of 1.0 to 10 mg/kg of an individual's body weight. The dose is administered as at least a first and second dose. The second dose is lower or higher than the first dose.

The multiple variable dose regimen for the treatment of primary hemophagocytic lymphohistiocytosis (HLH) in a human subject includes an induction or first dose and a therapeutic or second dose of an anti-IFNy monoclonal antibody. The individual is an adult individual or a child individual.

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 of the antibody 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 of the antibody is 3.0 mg/kg body weight and the second dose is 6.0 or 10.0 mg/kg body weight.

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

The second dose is administered over one or more periods of treatment. The second dose is administered during the first period of treatment. During the first treatment period, a second dose is administered every three days after the first dose. The first treatment period lasts approximately 1, 2, 3, 4, 5, 6 or more weeks. Preferably, the first treatment period is 2 weeks. Optionally, the second dose is administered during the second treatment period following the completion of the first treatment period. During the second treatment period, the second dose is administered twice a week. The second treatment period lasts approximately 1-20 weeks, 2-20 weeks, 3-20 weeks, 4-20 weeks, 5-20 weeks, 6-20 weeks, 1-10 weeks, 2-10 weeks, 3-10 weeks, 410 weeks, 5-10 weeks, 6-10 weeks. The second treatment period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks.

In some embodiments, the second dose is increased or decreased during the first or second treatment period. The increased or reduced dose is called the third dose. The third dose may be 1.0, 3.0 or 6.0 mg/kg body weight. The third dose is administered during the remainder of the first and second treatment periods. Alternatively, the third dose is administered during the third treatment period. In some embodiments, the third dose is referred to as a maintenance dose and the third treatment period is referred to as a maintenance period.

The multiple variable dose regimen for the treatment of secondary hemophagocytic lymphohistiocytosis (HLH) in children includes an induction or first dose and a therapeutic or second dose of an anti-IFNy monoclonal antibody.

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

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

The second dose is administered over one or more periods of treatment. The second dose is administered during the first period of treatment. During the first treatment period, a second dose is administered every three days after the first dose. The first treatment period lasts approximately 1, 2, 3, 4, 5, 6 or more weeks. Preferably, the first treatment period is 2 weeks. Optionally, the second dose is administered during the second treatment period following the completion of the first treatment period. During the second period of treatment, the second dose is administered twice a week. The second treatment period lasts approximately 1-20 weeks, 2-20 weeks, 3-20 weeks, 4-20 weeks, 5-20 weeks, 6-20 weeks, 1-10 weeks, 2-10 weeks, 3-10 weeks, 410 weeks, 5-10 weeks, 6-10 weeks. The second treatment period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks.

In some embodiments, the second dose is increased or decreased during the first or second treatment period. The increased or reduced dose is called the third dose. Preferably, the third dose is 6.0 mg/kg body weight. The third dose is administered during the remainder of the first and second treatment periods. Alternatively, the third dose is administered during the third treatment period. In some embodiments, the third dose is referred to as a maintenance dose and the third treatment period is referred to as a maintenance period.

The multiple variable dose regimen for the treatment of secondary hemophagocytic lymphohistiocytosis (HLH) in adult humans includes an induction or first dose and a therapeutic or second dose of an anti-IFNy monoclonal antibody.

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

The second dose is administered over one or more periods of treatment. The second dose is administered during the first period of treatment. During the first treatment period, a second dose is administered every three days after the first dose. The first treatment period lasts approximately 1, 2, 3, 4, 5, 6 or more weeks. Preferably, the first treatment period is 2 weeks. Optionally, the second dose is administered during the second treatment period following the completion of the first treatment period. During the second treatment period, the second dose is administered twice a week. The second treatment period lasts approximately 1-20 weeks, 2-20 weeks, 3-20 weeks, 4-20 weeks, 5-20 weeks, 6-20 weeks, 1-10 weeks, 2-10 weeks, 3-10 weeks, 410 weeks, 5-10 weeks, 6-10 weeks. The second treatment period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks.

In some embodiments, the second dose is increased or decreased during the first or second treatment period. The increased or reduced dose is called the third dose. The third dose may be 1.0, 3.0 or 6.0 mg/kg body weight. The third dose is administered during the remainder of the first and second treatment periods. Alternatively, the third dose is administered during the third treatment period. In some embodiments, the third dose is referred to as a maintenance dose and the third treatment period is referred to as a maintenance period.

A multiple variable dose regimen for treating a disease state in a human subject includes an induction or first dose and a therapeutic or second dose of an anti-IFNy monoclonal antibody. The individual is an adult individual or a child individual. The condition is associated with elevated levels of IFNy. The condition is transplant rejection such as solid organ transplant rejection or acute bone marrow transplant rejection, graft-versus-host disease, paraneoplastic cerebellar degeneration, hemorrhagic fever, sarcoidosis, acquired Still's disease. In other embodiments, the implementation of the dosing regimen is applied to the individual after receiving CART cell therapy.

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

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

The second dose is administered over one or more periods of treatment. The second dose is administered during the first period of treatment. During the first treatment period, a second dose is administered every three days after the first dose. The first treatment period lasts approximately 1, 2, 3, 4, 5, 6 or more weeks. Preferably, the first treatment period is 2 weeks. Optionally, the second dose is administered during the second treatment period following the completion of the first treatment period. During the second treatment period, the second dose is administered twice a week. The second period of treatment lasts approximately 1-20 weeks,

2-20 weeks, 3-20 weeks, 4-20 weeks, 5-20 weeks, 6-20 weeks, 1-10 weeks, 2-10 weeks, 3-10 weeks, 410 weeks, 5-10 weeks, 6- 10 weeks. The second treatment period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks.

In some embodiments, the second dose is increased or decreased during the first or second treatment period. The increased or reduced dose is called the third dose. The third dose may be 1.0, 3.0, 6.0 or 10.0 mg/kg body weight. The third dose is administered during the remainder of the first and second treatment periods. Alternatively, the third dose is administered during the third treatment period. In some embodiments, the third dose is referred to as a maintenance dose and the third treatment period is referred to as a maintenance period.

In the multiple variable dose regimen of the invention, the first and second time periods for the second dose may be adjusted from time to time, taking into account, for example, the health of the individual. For example, the interval between doses can be adjusted to ±1, 2, 3 or 4 days. Preferably ±2 days.

In the multiple variable dose regimen of the invention, the dose is administered over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours. Preferably, doses are administered within an hour. The time period for infusion dose depends on a number of factors known in this field, for example, age, body weight, physical condition of the individual or the presence of an adverse reaction to a therapeutic agent. The time period for dose infusion is generally determined by the highest infusion rate that an individual can tolerate (ie, that does not cause an adverse reaction).

The dose is administered as a single injection. The antibody is administered as monotherapy or as part of combination therapy for the disease or condition being treated. For example, a second agent is administered to an individual. The second agent is, for example, an anti-inflammatory agent and/or an immunosuppressive agent.

Optionally, the individual was administered dexamethasone immediately prior to antibody administration. Dexamethasone is administered at a dose of at least 10 mg/m ² . Alternatively, dexamethasone is administered at a dose of at least 5 mg/m ² .

In some embodiments, the individual has not previously received HLH treatment.

Pharmaceutical compositions

Antibodies or soluble chimeric polypeptides of the invention (also referred to herein as active compounds), as well as derivatives, fragments, analogs and homologues thereof, can be included in pharmaceutical compositions suitable for administration. Such compositions typically contain an antibody or soluble chimeric polypeptide and a pharmaceutically acceptable carrier. As used herein, the term pharmaceutically acceptable carrier includes any and all diluents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, the standard reference guide in the art, the contents of which are 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. Liposomes and non-aqueous media such as fixed oils can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active compound, their use in the compositions is contemplated. Auxiliary active compounds may also be included in the compositions.

The 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 routes of administration. Solutions or suspensions used for parenteral, intradermal or subcutaneous administration may contain the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; and agents for adjusting tonicity such as sodium chloride or dextrose. The pH value can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. The parenteral formulation can be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

The preferred route of administration is by injection, eg intravenous injection. Intravenous injection may be a rapid or slow infusion. For example, infusions are performed over a time period of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours. The time period for infusion depends on a number of factors known in the art, for example, the age, body weight, physical condition of the individual, or the presence of an adverse reaction to a therapeutic agent. The time period for infusion is generally determined by the highest infusion rate that an individual can tolerate (ie, that does not cause an adverse reaction).

Pharmaceutical compositions suitable for injection include sterile aqueous solutions (in the case of water-soluble compounds) or dispersions and sterile powders for preparing sterile injectable solutions or dispersions immediately prior to administration. Suitable intravenous vehicles include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid to the extent that it can be easily injected with a syringe. It must be stable under the conditions of manufacture and storage and must be protected from contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol, and the like), as well as their respective mixtures. Proper fluidity can be maintained, for example, by using coatings such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using surfactants. Protection against the action of microorganisms can be achieved through the use of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases it will be preferable to include isotonic agents such as sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition of an agent that slows down the absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be obtained by incorporating the active compound in the required amount of an appropriate solvent with one or a combination of the ingredients listed above, as needed, followed by filtered sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile medium which contains the base dispersion medium and other necessary ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze drying, obtaining a powder of the active ingredient with any additional desired ingredient from their solution, previously sterilized by filtration.

Preferred injectable pharmaceutical compositions contain excipients such as L-histidine, L-histidine monohydrochloride, monohydrate, sodium chloride (NaCl), polysorbate 80.

The pH value of the injectable pharmaceutical composition is about 5.8-6.2. Preferably the pH of the injectable pharmaceutical composition is 6.0.

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

In some aspects, the pharmaceutical composition is packaged as a unit dose. The standard dose is contained in a container. The container is a glass or plastic container. The container is a syringe, vial, infusion bottle, ampoule or carpule. The container can hold a volume of 1-25ml. For example, the container may hold a volume of 2, 5, 10, or 20 ml.

The standard dose of a fully human anti-interferon gamma (IFNy) monoclonal antibody (eg, NI-501) is 5-25 mg/mL. Preferably, the unit dose is 5 mg/ml or 25 mg/ml. The antibody is in solution (for example, water for injection) containing per ml: 1.55 mg L-histidine, 3.14 mg L-histidine monohydrochloride, monohydrate, 7.31 mg sodium chloride (NaCl) and 0. 05 mg polysorbate 80.

In some embodiments, the invention relates to a unit dose container containing 20 ml of a solution of a fully human monoclonal antibody against interferon-gamma (IFNy) at a concentration of 5 mg/ml or 25 mg/ml, the pH of the solution being from 5.8 to 6, 2. The antibody is solubilized in solution such that the solution is clear, colorless and free of precipitate. Preferably, the antibody is in a solution (eg, water for injection) containing per ml: 1.55 mg L-histidine, 3.14 mg L-histidine monohydrochloride, monohydrate, 7.31 mg sodium chloride (NaCl) and 0.05 mg polysorbate 80.

In other embodiments, the invention relates to a unit dose container containing 10 ml or 20 ml of a solution of a fully human monoclonal antibody against interferon-gamma (IFNy) at a concentration of 25 mg/ml, the pH of the solution being from 5.8 to

- 30 042261

6.2. The antibody is solubilized in solution such that the solution is clear, colorless and free of precipitate. Preferably, the antibody is in solution (eg water for injection) containing per ml: 1.55 mg L-histidine, 3.14 mg L-histidine monohydrochloride, monohydrate,

7.31 mg sodium chloride (NaCl) and 0.05 mg polysorbate 80.

In other embodiments, the invention relates to a unit dose container containing a 2 ml or 10 ml solution of a fully human anti-interferon-gamma (IFNy) monoclonal antibody at a concentration of 5 mg/ml, the pH of the solution being from 5.8 to 6, 2. The antibody is solubilized in solution such that the solution is clear, colorless and free of precipitate. Preferably, the antibody is in a solution (eg, water for injection) containing per ml: 1.55 mg L-histidine, 3.14 mg L-histidine monohydrochloride, monohydrate, 7.31 mg sodium chloride (NaCl) and 0.05 mg polysorbate 80.

Oral compositions typically contain an inert diluent or edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For purposes of oral therapeutic administration, the active compound may be combined with excipients and used in the form of tablets, lozenges or capsules. Oral compositions can also be prepared using a liquid carrier for use as a mouthwash, wherein the compound in the liquid carrier is administered orally, rinsed in the mouth and spit or swallowed. Pharmaceutically acceptable binders and/or adjuvants may also be included in the composition. Tablets, pills, capsules, lozenges, and the like may contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; or a flavor such as peppermint, methyl salicylate, or orange flavor.

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

Systemic administration can also be carried out through the mucous membrane or through the skin. For mucosal or skin administration, penetrants suitable for the barrier through which penetration will occur are used in the composition. Such penetrants are generally known in the art and include, for example, transmucosal agents, detergents, bile salts, and fusidic acid derivatives. The introduction through the mucous membrane can be performed using nasal sprays or suppositories. For transdermal administration, the active compounds are formulated as ointments, salves, gels or creams, as is well known in the art.

The compounds may also be formulated in suppository form (eg, with conventional suppository bases such as cocoa butter and other glycerides) or in retention enemas for rectal delivery.

In one embodiment, the active compounds are combined with carriers that will protect the compound from rapid elimination from the body, eg formulated as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations are known to those skilled in the art. Materials may also be purchased from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including cell-targeted liposomes with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. They can be obtained by methods known to those skilled in the art, for example, as described in US patent No. 4522811.

It is particularly preferred to formulate oral or parenteral compositions in unit dosage form for ease of administration and dose uniformity. As used herein, the term unit dosage form means physically discrete units containing unit doses for an individual receiving treatment; wherein each unit contains a predetermined amount of the active compound, calculated to provide the desired therapeutic effect, together with the necessary pharmaceutical carrier. The specification for unit dosage forms of the invention is dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect that is intended to be achieved, as well as on the limitations of the field of creating formulations, for example, of the active compound, for the treatment of individuals.

Pharmaceutical compositions may be enclosed in a container, package or dispenser along with instructions for administration.

Detection of CXCL9 and other biomarkers

Levels of CXCL9 and other biomarkers are determined using any of a variety of standard detection methods. Detection means may be used to detect the presence of a particular target (or protein fragment thereof) in a sample. In some embodiments, the detection means comprises a detectable label. In some embodiments, the detection means is an antibody (or fragment thereof) or a probe. In some embodiments, the agent or probe is labeled. The term labeled, when applied to a probe or antibody, encompasses direct labeling of a probe or antibody by binding (i.e., physically connecting) a detectable substance to the probe or antibody, as well as indirect labeling of a probe or antibody by reacting with another reagent that is directly labeled. Examples of the use of indirect labeling include detection of the primary antibody with a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin so that it can be detected with a fluorescently labeled streptavidin.

The term biological sample should include tissues, cells, and body fluids obtained from an individual, as well as tissues, cells, and fluids found in the body of an individual. Thus, the term biological sample encompasses blood as well as a fraction or component of blood, including blood serum, blood plasma or lymph. Body fluids may be fluids derived from any part of the individual's body, preferably peripheral, including, but not limited to, e.g., blood, plasma, serum, synovial fluid, urine, sputum, cerebrospinal fluid, CSF, pleural fluid, fluid from the respiratory, digestive and urogenital tracts, saliva, organ fluid, ascitic fluid, tumor cystic fluid, amniotic fluid, and combinations thereof. The biological sample also includes artificially separated fractions of all of the listed fluids. Biological samples also include solutions or mixtures containing homogenized solid material such as feces, tissue, and biopsy specimens. The detection method of the invention can be used to detect an mRNA, protein or genomic DNA analyte in a biological sample in vitro as well as in vivo. For example, in vitro methods for detecting an mRNA analyte include Northern hybridization and in situ hybridization. In vitro methods for detecting a protein analyte include enzyme-linked immunosorbent assays (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. In vitro methods for detecting a genomic DNA analyte include Southern hybridization. Methods for conducting assays are described, for example, in ELISA: Theory and Practice: Methods in Molecular Biology, Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa, NJ, 1995; Immunoassay, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and Practice and Theory of Enzyme Immunoassays, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. In addition, in vivo methods for detecting a protein analyte involve administering a labeled antibody against the analyte protein to an individual. For example, an antibody can be labeled with a radioactive marker, the presence and location of which in an individual can be determined by standard imaging techniques.

Definitions:

Unless otherwise indicated, scientific and technical terms used in connection with the present invention shall have the meanings commonly given to them by those skilled in the art. In addition, unless the context dictates otherwise, singular terms shall include plural terms, and plural terms shall include singular terms. In general, the nomenclatures used, as well as cell and tissue culture techniques, molecular biology techniques, as well as protein, oligo- or polynucleotide chemistry, and hybridization techniques described herein are well known to those skilled in the art and commonly used in the art. For obtaining recombinant DNA, the synthesis of oligonucleotides, as well as for the cultivation and transformation of tissues (for example, electroporation, lipofection), standard methods are used. Enzymatic reactions and purification methods are performed according to the manufacturer's instructions, as is customary in the art or as described herein. These methods and techniques are generally used in the conventional manner well known in the art and as described in the various general or specific literature referenced and discussed throughout this specification. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclatures used, as well as the laboratory procedures and methods of analytical chemistry, organic synthesis chemistry, and medicinal and pharmaceutical chemistry described herein, are well known and widely used in this field. For chemical synthesis, chemical analyses, creation, formulation and delivery of pharmaceutical formulations, as well as for the treatment of patients, standard methods are used.

As used herein, the term dose refers to the amount of anti-IFNy antibody that is administered to an individual.

The term multiple variable doses means different doses of anti-IFNy antibodies that are administered to an individual for therapeutic treatment. The terms multiple variable dose regimen or multiple variable dose therapy describe a treatment regimen based on the administration of different amounts of anti-IFNy antibody at different times during the course of treatment. In one

- 32 042261 embodiment, the invention relates to a method of treatment using multiple varying doses, comprising an induction phase and a treatment phase, wherein the anti-IFNγ antibody is administered at a higher or lower dose during the induction phase than during the treatment phase. In another embodiment, the invention relates to a multiple variable dose treatment method comprising an induction phase, a first treatment phase, and a second treatment phase, wherein the anti-IFNy antibody is administered at a higher or lower dose during the induction phase than during the treatment. The anti-IFNy antibody may be administered at a higher or lower dose during the first phase of treatment than during the second phase of treatment. Preferably, the doses are the same in the first phase of treatment and in the second phase of treatment.

As used herein, the term "induction phase" or "loading phase" refers to a period of treatment involving the administration of an anti-IFNγ antibody to an individual in order to achieve a threshold level. During the induction phase, at least one induction dose of an anti-IFNy antibody is administered to an individual suffering from a disease in which IFNy is detrimental.

As used herein, the term threshold level refers to a therapeutically effective level of an anti-IFNy antibody in an individual. The threshold level is reached by administering at least one induction dose during the induction phase of treatment. You can enter any number of induction doses to achieve a threshold level of anti-IFNy antibodies. After reaching the threshold level, the treatment phase begins.

The term induction dose, or the term loading dose used interchangeably, means the first dose of an anti-IFNy antibody that is either higher or lower than the maintenance or treatment dose. The induction dose may be a single dose or, alternatively, a series of doses. An induction dose is often used to bring the amount of drug in the body to a steady state, and can be used to quickly achieve steady state levels of the drug. The induction dose is followed by the administration of a smaller or larger dose of anti-IFNy antibody, ie, a treatment dose. The induction dose is administered during the induction phase of the course of therapy.

As used herein, the term treatment phase, or maintenance phase, refers to the period of treatment involving the administration of an anti-IFNy antibody to an individual in order to maintain the desired therapeutic effect. The treatment phase follows the induction phase and therefore begins after the threshold level has been reached. More than one treatment phase may be used, that is, the treatment phases are shortened or extended to maintain a certain threshold or to achieve a desired clinical outcome. Preferably, in the treatment phase, the substance is administered every 3 days after the administration of the induction dose (eg initial dose). Alternatively, the substance is administered once or twice a week.

The term treatment dose, or maintenance dose, means the amount of anti-IFNy antibody received by an individual to maintain or maintain the desired therapeutic effect. The therapeutic dose is administered after the induction dose. The treatment dose may be a single dose or, alternatively, a series of doses. The therapeutic dose is administered during the treatment phase of the course of therapy. Therapeutic doses are lower or higher than the induction dose and may be the same when administered sequentially. If more than one treatment phase is used, more than one treatment dose may also be used.

The term dosing regimen, or dosing regimen, means a treatment regimen based on the administration of a specific series of doses.

As used herein, the term dosing refers to the administration of a substance (eg, an anti-IFNy antibody) to achieve a therapeutic goal (eg, treatment of an IFNy-associated disease).

As used herein, the terms biweekly dosing regimen, biweekly dosing, and biweekly administration refer to the schedule of administration of a substance (e.g., anti-IFNy antibody) to an individual to achieve a therapeutic goal (e.g., treatment of an IFNy-related disease). A biweekly dosing regimen should not include a once a week dosing regimen.

The term combination, for example, in the expression first agent in combination with a second agent, means the joint administration of the first agent and the second agent, which, for example, can be dissolved or mixed in the same pharmaceutically acceptable carrier, or the administration of the first agent, and then the second means, or the introduction of the second agent, and then the first agent. Thus, the present invention includes combination therapy methods and combined pharmaceutical compositions.

The term concomitant, for example in the expression concomitant therapy, means the administration of an agent in the presence of a second agent. The concomitant therapy method includes methods in which the first, second, third, or additional agents are administered together. The concomitant therapy method also includes methods in which the first or additional agents are administered in the presence of the second

- 33 042261 or additional funds, while the second or additional funds, for example, can be entered in advance. The method of concomitant therapy can be carried out in stages by different operators. For example, one operator may administer the first agent to the individual, and a second operator may administer the second agent to the individual, and the administration steps may be performed simultaneously, or nearly simultaneously, or at different points in time, provided that the first agent (and additional agents) after administration will be in the presence of the second agent (and additional agents). The operator and the individual may belong to the same category (for example, be human).

As used herein, the term combination therapy means the administration of two or more therapeutic agents, for example an anti-IFNγ antibody and another drug such as a DMARD or NSAID. The other drug(s) may be administered during, before or after administration of the anti-IFNγ antibody.

Embodiments

In the compositions and methods provided herein, the NI-0501 antibody is formulated as a sterile infusion concentrate (per ml). In some embodiments, NI-0501 is formulated as follows: 5 mg NI-051, 1.55 mg L-histidine, 3.14 mg L-histidine monohydrochloride monohydrate, 7.31 mg sodium chloride (NaCl), and 0.05 mg polysorbate 80 , while the pH value is from 5.8 to 6.2. In some embodiments, NI-0501 is formulated as follows: 5 mg NI-051, 1.55 mg L-histidine, 3.14 mg L-histidine monohydrochloride, monohydrate, 7.31 mg sodium chloride (NaCl), and 0.05 mg polysorbate 80, while the pH value is 6.0.

In the compositions and methods provided herein, NI-0501 is administered to an individual in need thereof to treat, prevent, and/or delay the onset or progression or amelioration of a symptom associated with HLH. In some embodiments, NI-0501 is administered to an individual in need thereof by intravenous infusion over one hour at an initial dose of 1 mg/kg. In some patient populations, such as those with low body weight and/or very young patients, an IV infusion may be performed over more than one hour, such as at least 90 minutes, at least 2 hours, or at least 3 hours or more.

In some embodiments, NI-0501 is administered to an individual in need thereof by at least one additional IV infusion following the initial IV infusion over one hour at an initial dose of 1 mg/kg. In some embodiments, at least one additional IV infusion is performed at a dose that is 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 performed at least three days after the initial IV infusion. In some embodiments, at least one additional IV infusion is performed at a time selected from the group consisting of 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after initial IV infusion, 12 days after initial infusion, and 15 days after initial infusion. In some embodiments, at least one additional IV infusion is given 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial IV infusion and 15 days after the initial infusion.

In some embodiments, NI-0501 is administered to an individual in need thereof by at least one series of additional IV infusions following the initial IV infusion for one hour at an initial dose of 1 mg/kg, with the series of additional IV infusions infusions includes at least one series of intravenous infusions twice a week. In some embodiments, at least one series of intravenous infusions twice a week is performed at a dose higher than the initial dose of 1 mg/kg. In some embodiments, at least one series of intravenous infusions twice a week is performed at a dose of 3 mg/kg. In some embodiments, at least one additional IV infusion is performed at least three weeks after the initial IV infusion. In some embodiments, at least one additional IV infusion is performed at a time selected from the group consisting of 3 weeks after the initial IV infusion, 4 weeks after the initial IV infusion, 5 weeks after initial IV infusion, 6 weeks after initial infusion, 7 weeks after initial infusion, and 8 weeks after initial infusion. In some embodiments, at least one additional IV infusion is given 3 weeks after the initial IV infusion, 4 weeks after the initial IV infusion, 5 weeks after the initial IV infusion, 6 weeks after the initial IV infusion , 7 weeks after the initial infusion and 8 weeks after the initial infusion.

In some embodiments, NI-0501 is administered to an individual 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 performed at a dose greater than

- 34 042261 higher than the initial dose of 1 mg/kg. In some embodiments, the first additional IV infusion and the second additional IV infusion are performed at the same dose. In some embodiments, the first additional IV infusion and the second additional IV infusion are performed at the same dose, which is higher than the initial dose. In some embodiments, at least one of the first and second additional IV infusions is performed at a dose of 3 mg/kg. In some embodiments, the first additional IV infusion is performed at least three days after the initial IV infusion. In some embodiments, the first additional IV infusion is performed at a time selected from the group consisting of 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, per infusion, 12 days after the initial infusion, and 15 days after the initial infusion. In some embodiments, the first additional IV infusion is performed 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial IV infusion, and 15 days after the initial IV infusion. days after initial infusion. In some embodiments, the second additional IV infusion is performed at a time selected from the group consisting of 3 weeks after the initial IV infusion, 4 weeks after the initial IV infusion, 5 weeks after the initial IV infusion, at infusion, 6 weeks after initial infusion, 7 weeks after initial infusion, and 8 weeks after initial infusion. In some embodiments, the second additional IV infusion is performed 3 weeks after the initial IV infusion, 4 weeks after the initial IV infusion, 5 weeks after the initial IV infusion, 6 weeks after the initial IV infusion, 7 weeks after the initial infusion and 8 weeks after the initial infusion. In some embodiments, the first additional IV infusion is performed 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial IV infusion, and 15 days after the initial IV infusion. days after the initial infusion, and a second additional IV infusion is performed 3 weeks after the initial IV infusion, 4 weeks after the initial IV infusion, 5 weeks after the initial IV infusion, 6 weeks after the initial infusion, 7 weeks after the initial infusion and 8 weeks after the initial infusion.

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

- 35 042261 initial infusion and 8 weeks after the initial infusion.

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

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

In the compositions and methods provided herein, NI-0501 is administered to an individual in need thereof to treat, prevent, and/or delay the onset or progression or amelioration of a symptom associated with HLH. In some embodiments, NI-0501 is administered to an individual in need thereof by intravenous infusion over one hour at an initial dose of 1 mg/kg. In some embodiments, infusions are performed every 3 days after the initial dose for up to 15 days after the initial dose. In some embodiments, infusions are performed every 3 days after the initial dose for up to 15 days after the initial dose, followed by twice-weekly infusions starting at least 15 days after the initial dose. In some embodiments, the infusion dose is increased to 3 mg/kg at any time after the initial dose. In some embodiments, after a minimum of two infusions at a dose of 3 mg/kg, the dose of NI-0501 is increased to 6 mg/kg for up to four subsequent infusions.

In the compositions and methods provided herein, NI-0501 is administered to an individual in need thereof to treat, prevent, and/or delay the onset or progression or amelioration of a symptom associated with HLH. In some embodiments, NI-0501 is administered to an individual in need thereof by intravenous infusion at a dose greater than 6 mg/kg. In some embodiments, NI-0501 is administered to an individual in need thereof by iv infusion following an initial dose in a second dose greater than 6 mg/kg. 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 administered daily. In some embodiments, the second dose is 10 mg/kg administered daily for 1 week. In some embodiments, the second dose is 10

- 36 042261 mg/kg, while it is administered daily for 2 weeks. In some embodiments, the second dose is 10 mg/kg administered daily for more than 2 weeks.

In some embodiments, NI-0501 is administered to an individual in need thereof to treat, prevent and/or delay the onset or progression of, or alleviate a symptom associated with secondary HLH. In some embodiments, NI-0501 is administered to an individual in need thereof to treat, prevent, and/or delay the onset or progression of, or relieve a symptom associated with secondary HLH in sJIA. In some embodiments, NI-0501 is administered to an individual in need thereof at an initial dose of 6 mg/kg. In some embodiments, NI-0501 treatment is continued using the next dose of NI-0501. In some embodiments, treatment with NI-0501 is continued using the next dose of NI-0501 of 3 mg/kg every 3 days for at least 4 weeks (ie, until SD27).

In some embodiments, treatment with NI-0501 is suspended, stopped, or otherwise reduced after the desired clinical outcome has been achieved. In some embodiments, treatment with NI-0501 is halted after there is evidence of a complete clinical response, ie, remission of MAS.

In some embodiments, after 4 weeks, treatment with NI-0501 is continued for up to an additional 4 weeks (ie, up to SD56) as maintenance therapy, as needed, until MAS remission is achieved. In some embodiments, after 4 weeks, treatment with NI-0501 is continued for up to an additional 4 weeks (i.e., up to SD56) as maintenance therapy, as needed, until MAS remission is achieved, with the option of lowering the dose to 1 mg/day. kg and increase the interval between infusions to the introduction of once a week.

In the compositions and methods provided herein, NI-0501 is administered to an individual in need thereof to treat, prevent and/or delay the onset or progression of, or alleviate a symptom associated with HLH in cases where the individual has previously been administered dexamethasone. In some embodiments, the individual is a previously untreated patient (ie, who has not previously received HLH treatment), and dexamethasone is administered at a dose of at least 10 mg/m ² . In some embodiments, the individual is receiving NI-0501 as second line therapy for HLH and dexamethasone is administered at a dose ranging from 10 to 5 mg/m ² . In some embodiments, the individual is receiving NI-0501 as second line therapy for HLH and dexamethasone is administered at a dose of at least 5 mg/m ² . In some embodiments, the individual is receiving NI-0501 as second line therapy for HLH and dexamethasone is administered at a dose of less than 5 mg/m ² .

In some embodiments, NI-0501 is administered before and/or during and/or after treatment in combination with one or more additional agents such as, but not limited to, a therapeutic agent, an anti-inflammatory agent, and/or an immunosuppressive agent. In some embodiments, the second agent is an agent known to be used to treat HLH. In some embodiments, the additional agent comprises at least etoposide. In some embodiments, NI-0501 and the additional agent are formulated in the same therapeutic composition, and NI-0501 and the additional agent are administered simultaneously. Alternatively, NI-0501 and the additional agent are separate from each other, for example, each is formulated in a separate therapeutic composition and NI-0501 and the additional agent are administered simultaneously, or NI-0501 and the additional agent are administered at different times during treatment. For example, NI-0501 is administered before the adjuvant is administered, NI-0501 is administered after the adjunct is administered, or NI-0501 and the adjuvant is administered alternately. As described herein, NI-0501 and an additional agent are administered in single doses or in multiple doses.

In some embodiments, NI-0501 and additional agent(s) are administered simultaneously. For example, NI-0501 and additional agent(s) may be formulated in the same composition or 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 time points during treatment.

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

In some embodiments, the additional agent is intrathecally administered methotrexate and/or glucocorticoids. In some embodiments, the individual received intrathecal methotrexate and/or glucocorticoids prior to initiation of NI-0501 administration.

In some embodiments, the additional agent is an intravenous immunoglobulin (IV IG). In some embodiments, IV IG is administered as replacement therapy to an individual with established immunoglobulin deficiency. In some embodiments, when the individual has an established immunoglobulin deficiency, IV IG 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 analgesics, transfusion blood products, infusion electrolytes and glucose, antibiotics, antifungal and antiviral agents, and/or general maintenance drugs.

The compositions and methods provided herein are useful for treating, preventing and/or delaying the onset or progression of, or alleviating a symptom associated with graft failure, graft rejection, and/or inflammatory disease associated with graft rejection. The compositions and methods provided herein are useful for treating, suppressing, delaying progression, or otherwise alleviating a symptom of graft-versus-host disease (GvHD) in an individual who has received or is receiving a graft containing biological material or a series of grafts containing biological material. The compositions and methods provided herein are useful for extending the life of transplanted biological material.

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

Using the compositions and methods provided herein, NI-0501 is administered to an individual in need thereof to treat, prevent and/or delay the onset or progression of, or alleviate a symptom associated with graft failure, graft rejection, and/or inflammatory disease associated with transplant rejection. 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 provided herein, the NI-0501 antibody is formulated as a sterile infusion concentrate (per ml). In some embodiments, NI-0501 is formulated as follows: 5 mg NI-051, 1.55 mg L-histidine, 3.14 mg L-histidine monohydrochloride monohydrate, 7.31 mg sodium chloride (NaCl), and 0.05 mg polysorbate 80 , while the pH value is from 5.8 to 6.2. In some embodiments, NI-0501 is formulated as follows: 5 mg NI-051, 1.55 mg L-histidine, 3.14 mg L-histidine monohydrochloride, monohydrate, 7.31 mg sodium chloride (NaCl), and 0.05 mg polysorbate 80, while the pH value is 6.0.

In some embodiments, transplant rejection is chronic. In some embodiments, NI-0501 is administered to an individual in need thereof by intravenous infusion over one hour at an initial dose of 1 mg/kg. In some patient populations, e.g., low body weight patients and/or very young patients, IV infusion may be performed over more than 1 hour, such as at least 90 minutes, at least 2 hours, or at least 3 hours. , or more.

In some embodiments, NI-0501 is administered to an individual in need thereof by at least one additional IV infusion following the initial IV infusion over 1 hour at an initial dose of 1 mg/kg. In some embodiments, at least one additional IV infusion is performed at a dose that is 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 performed at least three days after the initial IV infusion. In some embodiments, at least one additional IV infusion is performed at a time selected from the group consisting of 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after initial IV infusion, 12 days after initial infusion, and 15 days after initial infusion. In some embodiments, at least one additional IV infusion is given 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial IV infusion and 15 days after the initial infusion.

In some embodiments, NI-0501 is administered to an individual in need thereof by at least one additional IV infusion series following the initial IV infusion for 1 hour at an initial dose of 1 mg/kg, with the additional IV infusion series infusions includes at least one series of intravenous infusions twice a week. In some embodiments, at least one IV infusion series twice a week is administered at a dose higher than the initial dose of 1 mg/kg. In some embodiments, at least one series of intravenous infusions twice a week is performed at a dose of 3 mg/kg. In some embodiments, at least one additional IV infusion is performed at least three weeks after the initial IV infusion. In some embodiments, at least one additional IV infusion is performed at a time selected from the group consisting of 3 weeks after the initial IV infusion, 4 weeks after the initial IV infusion, 5 weeks after initial IV infusion, 6 weeks after initial infusion, 7 weeks after initial infusion, and 8 weeks after initial infusion. In some embodiments, at least one additional IV infusion is given 3 weeks after the initial IV infusion, 4 weeks after the initial IV infusion, 5 weeks after the initial IV infusion, 6 weeks after the initial IV infusion , 7 weeks after the initial infusion and 8 weeks after the initial infusion.

In some embodiments, NI-0501 is administered to an individual 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 performed at a dose higher than the initial dose of 1 mg/kg. In some embodiments, the first additional IV infusion and the second additional IV infusion are performed at the same dose. In some embodiments, the first additional IV infusion and the second additional IV infusion are performed at the same dose, which is higher than the initial dose. In some embodiments, at least one of the first and second additional IV infusions is performed at a dose of 3 mg/kg. In some embodiments, the first additional IV infusion is performed at least three days after the initial IV infusion. In some embodiments, the first additional IV infusion is performed at a time selected from the group consisting of 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, in infusion, 12 days after the initial infusion, and 15 days after the initial infusion. In some embodiments, the first additional IV infusion is performed 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial IV infusion, and 15 days after the initial IV infusion. days after initial infusion. In some embodiments, the second additional IV infusion is performed at a time selected from the group consisting of 3 weeks after the initial IV infusion, 4 weeks after the initial IV infusion, 5 weeks after the initial IV infusion, at infusion, 6 weeks after initial infusion, 7 weeks after initial infusion, and 8 weeks after initial infusion. In some embodiments, the second additional IV infusion is performed 3 weeks after the initial IV infusion, 4 weeks after the initial IV infusion, 5 weeks after the initial IV infusion, 6 weeks after the initial IV infusion, 7 weeks after the initial infusion and 8 weeks after the initial infusion. In some embodiments, the first additional IV infusion is performed 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial IV infusion, and 15 days after the initial IV infusion. days after the initial infusion, and a second additional IV infusion is performed 3 weeks after the initial IV infusion, 4 weeks after the initial IV infusion, 5 weeks after the initial IV infusion, 6 weeks after the initial infusion, 7 weeks after the initial infusion and 8 weeks after the initial infusion.

In some embodiments, the first additional IV infusion and the second additional IV infusion are performed at different doses. In some embodiments, the first additional IV infusion and the second additional IV infusion are administered at different doses, with the dose of the second additional IV infusion being higher than the dose of the first additional IV infusion. In some embodiments, the first additional IV infusion and the second additional IV infusion are administered at different doses, wherein the dose of the second additional IV infusion is higher than the dose of the first additional IV infusion, and the doses of both the first and and second, an additional IV infusion higher than the initial dose. In some embodiments, at least one of the first and second additional IV infusions is performed at a dose of 3 mg/kg. In some embodiments, the first additional IV infusion is performed at a dose of 3 mg/kg and the second additional IV infusion is performed at a dose of 6 mg/kg. In some embodiments, the first additional IV infusion is performed at least three days after the initial IV infusion. In some embodiments, the first additional IV infusion is performed at a time selected from the group consisting of 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, in infusion, 12 days after the initial infusion, and 15 days after the initial infusion. In some embodiments, the first additional IV infusion is performed 3 days after the initial IV.

- 39 042261 infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial infusion, and 15 days after the initial infusion. In some embodiments, the second additional IV infusion is performed at a time selected from the group consisting of 3 weeks after the initial IV infusion, 4 weeks after the initial IV infusion, 5 weeks after the initial IV infusion, at infusion, 6 weeks after initial infusion, 7 weeks after initial infusion, and 8 weeks after initial infusion. In some embodiments, the second additional IV infusion is performed 3 weeks after the initial IV infusion, 4 weeks after the initial IV infusion, 5 weeks after the initial IV infusion, 6 weeks after the initial IV infusion, 7 weeks after the initial infusion and 8 weeks after the initial infusion. In some embodiments, the first additional IV infusion is performed 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial IV infusion, and 15 days after the initial IV infusion. days after the initial infusion and a second additional IV infusion is performed 3 weeks after the initial IV infusion, 4 weeks after the initial IV infusion, 5 weeks after the initial IV infusion, 6 weeks after the initial IV infusion, after 7 weeks after initial infusion and 8 weeks after initial infusion.

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

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

The invention also relates to compositions and methods useful for identifying or more accurately defining a group of patients suffering from a disease in which the patient has an elevated level of CXCL9, alone or in combination with one or more additional interferon-y (IFNy) associated biomarkers. In particular, the invention relates to compositions and methods for determining levels of CXCL9 as a biomarker for IFNy production in patients suffering from

- 40 042261 giving, or suspected suffering from, hemophagocytic lymphohistiocytosis (HLH). In particular, the invention relates to compositions and methods for determining levels of CXCL9 as a biomarker for IFNy production in patients suffering from or suspected of suffering from secondary hemophagocytic lymphohistiocytosis (HLH). In some embodiments, the compositions and methods are used to determine levels of CXCL9 as a biomarker for IFNy production in patients suffering from, or suspected to be suffering from, macrophage activation syndrome (MAS). In some embodiments, the compositions and methods are used to determine levels of CXCL9 as a biomarker for IFNy production in patients suffering from, or suspected to be suffering from, MAS in the context of an autoimmune disease or inflammatory disease. In some embodiments, the compositions and methods are used to determine levels of CXCL9 as a biomarker for IFNy production in patients suffering from, or suspected of suffering from, MAS in the context of a systemic autoimmune disease or inflammatory disease. In some embodiments, the compositions and methods are used to determine levels of CXCL9 as a biomarker for IFNy production in patients suffering from, or suspected to be suffering from, MAS in the context of systemic juvenile idiopathic arthritis (sJIA). In some embodiments, the compositions and methods are used to determine levels of CXCL9 as a biomarker for IFNy production in patients suffering from, or suspected to be suffering from, MAS in the context of systemic lupus erythematosus (SLE).

Patients identified as having elevated levels of CXCL9 are considered suitable candidates for treatment with an agent (eg, antibodies or other polypeptide-based therapeutics, peptide-based therapeutics, small molecule inhibitors, nucleic acid-based therapeutics, and derivatives thereof) that prevents or is an antagonist of one or more IFNy biological activities, such as, for example, IFNy signaling, and neutralizes at least one IFNy biological activity.

In some patients with or suspected of having a disease, body fluids and other biological samples contain elevated levels of CXCL9 alone or in combination with other IFNy-associated biomarkers such as, for example, CXCL10 and/or CXCL11.

CXCL9 and these other biomarkers are in vivo indicators of IFNy production. Thus, the use of an anti-IFNy antagonist that interferes with, inhibits, reduces, or otherwise antagonizes IFNy signaling, for example, an anti-IFNy neutralizing antibody or other polypeptide-based therapeutic, a peptide-based therapeutic, a small molecule inhibitor, based on nucleic acids and their derivatives, leads to blocking or other inhibition of IFNy activity. Thus, the compositions and methods are useful for treating, slowing the progression, or ameliorating a symptom of a disease that is dependent on, caused by, associated with, or otherwise affected by aberrant, e.g., increased expression and/or activity of IFNy, aberrant production of pro-inflammatory cytokines, and/or their combinations, by administering an anti-IFNy antagonist, e.g., an anti-IFNy neutralizing antibody or other polypeptide-based therapeutic agent, peptide-based therapeutic agent, small molecule inhibitor, nucleic acid-based therapeutic agent and their derivatives, to patients having elevated levels of expression of CXCL9 and /or other biomarkers. Patients who are likely to be suitable candidates for treatment with an anti-IFNy antagonist, such as a neutralizing anti-IFNy antibody, such as those described herein, are identified by determining the level of CXCL9, alone or in combination with one or more associated with IFNy ligands or other biomarkers. In some embodiments, patients who do not have elevated levels of CXCL9, alone or in combination with other IFNy-associated biomerkers, may also be treated with anti-IFNy antagonists, including any of the anti-IFNy neutralizing antibodies described herein or other therapeutic agents for based on polypeptides, therapeutic agents based on peptides, small molecule inhibitors, therapeutic agents based on nucleic acids and their derivatives.

Patients having elevated levels of CXCL9, alone or in combination with one or more additional IFNy-associated biomarkers, are identified as suitable candidates for therapy with one or more anti-IFNy antagonists, such as the anti-IFNy neutralizing antibody described herein. As used herein, the expression elevated expression level means an expression level that is higher than the baseline expression level of CXCL9, alone or in combination with one or more additional biomarkers, in a sample from a patient who does not, or is not suspected to suffer, from a primary or secondary HLH or HLH related disease or other control sample. In some embodiments, an increased expression level of CXCL9 and/or another biomarker is a significant

- 41 042261 significantly elevated level.

Determining the level of CXCL9, alone or in combination with one or more other IFNy-associated biomarkers, is useful for refining or stratifying a patient population. In some embodiments, the level determined is used to determine the dose of anti-IFNy antagonist to be administered to a particular patient. In some embodiments, the determined level is used to categorize or stratify a patient population. For example, patients may be classified as having severe or severe MAS, or vice versa, non-severe or mild MAS, based on the level of CXCL9 measured.

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

In addition to detectable levels of IFNy and/or other IFNy-related biomarkers, patients suitable for treatment with anti-IFNy antagonists can also be identified by assessing any of a number of additional biological and clinical parameters that will increase the sensitivity and specificity of biomarkers to identify or clarification of the patient population. Alternatively, these additional biological and clinical indicators can be used alone as a means to identify patients who are suitable candidates for treatment with an anti-IFNy antagonist or other appropriate therapeutic agent. These biological and clinical indicators include, as a non-limiting example, any of the following: ferritin levels, neutrophil counts, platelet counts, alanine aminotransferase levels, and/or lactate dehydrogenase levels.

Diseases that may be treated with the compositions and methods of the invention include any disease in which there is aberrant, eg, increased expression and/or activity of IFNy, in particular HLH, including secondary HLH, MAS, and/or sJIA.

As non-limiting examples, the methods and compositions provided herein are suitable for the diagnosis and/or treatment of diseases such as primary and/or secondary HLH. Suitable autoimmune and/or inflammatory diseases include, by way of non-limiting example, primary and/or secondary HLH associated with aberrant IFNy expression and/or activity.

After identifying patients as having elevated levels of CXCL9, alone or in combination with one or more IFNy-associated biomarkers, they are treated with an anti-IFNy antagonist. For example, an anti-IFNy antagonist is a neutralizing anti-IFNy antibody, or an immunologically active (eg, antigen-binding) fragment thereof. Suitable neutralizing anti-IFNy antibodies include any of the anti-IFNy antibodies described herein.

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

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

ID NO: 5); and a VL CDR3 region containing the amino acid sequence QSYDGSNRWM (SEQ ID NO: 6).

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

In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises a light chain comprising a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination is a combination of three light chain CDR sequences (VL CDR1, VL CDR2, VL CDR3), given in one row in table. 1B.

In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises a heavy chain comprising a combination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence, wherein the combination is a combination of three heavy chain CDR sequences (VH CDR1, VH CDR2, VH CDR3) shown in one row in Table 1A, and a light chain containing a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, the combination being a combination of three light chain CDR sequences (VL CDR1, VL CDR2, VL CDR3) given in one row in table. 1B.

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

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

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

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

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

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

In some embodiments, the anti-IFNy antibody or immunologically active fragment thereof contains an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of the heavy chain corresponding to amino acid sequence of SEQ ID NO: 44, and an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of the light chain corresponding to the amino acid the sequences of SEQ ID NO: 46.

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

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

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

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

In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 50, 54, 58, 62, 66, 70, 74, 78, 82, 86 , 90, 94, 98 and 102.

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

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

In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof is administered in a therapeutically effective amount. A therapeutically effective amount of an antibody of the invention generally means the amount necessary to achieve the therapeutic goal. This therapeutic target may be a binding interaction between an antibody and its target antigen, which in some cases interferes with the functioning of the target. The generally accepted range of therapeutically effective doses of an antibody or antibody fragment of the invention may be, as a non-limiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight. The generally accepted frequency of dosing may be in the range, for example, from administration twice a day to administration once a week.

In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof is administered at an initial dose, i.e., a loading dose, ranging from about 0.5 to about 2 mg/kg, for example, in the range from about 0.5 to about 1.5 mg/kg and/or from about 0.5 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, the anti-IFNγ antibody or immunologically active fragment thereof is administered at an initial loading dose followed by one or more maintenance doses. In some embodiments, the one or more maintenance doses are a dose that is substantially the same as the initial loading dose. In some embodiments, one or more maintenance doses is a dose that is less than the initial loading dose. In some embodiments, one or more maintenance doses is a dose that is greater than the initial loading dose.

In some embodiments, one or more maintenance doses comprise at least

- 44 042261 at least two or more doses, with all maintenance doses being the same dose. In some embodiments, two or more maintenance doses are substantially the same as the initial loading dose. In some embodiments, two or more maintenance doses are greater than the initial loading dose. In some embodiments, two or more maintenance doses are less than the initial loading dose.

In some embodiments, the implementation of one or more maintenance doses include at least two or more doses, while all maintenance doses are not the same dose. In some embodiments, two or more maintenance doses are administered in order of increasing dose. In some embodiments, two or more maintenance doses are administered in descending order of dose.

In some embodiments, the implementation of one or more maintenance doses include at least two or more doses, with each maintenance dose being administered periodically, at certain intervals of time. In some embodiments, two or more doses are administered at increasing time intervals. In some embodiments, two or more doses are administered at decreasing time intervals.

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

Pharmaceutical compositions of the invention may contain an anti-IFNy antibody of the invention and a carrier. These pharmaceutical compositions may be included in kits such as, for example, diagnostic kits.

The invention also relates to kits for practicing any of the methods proposed herein. For example, in some embodiments, the kits include a detection reagent specific for CXCL9 alone or in combination with one or more of the IFNy-associated biomarkers, as well as a means for detecting the detection reagent.

The invention will now be described by way of examples, which do not limit the scope of the invention as set forth in the claims.

Examples

Example 1 CXCL9 levels as a biomarker for IFNy production in macrophage activation syndrome (MAS)

The studies presented herein were designed to correlate serum levels of IFNy and the three IFNy-related chemokines with each other and with laboratory measures of disease activity in patients with active MAS in order to search for a potential biomarker for in vivo IFNy production.

Circulating levels of IFNy, CXCL9, CXCL10, CXCL11, and IL-6 were measured using the Luminex multiplex assay in sJIA patients (n=54), of whom 20 had MAS at the time of sample collection. The association of these circulating levels with indicators of disease activity was assessed, as well as the correlation of IFNy levels with levels of CXCL9, CXCL10 and CXCL11.

Levels of IFNy and 3 IFNy-related chemokines (CXCL9, CXCL10 and CXCL11) were significantly elevated with active MAS compared with active sJIA without MAS at the time of sample collection (all values <0.005). In active MAS, laboratory measures of disease severity (ferritin, neutrophils, platelets, alanine aminotransferase, and lactate dehydrogenase levels) correlated significantly with IFNy and CXCL9 levels, and to a lesser extent with CXCL10 and CXCL11 levels; no correlation was found with IL-6 levels. In patients with active sJIA without MAS, there was no significant correlation between laboratory values and cytokine levels, as shown in Table 1. 7 below. In active MAS, IFNy levels correlated significantly with CXCL9 levels (r=0.69; ^r2 =0.47; p=0.001), to a lesser extent with CXCL10 levels (r=0.53; ^r2 =0.28 ; p=0.015) and did not correlate with CXCL11 levels (r=-0.04; p=0.886).

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Table 7. Correlation of laboratory indicators of disease activity with the levels of IFNy, CXCL9,

CXCL10, CXCL11 and IL-6 in MAS patients and in patients with active sJIA

macrophage activation syndrome IFNy CXCL9 CXCL10 g*r CXCL11 IL-6 G* R G* R G* R G* R ferritin 8000 (3159-13174) 0.57 0.014 0.49 0.041 0.66 0.002 0.62 0.023 0.17 >0, 1 N 6.9 (3.4-13.9) -0.64 0.005 -0.61 0.010 -0.37 >0.1 -0.08 >0.1 0.09 >0.1 PLT 198(115-392) -0.53 0.017 -0.52 0.022 -0.58 0.008 -0.22 >0.1 -0.02 >0.1 ALT 46 (18-164) 0.49 0.045 0.49 0.044 0.51 0.038 0.06 >0.1 -0.44 0.080 LDH 1152(722-2135) 0.45 0.095 0.62 0.013 0.64 0.010 0.64 0.048 0.09 >0.1 systemic juvenile IFNy CXCL9 CXCL10 CXCL11 IL- 6 idiopathic arthritis G* R G* R G* R G* R G* R ferritin 215(38-1669) -0.27 >0.1 0.28 >0.1 0.27 >0.1 0.29 >0.1 -0.12 >0, 1 N 8.4 (5.2-14.5) 0.30 >0.1 0.40 0.061 0.32 >0.1 0.40 0.067 0.28 >0.1 PLT 444 (353-544) 0.21 >0.1 -0.14 >0.1 -0.13 >0.1 0.27 >0.1 0.35 0.064 ALT 16 (11-24) 0.29 >0.1 0.42 0.049 0.50 0.011 0.44 0.039 0.04 >0.1 LDH 506(456-851) 0.07 >0.1 0.49 >0.1 0 >0.1 0.26 >0.1 0 >0.1

^number of neutrophils; PLT=platelet count; ALT=alan aminotransferase, 1=median (IQR); r*=r Spearman

High levels of IFNy and CXCL9 observed in patients with active MAS correlated significantly with laboratory measures of disease severity. In patients with active MAS, IFNy and CXCL9 levels are closely correlated. Since CXCL9 has been shown to be induced only by IFNy and no other interferons (see e.g. Groom J.R. and Luster A.D. Immunol Cell Biol 2011, Feb; 89(2):207-15), these data demonstrate that CXCL9 is a biomarker for IFNy production. at MAS.

Example 2 Correlation of CXCL9 and IFNy Levels in Patients with Primary Hemophagocytic Lymphohistiocytosis (HLH)

The data reported herein are derived from the current phase 2 pilot study in patients with primary HLH who were administered the NI-0501 antibody and in patients who were given the NI-0501 antibody on humanitarian grounds.

As shown in FIG. 1, serum levels of CXCL9 and IFNy were measured using Luminex and Meso Scale Discovery (MSD) technology, respectively, in samples obtained from 6 patients with primary HLH and from 3 patients treated for humanitarian reasons. Correlations were made between the concentrations of CXCL9 and total IFNy. Statistical data processing was carried out and p-values were obtained using Spearman's test.

As shown in FIG. 2, pre-dose serum levels of CXCL9 and IFNy were measured using Luminex and MSD technology, respectively, in samples from 6 patients with primary HLH and from 3 humane treated patients. Correlations were made between the concentrations of CXCL9 and total IFNy. Statistical data processing was carried out and p-values were obtained using Spearman's test.

Example 3 Correlation of CXCL9 and IFNy Levels in Patients with Secondary Hemophagocytic Lymphohistiocytosis (HLH)

The data reported herein are derived from an observational study of patients with secondary HLH who received the NI-0501 antibody and from patients who received the NI-0501 antibody on humanitarian grounds.

Specifically, the study involved patients with systemic juvenile idiopathic arthritis (sJIA) who developed macrophage activation syndrome (MAS, a form of secondary HLH). In these patients, a correlation was also found between CXCL9 or IFNy levels and measures of disease activity such as ferritin level, platelet count (PLT), neutrophil count (Neu), and alanine aminotransferase (ALT) level.

As shown in FIG. 3A and 3B, serum levels of CXCL9 and IFNy were measured in multiplex analysis using Luminex technology in samples obtained from 19 patients with MAS secondary to sJIA and 24 patients with active sJIA at the time of sample collection. Correlations were made between CXCL9 and IFNy concentrations. Statistical data processing was carried out and p-values were obtained using Spearman's test.

As shown in FIG. 4A-1, 4A-2, 4B-1, 4B-2, 4C-1, 4C-2, 4D-1, and 4D-2, serum levels of CXCL9 and IFNy were measured in multiplex assays using Luminex technology in patient samples. with MAS secondary to sJIA and patients with active sJIA at the time of sample collection. Correlations have been made between IFNy or CXCL9 levels and ferritin levels, platelet counts, neutrophil counts, or ALT (alanine aminotransferase) levels. Statistical data processing was carried out and p-values were obtained using Spearman's test.

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Example 4 Correlation of CXCL9 and IFNy Levels in a Patient with Severe Hemophagocytic Lymphohistiocytosis (HLH)

The data provided herein is from a patient to whom the NI-0501 antibody was provided on humanitarian grounds. This patient exhibited symptoms of NLRC4-associated disease and severe hemophagocytic lymphohistiocytosis (HLH). Mutations in the NLRC4 gene have recently been reported to cause recurrent macrophage activation syndrome and increased production of IL-18, which is known to induce IFNy.

The patient in this study had the following characteristics: onset at 20 weeks of age with symptoms of fever, rash, severe hepatosplenomegaly, pancytopenia, hypofibrinogenemia, hypertriglyceridemia, markedly elevated ferritin and SCD25 levels. After the development of multiple organ failure, hospitalization in the intensive care unit was required. The diagnosis of HLH was based on 6 of the 8 criteria of the HLH-2004 guidelines. The results of the analysis of genes causing primary HLH (PRF1, UNC13D, STXBP2, STX11, RAB27A, XIAP) and functional tests (perforin expression, degranulation and cytotoxicity) were negative. In / in the introduction of high-dose glucocorticoids and / in the introduction of cyclosporine-A, with a progressive improvement in the general condition and laboratory parameters. HLH reactivation initiated by infections (Candida albicans and Klebsiella pneumoniae, sepsis), rapid deterioration of general condition and re-admission to the intensive care unit. Treatment with etoposide and/or ATG was not considered due to the presence of active infections in an already immunocompromised individual.

Measurable IFNy levels and high serum levels of the IFNy-inducible chemokines CXCL9 and CXCL10, as well as markedly elevated serum levels of IL-18, were recorded (Table 8).

Table 8 Levels of IFNy, IFNy-associated chemokines and IL-18 at initiation of NI-0501 treatment and during NI-0501 treatment

Before treatment 1 month after treatment 2 months after treatment 3 months after treatment 4 months after treatment sJIA* inactive Free IFNy (pg/ml) 6.02 But But But But 4.2 (3.2-9.3) CXCL9 (pg/ml) 5670 495.16 207.4 207.4 207.4 901 (466-1213) CXCL10 (pg/ml) 4400 529.54 201.12 147.84 138 235 (172-407) CXCL11 (pg/ml) 188.68 But But But But 111 (63-187) IL18 (pg/ml) >300000 95000 34000 27000 32000 -

*Median (interquartile range)

Treatment with NI-0501, for humanitarian reasons, was started with dexamethasone (13.6 mg/m ² ) and intravenous cyclosporine-A. NI-0501 was administered every 3 days, and then every 7 days thereafter, according to pharmacokinetics. Infusion reactions were not observed. N10501 was well tolerated. Clinical signs and laboratory parameters of HLH improved progressively. Active current infections quickly disappeared. After 5 months of treatment, the patient remained in excellent condition. The patient continued to receive oral cyclosporine-A (6 mg/kg) and prednisone (0.3 mg/kg, equivalent to 0.9 mg/m ² dexamethasone). All indicators characteristic of HLH returned to normal.

The individual still had unexplained episodes of inflammation. NLRC4 analysis showed a de novo missense mutation (T337N). Elevated serum levels of IL-18 were noted, confirming an important role for the NLRC4 mutation. High levels of IFNy production have been demonstrated by having high concentrations of IFNy in complex with NI-0501. IFNy was completely neutralized as evidenced by the absence of detectable levels of IFNy-induced chemokines (FIG. 5 and Table 8). Circulating levels of IL-18 were consistently elevated.

In summary, the results of this study show that in a patient with severe refractory HLH (due to an NLRC4 mutation), IFNy blocking with NI-0501 was well tolerated, did not cause safety concerns, controlled all signs of HLH, allowed rapid tapering of glucocorticoids, and associated with resolution of current active infections.

Example 5 Targeted approach to the treatment of hemophagocytic lymphohistiocytosis (HLH) using NI-0501

The data reported herein are from a phase 2 pilot study in children with primary HLH. Primary HLH (pHLH) is a rare immune regulatory disorder that is notoriously fatal if left untreated. It results from abnormal immune activation leading to fever, splenomegaly, cytopenias, and coagulopathy, which can lead to multiple organ failure and death. Based on data from primary and secondary HLH (sHLH) models in mice treated with an anti-IFNy antibody, as well as observational studies in patients with HLH, high levels of IFNy production are considered to be a critical factor driving the development of the disease. Immuno-chemotherapy, mainly etoposide-based regimens, are currently considered the only pharmacological approaches to control HLH and prepare patients for healing allogeneic hematopoietic stem cell transplantation (alloHSCT). Despite recent attempts to further intensify treatment regimens, mortality and morbidity remain high, in part due to drug-related toxicity.

As described above, NI-0501 is a fully human high affinity anti-IFNy mAb that binds and neutralizes human IFNy and is at the heart of a new and targeted approach to the control of HLH.

Methods: An open-label phase 2 clinical trial was conducted in the US 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 a possible increase in dose depending on the PK data and/or clinical response in each patient, against the background of an initial dose of 5-10 mg/m ² dexamethasone. The treatment lasted from 4 to 8 weeks. The possibility of switching to allo-HSCT, relevant indicators of HLH severity, and 8-week survival rates were assessed.

Study Population: A total of 13 patients were included in the study: 8F/5M, mean age 1.0 years (range 2.5 months - 13 years). Twelve patients received NI-0501 as second-line therapy after receiving conventional therapy in the event of either reactivation of the disease, lack of a satisfactory response to therapy, or intolerance to therapy. One patient received NI-0501 as 1st line therapy. Nine patients had a known genetic defect for HLH (3 FHL2, 2 FHL3, 2 GS-2, 1 XLP1, 1 XLP2). Most of the patients had severe HLH, poor general condition, and severe toxicity associated with previous HLH treatments. Ferritin levels were elevated in 12/13 patients and SCD25 in 8, cytopenias occurred in 10 patients, splenomegaly in 8, hypofibrinogenemia and hypertriglyceridemia in 9. Liver and CNS dysfunction occurred in 7 and 3 patients, respectively.

Results: Overall, treatment with NI-0501 resulted in a significant improvement in HLH activity scores (FIG. 6) and 9 out of 13 patients achieved a satisfactory response. Transition to HSCT became possible for six patients. Two patients with well-controlled HLH were scheduled for HSCT once a matched donor was found. For one patient (who achieved disease control with NI-0501 as 1st-line therapy), HSCT is not yet planned, given the absence of a gene mutation causing HLH. Eleven of 13 patients were alive for 8 weeks. Signs and symptoms of CNS dysfunction were resolved in 2 evaluable patients. Greater than 50% dose reduction of dexamethasone was possible in 50% of patients during the first 4 weeks of treatment with NI-0501.

Evaluation of biomarkers, in particular CXCL9, a chemokine known to be exclusively induced by IFNy, not only demonstrated complete neutralization of IFNy, but also provided a new indicator for diagnosing HLH correlated with IFNy production (FIGS. 7A and 7B).

NI-0501 was well tolerated and no safety issues were identified. No infections known to be promoted by IFNy neutralization have been reported, and no infections have been reported in chemotherapy-naive patients. Seven patients reported at least one SAE, all assessed by an independent data monitoring committee (DMC) as not related to NI-0501 administration. No unexpected events attributable to off-target effects of NI-0501 (eg, myelotoxicity, hemodynamic effects) were observed.

Conclusions: Targeted neutralization of IFNy using NI-0501 is an innovative and potentially less toxic approach to control HLH. The results of this study show that administration of NI-0501 is a safe and effective treatment option for patients with primary HLH who do not respond satisfactorily to conventional therapy or are unable to tolerate it. Moreover, NI-0501 therapy is not associated with any of the typical short-term or long-term toxicity associated with etoposide-based treatment regimens. Evaluation of NI-0501 as a 1st line therapy in patients with pHLH is ongoing and it is expected that similar significant clinical successes can be achieved.

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Example 6 Elevated circulating levels of interferon-γ and interferon-induced chemokines are characteristic of patients with macrophage activation syndrome, which is a complication of systemic JIA

Interferon-gamma (IFNy) is the main mediator in mouse models of primary hemophagocytic lymphohistiocytosis (HLH). Given the similarities between primary and secondary HLH (sec HLH), including macrophage activation syndrome (MAS), !ΗΝγ levels and its biological activity were analyzed in patients with systemic juvenile idiopathic arthritis (sJIA) and MAS.

The studies described herein used the Luminex multiplex assay to assess serum levels of IL-1e, IL-6, !ΗΝγ, and IFN-induced and/or IFN-related chemokines CXCL9, CXCL10, and CXCL11 in patients with sec-HLH (n= 11) and in patients with sJIA (n=54), of whom 20 had MAS at the time of sample collection. Expression of IFNγ-inducible chemokines (mRNA levels of CXCL9 and CXCL10 in the liver and spleen), as well as the correlation of their levels with serum levels of ferritin, was evaluated in an IL-6 transgenic mouse model in which signs of MAS were induced by stimulation of TLR4 with LPS.

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

Thus, the results of the studies below show that high levels of IFNy and IFN-inducible chemokines, as well as their correlation, in particular in the case of CXCL9, with the degree of laboratory abnormality of MAS, indicate that IFNy plays a key role in MAS. . Elevated circulating levels of interferon-y and interferon-inducible chemokines are characteristic of patients with macrophage activation syndrome, which is a complication of systemic JIA.

Materials and methods

Patients and samples.

Peripheral blood samples were collected from patients suffering from sJIA with or without MAS at 3 pediatric rheumatology centers: Ospedale Pediatrico Bambino Gesu in Rome, Istituto Giannina Gaslini in Genoa, and Cincinnati Children's Hospital Medical Center. Fifty-four patients with sJIA (age at onset 7.9 years, interquartile range 4.6-13.6 years; 48% female) who met the ILAR classification criteria for systemic arthritis were studied (Petty, R.E., et al., International League of Associations for Rheumatology classification of juvenile idiopathic arthritis: second revision, Edmonton, 2001. J Rheumatol, 2004. 31(2): p. 390-2). Twenty of the sJIA patients were sampled during episodes of active, severe MAS that were diagnosed by the attending physicians at each of the three centers. Post hoc analysis showed that 17 of these 20 episodes (85%) met the new proposed MAS qualifying criteria (Minoia F, Davi S, Bovis F, et al. Development of new classification criteria for macrophage activation syndrome complicating systemic juvenile idiopathic arthritis. Pediatric Rheumatology 2014, 12 (Suppl 1):01.). Samples were available from twenty-eight patients with active sJIA without evidence of MAS. Thirty-five samples were available from 35 patients with sJIA (both with and without MAS in their medical history) in clinically inactive disease, defined according to the Wallace criteria (Wallace, C.A., et al., Preliminary criteria for clinical remission for select categories of juvenile idiopathic arthritis J Rheumatol, 2004. 31(11): p. 2290-4).

Since IFNy levels were shown to be elevated in patients with sec-HLH (rheumatic disease was ruled out), samples were also collected from 11 patients (age at onset 8.6 years, interquartile range 4.1-12.9 years; 36% female) with sec-HLH observed in Ospedale Pediatrico Bambino Gesu and used as positive controls. All patients with secondary HLH met the criteria of the HLH-2004 diagnostic guidelines (Henter J.I., et al., HLH-2004: Diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer, 2007. 48(2): p. 124-31): 6 patients met 5 criteria and 5 patients met 4 criteria. It should be noted that SCD25 levels expressed in U/mL could not be determined because this test is not normally performed at the healthcare facility where these patients were seen. The diagnosis of primary HLH was ruled out based on no family history of disease, the absence of pathogenic mutations in genes known to cause HLH, and the presence of normal functional test results (including NK cell activity, perforin expression, and CD107 degranulation). One sample was collected from all 11 patients with sec-HLH, all of which were obtained during active disease.

Clinical and laboratory parameters of all patients relevant to diagnosis and determined at the time of sample collection were collected in a centralized web-based database by investigators at each center. Of the 20 MAS patients sampled during active disease, 6 were not receiving any treatment at the time of sample collection, while the remaining 14 patients were already receiving one of the MAS-specific therapies, including intermittent glucocorticoids, cyclosporine Ah, anakinra or cyclophosphamide. Six of the 11 patients with active sec-HLH were not receiving specific treatment at the time of sample collection, while the remaining 5 patients were already receiving at least one of the above therapeutic agents. The study was approved by the Ethics Committee of Ospedale Pediatrico Bambino Gesu. Written consent was obtained for all study participants.

Quantification of cytokines

IL-6, IL-Ιβ, IFNy, CXCL9, CXCL10 and CXCL11 levels were analyzed using Luminex® Bead Multiplex Assay Technology. Reagents were purchased from Millipore and all reagents were provided with Milliplex® MAP kits. The reagents were prepared according to the manufacturer's protocol. 25 µl/well of standards, blanks, and quality control samples were added in duplicate to a 96-well Milliplex MAP plate, followed by the 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 were added in duplicate or triplicate, depending on the available sample volume. The plate was read on a Luminex 200® system (Luminex Corp.). Primary data were obtained using the x PONENT software version 3.1 (Luminex Corp.) and the data was analyzed using the Milliplex Analyst software version 3.5.5.0 (Millipore). The raw data obtained with the Milliplex Analyst program was then further analyzed in a special Luminex analysis macro program (NI-Sc-ESM-MAC-012-v01 and Sc-ESM-MAC-013-v01).

Animal experiments

The production and phenotype of IL-6 transgenic mice, as well as signs of a MAS-like syndrome caused by administration of TLR ligands, have been described previously (Strippoli, R., et al., Amplification of the response to Toll-like receptor ligands by increased exposure to interleukin -6 in mice: implication for the pathogenesis of macrophage activation syndrome Arthritis Rheum, 2012. 64(5): p. 1680-8). Mice were kept under specific pathogen free conditions and handled in accordance with national policy in this area. The study protocol was approved by the local ethics committee. All experiments were performed on mice aged 10-14 weeks. Mice were injected intraperitoneally with a single dose of 5 μg/g body weight of lipopolysaccharide (LPS, E. coli serotype 055:B5; Sigma-Aldrich). Mice were sacrificed after 30 hours. Total RNA was extracted from spleen and liver tissues using Trizol (Life technologies). cDNA was obtained using the Superscript Vilo kit (Invitrogen). Real-time PCR analyzes were performed using the TaqMan Universal PCR Master Mix (Applied Biosystems) with mouse Cxcl9 and Cxcl10 gene expression assay kits (Applied Biosystems). Gene expression data were normalized to mouse Hprt gene expression (Applied Biosystems). Data are expressed in arbitrary units (PU) determined using the 2' ^Act method. Serum ferritin concentrations were determined using a commercially available 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 medians and interquartile ranges (IQR) and compared using the Mann-Whitney U-test. The Wilcoxon signed-rank test was used to compare two paired groups, not assuming that the before-after difference distribution followed a Gaussian distribution. Spearman's rank correlation coefficient was used to assess association with laboratory parameters. A p-value of <0.05 was considered to be statistically significant.

Results:

Elevated levels of IFNy and IFNy-induced chemokines in patients with MAS. When comparing patients with active sJIA without MAS at the time of sample collection with patients from whom samples were collected during clinically inactive disease, it was found as expected (de Benedetti, F., et al., Correlation of serum interleukin-6 levels with joint involvement and thrombocytosis in systemic juvenile rheumatoid arthritis Arthritis Rheum, 1991. 34(9): p.1158-63) that IL-6 levels were significantly higher in patients with active sJIA (p<0.01) compared to levels in patients with clinically inactive disease. As found in several previous studies of active SJIA, serum levels of IL-1e were below the detection limit in most patients, regardless of disease activity status. Of note, there were no differences in levels of IFNy and the three IFNy-induced chemokines between patients with clinically active sJIA and patients with clinically inactive disease.

When comparing patients with MAS at the time of sample collection with those with active sJIA without MAS at the time of sample collection, IL-1e and IL-6 levels were comparable, indicating that

- 50 042261 levels of two cytokines known to play a key role in active sJIA do not increase during a pronounced MAS. Of note, circulating levels of IL-1e were below the limit of quantitation (ie, 3.5 pg/mL) in most sJIA patients with or without MAS. In contrast, circulating levels of IFNy were significantly higher in patients with active MAS compared to patients with active sJIA without MAS at the time of sample collection. Levels of the three IFNy-related chemokines CXCL9, CXCL10, and CXCL11 were also significantly higher in patients with active MAS compared with active sJIA patients without MAS at the time of sample collection. This difference was particularly evident in the case of CXCL9, whose median levels were about 15 times higher in patients with MAS than in patients with active sJIA without MAS.

In sec-HLH patients, IFNy levels as well as three IFNy-related chemokines were markedly elevated. Levels of IFNy and IFNy-related chemokines were largely indistinguishable from levels in patients with MAS, and the differences were not statistically significant. Incidentally, in patients with active MAS and in patients with active sec-HLH, levels of IFNy and the three IFNy-inducible chemokines were comparable in untreated and already treated patients.

IFNy levels, as well as CXCL9, CXCL10 and CXCL11, have been associated with the presence of MAS in individual patients. In FIG. 8A-8D show IFNy levels as well as CXCL9, CXCL10 and CXCL11 in individual patients from whom paired samples were obtained during active MAS and during active sJIA without MAS. Consistent with the results obtained in the crossover analysis, the levels of IFNy and the three IFNy inducible chemokines were significantly higher in the samples obtained during the MAS when pairwise analysis of the samples. In addition, samples from some patients were obtained both before and after episodes of MAS and it was shown that the levels of IFNy and IFNy-induced chemokines returned to normal with resolution of the clinical symptoms of MAS. For example, one patient in this study experienced three episodes of MAS, and serum samples were obtained from him during these episodes, as well as during phases of the disease without MAS at the time of sample collection. Additional evidence for an association of increased production of IFNy and the three IFNy-induced chemokines with active MAS was that, in this patient, elevated levels of IFNy and the three IFNy-related chemokines were found only during episodes of MAS (FIGS. 9A-9B).

Levels of IFNy and IFNy-related chemokines correlate with abnormal laboratory MAS values. Subsequently, the correlation of levels of IFNy and three IFNy-induced chemokines with laboratory MAS values at the time of sample collection was studied. In patients with active sJIA without MAS, levels of IFNy and three IFNy-inducible chemokines were not associated with laboratory MAS values, with one exception: CXCL9, CXCL10, and CXCL11 levels were weakly correlated with ALT levels, with an ^r2 value ranging from 0.17 to 0. 25 (Table 2). The significance of this connection remains unclear; however, it should be noted that ALT levels were within the normal range in all patients with active sJIA without MAS. In patients with MAS, no significant correlation of laboratory MAS values with IL-1 and IL-6 levels was found at the time of sample collection. In contrast, in MAS patients at the time of sample collection, levels of IFNy and IFNy-inducible chemokines were associated with ferritin levels, neutrophil and platelet counts, and elevated LDH and ALT levels, all of which tend to be abnormal in MAS patients. (Table 2). Correlations with abnormal laboratory values were particularly evident for IFNy and for CXCL9, with the sole exception of the correlation of IFNy with LDH, which was not statistically significant (Table 2 and Figures 10A-10J). Again, as mentioned above, these correlations were absent in patients with active sJIA without MAS at the time of sample collection. One patient in this group had significantly elevated levels of IFNy (336.2 pg/ml), CXCL9 (549400 pg/ml) and CXCL10 (35066 pg/ml). This patient had a particularly severe form of MAS and was hospitalized in the intensive care unit with severe impairment of the functions of the central nervous system. This observation further supports the hypothesis that there is a strong relationship between IFNy and CXCL9 levels and disease severity. Taken together, these results indicate that increased production of IFNy and IFNy-related chemokines is a hallmark of active MAS that strongly correlates with the severity of MAS laboratory abnormalities.

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Table 2. Serum levels of IL-1e, IL-6, IFNy, and the three IFNy-related chemokines CXCL9, CXCL10, and CXCL11 in active secondary HLH patients with active MAS at sample collection, active sJIA without MAS at sample collection, and with clinically inactive sJIA

sec—HLH Active sJIA Second-HLH vs. MAS MAS vs active sJIA without MAS MAS at time of sample collection without MAS Inactive sJIA (n=11) (n=20) (n=28) (n=35) ^{p_value p_value} IL-Ιβ (pg/ml) <3.5 (<3.510.7) <3.5 "3.5- 6.1) <3.5 "3.5- 3.8) <3.5 (<3.5-3.5) 0.69 0.86 IL-6 (pg/ml) 11.4 (3.249.3) 22.9 (5.545.6) 20.3* (5.9- 54.9) 3.2 (3.2-7.9) 0.56 0.43 IFNy (pg/ml) 34.7 (23.9170.1) 15.4 (5.152.6) 4.9 (3.2-8.6) 4.2 (3.2-9.3) 0.12 0.03 CXCL9 (pg/ml) 33598 (3083- 127687) 13392 (216335452) 837 (4712505) 901(466-1213) 0.23 0.005 CXCL10 (pg/ml) 4420 (7 9 98226) 1612 (42 54309) 307(199-694) 235(172-407) 0.19 0.0016 CXCL11 (pg/ml) 1327 (18 92000) 565 (1981007) 122(62-197) 111 (63-187) 0.30 0.003

Values are presented as median (interquartile range) *Active sJIA vs clinically inactive sJIA: p<0.01

Table 3. Correlation of Laboratory Measures of Disease Activity with IFNy, CXCL9, CXCL10, CXCL11, and IL-6 Levels in MAS Patients and Active sJIA Non-MAS Patients at Sample Collection

MYSELF IFNy CXCL9 CXCL10 CXCL11 IL-6 G* R G* R G* R G* R G* R ferritin 8000(3158-13174)1 0.57 0.014 0.49 0.041 0.66 0.002 0.62 0.023 0.17 >0.1 NEU 6.9(3.4-13.9)1 -0.64 0.005 -0.61 0.010 -0.37 >0.1 -0.08 >0, 1 0.09 >0.1 PLT 197(114-392)1 -0.53 0.017 -0.52 0.022 -0.58 0.008 -0.22 >0.1 -0.02 >0.1 ALT 46(18-164)1 0.49 0.045 0.49 0.044 0.51 0.038 0.06 >0, 1 -0.44 0.080 LDH 1152 (722-2135)1 0.45 0.095 0.62 0.013 0.64 0.001 0.64 0.048 0.08 >0.1

Active sJIA without CAM r' P r* P r* P r‘ P r* P ferritin 214(37-1669)1 -0.27 >0.1 0.28 >0.1 0.27 >0.1 0.29 >0.1 -0.12 >0.1 N 8.4(5.2-14.5)1 0.30 >0, 1 0.40 0.061 0.32 >0.1 0.40 0.067 0.28 >0.1 PLT 444(353-544) ¹ 0.21 >0.1 -0.14 >0.1 -0.13 >0.1 0.27 >0.1 0.35 0.064 ALT 16(11-24)1 0.29 >0, 1 0.42 0.049 0.50 0.011 0.44 0.039 0.04 >0.1 LDH 506(455-851) ¹ 0.07 >0, 1 0.49 >0.1 0 >0.1 0.26 >0, 1 0 >0.1

NEU=neutrophil count; PLT=platelet count; ALT=alanine aminotransferase; LDH=lactate dehydrogenase;

1= Median (IQR);

r* = r Spearman.

Correlation of disease activity laboratory parameters with IFNy, CXCL9, CXCL10, CXCL11, and IL-6 levels in patients with MAS and in patients with active sJIA.

Correlation of IFNy levels with levels of IFNY-induced chemokines in patients with MAS. To further elucidate the relationship between IFNy and the three IFNy-induced chemokines in MAS patients, the correlation of IFNy levels with levels of each individual chemokine was assessed. Notably, CXCL9 appears to be primarily and specifically induced by IFNy, while CXCL10 and CXCL11 also appear to be induced by type I interferons. Consistent with this is the fact that in patients with active MAS, circulating IFNy levels correlated significantly with CXCL9 levels (r=0.693; ^r2 =0.48; p=0.001), but correlated to a lesser extent with CXCL10 levels (r= 0.535; r ² =0.29; p=0.015) (FIGS. 11A-11F). The correlation with CXCL11 levels was also weaker and did not reach the level of statistical significance (r=0.447; ^r2 =0.20; p=0.08) (not shown).

Levels of IFNy-induced chemokines correlate with disease activity in a mouse model of MAS. To further explore the association of IFNy-induced chemokine production with MAS, the expression of these chemokines in target tissues (liver and spleen) was studied in a mouse model of MAS. IN

In this model, clinical and laboratory signs of MAS are induced by mimicking acute infection using the TLR4 agonist lipopolysaccharide (LPS) against high levels of IL-6 in IL-6 transgenic mice (Strippoli et al., Arthritis Rheum 2012). This approach mimics what happens in patients with sJIA: infection can trigger MAS/HLH in the presence of active disease, which is indeed characterized by high levels of IL-6. After induction with LPS, high levels of mRNA for CXCL9 and CXCL10 were found in the liver and spleen of IL-6 transgenic mice. Notably, serum ferritin levels correlated significantly with CXCL9 expression levels in the spleen and liver, and CXCL10 in the liver, demonstrating an association between IFNy-associated antecedent events in target tissues (i.e., CXCL9 and CXCL10 production in the liver and spleen) and typical subsequent laboratory abnormalities such as high ferritin levels. Taken together, data from patients with MAS and from a mouse model of MAS indicate a clear association of increased IFNy production with increased expression of CXCL9 and, to a lesser extent, CXCL10, as well as abnormal laboratory values in MAS.

Studies in both patient and mouse models of n-HLH have demonstrated the central role of IFNy in the pathogenesis of the disease. However, the role of IFNy in sec-HLH, including MAS in the setting of sJIA, remained unclear. This study convincingly demonstrated that high levels of IFNy and IFNy-inducible chemokines occurred in patients with MAS arising from sJIA. In addition, IFNy, CXCL9, and CXCL10 levels were strongly correlated with laboratory measures of MAS severity. In this study, serum levels of IFNy and the three IFNy-related chemokines were found to be comparable in patients with active sJIA and patients with clinically inactive disease. This result contradicts the opinion about the pathogenic role of IFNy in sJIA and, indeed, is consistent with a number of observations made by other authors. Three gene expression studies failed to detect a characteristic IFNy-induced gene expression profile in peripheral blood mononuclear cells (PBMCs) of patients with active sJIA without MAS at the time of sample collection (Fall N., et al., Gene expression profiling of peripheral blood from patients with untreated new-onset systemic juvenile idiopathic arthritis reveals molecular heterogeneity that may predict macrophage activation syndrome Arthritis Rheum, 2007. 56(11): p. 3793-804; Ogilvie, E. M., et al., Specific gene expression profiles in systemic juvenile Pascual, V., et al., Role of interleukin-1 (IL-1) in the pathogenesis of systemic onset juvenile idiopathic arthritis and clinical response, idiopathic arthritis Arthritis Rheum, 2007. 56(6): p. to IL-1 blockade J Exp Med, 2005. 201(9): pp. 1479-86). After ex vivo PBMC stimulation, the number of IFNy-producing cells in patients with active sJIA was similar to that in controls (Lasiglie D., et al., Role of IL-1 beta in the development of human T(H)17 cells: lesson from NLPR3 mutated patients, PLoS One, 2011, 6(5): p.e20014). Patients with both active and inactive sJIA did not consistently show elevated serum or synovial fluid levels of IFNy (de Jager, W., et al., Blood and synovial fluid cytokine signatures in patients with juvenile idiopathic arthritis: a crosssectional study Ann Rheum Dis, 2007. 66(5): pp. 589-98). In support of the lack of involvement of IFNy in joint inflammation in sJIA, CXCL9 and CXCL10 were almost unmeasurable in the synovial tissues of sJIA patients, while high levels of these chemokines can be found in the synovial tissues of patients with oligoarticular or polyarticular JIA (Sikora K.A. , et al., The limited role of interferon-gamma in systemic juvenile idiopathic arthritis cannot be explained by cellular hyporesponsiveness Arthritis Rheum, 2012. 64(11): p. 3799-808). Recent results in mice have demonstrated that immune stimulation of IFNy knockout mice with complete Freund's adjuvant induces the development of a systemic inflammatory syndrome that includes the characteristic features of sJIA, also suggesting a limited role for IFNy 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): p. 1340-51.

In sharp contrast, this study demonstrated markedly elevated levels of IFNy and IFNy-related chemokines in patients with active MAS at the time of sample collection compared to levels in patients with active sJIA without MAS at the time of sample collection. This has also been confirmed in the case of individual patients who were serially sampled both during active MAS and during active sJIA without MAS. Incidentally, in this study, patients from whom samples were collected during MAS did not show a significant increase in IL-6 or IL-1e levels, nor any association with laboratory MAS values, indicating that these cytokines , although they are seriously involved in the pathogenic mechanism of sJIA (De Benedetti F., et al., Randomized trial of tocilizumab in systemic juvenile idiopathic arthritis. N Engl J Med, 2012. 367(25): p. 2385-95; Ruperto N. , et al., Two randomized trials of canakinumab in systemic juvenile idiopathic arthritis, N Engl J Med, 2012. 367(25): p. 2396-406), may not play a key role in maintaining MAS. These data on elevated levels of IFNy and IFNy-associated chemokines are consistent with some previous results. Shimizu et al reported that levels of neopterin, a catabolite of guanosine tri

- 53 042261 phosphate synthesized by human macrophages upon IFNy stimulation was higher in patients with MAS during sJIA than in patients with active sJIA without MAS (Shimizu M., et al., Distinct cytokine profiles of systemic-onset juvenile idiopathic arthritis- associated macrophage activation syndrome with particular emphasis on the role of interleukin-18 in its pathogenesis Rheumatology (Oxford), 2010. 49(9): pp. 1645-53. More recently, Put et al. reported elevated levels of IFNy and CXCL10 in 5 patients with both primary and secondary HLH, 3 of whom had MAS during sJIA (Put K., et al., Cytokines in systemic juvenile idiopathic arthritis and haemophagocytic lymphohistiocytosis: tipping the balance between interleukin-18 and interferon-gamma Rheumatology (Oxford), 2015. According to these results, 5 patients with active sJIA without MAS had markedly lower levels of IFNy and CXCL10 at the time of sample collection (Put et al., Rheumatology 2015).

Interestingly, the present study found that not only were levels of IFNy and IFNy-related chemokines markedly elevated, but also that these levels, in particular CXCL9 levels, clearly correlated with laboratory MAS values, indicating an association with the severity of the disease. As further evidence of an association with disease severity, this study found markedly elevated levels of IFNy, as well as levels of CXCL9 and CXCL10, in one patient suffering from severe disease with multiple organ failure and involvement of the central nervous system, with generalized seizures requiring prolonged stay in intensive care unit.

In MAS patients, of the three IFNy-induced chemokines, CXCL9 was found to show the strongest correlation with IFNy levels. This observation is consistent with the established fact that CXCL9 production appears to be specifically and exclusively induced by IFNy, in contrast to CXCL10 and CXCL11 production, which can also be induced by type I interferons (Groom, J.R. and A.D. Luster, CXCR3 ligands: redundant, collaborative and antagonistic functions Immunol Cell Biol, 2011 89(2): pp. 207-15). This suggests that CXCL9 levels may serve as a sensitive and specific biomarker for MAS activity. Indeed, in the present study, using a mouse model of MAS that mimics MAS initiation by an infectious stimulus in the presence of high levels of IL-6 (Strippoli et al., Arthritis Rheum 2012), it was also found that liver and spleen CXCL9 expression levels are significantly correlated with circulating ferritin levels. For CXCL10 expression, this correlation only occurred for liver levels and not for spleen levels. This is also supported by the results obtained in patients with MAS, in which CXCL9 levels clearly correlated with all laboratory parameters of MAS. Taken together, these human and mouse results demonstrate that CXCL9 levels strongly correlate with MAS features and IFNy production, further supporting the hypothesis that IFNy overproduction plays a major pathogenic role in MAS. These observations are also consistent with the immunohistochemical data obtained by Put et al in studying serial lymph node biopsies from the same patient with sJIA, which were obtained during active sJIA without MAS, as well as during MAS. These authors report that CXCL10 and indolamine 2,3-dioxygenase, both IFNy-inducible proteins, were detected at high levels by immunohistochemistry in tissue collected during MAS but not in tissue collected during active sJIA without MAS ( Put et al., Rheumatology 2015).

These results for MAS and for sec-HLH, together with the results available in the literature in patients with n-HLH, support the hypothesis that increased levels of IFNy and IFNy-related chemokines, in particular CXCL9, are a hallmark of HLH regardless of underlying cause. In this regard, it is interesting to note that high levels of CXCL9 have been found in a patient with recurrent MAS induced by a gain-of-function mutation in the NLRC4 gene (Canna S.W., et al., An activating NLRC4 inflammasome mutation causes autoinflammation with recurrent macrophage activation syndrome. Nat Genet, 2014. 46(10): pp. 1140-6), this indicated that even in the case of HLH induced by inflammasome dysregulation alone, there may be overproduction of IFNy.

Data obtained in animal models of n-HLH in both perforin knockout and Rab27a knockout mice clearly demonstrate the pathogenic role of IFNy. Similarly, recent data in a TLR9-induced model of HLH, a model of HLH secondary to infection, also demonstrated an important role for increased IFNy production (Behrens, E.M., et al., Repeated TLR9 stimulation results in macrophage activation syndrome-like disease in mice. J Clin Invest, 2011. 121(6): pp. 2264-77 and Bautois et al., forthcoming). Recent studies using the mouse model of MAS described above have shown that treatment with an anti-IFNy antibody results in improved survival rates and a return to normal of abnormal clinical and laboratory values characteristic of MAS (Prencipe et al., forthcoming). Taken together, the results of this study and the animal data support the deprecation of IFNy as a therapeutic approach in MAS.

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Example 7 Evaluation of the safety, tolerability, pharmacokinetics and efficacy of repeated intravenous administration of anti-IFNy monoclonal antibodies against interferon-gamma in pediatric patients with primary hemophagocytic lymphohistiocytosis (HLH)

The studies described herein were designed to determine the safety and tolerability profile of multiple intravenous (IV) administration of an anti-IFNy antibody, referred to herein as NI-0501; to determine the efficacy and benefit/risk ratio of NI-0501 in patients with HLH; to describe the pharmacokinetic (PK) profile of NI-0501 in patients with HLH; to determine an appropriate dosing regimen for NI-0501 in HLH; as well as to assess the immunogenicity of NI-0501.

Preclinical studies:

Previous studies have shown that NI-0501 shows similar binding affinity and blocking activity for IFNy from non-human primate species, including rhesus and cynomolgus monkeys, but not dog, cat, pig, rabbit, rat, or mouse IFNy. Toxicity and safety studies in cynomolgus monkeys showed no off-target toxicity when NI-0501 was administered; weekly administration of NI-0501 was well tolerated; there was no need for prophylactic antibiotics and no abnormal histopathological or behavioral features were observed during these preliminary studies.

Because of the ability of NI-0501 to bind free and IFNyR1-bound IFNy, studies were conducted to investigate the potential ability of NI-0501 to mediate ADCC and CDC activities in the presence of a target. No ADCC activity was demonstrated and no induction of CDC activity was noted.

Phase I clinical trial:

Randomized, double-blind, placebo-controlled, phase 1, single incremental dose study in 20 healthy adult volunteers to investigate the safety, tolerability, and pharmacokinetic profiles of single intravenous (IV) administration of NI0501. During this study, 6 individuals received a placebo, and 3, 3, 4, and 4 individuals (a total of 14 individuals) received doses of 0.01, 0.1, 1, and 3 mg/kg of NI-0501, respectively.

The NI-0501 PK assay showed the expected profile for IgG1 with a long half-life (approximately 22 days), slow elimination (<0.007 L/h) and low volume of distribution (<6 L on average).

A total of 41 adverse events (AEs) were observed after the start of drug infusion in 14 of 20 individuals (70%), 10 of which were reported by 4 individuals receiving placebo. Thirty-six (87.8%) AEs were of mild intensity and 5 (12.2%) were of moderate intensity. No severe or life-threatening AEs were noted. Twenty-three AEs (56.1%) in 10 out of 14 individuals who experienced AEs were considered drug-related (at least with a reasonable probability). Most of the AEs were single events and there was no association with increasing doses of NI-0501. All NI-0501 infusions were uncomplicated.

In summary, the NI-0501 infusion was well tolerated and the effects observed during the 8 weeks of monitoring after drug infusion did not reveal any major or unexpected safety issues due to off-target effects or drug immunogenicity.

Phase 2/3 clinical trial.

Materials and methods:

This study includes patients with primary HLH. The study is divided into three parts: screening, treatment and follow-up. The general scheme is shown in Fig. 12.

Eligible patients for this study include patients who have not previously received HLH treatment (herein also referred to as first-line patients) or patients who have previously received conventional therapy for HLH (herein also referred to as second-line patients) but have a satisfactory response to therapy. was not achieved, for example, according to the opinion of the attending physician, or there were signs of intolerance to the therapy. Patients treated with NI-0501 after failure or intolerance to conventional HLH therapy represent a key study group to demonstrate the efficacy of NI-0501 as a second-line therapy for primary HLH. Previously untreated patients were included in the study to collect data on efficacy and safety in the setting of first-line therapy.

The following patients are excluded from the study: patients who have been diagnosed with secondary HLH due to confirmed rheumatic or neoplastic disease; patients previously treated with any T-cell depleting agent (such as, for example, anti-thymocyte globulin (ATG), anti-CD52 therapeutic agents) within the previous 2 weeks prior to screening, or treated with any other biologic drug within the 5 times its defined half-life (with the exception of rituxim

- 55 042261 ba in the case of a documented infection of B-cells with the EBV virus); patients with active infections caused by mycobacteria, Histoplasma Capsulation, Shigella, Salmonella, Campylobacter and Leishmania; patients with signs of past tuberculosis or latent tuberculosis; patients with positive serological test results for antibodies to HIV, antibodies to the surface antigen of the hepatitis B virus or hepatitis C; patients with a malignant neoplasm; patients with other concomitant disease or congenital malformation with a serious effect on the function of the cardiovascular system, lungs, liver or kidneys; patients with a history of hypersensitivity or allergy to any component used in the study; Patients marked with a live or attenuated live (including BCG) vaccine within the preceding 12 weeks prior to screening; and/or pregnant or lactating female patients.

The study described herein uses anti-interferon-gamma antibody NI-0501, a fully human IgG1 monoclonal antibody (mAb) directed against human IFNy. NI-0501 is provided as a sterile infusion concentrate (per ml) as shown in Table 1 below. 4.

Table 4. Preparation NI-0501

Ingredient Quantity (per ml) NI-0501 5 mg L-histidine 1.55 mg L-histidine monohydrochloride, monohydrate 3.14 mg Sodium chloride (NaCl) 7.31 mg Polysorbate 80 0.05 mg pH 6.0±0.2

In this study, NI-0501 was administered by IV infusion over 1 hour at an initial dose of 1 mg/kg. This dose is expected to inhibit for 3 days at least 99% of the effect of IFNy in patients with baseline IFNy concentrations less than or equal to 3400 pg/mL. Infusions are performed every 3 days until day 15 of the study (SD15) (infusion #6), and then twice a week. The dose of NI-0501 is increased to 3 mg/kg, if possible, according to predetermined criteria, guided by clinical and laboratory results in each patient (as described in Table 5, below), at any time during the study. After a minimum of two infusions at a dose of 3 mg/kg, if after re-evaluation it is found that the clinical and laboratory criteria that qualify the patient for a dose of 3 mg/kg of NI-0501 remain valid, the dose of NI-0501 may be increased to 6 mg /kg for up to four infusions, with regular monitoring of clinical and laboratory indicators of HLH. Depending on changes in these parameters, the dose of NI-0501 may either be i) reduced again to 3 mg/kg or ii) maintained at 6 mg/kg for additional IV infusions (or increased to more than 6 mg/kg ), if the PK and PD indicators indicate excessively high production of IFNy and, consequently, rapid elimination of NI-0501. The dose can be increased at any time during the study if the indicators meet the clinical and laboratory criteria set out in this document.

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Table 5. Clinical and laboratory criteria for the decision to increase the dose

Study Day (SD) Dose NI-0501 In SD0 Initial dose 1 mg/kg In SD3 Increase to 3 mg/kg Required criteria: - if fever persists or recurs (if present at baseline) or - if the clinical condition worsens significantly From SD6 onwards ³ Increase to 3 mg/kg ^b Required criteria: - if there is no satisfactory improvement in the clinical condition and - at least 1 of the following occurs: Platelet count (x10 ³ /µl) if ref. quantity <50 A no improvement to >50 ref. quantity 50-100 improvement less than 30% ref. amount >100 A any reduction to <100 AKN (amount/µl) if ref. quantity <500 A no improvement to >500 ref. quantity 500-1000 A any reduction to <500 ref. amount >1000 A any reduction to <1000 Ferritin (ng/µl) if ref. levels >3000 -> no improvement (<20% reduction) ref. levels <3000 A any change up to >3000 Splenomegaly A worsening (by clinical examination or ultrasound) Coagulopathy (should also consider D-dimer, From SD9 or SD12 onwards ³ Increase to 3 mg/kg ^a and fibrinogen) D-dimer if abnormal at baseline A no improvement fibrinogen if ref. levels <100A no improvement ref. levels >100A any reduction to <100 Required criteria: - if, after a minimum of two infusions at a dose of 3 mg/kg, a re-evaluation is carried out and it is found that the above criteria are still met

^a Dose of NI-0501 should be increased from 1 to 3 mg/kg if eligibility occurs after SD6.

^b If the dose of NI-0501 has already been increased on SD3, at least two infusions of 3 mg/kg must be completed before eligibility is reassessed.

^c Depending on whether dose increase to 3 mg/kg occurred in SD3 or SD6.

^d For a maximum of four infusions.

Abbreviations: ref.=original(s);

ANC=absolute neutrophil count; Ultrasound = ultrasound.

In these studies, NI-0501 is administered for 8 weeks and the treatment period will be divided into 2

- 57042261 unit periods: treatment periods 1 and 2 as shown in FIG. 12.

After NI-0501 has been administered for 8 weeks, a conditioning process in preparation for hematopoietic stem cell transplantation (HSCT) can begin. The expected duration of treatment may be reduced, albeit to at least 4 weeks, if the patient's condition and the availability of a donor allow transplantation. In the event that a suitable donor has not been found by week 8, or if transplantation has to be delayed for reasons unrelated to NI0501 administration, NI-0501 treatment may be continued in the context of a long-term follow-up study, provided a favorable benefit/risk balance for the patient is established. .

In these studies, NI-0501 is administered in combination with dexamethasone, the dose of which may be reduced depending on the patient's condition. For previously untreated patients, NI-0501 will be administered in conjunction with dexamethasone at a dose of 10 mg/m ² . In a patient receiving NI-0501 as second-line therapy for HLH, dexamethasone should be administered at a dose of at least 5 mg/m ² or the dose given prior to screening if higher. Patients should receive dexamethasone starting at SD-1.

Doses of dexamethasone may be reduced depending on the condition of patients at the discretion of the attending physician. The dose reduction regimen may be chosen by the attending physician, provided that the dose of dexamethasone at each stage is reduced by no more than half, and the frequency of changes does not exceed once a week.

If the disease worsens after a dose reduction of dexamethasone, the dose of dexamethasone can be increased and maintained until a satisfactory response is achieved in the opinion of the attending physician.

As recommended in the HLH treatment guidelines, patients receive prophylactic treatment for Pneumocystis jiroveci, fungal infection, and Herpes zoster from the day prior to initiation of NI-0501 treatment until the end of the study. Patients receive prophylactic treatment from the day prior to initiation of NI-0501 (ie, SD-1) treatment until the end of the study. For example, to prevent Pneumocystis jiroveci infection, patients may receive, for example, 750 mg/m ² /day of sulfamethoxazole with 150 mg/m ² /day of trimethoprim, taken orally in equally divided doses twice daily for 3 consecutive days per week. To prevent fungal infection, patients may receive, for example, 12 mg/kg of fluconazole per day with a maximum daily dose of 400 mg. To prevent HZ infection, patients may receive, for example, 200 mg of acyclovir four times a day for children over two years of age, for children under two years of age 100 mg four times a day. These therapeutic agents will be administered orally when possible, otherwise intravenously.

Patients may also receive any of a variety of concomitant therapeutic agents, such as, for example, cyclosporine A, intrathecal methotrexate, and glucocorticosteroids, among others. The administration of cyclosporine A (CsA) can be continued if it is administered to the patient prior to the screening procedure. The introduction of CsA can be stopped at any time. CsA administration should not be initiated de novo during the study after NI-0501 administration has been initiated.

If the patient is receiving intrathecal methotrexate and glucocorticoids at the time of initiation of NI-0501 treatment, this treatment will be continued as needed. If symptoms of CNS dysfunction occur prior to initiation of NI-0501 treatment, therapy with intrathecal methotrexate and glucocorticoids should begin prior to the first administration of NI-0501.

In / in the introduction of immunoglobulins (in / in IG) is allowed only as replacement therapy in case of confirmed immunoglobulin deficiency. For example, in cases of confirmed immunoglobulin deficiency warranting replacement therapy, IV IG may be administered at a dose of 0.5 g/kg every 4 weeks, or more frequently, to maintain adequate IgG levels. Any infusion within the 4 weeks prior to screening, as well as any infusion during NI-0501 treatment, is acceptable.

Treatment with analgesics, transfusion of blood products, infusions of electrolytes and glucose, antibiotics, antifungal and antiviral drugs, and general strengthening drugs are acceptable. The use of additional anti-HLH drugs may be allowed in the event of unstable or limited attenuation of HLH after reaching the maximum dose level of NI-0501. As used herein, the term unstable HLH attenuation refers to patients who do not maintain at least 50% improvement from baseline 3 HLH scores (see Table 6, below). At least two consecutive measurements should confirm the absence of HLH attenuation. As used herein, limited HLH attenuation means less than a 50% change from baseline in at least 3 clinical and laboratory measures of HLH. Etoposide should be administered as an adjunct anti-HLH drug unless the patient's medical history indicates a lack of response to the drug or intolerance to the drug.

The following therapeutic agents should not be used concomitantly with NI-0501 administration: etoposide, T-cell depleting agents, or any other biologic agents are generally not allowed, except for the following: G-CSF in case of prolonged neutropenia

- 58 042261 Research Institute; rituximab for documented EBV B cell infection and additional anti-HLH therapeutics for unstable or limited attenuation of HLH (as defined herein) using the highest dose level of NI-0501. Etoposide should be administered unless the patient's medical history indicates a lack of response to the drug or intolerance to the drug. Vaccination with a live or attenuated (including BCG) vaccine should be avoided throughout the study, including the 4 week follow-up period. In the event that NI-0501 concentrations remain at therapeutic levels after completion of the study, the vaccination-free period should be extended until the concentration of NI-0501 is no longer measurable.

Changes in clinical signs (fever, splenomegaly, CNS symptoms) and laboratory parameters (levels of SHS, fibrinogen, ferritin, SCD25) characterizing the disease are used to assess the achievement of response and time to response. The main efficacy endpoint includes the overall response rate, that is, the achievement of either complete or partial response or attenuation of HLH at the end of the treatment period (EoT), as defined in Table 1. 6 below. Secondary efficacy measures include time to response at any point in time throughout the study; persistence of response, i.e., maintenance of the achieved response at any point in time during the study before and after EoT (including data obtained in any long-term follow-up study); the number of patients for whom it became possible to reduce the dose of glucocorticoids by 50% or more of the dose at the beginning of the study; the number of patients who were able to switch to HSCT if they were instructed to do so; survival at week 8 (or EoT) and end of study; serum concentration of NI-0501 to determine the pharmacokinetic (PK) profile of NI-0501; determination of pharmacodynamic (PD) effects, including levels of circulating total IFNy and markers of its neutralization, namely, CXCL9 and CXCL10; and determination of other biomarkers, for example, sCD25, IL-10.

Table 6. Answer definition

Overall response rate Full answer The answer is considered complete if: - no fever = body temperature <37.5°C - the spleen is of normal size, as determined by 3D abdominal ultrasound - no cytopenia = absolute neutrophil count >1.0 x 10 ⁹ /l and platelet count > 100 x 10 ⁹ /L [absence of G-CSF and maintenance transfusion must be documented at least 4 days prior to decision of no cytopenia] - no hyperferritinemia = serum level <2000 µg/L - no evidence of coagulopathy, i.e., normal levels of D- dimer and/or normal levels (>150 mg/dl) of fibrinogen - no neurological and CSF abnormalities associated with HLH - no sustained worsening of sCD25 scores (as evidenced by at least two consecutive measurements, with scores 52-fold higher than baseline) Partial A response is considered partial if:

- 59 042261

answer - at least 3 of the abnormal clinical and laboratory findings characteristic of HLH (including CNS abnormalities) meet the above criteria for a "complete response". In the case of "reactivated patients" who were included in the study with 3 abnormal, characteristic of HLH indicators, at least 2 indicators must meet the specified criteria - there is no progression of other pathological signs of HLH (such as, for example, jaundice, liver size, edema, clinical changes in the CNS) Weakening HLH - reduction (>50% change from baseline) in at least 3 abnormal clinical and laboratory parameters characteristic of HLH (including CNS abnormalities). In the case of "reactivated patients" who were included in the study with 2 abnormal HLH-specific measures, a change from baseline greater than 50% for both measures indicates attenuation of HLH. Limited Attenuation/No Attenuation/No Response - less than 50% change from baseline 3 or more of the above abnormal, HLH-specific clinical and laboratory parameters [in the case of "reactivated patients” who were enrolled in the study with 2 abnormal HLH-specific measures, a change from baseline of less than 50% would be sufficient for both measures to indicate limited disease attenuation] and - no apparent weakening of other pathological aspects of the disease

Reactivation

- Deterioration of two or more clinical and laboratory values of HLH with the following specifications: 1. Quantitative laboratory values* must become abnormal and deteriorate by more than 30% from the previous measurement on two consecutive assessments performed at least 1 day and maximum 1 week apart 2. Deterioration of clinical indicators must be confirmed by appropriate observations of deterioration over three consecutive days - the development of new, or recurrence, CNS symptoms is considered a monocriterion for reactivation.

* The following laboratory parameters are particularly taken into account when determining reactivation: - platelets - neutrophils - fibrinogen - ferritin

- soluble CD25 (sCD25; i.e., soluble IL-2 receptor). Evaluation of NK function, erythrocyte/hemoglobin and triglyceride levels should not be considered in determining reactivation.

Safety indicators that should be collected and assessed include the incidence, severity, causality and outcome of adverse events (AEs) (serious and non-serious), with particular attention given to infections; changes in laboratory parameters, such as

- 60 042261 complete blood cell count (CBC), especially red cells (hemoglobin), neutrophils and platelets, liver tests, kidney function tests and blood clotting; the number of patients withdrawn from the study for safety reasons; as well as other indicators such as the level (if any) of circulating antibodies against NI-0501 for immunogenicity determination (ADA).

The primary outcome measure (overall response rate) was assessed using a one-tailed binomial exact test with a significance level of 0.025. Time to response, persistence of response, and survival time are estimated from Kaplan-Meier curves with calculated medians, if possible. 95% confidence intervals are calculated for the median for each of these criteria. Additional criteria based on binary outcomes, including the number of patients for whom the dose of glucocorticoids was reduced by 50% or more and the number of patients who were able to switch to HSCT, will be proportioned and the corresponding 95% confidence intervals will be calculated. Statistical significance in terms of p-values will only be determined for the primary performance measure. All other criteria are considered to support the primary performance criterion and, therefore, no formal hierarchy of criteria is stated.

Administration of NI-0501 to patients results in a rapid resolution of fever within a few hours of the first infusion of NI-0501. In FIG. 13A and 13B show the effect of NI0501 infusion on body temperature in two patients with a body temperature >37.5°C at the start of NI-0501 treatment. In FIG. 14 is a series of graphs and a table showing the effect of NI0501 administration on neutrophil counts in patients. In FIG. 15 is a series of graphs and a table showing the effect of NI-0501 administration on platelet counts in patients. In FIG. 16 is a series of graphs and a table showing the effect of NI-0501 administration on serum ferritin levels in patients. In FIG. 17 is a series of graphs and a table showing the effect of NI0501 administration on glucocorticoid dose reduction in patients. In FIG. 18 is a graph showing that NI-0510 administration maintains IFNy neutralization until the time of HSCT. The change in HLH values in response to the introduction of NI-0501 also persists until transplantation. Patients were also assessed for any signs of CNS involvement following NI-0501 administration. Summary baseline measures of CNS involvement and status at the end of the study (EOT) are shown in Table 1 below. 10.

Table 10. Response to NI-0501 administration - CNS involvement

Study Start (SDO) EOT Patient No. 6 Dullness of sensitivity; hemiparesis Breaking the pace of development Eliminated Fully restored Increased content of protein and neopterin in CSF; Pleocytosis Eliminated Abnormal activation on MRI Adjusted Patient No. 3 Increased protein content in CSF; Pleocytosis Eliminated Patient No. 15 Loss of ability to walk restored Increased content of protein and neopterin in CSF; Pleocytosis Eliminated Patient No. 18* Pleocytosis; Increased protein content in CSF Adjusted Axial hypotension Adjusted Patient No. 2O ⁵ Increased content of protein and neopterin in CSF; Pleocytosis Initial Correction Paresis of the 6th nerve; ankle clonus Initial Correction Patient No. 4 ^L Increased protein content in CSF; Pleocytosis Can't be assessed Abnormal infiltrates on MRI Can't be assessed

Note ^: Patients received I/T therapy, except for patient #4, who was not treated with regular medical LP *Treatment ongoing

Of the ten patients who underwent hematopoietic stem cell transplantation (HSCT), all were successfully transplanted; One patient required additional CD34 stem cell transplantation due to mixed chimerism at D+145 after HSCT. Secondary graft rejection occurred in 1 patient, followed by HLH reactivation. This patient died at D+68 after HSCT due to acute respiratory failure and bacterial infections. Another patient died at D+47 after HSCT (septic shock in the context of severe GvHD). A moderate GvHD reaction occurred in the other 3 patients and was/is being eliminated.

- 61 042261

Neutralizing serum concentrations of NI-0501 during HSCT were found in 8 of 10 transplant patients, as evidenced by levels of CXCL9 (a chemokine induced exclusively by IFNy) below the limit of quantitation. Thus, these data indicate that NI-0501 avoids the short- or long-term toxicity seen with etoposide-based regimens. This means a reduced risk of complications associated with allo-HSCT.

These results demonstrate that treatment with NI-0501 results in attenuation and/or may result in resolution of relevant clinical and laboratory abnormalities characteristic of HLH, including signs and symptoms of CNS dysfunction. The response to NI-0501 is independent of the presence and type of disease-causing mutations and/or the presence and type of infectious trigger. NI-0501 was well tolerated. No safety concerns have arisen to date (eg, no myelotoxicity, no general immunosuppression). No pathogenic infections were found that are known to be stimulated by neutralization of IFNy. IFNy neutralization with the NI-0501 could be an innovative and targeted approach to HLH control.

Example 8 Safety, tolerability, pharmacokinetics, and efficacy of short-term intravenous administration of NI-0501, an anti-interferon-gamma (anti-IFNy) monoclonal antibody, in patients with systemic juvenile idiopathic arthritis (sJIA) associated with macrophage activation syndrome/secondary HLH (MAS/ in-HLH)

The study described herein is designed to demonstrate the efficacy and safety of NI-0501 for the treatment of MAS/b-HLH in patients with sJIA and is divided into two parts (i) a pilot study to evaluate the PK profile of NI-0501 and dosing strategy, and to preliminarily assess the benefit/risk ratio for NI-0501 in this patient population; and (ii) a pivotal study to demonstrate the efficacy and safety of NI-0501 (study will continue after approval of the dosing regimen and confirmation of a positive benefit/risk ratio for NI0501). In FIG. 19 is a schematic representation of the design of this study.

The main objectives of the pilot study are: (i) to determine an appropriate NI-0501 therapeutic dose regimen for patients suffering from sJIA with MAS/b-HLH; (ii) assessing the benefit/risk ratio for the use of NI-0501 in patients suffering from sJIA with MAS/b-HLH; and (iii) determination of the pharmacokinetic (PK) profile of NI-0501 in patients suffering from sJIA with MAS/b-HLH. The main objectives of the main study are: (i) to determine the efficacy of NI-0501 in patients suffering from sJIA with MAS/b-HLH; (ii) evaluate the safety and tolerability profile of short-term intravenous (IV) administration of NI-0501 in patients suffering from sJIA with MAS/b-HLH; (iii) confirmation of a positive benefit/risk ratio for the use of NI-0501 in patients suffering from sJIA with MAS/bHLH; (iv) performing a trial evaluation of the chemokines CXCL9 and CXCL10 as diagnostic biomarkers of MAS/b-HLH and as predictors of response to NI-0501 treatment; and (v) assessing the immunogenicity of NI0501 in patients suffering from sJIA with MAS/b-HLH.

The study population includes sJIA patients with MAS/b-HLH who have shown an inadequate response to high-dose glucocorticoid treatment. The inclusion criteria for the study are as follows: (i) gender: male and female; (ii) age: <16 years at the time of diagnosis of sJIA; (iii) the diagnosis of active MAS/b-HLH is confirmed by the attending rheumatologist, with at least 2 of the following laboratory and clinical criteria: (a) laboratory criteria: platelet count <262 x 109/l, white blood cell count (WBC) <4, 0x109/L, AST levels >59 U/L and/or fibrinogen levels <2.5 g/L; (b) clinical criteria: hepatomegaly, signs of bleeding and/or CNS dysfunction; (iv) a patient who demonstrates an inadequate response to treatment with high intravenous doses of glucocorticoids for at least 3 days (including, but not limited to, 30 mg/kg mPDN pulsed administration for 3 consecutive days), in accordance with the local standard of medical help; (v) high intravenous dose of glucocorticoids should not be less than 2 mg/kg/day mPDN equivalent in 2 divided daily doses, up to 60 mg/day. In the event of a rapid deterioration in the patient's health and / or laboratory parameters, he can be included in the study within less than 3 days from the start of a high intravenous dose of glucocorticoids; (vi) the consent of the patient (or the consent of his legal representative(s)) and (vii) taking contraceptive measures if the patient has reached puberty.

The exclusion criteria are as follows: (i) a diagnosis of suspected or confirmed primary HLH or HLH due to neoplastic disease; (ii) patients treated with: anakinra, tocilizumab, canakinumab, TNF inhibitors, rituximab, or any other biological drug within 5 times its defined half-life; (iii) active infections caused by mycobacteria (typical and atypical), Histoplasma capsulation, Shigella, Salmonella, Campylobacter and Leishmania; (iv) signs of latent tuberculosis; (v) positive serological test results for antibodies to HIV; (vi) the presence of malignant neoplasms; (vii) patients with other comorbid disease or malformation are seriously affected

- 62 042261 affecting cardiovascular, pulmonary, CNS, hepatic, or renal function that, in the investigator's opinion, could seriously affect the likelihood of response to treatment and/or the safety assessment of NI0501; (viii) a history of hypersensitivity or allergy to any component used in the study; (ix) record of vaccination with BCG vaccine within the preceding 12 weeks prior to screening; (x) record of vaccination with other live or attenuated live vaccines within the previous 6 weeks prior to screening and/or (xi) pregnant or breastfeeding female patients.

Dosing regimen, frequency of administration and duration of treatment

In this study, NI-0501 is used in the formulation described in Example 7. In Part 1 of the study, NI-0501 was administered at an initial dose of 6 mg/kg by infusion over 1 hour on SD0 day. Administration of NI-0501 is continued at 3 mg/kg every 3 days for 4 weeks (ie, until day SD27). The timing of NI0501 administration may be shortened after a complete clinical response (ie, MAS remission) has been achieved. After 4 weeks, NI-0501 may be continued for an additional period of up to 4 weeks (i.e., until SD56 day) as maintenance therapy, as needed, until MAS remission is achieved, with the option of dose reduction to 1 mg/kg and increasing the interval between infusions to weekly administration. If the PK profile shows unexpected target-mediated drug distribution (TMDD) (which is a signal of extremely strong IFNy production), the dose of NI-0501 can be increased up to 10 mg/kg, guided by the judgment of the attending physician and PK parameters. This dose increase can only be approved after a careful assessment of the benefit/risk ratio for this particular patient.

In Part 2, after confirming that the proposed dosing regimen is appropriate and demonstrating a positive benefit/risk ratio for the use of NI-0501, the study will proceed. Small modifications to the dosing regimen may be used, if necessary, based on the results obtained in Part 1.

Background therapy and concomitant drugs

NI-0501 is administered in conjunction with methylprednisolone (mPDN) at a dose of at least 2 mg/kg, equivalent to a dose of up to 60 mg/day (in patients weighing 30 kg or more), which may be tapered over the course of treatment depending on the patient's condition. Patients receive prophylactic treatment for Herpes zoster infection, starting preferably the day before (and in any case before) administration of NI-0501 until no more NI-0501 is detected in serum. Cyclosporine A (CsA) administration may be continued if started at least 3 days before the start of NI-0501 administration. Dose adjustment of CsA is acceptable to maintain therapeutic levels. CsA administration may be discontinued at any time during the course of the study at the discretion of the investigator. CsA administration must not be started de novo after the start of NI-0501 administration. If the patient is receiving intrathecal methotrexate and glucocorticoids at the time of initiation of NI-0501, this treatment may be continued as needed. Vaccination with a live or attenuated (including BCG) vaccine should be avoided for the duration of the study and, in any case, until no more NI-0501 is detected in serum. Treatment with analgesics, transfusion of blood products, infusions of electrolytes and glucose, antibiotics, antifungal and antiviral drugs, and general tonic drugs are acceptable.

Sample size:

Part 1 of the study will include at least 5 evaluable patients. Part 2 of the ongoing study will have at least 10 evaluable patients to reach a total of 15 evaluable patients. The sample size of 15 patients is not formally validated given the rare nature of the disease and the absence of any approved treatment. However, assuming that at least 50% of patients respond inadequately to systemic glucocorticoids alone, i.e. 50% of patients receiving glucocorticoids have MAS remission by week 8 post-treatment, this study would have a 70% power to detect improvement in condition from 50% to 77% using a one-sided significance level of 5%.

Duration of the study and determination of the end of the study:

The duration of the study will be 8 weeks for each patient (plus a screening period of up to 1 week). The end of the study is considered the day of the last visit of the last patient. All patients who receive at least one dose of NI-0501 will be offered participation in the NI-0501-05 study for a long-term follow-up period.

Study performance criteria

Part 1 (pilot) study evaluates the following indicators to confirm the dosing regimen in this patient population:

(i) benefit/risk ratio for NI-0501; (ii) PK profile NI-0501; (iii) levels of chemokines known to be induced by IFNy (eg, CXCL9, CXCL10, CXCL11); (iv) changes in the characteristic features of MAS: cytopenias, liver dysfunction and coagulopathy at 2, 4, 6 and 8 weeks after onset

- 63 042261 la administration of NI-0501 and (v) doses and duration of administration of NI-0501. In the Part 2 (core) study, the drug efficacy criteria in the study are as follows: (a) primary efficacy criterion: number of patients in MAS remission by week 8 after initiation of NI0501 administration; and (b) secondary efficacy measures: time to MAS remission; time to initial response as judged by the investigator; the number of patients for whom, at any time during the study, the dose of glucocorticoids could be gradually reduced to the same (or lower) dose that they were administered before the onset of MAS; time to the possibility of reducing the dose of glucocorticoids; survival at the end of the study and the number of patients withdrawn from the study due to lack of efficacy. In Part 2 (core) of the study, the safety criteria are as follows: (a) incidence, severity, causation, and outcome of AEs (serious and non-serious), with particular attention given to infections; changes in laboratory parameters, in particular SVS (primarily hemoglobin, neutrophils and platelets), liver function test results (LFT) and blood coagulation parameters; the number of patients withdrawn from the study for safety reasons; and levels (if any) of circulating anti-NI-0501 antibodies for immunogenicity determination (ADA).

Pharmacokinetics and pharmacodynamics are evaluated based on the PK profile of NI-0501; levels of circulating free IFNy before drug administration, as well as total IFNy (free and NI-0501 bound IFNy) after the start of NI-0501 administration; levels of chemokines known to be induced by IFNy (eg, CXCL9, CXCL10, CXCL11); correlations between chemokine levels (CXCL9, CXCL10) and levels of free NI-0501, free IFNy (before drug administration), and total IFNy; correlations between chemokine levels and total IFNy, as well as laboratory measures of MAS severity, eg ferritin levels, platelet counts, LFT results (exploratory analysis); and levels of other potential disease biomarkers (eg, sCD25, IL-10, IL-6, IL-18, TNFa, neopterin).

Other embodiments

Although the present document provides a detailed description of the invention, the foregoing description is intended to illustrate, but not to limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the appended claims.

Claims (23)

CLAIM 1. A method of treating symptoms of macrophage activation syndrome (MAS) or secondary hemophagocytic lymphohistiocytosis (HLH) in an individual, comprising administering an antibody that binds to interferon gamma (IFNy), wherein the antibody is administered i) at a first dose of 6.0 mg/kg based on the individual's body weight, and ii) at a second dose of 3.0 to 10 mg/kg based on the individual's body weight, and wherein the antibody comprises heavy chain variable region complementarity determining region 1 (VH CDR1 ) containing the amino acid sequence SYAMS (SEQ ID NO: 1); heavy chain variable region complementarity determining region 2 (VH CDR2) comprising the amino acid sequence AISGSGGSTYYADSVKG (SEQ ID NO: 2); heavy chain variable region complementarity determining region 3 (VH CDR3) comprising the amino acid sequence DGSSGWYVPHWFDP (SEQ ID NO: 3); light chain variable region complementarity determining region 1 (VL CDR1) containing the amino acid sequence TRSSGSIASNYVQ (SEQ ID NO: 4); light chain variable complementarity determining region 2 (VL CDR2) comprising the amino acid sequence EDNQRPS (SEQ ID NO: 5); and light chain variable region complementarity determining region 3 (VL CDR3) containing the amino acid sequence QSYDGSNRWM (SEQ ID NO: 6). 2. The method of claim 1, wherein the antibody is administered ii) at a second dose of 3.0 mg/kg based on the individual's body weight. 3. The method of claim 1, wherein the antibody comprises the heavy chain variable region amino acid sequence of SEQ ID NO: 47 and the light chain variable region amino acid sequence of SEQ ID NO: 48. 4. The method of claim 1, wherein the first dose of antibody is administered over 1 hour or more. 5. The method of claim 1, wherein the first dose of antibody is administered over a period of 1 to 2 hours. 6. The method of claim 1 wherein the second dose is administered during the first treatment period, wherein the second dose is administered every three days after the first dose during the first administration period. 7. The method of claim 6 wherein the first treatment period is about two weeks. 8. The method of claim 6 wherein the second dose is administered during the second treatment period following the completion of the first treatment period. 9. The method of claim 8, wherein the second dose is administered twice a week during the second administration period. - 64 042261 10. The method of claim 8, wherein the second treatment period is about two weeks. 11. The method of claim 5, wherein the second dose is administered at a time other than the second treatment period following the completion of the first treatment period. 12. The method of claim 11 wherein the first treatment period is about two weeks. 13. The method of claim 1 wherein the first dose and the second dose are administered intravenously. 14. The method according to claim 1, where the antibody is in the pharmaceutical composition at a concentration of 5 mg/ml. 15. The method according to claim 1, where the antibody is in the pharmaceutical composition at a concentration of 25 mg/ml. 16. The method according to claim 1, where the antibody is administered in the form of a pharmaceutical composition and where the pharmaceutical composition contains L-histidine, L-histidine monohydrochloride, monohydrate, sodium chloride (NaCl) and polysorbate 80, where the pH of the composition is from pH 5.8 to pH 6.2. 17. The method of claim 1 wherein the subject has systemic juvenile idiopathic arthritis (sLJIA). 18. The method of claim 17, wherein the subject has an inadequate response to treatment with high doses of glucocorticoids. 19. The method of claim 1 wherein the subject has an immune-related disorder such as an autoimmune disease or an inflammatory disease. 20. The method of claim 1, wherein the individual is suffering from adult Still's disease (AOSD). 21. The method of claim 1, wherein the individual is suffering from systemic lupus erythematosus (SLE). 22. The method of claim 1 wherein the individual is an adult. 23. The method of claim 1, wherein the individual is a child.