Methods and compositions for diagnosis and treatment of disorders in patients with elevated levels of CXCL9 and other biomarkers

Abstract

To provide methods and compositions for treating hemophagocytic lymphohistiocytosis (HLH); the disclosure also relating generally to methods and compositions for diagnosing and treating disorders associated with elevated levels of CXCL9, elevated levels of total IFNγ, and other biomarkers; the disclosure further relating to methods of treating, delaying the progression of, or otherwise ameliorating a symptom of a disorder in patients with elevated levels of CXCL9, patients with elevated levels of total IFNγ, and patients with other biomarkers.SOLUTION: Used are agents that interfere with or otherwise antagonize interferon gamma (IFNγ) signaling, including neutralizing anti-IFNγ antibodies.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 158,153, filed May 7, 2015; U.S. Provisional Application No. 62 / 221,393, filed September 21, 2015; and U.S. Provisional Application No. 62 / 246,949, filed October 27, 2015, the contents of each of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THE INVENTION The present disclosure generally relates to methods and compositions for treating hemophagocytic lymphohistiocytosis (HLH). It further provides methods and compositions for diagnosing and treating disorders associated with elevated levels of CXCL9, elevated levels of total IFNγ, and other biomarkers. The present disclosure also relates to methods for treating, slowing the progression of, or otherwise ameliorating symptoms of disorders in patients with elevated levels of CXCL9, elevated levels of total IFNγ, and other biomarkers using agents that interfere with or otherwise antagonize interferon gamma (IFNγ) signaling, including neutralizing anti-IFNγ antibodies. [Background technology]

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

[0004] IFNγ has been implicated in numerous disorders, and anti-IFNγ agents are under development as therapeutics. Thus, there is a need for compositions and methods for use in identifying biomarkers of IFNγ production in IFNγ-associated disorders. Summary of the Invention

[0005] The compositions and methods provided herein use a fully human IgG1 anti-interferon gamma (IFNγ) monoclonal antibody (mAb), herein designated NI-0501, which binds to and neutralizes IFNγ. NI-0501 binds to the soluble and receptor (IFNγR1)-bound forms of IFNγ. The compositions and methods provided herein are useful in the treatment of hemophagocytic lymphohistiocytosis (HLH).

[0006] The anti-IFNγ antibody referred to herein as NI-051 comprises a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO: 1); variable heavy chain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence of TIFF2025118898000002.tif4128; and variable heavy chain complementarity-determining region 3 (VH CDR3) containing the amino acid sequence of TIFF2025118898000003.tif4128; variable light chain complementarity-determining region 1 (VL CDR1) containing the amino acid sequence of TIFF2025118898000004.tif4128; a variable light chain complementarity-determining region 2 (VL CDR2) region comprising the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and NI-0501 contains a variable light chain complementarity determining region 3 (VL CDR3) region having the amino acid sequence of SEQ ID NO: 47 and a variable heavy chain amino acid sequence of SEQ ID NO: 48.

[0007] In the compositions and methods provided herein, NI-0501 is formulated as a sterile concentrate for injection (per mL). In some embodiments, NI-0501 is formulated as follows: 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, where the pH is 5.8-6.2. 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, where the pH is 6.0.

[0008] In the compositions and methods provided herein, NI-0501 is administered to a subject in need thereof to treat, prevent, and / or delay the onset or progression of HLH, or to alleviate symptoms associated therewith. In some embodiments, NI-0501 is administered to a subject in need thereof by IV infusion over a period of 1 hour at an initial dose of 1 mg / kg. In certain patient populations, such as patient populations with low body weight and / or very young patient populations, the IV infusion may last for more than 1 hour, for example, at least 90 minutes, at least 2 hours, or at least 3 hours or more.

[0009] In some embodiments, NI-0501 is administered to a subject in need thereof by an initial IV infusion over a period of 1 hour at an initial dose of 1 mg / kg, followed by at least one additional IV infusion. In some embodiments, the at least one additional IV infusion is at a dose higher than the initial dose of 1 mg / kg. In some embodiments, the at least one additional IV infusion dosage is 3 mg / kg. In some embodiments, the at least one additional IV infusion is administered at least 3 days after the initial IV infusion. In some embodiments, the at least one additional IV infusion is administered at a time selected from the group consisting of: 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial infusion, and 15 days after the initial infusion. In some embodiments, the at least one additional IV infusion is administered 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial infusion, and 15 days after the initial infusion.

[0010] In some embodiments, NI-0501 is administered to a subject in need thereof by an initial IV infusion over a period of 1 hour at an initial dose of 1 mg / kg, followed by at least one series of additional IV infusions, wherein the series of additional IV infusions includes at least one series of twice-weekly IV infusions. In some embodiments, the at least one series of twice-weekly IV infusions is administered at a dose higher than the initial dose of 1 mg / kg. In some embodiments, the at least one series of twice-weekly IV infusions is administered at a dose of 3 mg / kg. In some embodiments, the at least one additional IV infusion is administered at least 3 weeks after the initial IV infusion. In some embodiments, the at least one additional IV infusion is administered at a time selected from the group consisting of 3 weeks after the initial IV infusion, 4 weeks after the initial IV infusion, 5 weeks after the initial IV infusion, 6 weeks after the initial infusion, 7 weeks after the initial infusion, and 8 weeks after the initial infusion. In some embodiments, at least one additional IV infusion is administered 3 weeks after the 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.

[0011] In some embodiments, NI-0501 is administered to a subject in need thereof by at least two additional IV infusions after an initial IV infusion. In some embodiments, the at least two additional IV infusions are at doses higher than the initial dose of 1 mg / kg. In some embodiments, the first additional IV infusion and the second additional IV infusion are administered at the same dosage. In some embodiments, the first additional IV infusion and the second additional IV infusion are administered at the same dosage higher than the initial dose. In some embodiments, at least one of the first and second additional IV infusions is administered at a dosage of 3 mg / kg. In some embodiments, the first additional IV infusion is administered at least 3 days after the initial IV infusion. In some embodiments, the first additional IV infusion is administered at a time selected from the group consisting of 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial infusion, and 15 days after the initial infusion. In some embodiments, the first additional IV infusion is administered 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial infusion, and 15 days after the initial infusion. In some embodiments, the second additional IV infusion is administered at a time selected from the group consisting of 3 weeks after the initial IV infusion, 4 weeks after the initial IV infusion, 5 weeks after the initial IV infusion, 6 weeks after the initial infusion, 7 weeks after the initial infusion, and 8 weeks after the initial infusion. In some embodiments, the second additional IV infusion is administered 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, a first additional IV infusion is administered 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial infusion, and 15 days after the initial infusion, and a second additional IV infusion is administered 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.

[0012] In some embodiments, the first additional IV infusion and the second additional IV infusion are administered at different dosages. In some embodiments, the first additional IV infusion and the second additional IV infusion are administered at different dosages, wherein the second additional IV infusion dosage is higher than the first additional IV infusion. In some embodiments, the first additional IV infusion and the second additional IV infusion are administered at different dosages, wherein the second additional IV infusion dosage is higher than the first additional IV infusion, and wherein both the first and second additional IV infusion dosages are higher than the initial dosage. In some embodiments, at least one of the first and second additional IV infusions is administered at a dosage of 3 mg / kg. In some embodiments, the first additional IV infusion is administered at a dosage of 3 mg / kg, and the second additional IV infusion is administered at a dosage of 6 mg / kg. In some embodiments, the first additional IV infusion is administered at least 3 days after the initial IV infusion. In some embodiments, the first additional IV infusion is administered at a time selected from the group consisting of: 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial infusion, and 15 days after the initial infusion. In some embodiments, the first additional IV infusion is administered at a time selected from the group consisting of: 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial infusion, and 15 days after the initial infusion. In some embodiments, the second additional IV infusion is administered at a time selected from the group consisting of: 3 weeks after the initial IV infusion, 4 weeks after the initial IV infusion, 5 weeks after the initial IV infusion, 6 weeks after the initial IV infusion, 7 weeks after the initial infusion, and 8 weeks after the initial infusion. In some embodiments, the second additional IV infusion is administered at a time selected from the group consisting of: 3 weeks after the initial IV infusion, 4 weeks after the initial IV infusion, 5 weeks after the initial IV infusion, 6 weeks after the initial infusion, 7 weeks after the initial infusion, and 8 weeks after the initial infusion. In some embodiments, a first additional IV infusion is administered 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial infusion, and 15 days after the initial infusion, and a second additional IV infusion is administered 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.

[0013] In some embodiments, the first additional IV infusion comprises at least a first series of twice-weekly IV infusions, and the second additional IV infusion comprises at least a second series of twice-weekly IV infusions. In some embodiments, the first series of twice-weekly IV infusions and the second series of twice-weekly IV infusions are administered at doses higher than the initial dose of 1 mg / kg. In some embodiments, the first series of twice-weekly IV infusions is administered at a dose of 3 mg / kg, and the second series of twice-weekly IV infusions is administered at a dose of 6 mg / kg. In some embodiments, the first series of additional IV infusions is administered at least 3 days after the initial IV infusion. In some embodiments, the first series of additional IV infusions is administered at a time selected from the group consisting of 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial infusion, and 15 days after the initial infusion. In some embodiments, a first series of additional IV infusions is administered 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial infusion, and 15 days after the initial infusion. In some embodiments, a second series of additional IV infusions is administered at a time selected from the group consisting of 3 weeks after the initial IV infusion, 4 weeks after the initial IV infusion, 5 weeks after the initial IV infusion, 6 weeks after the initial infusion, 7 weeks after the initial infusion, and 8 weeks after the initial infusion. In some embodiments, a second series of additional IV infusions is administered 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, a 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 infusion, and 15 days after the initial infusion, and a second series of additional IV infusions is administered 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.

[0014] In some embodiments, infusions are administered every 3 days after the first dose for up to 15 days after the first dose. In some embodiments, infusions are administered every 3 days after the first dose for up to 15 days after the first dose, followed by twice-weekly infusions starting at least 15 days after the first dose. In some embodiments, the infusion dosage is increased to 3 mg / kg at any time after the first dose. In some embodiments, after a minimum of two infusions at 3 mg / kg, the dose of NI-0501 is increased to 6 mg / kg for up to four infusions.

[0015] In the compositions and methods provided herein, NI-0501 is administered to a subject in need thereof to treat, prevent, and / or delay the onset or progression of, or alleviate symptoms associated with, HLH. In some embodiments, NI-0501 is administered to a subject in need thereof by IV infusion over a period of 1 hour at an initial dose of 1 mg / kg. In some embodiments, the infusion is administered every 3 days after the initial dose for up to 15 days after the initial dose. In some embodiments, the infusion is administered 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 dosage is increased to 3 mg / kg at any time after the initial dose. In some embodiments, after at least two infusions at 3 mg / kg, the dose of NI-0501 is increased to 6 mg / kg for up to four infusions.

[0016] In the compositions and methods provided herein, NI-0501 is administered to a subject in need thereof to treat, prevent, and / or delay the onset or progression of, or alleviate symptoms associated with, HLH. In some embodiments, NI-0501 is administered to a subject in need thereof by IV infusion for a dosage greater than 6 mg / kg. In some embodiments, after the initial dosage, NI-0501 is administered to a subject in need thereof by IV infusion for a second dosage greater than 6 mg / kg. In some embodiments, the second dosage is at least 10 mg / kg. In some embodiments, the second dosage is 10 mg / kg. In some embodiments, the second dosage is 10 mg / kg repeated daily. In some embodiments, the second dosage is 10 mg / kg repeated daily for one week. In some embodiments, the second dosage is 10 mg / kg repeated daily for two weeks. In some embodiments, the second dosage is 10 mg / kg repeated daily for more than two weeks.

[0017] In some embodiments, NI-0501 is administered to a subject in need thereof to treat, prevent, and / or delay the onset or progression of, or alleviate symptoms associated with, secondary HLH. In some embodiments, NI-0501 is administered to a subject in need thereof to treat, prevent, and / or delay the onset or progression of, or alleviate symptoms associated with, secondary HLH in the setting of sJIA. In some embodiments, NI-0501 is administered to a subject in need thereof as an initial dose of 6 mg / kg. In some embodiments, NI-0501 treatment is continued with a continuous NI-0501 dose. In some embodiments, NI-0501 treatment is continued with a continuous NI-0501 dose of 3 mg / kg every three days for at least four weeks (i.e., up to SD27).

[0018] In some embodiments, NI-0501 treatment is tapered, stopped, or otherwise shortened upon achievement of the desired clinical outcome, hi some embodiments, NI-0501 treatment is shortened based on evidence of a complete clinical response, i.e., remission of MAS.

[0019] In some embodiments, after 4 weeks, NI-0501 treatment is continued as maintenance as needed until MAS remission is achieved, for up to an additional 4 weeks (i.e., up to SD56). In some embodiments, after 4 weeks, NI-0501 treatment is continued as maintenance as needed until MAS remission is achieved, for up to an additional 4 weeks (i.e., up to SD56), and the dose may be reduced to 1 mg / kg and the interval between injections may be extended to weekly dosing.

[0020] In the compositions and methods provided herein, NI-0501 is administered to a subject in need thereof to treat, prevent, and / or delay the onset or progression of, or alleviate symptoms associated with, HLH, and the subject is receiving background dexamethasone. In some embodiments, the subject is a treatment-naive patient (i.e., not previously treated for HLH), and the dexamethasone is administered at a dose of at least 10 mg / m 2 In some embodiments, the subject receives NI-0501 as a second-line HLH treatment and dexamethasone is administered at a dose of 10 mg / m 2 ~5 mg / m 2 In some embodiments, the subject receives NI-0501 as a second-line HLH treatment and dexamethasone is administered at a dose within the range of at least 5 mg / m 2 In some embodiments, the subject receives NI-0501 as a second-line HLH treatment and dexamethasone is administered at a dose of 5 mg / m 2 It is administered in doses less than 100 mg / kg.

[0021] In some embodiments, NI-0501 is administered before, during, and / or after treatment in combination with one or more additional agents, such as, but not limited to, therapeutic agents, anti-inflammatory agents, and / or immunosuppressants. In some embodiments, the second agent is an agent known to be used in the treatment of HLH. In some embodiments, the additional agent includes at least etoposide. In some embodiments, NI-0501 and the additional agent are formulated in a single therapeutic composition, and NI-0501 and the additional agent are administered simultaneously. Alternatively, NI-0501 and the additional agent are separated from each other, e.g., 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 the treatment regimen. For example, NI-0501 is administered before administration of the additional agent, NI-0501 is administered after administration of the additional agent, or NI-0501 and the additional agent are administered alternately. As described herein, NI-0501 and the additional agent are administered in a single dose or in multiple doses.

[0022] In some embodiments, NI-0501 and additional agent are administered simultaneously.For example, NI-0501 and additional agent can be formulated in a single composition, or can be administered as two or more separate compositions.In some embodiments, NI-0501 and additional agent are administered sequentially, or NI-0501 and additional agent are administered at different times during the course of treatment.

[0023] In some embodiments, the additional agent is an immunosuppressant. In some embodiments, the immunosuppressant is cyclosporine A (CsA). In some embodiments, the subject is a subject who received CsA prior to administration of NI-0501. In some embodiments, the additional agent comprises at least etoposide. In some embodiments, the subject is a subject who received etoposide prior to administration of NI-0501.

[0024] In some embodiments, the additional agent is intrathecal methotrexate and / or a glucocorticoid. In some embodiments, the subject was receiving intrathecal methotrexate and / or a glucocorticoid prior to administration of NI-0501.

[0025] In some embodiments, the additional agent is IV immunoglobulin (IVIG). In some embodiments, IVIG is administered as replacement therapy in subjects with documented immunoglobulin deficiency. In some embodiments, in subjects with documented immunoglobulin deficiency, IVIG is administered at a dose of 0.5 g / kg every 4 weeks or more frequently to maintain adequate IgG levels.

[0026] In some embodiments, the one or more additional medications are analgesic therapy, blood product transfusion, electrolyte and glucose infusion, antibiotic, antifungal and antiviral therapy, and / or systemic supportive therapy.

[0027] The present disclosure also provides compositions and methods useful in identifying or otherwise refining patient populations suffering from disorders in which patients have elevated levels of CXCL9, alone or in combination with one or more additional interferon-gamma (IFNγ)-related biomarkers. In particular, the present disclosure provides compositions and methods for detecting CXCL9 levels as a biomarker for IFNγ production in patients suffering from or suspected of suffering from hemophagocytic lymphohistiocytosis (HLH). In particular, the present disclosure provides compositions and methods for detecting CXCL9 levels as a biomarker for IFNγ production in patients suffering from or suspected of suffering from secondary hemophagocytic lymphohistiocytosis (HLH). In some embodiments, the compositions and methods are used to detect CXCL9 levels as a biomarker for IFNγ production in patients suffering from or suspected of suffering from macrophage activation syndrome (MAS). In some embodiments, the compositions and methods are used to detect CXCL9 levels as a biomarker for IFNγ production in patients with or suspected of having MAS in the context of an autoimmune disease or inflammatory disorder. In some embodiments, the compositions and methods are used to detect CXCL9 levels as a biomarker for IFNγ production in patients with or suspected of having MAS in the context of a systemic autoimmune disease or inflammatory disorder. In some embodiments, the compositions and methods are used to detect CXCL9 levels as a biomarker for IFNγ production in patients with or suspected of having MAS in the context of systemic juvenile idiopathic arthritis (sJIA). In some embodiments, the compositions and methods are used to detect CXCL9 levels as a biomarker for IFNγ production in patients with or suspected of having MAS in the context of systemic lupus erythematosus (SLE).

[0028] Patients identified as having elevated levels of CXCL9 are identified as suitable candidates for treatment with agents (e.g., antibodies or other polypeptide-based therapeutics, peptide-based therapeutics, small molecule inhibitors, nucleic acid-based therapeutics and derivatives thereof) that interfere with or otherwise antagonize one or more biological activities of IFNγ (e.g., IFNγ signaling) and neutralize at least one biological activity of IFNγ.

[0029] In some patients suffering from or suspected of suffering from the disorder, body fluids and other biological samples contain elevated levels of CXCL9, alone or in combination with other IFNγ-related biomarkers, such as CXCL10 and / or CXCL11.

[0030] CXCL9 and these other biomarkers are indicators of in vivo IFNγ production. Thus, the use of anti-IFNγ antagonists, e.g., neutralizing anti-IFNγ antibodies or other polypeptide-based therapeutics, peptide-based therapeutics, small molecule inhibitors, nucleic acid-based therapeutics, and derivatives thereof, that interfere with, inhibit, reduce, or otherwise antagonize IFNγ signaling blocks or otherwise inhibits IFNγ activity. Thus, the present compositions and methods are useful in treating, slowing the progression of, or otherwise ameliorating the symptoms of disorders that depend on, are driven by, are associated with, or are otherwise affected by aberrant, e.g., elevated, IFNγ expression and / or activity, aberrant inflammatory cytokine production, and / or a combination thereof, by administering anti-IFNγ antagonists, e.g., neutralizing anti-IFNγ antibodies or other polypeptide-based therapeutics, peptide-based therapeutics, small molecule inhibitors, nucleic acid-based therapeutics, and derivatives thereof, to patients who exhibit elevated CXCL9 expression levels and / or other biomarkers. Patients who are likely suitable candidates for treatment with an anti-IFNγ antagonist, such as a neutralizing anti-IFNγ antibody as described herein, are identified by detecting the level of CXCL9, alone or in combination with one or more IFNγ-related ligands or other biomarkers. In some embodiments, patients who do not have elevated levels of CXCL9, alone or in combination with other IFNγ-related biomarkers, can still be treated with an anti-IFNγ antagonist, including a neutralizing anti-IFNγ antibody or other polypeptide-based therapeutic, peptide-based therapeutic, small molecule inhibitor, nucleic acid-based therapeutic, or any of their derivatives, as described herein.

[0031] Patients with elevated levels of CXCL9, alone or in combination with one or more additional IFNγ-related biomarkers, are identified as suitable candidates for therapy with one or more anti-IFNγ antagonists, such as the neutralizing anti-IFNγ antibodies described herein.As used herein, the phrase "elevated expression level" refers to an expression level that is greater than the baseline expression level of CXCL9 (alone or in combination with one or more additional biomarkers) in a sample from a patient who is not suffering from or suspected of suffering from primary or secondary HLH or HLH-related disorders, or from another control sample.In some embodiments, the elevated expression level of CXCL9 and / or other biomarkers is a significantly elevated level.

[0032] The detection level of CXCL9 (alone or in combination with one or more other IFNγ-related biomarkers) is useful for refining or otherwise stratifying patient populations. In some embodiments, the detection level is used to determine the dosage of an anti-IFNγ antagonist to be administered to a given patient. In some embodiments, the detection level is used to categorize or otherwise stratify patient populations. For example, patients can be classified as having "severe" or high-grade MAS, or conversely, non-severe or low-grade MAS, based on the detection level of CXCL9.

[0033] The sample is, for example, blood or blood components, such as serum, plasma. In some embodiments, the sample is another body fluid, for example, but not limited to, urine, synovial fluid, bronchoalveolar fluid, cerebrospinal fluid, bronchoalveolar lavage fluid (BAL), and / or saliva. In some embodiments, the biological sample is CSF. In some embodiments, the biological sample is CSF from a HLH patient.

[0034] In addition to detecting levels of IFNγ and / or other IFNγ-related biomarkers, suitable patients for treatment with anti-IFNγ antagonists can also be identified by assessing any of a number of additional biological and clinical parameters that will improve the sensitivity and specificity of biomarkers for identifying or otherwise refining patient populations. Alternatively, these additional biological and clinical parameters can be used alone as a means to identify patients who are suitable candidates for treatment with anti-IFNγ antagonists or other suitable therapies. These biological and clinical parameters include, by way of non-limiting example, any of the following: ferritin levels, neutrophil counts, platelet counts, alanine aminotransferase levels, and / or lactate dehydrogenase levels.

[0035] Disorders that are useful in the compositions and methods of the invention include any disorder in which there is aberrant, e.g., elevated, IFNγ expression and / or activity, particularly HLH (including secondary HLH), MAS, and / or sJIA.

[0036] By way of non-limiting example, the methods and compositions provided herein are suitable for the diagnosis and / or treatment of disorders such as primary and / or secondary HLH disorders. Suitable autoimmune and / or inflammatory disorders include, by way of non-limiting example, primary and / or secondary HLH disorders associated with aberrant IFNγ activity and / or expression.

[0037] Once a patient is identified as having elevated levels of CXCL9, alone or in combination with one or more IFNγ-related biomarkers, the patient is then treated with an anti-IFNγ antagonist. For example, the anti-IFNγ antagonist is a neutralizing anti-IFNγ antibody or its immunologically active (e.g., antigen-binding) fragment. Suitable neutralizing anti-IFNγ antibodies include any of the anti-IFNγ antibodies described herein.

[0038] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises a variable heavy chain complementarity determining region 1 (VH CDR1) comprising an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SYAMS (SEQ ID NO: 1); a variable heavy chain complementarity determining region 2 (VH CDR2) comprising an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of TIFF2025118898000006.tif4128; and a variable heavy chain complementarity-determining region 3 (VH CDR3) comprising an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of TIFF2025118898000007.tif4128; a variable light chain complementarity-determining region 1 (VL CDR1) comprising an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of TIFF2025118898000008.tif4128; a variable light chain complementarity-determining region 2 (VL CDR2) comprising 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 It comprises a variable light chain complementarity determining region 3 (VL CDR3) comprising an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of TIFF2025118898000009.tif4128.

[0039] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises a VH CDR1 comprising the amino acid sequence of SYAMS (SEQ ID NO: 1); the VH CDR2 region comprising the amino acid sequence of TIFF2025118898000010.tif4128; and VH CDR3 region containing the amino acid sequence of TIFF2025118898000011.tif4128; the variable light chain complementarity-determining region 1 (VL CDR1) region containing the amino acid sequence of TIFF2025118898000012.tif4128; A VL CDR2 region comprising the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and It contains the VL CDR3 region containing the amino acid sequence of TIFF2025118898000013.tif4128.

[0040] 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 a single row in Table 1A.

[0041] 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) shown in a single row in Table 1B.

[0042] 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 the three heavy chain CDR sequences (VH CDR1, VH CDR2, VH CDR3) shown in a single row in Table 1A, and a light chain comprising a combination of a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the combination is a combination of the three light chain CDR sequences (VL CDR1, VL CDR2, VL CDR3) shown in a single row in Table 1B.

[0043] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to the heavy chain variable amino acid sequence of the amino acid sequence of SEQ ID NO:47.

[0044] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to the light chain variable amino acid sequence of the amino acid sequence of SEQ ID NO:48.

[0045] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to the heavy chain variable amino acid sequence of 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 light chain variable amino acid sequence of the amino acid sequence of SEQ ID NO: 48.

[0046] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises the heavy chain variable amino acid sequence of the amino acid sequence of SEQ ID NO:47.

[0047] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises the light chain variable amino acid sequence of the amino acid sequence of SEQ ID NO:48.

[0048] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises a heavy chain variable amino acid sequence of the amino acid sequence of SEQ ID NO:47 and a light chain variable amino acid sequence of the amino acid sequence of SEQ ID NO:48.

[0049] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to the heavy chain amino acid sequence of the amino acid sequence of SEQ ID NO:44.

[0050] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to the light chain amino acid sequence of the amino acid sequence of SEQ ID NO:46.

[0051] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to the heavy chain amino acid sequence of the amino acid sequence of SEQ ID NO:44 and an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to the light chain amino acid sequence of the amino acid sequence of SEQ ID NO:46.

[0052] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises the heavy chain amino acid sequence of the amino acid sequence of SEQ ID NO:44.

[0053] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises the light chain amino acid sequence of the amino acid sequence of SEQ ID NO:46.

[0054] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises a heavy chain amino acid sequence of the amino acid sequence of SEQ ID NO:44 and a light chain amino acid sequence of the amino acid sequence of SEQ ID NO:46.

[0055] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to a heavy chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, and 102.

[0056] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to a light chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, and 104.

[0057] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to a heavy chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, and 102, and an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical to a light chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, and 104.

[0058] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises a heavy chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, and 102.

[0059] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises a light chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, and 104.

[0060] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof comprises a heavy chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, and 102, and a light chain variable amino acid sequence selected from the group consisting of SEQ ID NOs: 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, and 104.

[0061] 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 present invention generally refers to the amount necessary to achieve a therapeutic goal. This therapeutic goal may be the binding interaction of the antibody with its target antigen, which may interfere with the function of the target. A typical range for a therapeutically effective dosage of an antibody or antibody fragment of the present invention may be, by way of non-limiting example, about 0.1 mg / kg body weight to about 50 mg / kg body weight. Typical dosing frequencies may range, for example, from twice daily to once weekly.

[0062] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof is administered at an initial dose or loading dose in the range of about 0.5 mg / kg to about 2 mg / kg, e.g., about 0.5 mg / kg to about 1.5 mg / kg, and / or about 0.5 mg / kg to about 1.0 mg / kg. In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof is administered at an initial dose of about 1.0 mg / kg.

[0063] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof is administered as an initial loading dose, followed by one or more maintenance doses. In some embodiments, the one or more maintenance doses are at a dosage substantially similar to the initial loading dose. In some embodiments, the one or more maintenance doses are at a dosage less than the initial loading dose. In some embodiments, the one or more maintenance doses are at a dosage greater than the initial loading dose.

[0064] In some embodiments, the one or more maintenance doses include at least two or more dosages, wherein each maintenance dose is the same dosage. In some embodiments, the two or more maintenance dosages are substantially similar to the initial loading dose. In some embodiments, the two or more maintenance dosages are greater than the initial loading dose. In some embodiments, the two or more maintenance dosages are less than the initial loading dose.

[0065] In some embodiments, the one or more maintenance doses include at least two or more dosages, wherein each maintenance dosage is not the same dosage. In some embodiments, the two or more maintenance dosages are administered in increasing dosages. In some embodiments, the two or more maintenance dosages are administered in decreasing dosages.

[0066] In some embodiments, the one or more maintenance doses include at least two or more dosages, wherein each maintenance dose is administered at periodic time intervals. In some embodiments, the two or more dosages are administered at increasing time intervals. In some embodiments, the two or more dosages are administered at decreasing time intervals.

[0067] In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof is administered at an initial loading dose of about 0.5 mg / kg to about 2 mg / kg, e.g., about 0.5 mg / kg to about 1.5 mg / kg, and / or about 0.5 mg / kg to about 1.0 mg / kg, followed by at least one, e.g., two or more, three or more, four or more, or five or more maintenance doses. In some embodiments, the anti-IFNγ antibody or immunologically active fragment thereof is administered at an initial loading dose of about 1.0 mg / kg, followed by at least one, e.g., two or more, three or more, four or more, or five or more maintenance doses.

[0068] Pharmaceutical compositions according to the present invention can include an anti-IFNγ antibody of the present invention and a carrier. These pharmaceutical compositions can be included in a kit, such as, for example, a diagnostic kit.

[0069] The present invention also provides kits for carrying out any of the methods provided herein. For example, in some embodiments, the kit includes a detection reagent specific for CXCL9 (alone or in combination with one or more IFNγ-related biomarkers) and a means for detecting the detection reagent. [Brief explanation of the drawings]

[0070] [Figure 1] 1 is a graph depicting the correlation between total IFNγ levels 24 hours after infusion of the NI-0501 antibody and pre-dose serum CXCL9 levels in an ongoing Phase 2 pilot study in patients with primary HLH. [Figure 2] 1 is a graph depicting the correlation between total IFNγ pre-dose levels and serum CXCL9 levels 24 hours after infusion of the NI-0501 antibody in an ongoing Phase 2 pilot study in patients with primary HLH. [Figure 3]Figures 3A and 3B are a series of graphs depicting the correlation between serum CXCL9 and IFNγ levels in patients with macrophage activation syndrome (MAS) secondary to systemic juvenile idiopathic arthritis (sJIA) and in patients with active sJIA. [Figure 4A] 1 is a series of graphs depicting the correlation of IFNγ and serum CXCL9 levels with clinical parameters in patients with active sJIA and MAS secondary to sJIA. [Figure 4B] 1 is a series of graphs depicting the correlation of IFNγ and serum CXCL9 levels with clinical parameters in patients with active sJIA and MAS secondary to sJIA. [Figure 4C] 1 is a series of graphs depicting the correlation of IFNγ and serum CXCL9 levels with clinical parameters in patients with active sJIA and MAS secondary to sJIA. [Figure 4D] 1 is a series of graphs depicting the correlation of IFNγ and serum CXCL9 levels with clinical parameters in patients with active sJIA and MAS secondary to sJIA. [Figure 5] 1 is a graph depicting complete neutralization of IFNγ as indicated by undetectable levels of IFNγ-induced chemokines. [Figure 6] Graph depicting improvement in HLH disease activity during NI-0501 treatment (2 weeks and end of treatment): percent of patients with platelet count >100 x 109 / L, neutrophil count >1 x 109 / L, fibrinogen >1.5 g / L, and at least 25% ferritin reduction. [Figure 7] Figures 7A and 7B are a series of graphs depicting the correlation between total IFNγ levels and pre-dose CXCL9 levels 24 hours after NI-0501 infusion. The inset shown in Figure 7B depicts examples of individual IFNγ and CXCL9 profiles during NI-0501 treatment. [Figure 8]Figures 8A, 8B, 8C, and 8D are a series of graphs depicting serum levels of IFNγ, and CXCL9, CXCL10, and CXCL11 in individual patients for whom paired samples were available during active MAS and active sJIA without MAS (Act sJIA) at the time of sampling. Significance levels (p) were obtained using the Wilcoxon rank test for paired samples. [Figure 9] Figures 9A and 9B are a series of graphs depicting changes in white blood cell (WBC) and platelet (PLT) counts and ferritin levels (Figure 9A), as well as changes in serum levels of IFNγ, CXCL9, CXCL10, and CXCL11 (Figure 9B) in one patient who had three episodes of MAS during the course of sJIA. [Figure 10-1] 1 is a series of graphs depicting the correlation of IFNγ and CXCL9 levels with ferritin levels, neutrophil and platelet counts, and LDH and ALT levels in patients with active MAS at the time of sampling (red circles) and in patients with active sJIA without MAS at the time of sampling (black triangles). Spearman correlation coefficients (Rs) and significance levels (p) for each correlation are shown in Table 3. [Figure 10-2] 1 is a series of graphs depicting the correlation of IFNγ and CXCL9 levels with ferritin levels, neutrophil and platelet counts, and LDH and ALT levels in patients with active MAS at the time of sampling (red circles) and in patients with active sJIA without MAS at the time of sampling (black triangles). Spearman correlation coefficients (Rs) and significance levels (p) for each correlation are shown in Table 3. [Figure 11] Figures 11A, 11B, 11C, 11D, 11E, and 11F are a series of graphs depicting the relationship between IFNγ and CXCL9 and CXCL10 production in MAS. Panel A: Correlation between levels of IFNγ and levels of CXCL9 and CXCL10 in patients with MAS at the time of sampling. Spearman correlation coefficients (Rs) and significance levels (p) for each correlation are shown in Table 3. [Figure 12]1 is a schematic representation of the screening, treatment and follow-up portions of the study presented in Example 7. [Figure 13] 13A and 13B are graphs depicting the effect of NI-0501 administration on body temperature in two patients with a temperature >37.5° C. at the start of NI-0501 treatment. [Figure 14] 1 is a series of graphs and tables depicting the effect of NI-0501 administration on neutrophil counts in patients. [Figure 15] 1 is a series of graphs and tables depicting the effect of NI-0501 administration on platelet counts in patients. [Figure 16] 1 is a series of graphs and tables depicting the effect of NI-0501 administration on serum levels of ferritin in patients. [Figure 17] 1 is a series of graphs and tables depicting the effect of NI-0501 administration on glucocorticoid tapering in patients. [Figure 18] 1 is a graph depicting that administration of NI-0510 maintained IFNγ neutralization until the time of HSCT. The HLH response to NI-0501 treatment also persisted until transplant. [Figure 19] 1 is a schematic representation of the screening, treatment, and follow-up portions of the study presented in Example 8. DETAILED DESCRIPTION OF THE INVENTION

[0071] Detailed Description of the Invention The compositions and methods provided herein use a fully human IgG1 anti-interferon gamma (IFNγ) monoclonal antibody (mAb), herein designated NI-0501, which binds to and neutralizes IFNγ. NI-0501 binds to both soluble and receptor (IFNγR1)-bound forms of IFNγ. Because NI-0501 is a human IgG1, it retains the characteristics of this immunoglobulin isotype, including the ability to associate with Fcγ receptors and fix complement. IFNγ is one of the most potent and versatile cytokines of the immune system. It is important for innate and adaptive immunity against viral and intracellular bacterial infections. After binding to its receptor, IFNγ acts to effect various physiological and cellular responses. Numerous studies over the past two decades have implicated IFNγ in the pathogenesis and maintenance of inflammatory diseases (see, e.g., Billiau A. "Interferon-gamma: biology and role in pathogenesis." Adv. Immunol. 1996;62:61-130; Schoenborn JR, Wilson CB. "Regulation of interferon-gamma during innate and adaptive immune responses." Adv. Immunol. 2007;96:41-101; and Zhang SY, Boisson-Dupuis S, Chapgier A et al. "Inborn errors of interferon (IFN)-mediated immunity in humans: insights into the respective roles of IFN-alpha / beta, IFN-gamma, and IFN-lambda in host defense." Immunol. Rev. 2008;226:29-40). IFNγ is primarily produced by natural killer (NK) and natural killer T (NKT) cells as part of the innate immune response, and by CD4 Th1 and CD8 cytotoxic T lymphocyte (CTL) effector T cells once antigen-specific immunity has developed.

[0072] The compositions and methods provided herein are useful in the treatment of hemophagocytic lymphohistiocytosis (HLH), a syndrome characterized by severe impairment or absence of cytotoxic function by NK cells and CD8+ T cells, accompanied by marked activation of the immune system.

[0073] HLH includes primary (genetic / familial) HLH and secondary HLH, both of which are clinically explained by dysregulation of the immune system, leading to severe hypercytokinemia with deleterious consequences for various tissues and organs (Henter JI, Elinder G, Soder O et al. "Hypercytokinemia in familial hemophagocytic lymphohistiocytosis." Blood 1991;78:2918-2922). HLH classification is shown in Table 9 below.

[0074] (Table 9) HLH classification TIFF2025118898000014.tif120152

[0075] Primary HLH is a heterogeneous autosomal recessive disorder. It is most commonly seen in infancy and early childhood, and its prevalence in Europe is estimated at 1 in 50,000 live births (Henter JI, Elinder G, Söder O, Ost A. Incidence in Sweden and clinical features of familial hemophagocytic lymphohistiocytosis. Acta Paediatr.Scand. 1991;80:428-435). If untreated, the disease is invariably fatal, with a median survival of less than two months after the onset of symptoms (Janka GE. Familial hemophagocytic lymphohistiocytosis. Eur. J. Pediatr. 1983;140:221-230; and Arico M, Janka G, Fischer A, Henter JI, Blanche S, Elinder G, Martinetti M, Rusca MP. Hemophagocytic lymphohistiocytosis Report of 122 children from the International Registry. FHL Study Group of the Histiocyte Society. Leukemia. 1996 Feb;10(2):197-203).

[0076] The cytotoxic dysfunction present in HLH leads to hypercytokinemia and hemophagocytosis, which in turn cause all of the classic symptoms of HLH (Dhote R, Simon J, Papo T et al. Reactive hemophagocytic syndrome in adult systemic disease: report of 26 cases and literature review. Arthritis Rheum. 2003;49:633-639; Risdall RJ, McKenna RW, Nesbit ME et al. Virus-associated hemophagocytic syndrome: a benign histiocytic proliferation distinct from malignant histiocytosis. Cancer 1979;44:993-1002; and Risdall RJ, Brunning RD, Hernandez JI, Gordon DH. Bacteria-associated hemophagocytic syndrome. Cancer 1984;54:2968-2972). Typical symptoms of HLH include, for example, prolonged fever, splenomegaly, hepatomegaly, cytopenia, hyperferritinemia, hypertriglyceridemia, hypofibrinogenemia, hemophagocytosis, hypercytokinemia, and / or lymphohistiocytic infiltration, bone marrow hypoplasia, and meningeal infiltration.

[0077] Cytokines that are elevated in patients with HLH include IFNγ, interleukin 6 (IL-6), IL-10, tumor necrosis factor (TNF)α, IL-8, macrophage colony-stimulating factor (MCSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0078] HLH can also occur during the course of infectious, rheumatic, or neoplastic diseases, in which case it is called secondary HLH. Secondary HLH exhibits the same signs and symptoms as primary HLH and can be equally severe. Current treatment of secondary HLH aims to identify the underlying cause. This is certainly true for HLH caused by infectious diseases such as leishmaniasis. Of note, the presence of certain infectious diseases, particularly viral infections such as those caused by CMV or EBV, is very often the trigger for the manifestation of primary HLH.This observation is also supported by evidence that in animal models of primary HLH, infection with lymphocytic choriomeningitis virus (LCMV) is required for the development of this 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 hemophagocytosis 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).

[0079] When HLH manifests during neoplastic disease, especially hematologic malignancies, the severity of the patient's condition often necessitates urgent treatment of HLH before the underlying disease can be specifically identified.

[0080] The presence of signs and symptoms of HLH in patients suffering from rheumatic diseases such as systemic juvenile idiopathic arthritis (sJIA) and systemic lupus erythematosus (SLE)—often referred to by rheumatologists as macrophage activation syndrome (MAS)—may precede the onset of the rheumatic disease itself. The majority of patients with MAS exhibit impaired NK and perforin function tests, and the majority of patients exhibit polymorphisms or heterozygous mutations in PRF1 and UNC13D. This is an extremely serious and life-threatening condition, but it usually resolves once appropriate treatment, consisting of corticosteroids and cyclosporine, is initiated. However, in approximately 15% of patients who present with MAS, the disease can be difficult to control and the use of etoposide can be considered (Minoia F, Davi S, Horne AC et al. Clinical Features, Treatment, and Outcome of Macrophage Activation Syndrome Complicating Systemic Juvenile Idiopathic Arthritis: A Multinational, Multicenter Study of 362 Patients. Arthritis & Rheumatism 2014; 66: 3160-3169).

[0081] Although primary HLH is primarily recognized as a pediatric disease, HLH can also be seen in adults, and increased awareness indicates that this may be more common than previously recognized. In the majority of adult patients, the disease occurs during malignancies (mainly non-Hodgkin's lymphoma), infections, autoinflammatory or autoimmune diseases, and iatrogenic immunodeficiencies.

[0082] Currently, there are no approved medications for the treatment of HLH. However, experts in the field have established guidelines for the management of patients with HLH (Henter JI, Horne AC, Arico M, Egeler RM, Filipovich AH, Imashuku S, Ladisch S, McClain K, Webb D, Winiarski J, and Janka Diagnostic and Therapeutic Guidelines for Hemophagocytic Lymphohistiocytosis Blood Cancer 2007;48:124-13.1; 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 to treat hemophagocytic lymphohistiocytosis. Blood 2011; 118:4041-4052).

[0083] Management of patients with primary HLH currently involves the following phases (Henter et al., Blood Cancer 2007): (i) 8 weeks of induction therapy with a combination of corticosteroids and immunosuppressants (e.g., etoposide, CsA, alemtuzumab, antithymocyte globulin); (ii) maintenance therapy until transplantation; and (iii) transplantation for all patients in whom a genetic defect is identified, and ultimately in very severe HLH cases without a disease-associated mutation.

[0084] The primary goal of induction therapy is to suppress the life-threatening inflammatory process that characterizes HLH and make transplantation possible in patients who need it (Horne A, Janka G, Maarten ER et al. Haematopoietic stem cell transplantation in heemophagocytic lymphohistiocytosis. Br.J.Haematol. 2005;129:622-630). Transplantation is the only curative treatment for HLH associated with high-penetrance genetic mutations (Henter et al., Blood 2002).

[0085] Despite the adoption of these guidelines, the overall mortality rate for primary HLH remains approximately 40-50% (Henter et al., Blood 2002; Trottestam H, Horne A, Arico M et al. Chemoimmunotherapy for hemophagocytic lymphohistiocytosis: long-term results of the HLH-94 treatment protocol. Blood 2011;118:4577-4584).

[0086] During the induction period, the need to use drugs associated with serious short-term and long-term safety issues further contributes to an already high mortality rate. The compositions and methods provided herein have been developed as targeted therapies that ensure efficacy with less toxicity.

[0087] Over the past few years, evidence for the central role of IFNγ in the development of HLH has been mounting (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 IFNγ defeats heemophagocytosis 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; Hemophagocytic Lymphohistiocytosis in Children J Pediatr 2011; and Risma K, Jordan MB.Hemophagocytic lymphohistiocytosis: updates and evolving concepts. Curr.Opin.Pediatr. 2012;24:9-15). .

[0088] The genetic mutations that characterize the primary form of HLH all affect proteins involved in the same process, ultimately impairing cytotoxic activity. Perforin mutations were first identified in patients with HLH.

[0089] Perforin knockout (KO) mice are considered an appropriate model for this human disease. Indeed, once infected with LCMV, these mice exhibit all diagnostic and many clinical and laboratory features of this human disease and die if untreated. For these reasons, perforin KO mice have been used to study the pathophysiology of HLH. The HLH-like pathology they exhibit depends on CD8+ T cells and IFNγ production in response to antigen stimulation.

[0090] It has been demonstrated that neutralization of high circulating levels of IFNγ by administration of anti-IFNγ antibodies not only reverses clinical and laboratory abnormalities but also dramatically improves survival, whereas removal of any other cytokine had no effect on survival (Jordan et al., Blood 2004; Pachlopnik et al., EMBO Mol. Med. 2009).

[0091] 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 overstimulation in healthy mice (i.e., with normal genetic characteristics of cytotoxic pathways) as a model of HLH secondary to infection. These mice do not necessarily die, but they exhibit typical clinical and laboratory features of HLH. When IFNγ is neutralized by administration of anti-IFNγ antibodies, the clinical and laboratory features of the disease are reversed. Interestingly, this model demonstrated that administration of anti-IFNγ antibodies also resulted in complete neutralization of IFNγ effects in relevant target tissues such as the liver and spleen (manuscript in preparation).

[0092] To study the physiopathology of secondary HLH in the context of rheumatic diseases, an animal model was created using IL-6 transgenic mice, which express high levels of IL-6, similar to those seen in patients with sJIA, the rheumatic disease most frequently associated with secondary forms of HLH. When triggered with Toll-like receptor (TLR) ligands, these mice die with many of the hallmarks of human disease (Strippolli R, Carvallo F, Scianaro R et al. Amplification of the response to Toll-like receptor ligands by prolonged exposure to interleukin-6 in mice: Implication for the pathogenesis of macrophage activation syndrome. Arthritis & Rheumatism 2012;64:1680-1688). When IFNγ was neutralized in these mice by administration of anti-IFNγ antibodies, survival was significantly improved and laboratory parameters were restored (Prencipe G et al., manuscript in preparation).

[0093] Further reinforcing the importance of IFNγ in HLH is the high circulating IFNγ levels in patients with primary HLH (Henter et al., Blood 1991; Xu et al., J Pedatr 2011). In a series of 71 patients monitored from HLH diagnosis through treatment and follow-up, IFNγ levels exceeded the upper limit of normal (17.3 pg / mL) in all patients, with 53.5% having levels above 1000 pg / mL. It has also been reported that IFNγ levels rise early and rapidly, and that effective treatment of HLH can reduce IFNγ levels from >5000 pg / mL to normal within 48 hours.

[0094] Recently, in an observational study of patients with secondary forms of HLH, high levels of IFNγ were demonstrated in both patients with HLH secondary to infection and in patients with HLH occurring in the setting of sJIA. The levels of three chemokines known to be induced by IFNγ, CXCL9, CXCL10, and CXCL11, were also significantly elevated. Notably, the levels of IFNγ and the three IFNγ chemokines were found to significantly correlate with laboratory parameters of disease severity, such as ferritin, platelet count, and transaminases (Bracaglia et al., submitted).

[0095] Because hypercytokinemia and organ infiltration by activated lymphocytes and histiocytes underlie all HLH symptoms and depend on CD8+ T cell hyperactivity and high IFNγ levels, neutralization of IFNγ is a logical therapeutic approach. Indeed, no drugs that specifically target CD8+ T cells are currently available, and targeting individual cytokines downstream of IFNγ may not always be feasible.

[0096] Therefore, based on data from animal models of primary and secondary HLH, and from observations made in patients with both primary and secondary HLH, which confirm the important role played by IFNγ in the pathogenesis of this disease, neutralization of IFNγ provides a solid rationale for developing targeted therapies for HLH that should be effective with no or limited toxicity.

[0097] The present disclosure also provides compositions and methods that are useful in identifying or otherwise refining patient populations suffering from disorders in which patients have elevated levels of CXCL9, alone or in combination with one or more additional interferon-gamma (IFNγ)-related biomarkers. In particular, the present disclosure provides compositions and methods for detecting CXCL9 levels as a biomarker for IFNγ production in hemophagocytic lymphohistiocytosis (HLH), in secondary HLH, and / or in macrophage activation syndrome (MAS).

[0098] A large body of evidence in animal models points to a central pathogenic role for IFNγ in primary hemophagocytic lymphohistiocytosis (HLH). High levels of IFNγ are also found in humans with HLH. High levels of IFNγ and three IFNγ-related chemokines, CXCL9, CXCL10, and CXCL11, have previously been reported to be observed in patients with active MAS, a form of secondary HLH that occurs in the setting of systemic juvenile idiopathic arthritis (sJIA) (see, e.g., Bracaglia C., Caiello I, De Graaf K., et al. Pediatric Rheumatology 2014,12(Suppl 1):O3). Indirect evidence in mice suggests that IFNγ is mostly produced in peripheral tissues and that blood concentrations may be relatively low.

[0099] The term macrophage activation syndrome (MAS) refers to a severe and potentially fatal complication of chronic inflammatory rheumatic diseases. It typically occurs in the setting of systemic juvenile idiopathic arthritis (sJIA), with 10–20% of patients developing this syndrome during the course of the disease. It can also occur, albeit more rarely, in systemic lupus erythematosus, Kawasaki disease, and other autoimmune and autoinflammatory disorders. In sJIA, MAS typically occurs during the active disease phase, including at disease onset. Infectious triggers can be identified in a high percentage of patients. Typical features of MAS include fever, splenomegaly, bleeding, and signs of liver, central nervous system, and kidney involvement, which can lead to multiple organ failure. Laboratory abnormalities include decreased white blood cells, platelets, and hemoglobin, hypertransaminases, markedly increased ferritin, and evidence of intravascular activation of the coagulation system (Ravelli, A., et al., Macrophage activation syndrome as part of systemic juvenile idiopathic arthritis: diagnosis, genetics, pathophysiology, and treatment. Genes Immun. 13(4): p. 289-98). MAS causes significant morbidity and mortality, accounting for a relevant proportion of deaths attributable to sJIA (Minoia, F., et al., Clinical features, treatment, and outcome of macrophage activation syndrome complicating systemic juvenile idiopathic arthritis: a multinational, multicenter study of 362 patients. Arthritis Rheumatol, 2014. 66(11): pp. 3160-9; Hashkes, PJ, et al., Mortality outcomes in pediatric rheumatology in the US. Arthritis Rheum, 2010. 62(2): pp. 599-608).A better understanding of the pathogenesis, together with the consequent identification of new therapeutic targets and possible development of targeted therapies, may lead to significant improvements in the management and outcome of MAS.

[0100] MAS shares the majority of clinical features and laboratory abnormalities with hemophagocytic lymphohistiocytosis (HLH), which is currently classified as secondary or reactive HLH (sec-HLH) (Jordan, MB, et al., How I treat hemophagocytic lymphohistiocytosis. Blood, 2011. 118(15): pp. 4041-52). The primary form of HLH (p-HLH) is caused by mutations in genes encoding proteins involved in granule exocytosis, including PRF1, UNC13D, STXBP2, STX11, RAB27A, and XIAP, which typically lead to defective cytotoxic activity of CD8+ lymphocytes and NK cells. According to the current classification, HLH is defined as secondary or reactive in the absence of an identifiable genetic cause and / or familial inheritance. Sec-HLH can occur in the absence of a demonstrable trigger or in the setting of infection, malignancy or rheumatic disease, the latter commonly referred to as MAS. The genetic basis of MAS is gradually becoming clearer, and numerous studies have demonstrated an association between MAS, and generally sec-HLH, and p-HLH with low-penetrance mutations or heterozygosity for mutations in the same causative gene (Kaufman, KM, et al., Whole-exome sequencing reveals overlap between macrophage activation syndrome in systemic juvenile idiopathic arthritis and familial hemophagocytic lymphohistiocytosis. Arthritis Rheumatol, 2014. 66(12): pp. 3486-95; Vastert, SJ, et al., Mutations in the perforin gene can be linked to macrophage activation syndrome in patients with systemic onset juvenile idiopathic arthritis. Rheumatology (Oxford), 2010).49(3): 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; and Zhang, M., et al., Genetic defects in cytolysis in macrophage activation syndrome. Curr Rheumatol Rep, 2014. 16(9): p. 439; and Bracaglia C, Sieni E, Da Ros M, et al. Mutations of familial hemophagocytic lymphohistiocytosis (FHL) related genes and abnormalities of cytotoxicity function tests in patients with macrophage activation syndrome (MAS) occurring in systemic juvenile idiopathic arthritis (sJIA). Pediatric Rheumatology 2014, 12(Suppl 1):P53). These similarities in genetic background between p-HLH and MAS further support a common pathogenesis mechanism.

[0101] Studies in patients with p-HLH and in mouse models of p-HLH support the hypothesis that defective cytotoxic activity and abnormalities in antigen-presenting cell (APC)-CD8+ T cell crosstalk result in incomplete silencing of the immune response and abnormal T cell activation. This leads to uncontrolled immune activation and proinflammatory cytokine production by T lymphocytes and macrophages, resulting in organ damage. Studies in animal models of p-HLH, conducted in perforin- and Rab27-deficient mice, point to the important role of interferon-gamma (IFNγ) produced by activated CD8+ T cells. In perforin-deficient mice, neutralization of IFNγ results in survival from an otherwise fatal syndrome, accompanied by reversal of biochemical and hematological abnormalities (Jordan, MB, et al., An animal model of hemophagocytic lymphohistiocytosis (HLH): CD8+ T cells and interferon gamma are essential for the disorder. Blood, 2004. 104(3): pp. 735-43; Pachlopnik Schmid, J., et al., Neutralization of IFNγ defeats heemophagocytosis in LCMV-infected perforin- and Rab27a-deficient mice. EMBO Mol Med, 2009. 1(2): pp. 112-24). In Rab27-deficient mice (where the disease is not fatal), neutralization of IFNγ results in a marked improvement in peripheral organ involvement, including the central nervous system (Pachlopnik 2009).High circulating IFN-γ levels have also been found in patients with HLH diagnosed according to the HLH 2004 diagnostic guidelines (My, LT, et al., Comprehensive analyses and characterization of heemophagocytic lymphohistiocytosis in Vietnamese children. Br J Haematol, 2010. 148(2): pp. 301-10; Takada, H., et al., Increased serum levels of interferon-gamma-inducible protein 10 and monokine induced by gamma interferon in patients with heemophagocytic lymphohistiocytosis. Clin Exp Immunol, 2003. 133(3): pp. 448-53; Tang, Y., et al., Early diagnostic and prognostic significance of a specific Th1 / Th2 cytokine pattern in children with heemophagocytic syndrome. Br J Haematol, 2008. 143(1): p. 84-91; Xu, XJ, 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 is not necessarily based on the presence of a genetic mutation. It should be noted that these studies included a variable but significant proportion of patients without a demonstrable genetic cause (ibid.).

[0102] The study provided herein is designed to evaluate the serum levels of IFNγ and three IFNγ-related chemokines in patients with active MAS, and their correlation with laboratory parameters of disease activity, in order to find biomarkers of IFNγ in vivo production.In particular, the circulating levels of IFNγ, CXCL9, CXCL10, CXCL11 and IL-6 were measured in patients with sJIA, and approximately 37% of patients (20 out of 54) had MAS at the time of sampling.As well as evaluating the correlation between IFNγ levels and CXCL9, CXCL10 and CXCL11 levels, the relationship between circulating levels and disease activity parameters was also evaluated.In some embodiments, the biomarker is total IFNγ level, which is useful as a pharmacodynamic biomarker.

[0103] As demonstrated herein, levels of IFNγ and three IFNγ-related chemokines, CXCL9, CXCL10, and CXCL11, were significantly elevated in patients with active MAS compared with those with active sJIA without MAS at the time of sampling. In active MAS, laboratory parameters of disease severity, such as ferritin, neutrophils, platelets, alanine aminotransferase, and lactate dehydrogenase, were significantly correlated with IFNγ and CXCL9, and to a lesser extent with CXCL10 and CXCL11; no correlation was observed with IL-6 levels. In patients with active sJIA without MAS, there was no significant correlation between laboratory parameters and cytokine levels. In active MAS, IFNγ levels were significantly correlated with CXCL9 levels, and to a lesser extent with CXCL10 levels, but not with CXCL11 levels.

[0104] High levels of IFNγ and CXCL9 present in patients with active MAS are significantly correlated with the test parameters of disease severity.In patients with active MAS, IFNγ and CXCL9 are closely correlated.Because CXCL9 has been shown to be induced only by IFNγ and not by other interferons (see, for example, Groom JR and Luster AD Immunol Cell Biol 2011, Feb;89(2):207-15), the findings disclosed herein demonstrate that CXCL9 is a biomarker of IFNγ production in MAS.

[0105] The studies provided herein also demonstrate that levels of IFNγ and chemokine (C-X-C motif) ligand 9 (CXCL9), CXL10, and CXCL11 (three chemokines known to be induced by IFNγ) are elevated in patients with sJIA with MAS, but not in patients with active sJIA without MAS. Furthermore, in these patients, levels of IFNγ, CXCL9, CXCL10, and CXCL11 correlated with laboratory parameters of disease severity.

[0106] Neutralizing anti-IFNγ antibodies of the present invention include, for example, the heavy chain complementarity-determining regions (CDRs) shown in Table 1A below, the light chain CDRs shown in Table 1B below, and combinations thereof. The amino acids encompassing the complementarity-determining regions (CDRs) as defined by Chothia et al., 1989; E.A. Kabat et al., 1991 are highlighted below in underlined italic text. (See Chothia, C., et al., Nature 342:877-883 (1989); Kabat, E.A., et al., Sequences of Protein of Immunological Interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)).

[0107] Table 1A. VH CDR sequences from antibody clones that bind and neutralize IFNγ TIFF2025118898000015.tif161150

[0108] Table 1B. VL CDR sequences from antibody clones that bind and neutralize IFNγ TIFF2025118898000016.tif161151

[0109] Exemplary antibodies of the invention include, for example, the anti-IFNγ antibodies described in PCT Publication No. WO 2006 / 109191, the contents of which are incorporated herein by reference in their entirety.

[0110] Exemplary antibodies of the invention include, for example, the antibody designated herein as NI-0501, which binds to human IFNγ. The heavy, light, variable heavy (VH), and variable light (VL) chain sequences of the NI-0501 antibody are shown below, with the CDR sequences underlined in the VH and VL amino acid sequences. TIFF2025118898000017.tif134147TIFF2025118898000018.tif216148

[0111] Suitable anti-IFNγ antibodies include those described in US Pat. No. 7,700,098, which is incorporated herein by reference in its entirety. Some exemplary antibodies include ARC1.2R3P2_A6 ("A6"), ARC1.2R3P2_B4 ("B4"), ARC1.2R3P2_B9 ("B9"), ARC1.2R3P2_C9 ("C9"), ARC1.2R3P2_C10 ("C10"), ARC1.2R3P2_D3 ("D3"), ARC1.2R3P2_D6 ("D6"), ARC1.2R3P2_D8 ("D8"), ARC1.2R3P2_E1 ("E1"), ARC1.2R3P2_F8 ("F8"), ARC1.2R3P2_F9 ("F9"), ARC1.2R3P2_G7 ("G7"), ARC1.2R3P2_G9 ("G9"), and ARC1.2R3P2_G10 ("G10") and antibodies therein.

[0112] The sequences of these antibodies are shown below. TIFF2025118898000019.tif122147TIFF2025118898000020.tif221147TIFF20251188980 00021.tif227147TIFF2025118898000022.tif221147TIFF2025118898000023.tif220147 TIFF2025118898000024.tif226147TIFF2025118898000025.tif226147TIFF20251188980 00026.tif226147TIFF2025118898000027.tif226147TIFF2025118898000028.tif208147

[0113] In some embodiments, the IFNγ antibody is formatted as an IgG isotype. In some embodiments, the IFNγ antibody is formatted as an IgG1 isotype.

[0114] In some embodiments, the IFNγ antibody of the present invention specifically binds to human and / or cynomolgus monkey IFNγ, wherein the antibody binds to the same epitope as the NI-0501 antibody, A6 antibody, B4 antibody, B9 antibody, C9 antibody, C10 antibody, D3 antibody, D6 antibody, D8 antibody, E1 antibody, F8 antibody, F9 antibody, G7 antibody, G9 antibody, and / or G10 antibody.

[0115] Definition: Scientific and technical terms used in connection with the present invention have meanings commonly understood by those of ordinary skill in the art unless otherwise defined. Furthermore, unless otherwise required by context, singular terms include plurals and plural terms include the singular. Generally, the nomenclature utilized in connection with cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein, and the techniques thereof, are those well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The techniques and procedures described above are generally performed according to conventional methods well known in the art and as described in the various general and more detailed references cited and discussed throughout this specification. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). The nomenclature utilized in analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, and the laboratory procedures and techniques thereof, are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, drug preparation, formulation and delivery, and treatment of patients.

[0116] Administration of anti-IFNγ antibody It will be appreciated that administration of therapeutic entities according to the present invention will be administered with suitable carriers, excipients, and other agents. These other agents may be incorporated into formulations to provide improved transfer, delivery, tolerance, etc. Many suitable formulations can be found in formularies known to all pharmaceutical chemists, such as Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 by Blaug and Seymour. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., Lipofectin™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsions, carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the above mixtures may be suitable in treatments and therapies according to the present invention, provided that the active ingredients in the formulation are not inactivated by the formulation and the formulation is physiologically compatible and tolerable to the route of administration.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 further information related to formulations, excipients, and carriers well known to pharmaceutical chemists.

[0117] Efficacy of treatment is determined with any known method for diagnosing or treating the particular immune-related disorder. Alleviation of one or more symptoms of the immune-related disorder indicates that the antibody confers clinical benefit.

[0118] The antibodies of the present invention, including polyclonal antibodies, monoclonal antibodies, humanized antibodies, and fully human antibodies, can be used as therapeutic agents. Such agents will generally be used to treat or prevent diseases or conditions associated with the abnormal expression or activation of a given target in a subject. An antibody preparation, preferably one with high specificity and high affinity for its target antigen, is administered to a subject and generally has an effect due to its binding to the target. Administration of the antibody can block, inhibit, or interfere with the signal transduction function of the target. Administration of the antibody can block, inhibit, or interfere with the binding of the target to an endogenous ligand to which the target naturally binds.

[0119] A therapeutically effective amount of an antibody of the present invention generally relates to the amount necessary to achieve a therapeutic goal. As discussed above, this may be the binding interaction of the antibody with its target antigen, which may interfere with the function of the target. The amount required for administration will further depend on the binding affinity of the antibody for its specific antigen and the rate at which the antibody dissipates from the free volume of the subject to which it is administered. A typical range for a therapeutically effective dosage of an antibody or antibody fragment of the present invention may be, by way of non-limiting example, about 0.1 mg / kg body weight to about 50 mg / kg body weight. Typical dosing frequencies may range, for example, from twice daily to once weekly.

[0120] The antibody or fragment thereof of the present invention can be administered to treat various diseases and disorders in the form of a pharmaceutical composition. The principles and considerations involved in the preparation of such compositions, as well as guidance in the selection of components, are provided, for example, in Remington: The Science and Practice of Pharmacy 19th ed. (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.

[0121] The formulations can also contain more than one active compound, e.g., an anti-IFNγ antagonist, as needed for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can include an agent that enhances its function, such as a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the intended purpose.

[0122] In one embodiment, the active compound, for example, an anti-IFNγ antagonist, is administered in combination therapy, i.e., in combination with one or more additional agents useful for treating a pathological condition or disorder. The term "in combination" in this context means that the agents are administered substantially contemporaneously, simultaneously, or sequentially. When administered sequentially, the first of the two compounds is preferably still detectable at effective concentrations at the site of treatment at the start of administration of the second compound.

[0123] For example, the combination therapy can include one or more neutralizing anti-IFNγ antibodies of the invention co-formulated and / or co-administered with one or more additional therapeutic agents, such as one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and / or cytotoxic or cytostatic agents, as described in more detail below. Such combination therapy may advantageously allow for the administration of lower dosages of the therapeutic agents, thus avoiding potential toxicities or complications associated with various monotherapies.

[0124] When antibody fragments are used, the smallest inhibitory fragment that specifically binds to the target protein's binding domain and / or interferes with or otherwise antagonizes IFNγ signaling is preferred. For example, peptide molecules that retain the ability to bind to the target protein sequence can be designed based on the sequence of the antibody's variable region. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology (see, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). Formulations can also contain more than one active compound, preferably those with complementary activities that do not adversely affect each other, as needed for the particular indication being treated. Alternatively, or in addition, the composition can include an agent that enhances its function, such as a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitor. Such molecules are suitably present in combination in amounts effective for the intended purpose.

[0125] The levels of CXCL9 and other biomarkers are detected using any of a variety of standard detection techniques. A detection agent can be used to detect the presence of a given target (or its protein fragment) in a sample. In some embodiments, the detection agent contains a detectable label. In some embodiments, the detection agent is an antibody (or a fragment thereof) or a probe. In some embodiments, the agent or probe is labeled. The term "labeling," in reference to a probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, and indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody, and end-labeling of a DNA probe with biotin so that the DNA probe can be detected with fluorescently labeled streptavidin.

[0126] The term "biological sample" is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids present within a subject. Thus, blood and blood fractions or components, including serum, plasma, or lymph, are included within the scope of the term "biological sample." The biological fluid may be a fluid isolated from anywhere within a subject's body, preferably a peripheral location, including, but not limited to, blood, plasma, serum, synovial fluid, urine, sputum, spinal fluid, cerebrospinal fluid, pleural fluid, respiratory, intestinal, and genitourinary tract fluids, saliva, organ system fluids, ascites, tumor cyst fluid, amniotic fluid, and combinations thereof. Biological samples also include experimentally isolated fractions of any of the aforementioned fluids. Biological samples also include solutions or mixtures containing homogenized solid materials, such as feces, tissues, and biopsy samples. The detection methods of the present invention can be used in vitro and in vivo to detect analytes such as mRNA, protein, or genomic DNA in biological samples. For example, in vitro techniques for detecting analyte mRNA include Northern hybridization and in situ hybridization. In vitro techniques for detecting analyte protein include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence. In vitro techniques for detecting analyte genomic DNA include Southern hybridization. Procedures for performing immunoassays are described, for example, in "ELISA: Theory and Practice: Methods in Molecular Biology", Vol. 42, J.R. Crowther (Ed.) Human Press, Totowa, NJ, 1995; "Immunoassay", E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and "Practice and Theory of Enzyme Immunoassays", P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985.Furthermore, in vivo techniques for detecting an analyte protein include introducing into a subject a labeled anti-analyte protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in the subject can be detected by standard imaging techniques.

[0127] The compositions and methods are useful in treating any of a variety of disorders associated with interferon-gamma (IFNγ) expression and / or activity, including abnormal IFNγ expression and / or activity. The compositions and methods of the present disclosure are useful in treating hemophagocytic lymphohistiocytosis (HLH). HLH is a rare, serious, and life-threatening disease of pathological immune activation characterized by clinical signs and symptoms of extreme inflammation (fever, splenomegaly, cytopenia, coagulopathy), leading to the development of abnormal immune-mediated pathology, which, through tissue damage, can ultimately 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 (hereditary / familial) HLH and secondary HLH.

[0128] Primary HLH is a heterogeneous autosomal recessive disorder that is primarily present in infancy and early childhood, with an estimated prevalence in Europe of 1 / 50,000 live births (Janka GE: Familial hemophagocytic lymphohistiocytosis. Eur. J. Pediatr. 1983, 140:221-230). If untreated, the disease is invariably fatal, with a median survival time of less than 2 months after symptom onset (Filipovich AH: Hemophagocytic lymphohistiocytosis (HLH) and related disorders. Hematology Am Soc Hematol Educ Program 2009:127-131).

[0129] All genetic defects in primary HHL affect genes involved in the cytotoxic pathways of NK cells and / or cytotoxic lymphocytes, which are required to eliminate activated macrophages and encode proteins for perforin synthesis, cytolytic granule maturation, granule exocytosis, and release granule exocytosis or function (Filipovich, A., K. McClain, and A. Grom. 2010. Histiocytic disorders: recent insights into pathophysiology and practical guidelines. Biol. Blood Marrow Transplant. 16(1 Suppl):S82-S89). In approximately 20-40% of patients with primary HLH, the cytotoxic dysfunction that characterizes the HLH syndrome results from mutations in the gene (PRF1) encoding perforin, a cytotoxic protein of cytotoxic granules that is a key regulator of T cell- and natural killer cell-mediated cytolysis. In approximately 10% of patients, this disease is caused by mutations in the UNC13D gene, which encodes a protein involved in the release of perforin into target cells.In addition, some immunodeficiency syndromes, such as Griscelli syndrome type 2 (GS-2) and Chediak-Higashi syndrome (CHS), frequently show HLH (Janka GE, Lehmberg K: Hemophagocytic lymphohistiocytosis: pathogenesis and treatment. Hematology Am Soc Hematol Educ Program 2013, 2013:605-611).

[0130] Secondary forms of HLH can occur during the course of infectious diseases, autoimmune / rheumatic diseases, or in association with malignancies. Secondary forms present with the same signs and symptoms as primary HLH and can be equally severe.

[0131] The compositions and methods of the present disclosure are useful in the treatment of secondary HLH.The compositions and methods of the present disclosure are useful in the treatment of macrophage activation syndrome (MAS).

[0132] MAS is a severe, potentially life-threatening complication of rheumatic diseases caused by excessive activation and expansion of T lymphocytes and macrophages. Uncontrolled expansion of these immune cells leads to marked hypercytokinemia and a hyperinflammatory state with fever, cytopenia, hepatosplenomegaly, liver dysfunction, coagulation abnormalities, and hyperferritinemia, which can progress to multiple organ failure and death (Schulert GS, Grom AA: Pathogenesis of macrophage activation syndrome and potential for cytokine-directed therapies. Annu. Rev. Med. 2015, 66:145-159).

[0133] MAS is classified as a secondary or acquired form of HLH due to its strong clinical and pathological similarity with HLH. In fact, it has recently been demonstrated that the majority of patients with MAS have impaired NK and perforin function tests, and a significant number of MAS patients show 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).

[0134] MAS occurs most frequently in patients with sJIA, more rarely in patients with systemic lupus erythematosus (SLE), and, even more rarely, has also been described in patients with vasculitis, particularly Kawasaki disease. Approximately 7-17% of patients with SJIA develop overt MAS (Sawhney S, Woo P, Murray KJ: Macrophage activation syndrome: a potentially fatal complication of rheumatic disorders. Arch. Dis. Child. 2001, 85:421-426; Moradinejad MH, Ziaee V: The incidence of macrophage activation syndrome in children with rheumatic disorders. Minerva Pediatr. 2011, 63:459-466), and some evidence suggests that asymptomatic MAS may be seen in as many as one-third of patients with active systemic disease (Behrens EM, Beukelman T, Paessler M, Cron RQ: Occult macrophage activation syndrome in patients with systemic juvenile idiopathic arthritis. J. Rheumatol. 2007, 34:1133-1138).

[0135] Because MAS can be fatal, timely diagnosis and immediate therapeutic intervention are essential for the appropriate management of this disease. With a reported mortality rate of 20-30%, MAS remains the leading cause of death in pediatric rheumatology (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 / nrrheum.2015.179).

[0136] Different sets of criteria have been proposed for the diagnosis of MAS in patients with sJIA. The HLH-2004 diagnostic guidelines, originally developed for primary (hereditary) forms of HLH (Henter J, Horne A, Arico M, Egeler RM, et al: HLH-2004: Diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer 2007, 48:124-131), were initially recommended. However, they present several limitations and may not apply to patients with sJIA. For example, criteria such as subthreshold cytopenias and hypofibrinogenemia required by HLH-2004 are only evident in the later stages of MAS, as these patients often have increased white blood cell and platelet counts and elevated serum levels of fibrinogen as part of the sJIA inflammatory response (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 a significant proportion of patients presenting with MAS (Minoia F, Davi S, Horne A, Demirkaya E, et al: Clinical features, treatment, and outcome of macrophage activation syndrome complicating systemic juvenile idiopathic arthritis: a multinational, multicenter study of 362 patients. Arthritis & rheumatology (Hoboken, NJ) 2014, 66:3160-3169). Furthermore, hemophagocytosis, NK cell activity, and sCD25 are not routinely assessed in the setting of MAS.

[0137] An alternative approach is based on the application of Preliminary Diagnostic Guidelines (PDGs) for sJIA with MAS, which were developed through the analysis of a cohort of patients with MAS compared with a group of patients with the inflammatory phase (flare) of sJIA1.

[0138] Recently, the HLH-2004 diagnostic guidelines and preliminary diagnostic guidelines for sJIA-associated MAS were compared for their ability to distinguish between sJIA / MAS and sJIA (without MAS) and systemic infection in a large patient population (Davi S, Minoia F, Pistorio A, Horne A, et al: Performance of current guidelines for diagnosis of macrophage activation syndrome complicating systemic juvenile idiopathic arthritis. Arthritis & rheumatology (Hoboken, NJ) 2014, 66:2871-2880). Although with some limitations due to its retrospective nature, this study appears to indicate that the preliminary MAS guidelines achieve the best balance between sensitivity and specificity, as well as the best agreement with the diagnoses made by primary care physicians. The sensitivity of the HLH-2004 criteria set was <30%. In any case, the proportion of patients who meet each PDG criterion varies widely, and some clinical features (e.g., CNS dysfunction and bleeding) appear in the later stages of MAS, suggesting that their sensitivity in early MAS may be low (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).

[0139] More recently, a diagnostic score (HScore) was developed and validated in a retrospective cohort of 312 patients, 162 of whom were determined to have reactive hemophagocytic syndrome (Fardet L, Galicier L, Lambotte O, Marzac C, Aumont C, Chahwan D, Coppo P, Hejblum G: Development and validation of the HScore, a score for the diagnosis of reactive hemophagocytic syndrome. Arthritis & rheumatology (Hoboken, NJ) 2014, 66:2613-2620). Nine variables (three clinical [i.e., known underlying immunosuppression, hyperthermia, organomegaly], five biological [i.e., triglycerides, ferritin, serum glutamic oxaloacetic transaminase, fibrinogen levels, and cytopenias], and one cytological [i.e., hemophagocytic features of bone marrow aspirate]) were retained in HScore, and the probability of having hemophagocytic syndrome ranged from <1% for an HScore of ≤90 to >99% for an HScore of ≥250.

[0140] Until final agreement on valid diagnostic criteria for MAS is reached, clinical diagnosis by experienced clinicians remains critical in distinguishing MAS from conditions that show overlapping features, such as the inflammatory phase of SJIA or sepsis-like syndrome.

[0141] Currently, there are no approved medications for the treatment of MAS. High-dose glucocorticoids are usually the first-line treatment for MAS. In patients who do not respond to glucocorticoids, cyclosporine A (CsA) has been proposed as an additional treatment (Stephan JL, Kone-Paut I, Galambrun C, Mouy R, Bader-Meunier B, Prieur AM: Reactive heemophagocytic syndrome in children with inflammatory disorders. A retrospective study of 24 patients. Rheumatology (Oxford, England) 2001, 40:1285-1292).

[0142] Administration of etoposide, part of the HLH-94 treatment protocol developed to treat pHLH, is also considered in patients who fail high-dose glucocorticoids. However, the drug's potential toxicity remains a major concern. Other current first-line HLH treatments include dexamethasone. However, treatments such as etoposide and / or dexamethasone are myelosuppressive and / or broadly immunosuppressive. Currently, there is no standard of care for second-line HLH treatment, and treatments such as alemtuzumab / ATG are highly immunosuppressive, and survival is thought to be very poor with these treatments.

[0143] The utility of biologics that inhibit the IL-1, IL-6R, or TNFα pathways in the treatment of MAS remains unclear. Biologics that inhibit these pathways have been reported to be effective in isolated cases, but few patients develop MAS in the setting of these treatments (Stern A, Riley R, Buckley L: Worsening of macrophage activation syndrome in a patient with adult onset Still's disease after initiation of etanercept therapy. J Clin Rheumatol 2001, 7:252-256; Ramanan AV, Schneider R: Macrophage activation syndrome following initiation of etanercept in a child with systemic 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 trials of canakinumab in systemic juvenile idiopathic arthritis. N. Engl. J. Med. 2012, 367:2396-2406), as well as reports of patients who do not respond to these treatments, indicate that inhibition of IL-1, IL-6R, or TNFα does not provide complete protection from the development of MAS or effective treatment of the full blown syndrome.

[0144] A large, retrospective, multicenter study examined the clinical, laboratory, and histopathological features of MAS / sJIA, as well as current treatment and outcomes, in a total of 362 patients (Minoia F, Davi S, Horne A, Demirkaya E, et al: Clinical features, treatment, and outcome of macrophage activation syndrome complicating systemic juvenile idiopathic arthritis: a multinational, multicenter study of 362 patients. Arthritis & rheumatology (Hoboken, NJ) 2014, 66:3160-3169). Approximately half of the patients developed MAS in the setting of active sJIA or during the inflammatory phase of sJIA, and 30% of these patients presented at disease onset. An infectious trigger was identified in one-third of patients. Of the 24 patients for whom the type of infection was reported, EBV was the most common causative agent (25%). In 11 patients (3.8%), MAS was considered to be related to treatment side effects: 8 of these were treated with biologic agents targeting the IL-6 (N = 4), IL-1 (N = 3), or TNFα (N = 1) pathways. Nearly all patients received glucocorticoids. Cyclosporine, biologic agents, and etoposide were given in 61%, 15%, and 12% of patients, respectively.

[0145] Therefore, identifying effective treatment regimens for MAS is an area of high unmet medical need. More than 50% of patients with sJIA and MAS do not respond to systemic glucocorticoids alone or may require long-term treatment at high doses with significant morbidity. When patients do not respond to glucocorticoids, sufficient evidence-based data are not available regarding the effectiveness of additional treatments such as CsA or etoposide. The course of MAS can rapidly become irreversible and lead to a fatal outcome. Current data suggest that the mortality rate for sJIA-associated MAS is 8%, and approximately one-third of patients require intensive care unit (ICU) admission. Recent findings regarding the central role of IFNγ in the pathogenesis of this disease suggest that IFNγ blockade may represent a novel therapeutic target.

[0146] The compositions (including NI-0501 compositions) and methods of the present disclosure offer advantages over current therapies for primary and secondary HLH.

[0147] MAS and HLH are characterized by persistent immune cell activation and a concomitant cytokine storm of proinflammatory cytokines, with excessive production of IFNγ, TNFα, IL-1, and IL-6 (Henter JI, Elinder G, Soder O, Hansson M, et al: Hypercytokinemia in familial hemophagocytic lymphohistiocytosis. Blood 1991, 78:2918-2922; Imashuku S, Hibi S, Fujiwara F, Todo S: Hyper-interleukin (IL)-6-nemia in heemophagocytic 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-γ. Rheumatology (Oxford) 2015).During the last few years, HLH (Jordan MB, Hildeman D, Kappler J, Marrack P: An animal model of hemophagocytic lymphohistiocytosis (HLH): CD8+ T cells and interferon gamma are essential for the disorder. Blood 2004, 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, Accumulating evidence supports a central role for IFNγ in the pathogenesis of both macrophage activation syndrome (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).

[0148] For primary HLH, perforin knockout mice are considered an appropriate model because, once infected with LCMV, they exhibit all the diagnostic and many clinical and laboratory features of this human disease. The HLH-like disease they exhibit is dependent on CD8+ T cells and IFNγ production in response to antigen stimulation (Imashuku S, Hibi S, Fujiwara F, Todo S: Hyper-interleukin (IL)-6-anemia in heemophagocytic lymphohistiocytosis. Br. J. Haematol. 1996, 93:803-807). Neutralization of high circulating levels of IFNγ by administration of anti-IFNγ antibodies not only reversed clinical and laboratory abnormalities but also dramatically improved survival. In contrast, removal of many other cytokines had no effect on survival (Imashuku S, Hibi S, Fujiwara F, Todo S: Hyper-interleukin (IL)-6-naemia in heemophagocytic 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).Further reinforcing the importance of IFNγ in HLH is the high circulating IFNγ levels observed in these patients (Henter JI, Elinder G, Soder O, Hansson M, et al.: Hypercytokinemia in familial hemophagocytic lymphohistiocytosis. Blood 1991, 78:2918-2922; Xu X, Tang Y, Song H, Yang S, et al.: Diagnostic accuracy of a specific cytokine pattern in hemophagocytic lymphohistiocytosis in children. J. Pediatr. 2012, 160:984-90.e1). In a series of 71 patients monitored from HLH diagnosis through treatment and follow-up, IFNγ levels exceeded the upper limit of normal (17.3 pg / mL) in all patients, with 53.5% having levels above 1000 pg / mL. It has also been reported that IFNγ levels rise early and rapidly and can fall from >5000 pg / mL to normal in 48 hours with effective treatment of HLH.

[0149] Two animal models of secondary HLH were investigated in the context of the NI-0501 development program to elucidate the potential pathogenic role of IFNγ. First, in a mouse model mimicking infection-driven HLH, repeated administration of CpG induced hypercytokinemia via TLR9 activation, resulting in clinical (e.g., weight loss, splenomegaly) and laboratory (e.g., cytopenia, hyperferritinemia) features of HLH33. When IFNγ was neutralized by administration of anti-IFNγ antibodies, the clinical and laboratory features of the disease were reversed. IFNγ neutralization was also shown to be complete in relevant target tissues such as the liver and spleen. Interestingly, administration of anti-IFNγ antibodies revealed IFNγ levels 500-2,000-fold higher than those measured in the blood, likely reflecting IFNγ production in tissues. Two IFNγ-inducible chemokines (CXCL9 and CXCL10) were upregulated in both the blood and liver after TLR9 stimulation, and a significant correlation was observed between serum levels of IFNγ and serum concentrations of CXCL9 and CXCL10. Neutralization of IFNγ induced a significant decrease in serum CXCL9 and CXCL10 and their mRNA levels in the liver (Buatois V, Chatel L, Cons L, Lory S, et al: IFNγ drives disease in the TLR9-mediated secondary HLH in mice: rationale for a new therapeutic target in secondary HLH, in preparation).

[0150] Second, an animal model of IL-6 transgenic mice expressing high levels of IL-6 has been studied to mimic the condition of patients with sJIA, a rheumatic disease most frequently associated with secondary forms of HLH. When triggered with Toll-like receptor (TLR) ligands, increased mortality, increased inflammatory cytokine production, and hyperactivation of inflammatory signaling pathways were observed. Furthermore, these mice exhibited reduced platelet and neutrophil counts and increased levels of sCD25, ferritin, and LDH, resembling many of the features typically present in patients with MAS (Strippoli R, Carvello F, Scianaro R, De Pasquale L, et al: Amplification of the response to Toll-like receptor ligands by prolonged exposure to interleukin-6 in mice: implication for the pathogenesis of macrophage activation syndrome. Arthritis Rheum. 2012, 64:1680-1688). When IFNγ is neutralized in these mice by administration of anti-IFNγ antibodies, survival is significantly improved and laboratory parameters are restored (Prencipe G et al, manuscript in preparation).

[0151] Similar evidence has been gathered more recently in observational studies conducted in patients with secondary forms of HLH secondary to infection or of unknown origin (pHLH was ruled out by normal cytotoxic activity, lack of mutations in known genes causing pHLH, and lack of family history), or with MAS occurring in the setting of sJIA.

[0152] In 14 patients with secondary HLH (seven of whom had an identifiable underlying infection), serum samples were analyzed during active, end-stage disease and during disease remission. IFNγ, CXCL9, and CXCL10 levels were significantly higher in active disease compared with disease remission (IFNγ: 34.7 vs. <3.5 pg / ml; CXCL9: 33598 vs. 745 pg / ml; CXCL10: 4420 vs. 132 pg / ml; median values). IFNγ levels correlated significantly with CXCL9 levels (p=0.0018) and, to a lesser extent, with CXCL10 levels (p=0.014). Levels of IFNγ and chemokines (especially CXCL9) significantly correlated with parameters of disease severity such as neutrophil and platelet counts, ferritin and ALT, supporting the pathogenic role of IFNγ in secondary HLH and the potential use of chemokines as suitable biomarkers of this disease (Buatois V, Chatel L, Cons L, Lory S, et al: IFNγ drives disease in the TLR9-mediated secondary HLH in mice: rationale for a new therapeutic target in secondary HLH).

[0153] Similar findings were observed in patients with MAS, which occurs in patients with sJIA. Serum concentrations of IFNγ, IFNγ-inducible chemokines (CXCL9, CXCL10, CXCL11), and IL-6 were measured in 54 patients with sJIA, 20 of whom had MAS. IL-6 levels were comparable in patients with end-stage MAS at the time of sampling and in patients with active sJIA but no MAS. In contrast, circulating IFNγ and chemokine levels, particularly for CXCL9, were significantly higher in MAS patients, with median levels approximately 15-fold higher in patients with active sJIA but no MAS (13,392 vs. 837 pg / mL; p=0.005). Of note, significant correlations between CXCL9 levels and typically abnormal parameters such as ferritin (p = 0.041), neutrophil (p = 0.010) and platelet (p = 0.022) counts, ALT (p = 0.044), and LDH (p = 0.013) were demonstrated only in patients with MAS. IFNγ levels also correlated with laboratory parameters of disease severity, with the exception of LDH, which did not achieve statistical significance (Bracaglia et al., manuscript in preparation).

[0154] Collectively, these data provide a solid rationale for neutralization of IFNγ as a targeted therapy for secondary HLH and MAS and for its investigation in the clinical setting.

[0155] The disclosed compositions (including NI-0501 compositions) and methods offer advantages over current therapies for sJIA. For example, the disclosed compositions (including NI-0501 compositions) and methods are useful in treating MAS / sHLH in sJIA patients, with the primary goal of achieving MAS remission.

[0156] The rationale for identifying this patient population as benefiting from treatment with NI-0501 and for evaluating its efficacy in MAS / sHLH is based on multiple factors. First, preclinical data obtained in relevant animal models of MAS in sJIA demonstrated that IFNγ neutralization significantly improved survival and reversed changes in laboratory parameters. Second, observational data in patients with MAS / sHLH demonstrate the presence of high levels of IFNγ and, more importantly, highly elevated levels of the IFNγ-inducible chemokines CXCL9, CXCL10, and CXCL11. Third, in patients with MAS / sHLH, concentrations of IFNγ and CXCL9 significantly correlate with disease parameters such as ferritin, platelet count, and transaminases. Second, confirming the observations made in healthy volunteers, a favorable tolerability profile and a lack of associated safety concerns were observed in pHLH patients in a previous study in which all administered infusions were well tolerated. No infections caused by pathogens known to be favored by IFNγ neutralization were reported, and none of the infections that occurred in a subset of pHLH patients were considered to be related to NI-0501 treatment, but rather to immune status, disease duration, and previous or concurrent treatments. Fifth, preliminary data from a previous clinical study indicate a favorable impact on disease parameters, with a significant onset of effect within the first few days of treatment: typical clinical signs and symptoms of HLH began to improve rapidly after the first dose of NI-0501 (fever within hours, splenomegaly / hepatomegaly within days); and of 18 evaluable patients at the cutoff point, treatment with NI-0501 enabled 10 patients to proceed to HSCT. Next, evidence from PK modeling and simulation approaches demonstrates a predictable pharmacokinetic profile of NI-0501, and that IFNγ neutralization is achieved and maintained. Finally, conventional therapy (e.g., CsA) can be initiated immediately without the need for a drug holiday if NI-0501 does not adequately control the disease.

[0157] In conclusion, based on preclinical and clinical evidence, there is a strong rationale for neutralizing IFNγ in MAS / sHLH secondary to rheumatic diseases, and preliminary data in patients with pHLH indicate a favorable benefit-risk profile of NI-0501, with significant improvement towards normalization of HLH features.

[0158] NI-0501 therefore represents an innovative and effective therapeutic approach in the management of complications of this serious and life-threatening rheumatic disease, potentially limiting the side effects from long-term, high-dose glucocorticoid treatment.

[0159] Pharmaceutical Compositions The antibodies or soluble chimeric polypeptides of the present invention (also referred to herein as "active compounds"), as well as their derivatives, fragments, analogs, and homologs, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the antibodies or soluble chimeric polypeptides and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles, 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. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0160] The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous administration can contain the following components: a sterile diluent, e.g., water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent, e.g., benzyl alcohol or methylparaben; an antioxidant, e.g., ascorbic acid or sodium bisulfite; a chelating agent, e.g., ethylenediaminetetraacetic acid (EDTA); a buffer, e.g., acetate, citrate, or phosphate, and an agent for adjusting tonicity, e.g., sodium chloride or dextrose. pH can be adjusted with acids or bases, e.g., hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes, or multiple-dose vials.

[0161] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, such as sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin in the composition.

[0162] Sterile injectable solution can be prepared by incorporating the active compound in the required amount in suitable solvent with one or combination of the above-listed components as needed, and then sterilize by filtration.Generally, dispersion is prepared by incorporating the active compound into the sterile vehicle that contains the basic dispersion medium and the other components that are required from the above-listed components.For the sterile powder that is used to prepare sterile injectable solution, preparation method is vacuum drying and freeze-drying, thereby making the powder of active ingredient and any additional desired components from the solution that has been previously sterilized and filtered.

[0163] Oral compositions generally contain an inert diluent or an edible carrier. These can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with an excipient and used in the form of tablets, lozenges, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, in which case the compound in the fluid carrier is orally applied, swirled in the mouth, and then expectorated or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches, etc. can 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 disintegrant such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0164] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0165] Systemic administration can also be by transmucosal or transdermal means.For transmucosal or transdermal administration, penetrants suitable for the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and for example, for transmucosal administration, include surfactants, bile salts and fusidic acid derivatives.Transmucosal administration can be achieved by using nasal sprays or suppositories.For transdermal administration, active compound is formulated into ointments, salves, gels or creams that are generally known in the art.

[0166] The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0167] In one embodiment, the active compound is prepared with a carrier that protects the compound from rapid elimination from the body, such 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 clear to those skilled in the art. Materials can also be commercially obtained from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells containing monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0168] For ease of administration and uniform dosage, it is particularly advantageous to prepare oral or parenteral compositions in dosage unit form.Dosage unit form used herein refers to a physically separate unit that is suitable as a unit dosage for the subject to be treated; each unit contains a predetermined amount of active compound that is calculated to produce desired therapeutic effect together with required pharmaceutical carrier.The specification of dosage unit form of the present invention is determined by and directly depends on the specific characteristics of active compound and the specific therapeutic effect that is achieved, and the inherent limitations in the technical field of preparing this active compound for treating individuals.

[0169] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0170] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. [Example]

[0171] Example 1 CXCL9 levels as a biomarker for IFNγ production in macrophage activation syndrome (MAS) The study presented in this example was designed to evaluate serum levels of IFNγ and three IFNγ-related chemokines and their correlation with laboratory parameters of disease activity in patients with active MAS in order to search for potential biomarkers of IFNγ in vivo production.

[0172] Circulating levels of IFNγ, CXCL9, CXCL10, CXCL11, and IL-6 were measured using Luminex multiplexing assays in patients with sJIA (n=54), 20 of whom had MAS at the time of sampling. The correlation between IFNγ levels and CXCL9, CXCL10, and CXCL11 levels, as well as the relationship between these circulating levels and disease activity parameters, were evaluated.

[0173] Levels of IFNγ and three IFNγ-related chemokines (CXCL9, CXCL10, and CXCL11) were significantly elevated in active MAS compared with active sJIA without MAS at the time of sampling (all p values < 0.005). In active MAS, laboratory parameters of disease severity (ferritin, neutrophils, platelets, alanine aminotransferase, and lactate dehydrogenase) correlated significantly with IFNγ and CXCL9, and to a lesser extent with CXCL10 and CXCL11; no correlation was found with IL-6 levels. In patients with active sJIA without MAS, there was no significant correlation between laboratory parameters and cytokine levels, as shown in Table 7 below. In active MAS, IFNγ levels correlated significantly with CXCL9 levels (r = 0.69; r 2 =0.47; p=0.001), and to a lesser extent correlated with levels of CXCL10 (r=0.53; r 2 =0.28; p=0.015), but did not correlate with CXCL11 levels (r=-0.04; p=0.886).

[0174] Table 7. Correlations of disease activity laboratory parameters with IFNγ, CXCL9, CXCL10, CXCL11, and IL-6 in patients with MAS and active sJIA. TIFF2025118898000029.tif66153N = neutrophil count; PLT = platelet count; ALT = alanine aminotransferase; 1 = median (IQR); r* = Spearman r

[0175] High levels of IFNγ and CXCL9 present in patients with active MAS correlate significantly with laboratory parameters of disease severity. IFNγ and CXCL9 are closely correlated in patients with active MAS. CXCL9 has been shown to be induced only by IFNγ but not by other interferons (see, e.g., Groom JR and Luster AD Immunol Cell Biol 2011, Feb;89(2):207-15). These findings demonstrate that CXCL9 is a biomarker for IFNγ production in MAS.

[0176] Example 2 Correlation between CXCL9 and IFNγ levels in patients with primary hemophagocytic lymphohistiocytosis (HLH) The studies presented in this example are derived from an ongoing phase 2 pilot study in patients with primary HLH who received the NI-0501 antibody, as well as patients who received the NI-0501 antibody in compassionate use.

[0177] As shown in Figure 1, serum levels of CXCL9 and IFNγ were measured by Luminex and Meso Scale Discovery (MSD) technology, respectively, in samples obtained from six primary HLH patients and three compassionate use patients. Correlations were examined between CXCL9 and total IFNγ concentrations. Statistical tests were performed using the Spearman test, and p values were obtained.

[0178] As shown in Figure 2, pre-treatment serum levels of CXCL9 and IFNγ were measured by Luminex and MSD technology, respectively, in samples obtained from six primary HLH patients and three compassionate use patients. Correlations were examined between CXCL9 and total IFNγ concentrations. Statistical testing was performed using the Spearman test, and p values were obtained.

[0179] Example 3 Correlation between CXCL9 and IFNγ levels in patients with secondary hemophagocytic lymphohistiocytosis (HLH) The studies presented in this example are derived from observational studies in patients with secondary HLH who received NI-0501 antibody, as well as patients who received NI-0501 antibody in compassionate use.

[0180] In particular, these patients have systemic juvenile idiopathic arthritis (sJIA) that has developed macrophage activation syndrome (MAS, a form of secondary HLH). For these patients, there is also a correlation between CXCL9 or IFNγ and disease parameters such as ferritin, platelet count (PLT), neutrophil count (Neu), and alanine aminotransferase (ALT).

[0181] As shown in Figures 3A and 3B, serum levels of CXCL9 and IFNγ were measured by multiplex assay using Luminex technology from samples obtained from 19 patients with MAS secondary to sJIA and 24 patients with active sJIA at the time of sampling. Correlations were examined between CXCL9 and IFNγ concentrations. Statistical testing was performed using the Spearman test, and p values were obtained.

[0182] As shown in Figures 4A-1, 4A-2, 4B-1, 4B-2, 4C-1, 4C-2, 4D-1, and 4D-2, serum levels of CXCL9 and IFNγ were measured by multiplex assay using Luminex technology from patients with MAS secondary to sJIA and patients with active sJIA at the time of sampling. Correlations were examined between IFNγ or CXCL9 levels and ferritin, platelet count, neutrophil count, or ALT (alanine aminotransferase). Statistical tests were performed using the Spearman test, and p values were obtained.

[0183] Example 4 Correlation between CXCL9 and IFNγ levels in patients with severe hemophagocytic lymphohistiocytosis (HLH) The study presented in this example is from a patient who received the NI-0501 antibody under compassionate use. This patient showed symptoms of NLRC4-related disease and severe hemophagocytic lymphohistiocytosis (HLH). Recently, mutations in the NLRC4 gene have been reported to cause recurrent macrophage activation syndrome and increased production of IL-18, which is known to induce IFNγ.

[0184] The patient in this study had the following characteristics: onset of illness at 20 days of age was accompanied by fever, rash, marked hepatosplenomegaly, pancytopenia, hypofibrinogenemia, hypertriglyceridemia, and marked increases in ferritin and sCD25. Multiple organ failure subsequently occurred, necessitating intensive care unit (ICU) admission. The diagnosis of HLH was based on six of the eight HLH-2004 criteria. Genetic testing for primary HLH (PRF1, UNC13D, STXBP2, STX11, RAB27A, XIAP) and functional testing (perforin expression, degranulation, and cytotoxicity) were negative. High-dose intravenous glucocorticoids and intravenous cyclosporine A were associated with progressive improvement in performance status and laboratory abnormalities. HLH reactivation was precipitated by infection (Candida albicans and Klebsiella pneumoniae sepsis), leading to a rapid deterioration in the patient's general condition and new admission to the ICU. Due to the presence of active infection in an already immunocompromised subject, treatment with etoposide and / or ATG was not considered.

[0185] Measurable serum levels of IFNγ and high serum levels of the IFNγ-inducible chemokines CXCL9 and CXCL10, as well as markedly elevated serum levels of IL-18 were demonstrated (Table 8).

[0186] Table 8. Levels of IFNγ, IFNγ-related chemokines, and IL-18 at the start of NI-0501 treatment and during treatment with NI-0501 TIFF2025118898000030.tif67154 * Median (interquartile range)

[0187] Compassionate use treatment with NI-0501 was performed with dexamethasone (13.6 mg / m 2 ) and iv cyclosporine-A. NI-0501 was administered every 3 days, then every 7 days, according to pharmacokinetics. No infusion reactions were observed. NI-0501 was well tolerated. HLH clinical features and laboratory abnormalities gradually improved. The ongoing active infection rapidly resolved. After 5 months of treatment, the patient remained in good condition. The patient continued to receive oral cyclosporine-A (6 mg / kg) and prednisone (0.3 mg / kg, equivalent to 0.9 mg / m2 dexamethasone). All HLH parameters normalized.

[0188] The subject continued to experience episodes of inflammation of unknown etiology. Analysis of NLRC4 revealed a de novo missense mutation (T337N). Elevated serum IL-18 was supported, confirming the association of the NLRC4 mutation. High production of IFNγ was demonstrated by high levels of IFNγ complexed with NI-0501. IFNγ was completely neutralized, as indicated by undetectable levels of IFNγ-inducible chemokines (Figure 5 and Table 8). Circulating levels of IL-18 were persistently elevated.

[0189] Thus, this study demonstrates that in patients with severe treatment-resistant HLH (due to NLRC4 mutations), IFNγ blockade with NI-0501 was well tolerated without safety concerns, allowed control of all HLH features, permitted rapid glucocorticoid tapering, and was associated with resolution of ongoing active infection.

[0190] Example 5 A targeted approach to the treatment of hemophagocytic lymphohistiocytosis (HLH) with NI-0501 The research presented in this example is derived from a pilot phase 2 study in children with primary HLH. Primary HLH (pHLH) is a rare immunoregulatory disorder that is invariably fatal if untreated. It is driven by pathological immune activation, leading to the development of fever, splenomegaly, cytopenias, and coagulopathy, which can cause multiple organ failure and death. Based on data from mouse models of primary and secondary HLH (sHLH) treated with anti-IFNγ antibodies and observational studies in patients with HLH, high IFNγ production is thought to be a key factor driving the development of this disease. Immunochemotherapy, primarily etoposide-based regimens, is currently the only pharmacological approach to control HLH and guide patients toward curative allogeneic hematopoietic stem cell transplantation (allo-HSCT). Despite recent attempts to further intensify treatment regimens, mortality and morbidity remain high, in part due to drug-related toxicity.

[0191] As described above, NI-0501 is a fully human high-affinity anti-IFNγ mAb that binds to and neutralizes human IFNγ, providing a novel and targeted approach for the control of HLH.

[0192] METHODS: An open-label, phase 2 study was conducted in the United States and Europe to evaluate the safety and efficacy of NI-0501 in children with confirmed or suspected pHLH. Initial background dexamethasone 5-10 mg / m 2 In this study, NI-0501 was administered at an initial dose of 1 mg / kg every 3 days, with dose escalation occasionally guided by individual patient pharmacokinetic data and / or clinical response. Treatment duration ranged from 4 to 8 weeks. Ability to proceed to allo-HSCT, relevant HLH disease parameters, and 8-week survival were assessed.

[0193] Study Population: A total of 13 patients were enrolled: 8 women / 5 men (8F / 5M), median age 1.0 years (range 2.5 months to 13 years). Twelve patients (pts) received NI-0501 as second-line treatment after receiving conventional therapy and either reactivated, had an inadequate response, or were intolerant to therapy. One patient was treated with NI-0501 in the first-line setting. Nine patients had known HLH genetic defects (3 FHL2, 2 FHL3, 2 GS-2, 1 XLP1, and 1 XLP2). The majority of patients were on the severe end of the HLH spectrum, had impaired performance status, and experienced significant toxicity from previous HLH treatment. Of the 13 patients, ferritin was elevated in 12, sCD25 was elevated in 8, cytopenias were present in 10, splenomegaly was present in 8, and hypofibrinogenemia and hypertriglyceridemia were present in 9. Liver damage and CNS involvement were present in 7 and 3 patients, respectively.

[0194] Results: Overall, NI-0501 treatment significantly improved HLH disease activity parameters (Figure 6), with 9 of 13 patients achieving a satisfactory response. Six patients proceeded to HSCT. Two patients with good HLH control plan to proceed to HSCT as soon as a suitable donor is identified. In one patient (who achieved disease control with first-line NI-0501), HSCT is not yet planned given the lack of a causative HLH gene mutation. Eleven of 13 patients were alive at 8 weeks. CNS signs and symptoms resolved in two evaluable patients. A greater than 50% reduction in dexamethasone dose was possible in 50% of patients during the first 4 weeks of NI-0501 treatment.

[0195] Biomarker Evaluation In particular, CXCL9, a chemokine known to be exquisitely induced by IFNγ, not only enabled the demonstration of complete IFNγ neutralization but also emerged as a new parameter for the diagnosis of HLH that correlated with IFNγ production (Figures 7A and 7B).

[0196] NI-0501 was well tolerated, and no safety concerns were identified. None of the infections known to be favored by IFNγ neutralization were reported, and no infections occurred in patients who had not previously received chemotherapy. Seven patients reported at least one SAE, all of which were assessed by the DMC as unrelated to NI-0501 administration. No unexpected events (e.g., bone marrow toxicity, hemodynamic effects) attributed to "off-target" effects of NI-0501 were observed.

[0197] Conclusions: Targeted neutralization of IFNγ with NI-0501 offers an innovative and potentially less toxic approach to HLH management. The results of this study demonstrate that NI-0501 is a safe and effective treatment option in patients with primary HLH who have responded inadequately to or are intolerant to conventional therapies. Furthermore, therapy with NI-0501 was not associated with any of the typical short- or long-term toxicities associated with etoposide-based regimens. Evaluation of NI-0501 as first-line treatment in patients with pHLH is ongoing, and it is anticipated that similar significant clinical benefits may be achieved.

[0198] Example 6 Elevated circulating levels of interferon-γ and interferon-inducible chemokines characterize patients with systemic JIA complicated by macrophage activation syndrome. Interferon gamma (IFNγ) is a central mediator in a murine model of primary hemophagocytic lymphohistiocytosis (HLH). Given the similarities between primary HLH and secondary HLH (sec-HLH), including macrophage activation syndrome (MAS), we analyzed IFNγ levels and its biological activity in patients with systemic juvenile idiopathic arthritis (sJIA) and MAS.

[0199] In the study provided in this example, a Luminex multiplexing assay was used to assess serum levels of IL-1β, IL-6, IFNγ, and the 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), 20 of whom had MAS at the time of sampling. Expression of IFNγ-induced chemokines (CXCL9 and CXCL10 mRNA levels in the liver and spleen) and their correlation with serum ferritin levels were assessed in an IL-6 transgenic mouse model in which MAS features were induced by TLR4 stimulation with LPS.

[0200] As shown in more detail below, circulating levels of IFNγ and IFN-induced chemokines were significantly elevated during MAS (also referred to herein as active MAS) and sec-HLH. IFNγ and IFN-induced chemokine levels were significantly higher in patients with MAS compared with patients with active sJIA but without MAS. In this latter group, IFNγ and IFNγ-induced chemokines were comparable to those in patients with clinically inactive sJIA. During MAS, laboratory abnormalities characterizing this syndrome, including ferritin and alanine transferase levels and neutrophil and platelet counts, significantly correlated with IFNγ and CXCL9 levels. In a mouse model of MAS, serum ferritin levels significantly correlated with CXCL9 mRNA levels in the liver and spleen.

[0201] Thus, in the studies presented below, we show that high levels of IFNγ and IFN-induced chemokines, and their correlation, particularly for CXCL9, with the severity of laboratory abnormalities in MAS, suggest that IFNγ plays a central role in MAS. Elevated circulating levels of interferon-γ and interferon-induced chemokines characterize patients with systemic JIA complicated by macrophage activation syndrome.

[0202] Materials and Methods: Patients and Samples: Peripheral blood samples were collected from patients with sJIA, with or without MAS, at three 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 who fulfilled the ILAR classification criteria for systemic arthritis (age at onset 7.9 years, interquartile range 4.6-13.6 years; 48% female) were studied (Petty, RE, et al., International League of Associations for Rheumatology classification of juvenile idiopathic arthritis: second revision, Edmonton, 2001. J Rheumatol, 2004. 31(2): pp. 390-2). For 20 SJIA patients, samples were collected during episodes of active, end-stage MAS diagnosed by the attending physician at each of three centers. Post-hoc analysis showed that 17 of these 20 episodes (85%) met the newly proposed MAS classification criteria (Minoia F, Davi S, Bovis F, et al. Development of new classification criteria for macrophage activation syndrome complicating systemic juvenile idiopathic arthritis. Pediatric Rheumatology 2014, 12(Suppl 1):O1.). Samples were available from 28 patients with active SJIA without evidence of MAS.Thirty-five samples were available from 35 sJIA patients (both with and without a history of MAS) during clinically inactive disease as defined by the Wallace criteria (Wallace, CA, et al., Preliminary criteria for clinical remission for select categories of juvenile idiopathic arthritis. J Rheumatol, 2004. 31(11): p. 2290-4).

[0203] Because IFNγ has been shown to be elevated in patients with sec-HLH (excluding rheumatic diseases), samples were also collected from 11 patients with sec-HLH (age at onset: 8.6 years, interquartile range: 4.1–12.9 years; 36% female) seen at Ospedale Pediatrico Bambino Gesu and used as positive controls. All sec-HLH patients fulfilled the 2004 HLH diagnostic guidelines (Henter, JI, et al., HLH-2004: Diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer, 2007. 48(2): pp. 124–31): six patients fulfilled five criteria, and five patients fulfilled four criteria. Note that sCD25 levels in U / ml are not available because testing is not routinely performed at the institution where these patients were recruited. The diagnosis of primary HLH was excluded based on the absence of family history, the absence of pathogenic mutations in genes known to cause HLH, and the presence of normal functional studies (including NK activity, perforin expression, and CD107 degranulation). All 11 patients with sec-HLH gave one sample each obtained during active disease.

[0204] Clinical and laboratory characteristics of all patients at diagnosis and at the time of sampling were collected in a centralized web database by the investigators at each institution. Of the 20 MAS patients sampled during active disease, 6 were not receiving any treatment at the time of sampling, while the remaining 14 patients were already receiving one of the MAS-specific treatments, including glucocorticoid pulses, cyclosporine A, anakinra, or cyclophosphamide. Of the 11 patients with sec-HLH during active disease, 6 were not yet receiving specific treatment at the time of sampling, while the remaining 5 patients were already receiving at least one of the above-mentioned treatments. The Ethics Committee of the Ospedale Pediatrico Bambino Gesu approved the study. Consent forms were collected from all participants.

[0205] Cytokine quantification: IL-6, IL-1β, IFNγ, CXCL9, CXCL10, and CXCL11 levels were analyzed using Luminex® multiplexing bead technology. Reagents were purchased from Millipore, and all reagents were provided with the Milliplex® MAP kit. Reagents were prepared according to the manufacturer's protocol. 25 μl / well of standard, blank, and quality check samples were added in duplicate to a Milliplex® MAP 96-well plate, followed by 25 μl of serum matrix. 25 μl of assay buffer was added to each sample well, followed by 25 μl of sample. Depending on the available sample volume, samples were added in duplicate or triplicate. Plates were measured using a Luminex 200® system (Luminex Corp.). Raw data were obtained using PONENT software version 3.1 (Luminex Corp.), and data were analyzed using Milliplex Analyst software version 3.5.5.0 (Millipore). The raw data obtained with Milliplex Analyst software was then further analyzed with dedicated macros for Luminex analysis (NI-Sc-ESM-MAC-012-v01 and Sc-ESM-MAC-013-v01).

[0206] Animal Experiments: The generation and phenotype of IL-6 transgenic mice and the characteristics of the MAS-like syndrome induced by TLR ligand administration have been previously described (Strippoli, R., et al., Amplification of the response to Toll-like receptor ligands by prolonged exposure to interleukin-6 in mice: Implication for the pathogenesis of macrophage activation syndrome. Arthritis Rheum, 2012. 64(5): pp. 1680-1688). Mice were maintained under specific pathogen-free conditions and handled in accordance with national police regulations. The study protocol was approved by the institutional ethics committee. All experiments were performed on mice aged 10–14 weeks. Mice were intraperitoneally injected with a single dose of 5 μg / g body weight of lipopolysaccharide (LPS, Escherichia coli serotype 055:B5; Sigma-Aldrich). Mice were sacrificed 30 hours later. Total RNA was extracted from spleen and liver tissues using Trizol (Life Technologies). cDNA was obtained using the Superscript Vilo kit (Invitrogen). Real-time PCR assays were performed using TaqMan Universal PCR Master Mix (Applied Biosystems) with mouse Cxcl9 and Cxcl10 gene expression assays (Applied Biosystems). Gene expression data were normalized using mouse Hprt (Applied Biosystems). -Δct Data are expressed as arbitrary units (AU) determined using the method Serum ferritin concentrations were determined using a commercially available ELISA kit (ALPCO Diagnostics) according to the manufacturer's instructions.

[0207] Statistical analysis: Statistical analysis was performed using GraphPad Prism 5 software. Continuous variables (quantitative demographic, clinical, and laboratory data) were expressed as median and interquartile range (IQR) and compared using the Mann-Whitney U test. The Wilcoxon signed-rank test was used to compare two paired groups without assuming that the distribution of pre- and post-treatment differences followed a Gaussian distribution. Spearman rank correlation was used to evaluate the relationship with laboratory parameters. A p-value <0.05 was considered statistically significant.

[0208] Results: Increased levels of IFNγ and IFNγ-inducible chemokines in patients with MAS. When patients with active sJIA without MAS at the time of sampling were compared with patients sampled during clinically inactive disease, we 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): pp. 1158-63), that IL-6 levels were significantly higher (p<0.01) in patients with active sJIA compared with those in patients with clinically inactive disease. As reported in several previous studies of active SJIA, serum IL-1β levels were below the limit of detection in the majority of patients, regardless of disease activity status. It is noteworthy that there were no differences in the levels of IFNγ and the three IFNγ-induced chemokines between patients with clinically active sJIA and those with clinically inactive disease.

[0209] When patients with MAS at the time of sampling were compared with patients with active sJIA without MAS at the time of sampling, IL-1β and IL-6 levels were comparable, suggesting that the levels of these two cytokines, known to play a central role in active sJIA, are not elevated during end-stage MAS. It is noteworthy that circulating IL-1β levels were below the limit of quantification (i.e., 3.5 pg / ml) in the majority of patients with sJIA, with or without MAS. In contrast, circulating IFNγ levels were significantly higher in patients with active MAS compared with patients with active sJIA without MAS at the time of sampling. Levels of the three IFNγ-related chemokines CXCL9, CXCL10, and CXCL11 were also significantly higher in patients with active MAS compared with patients with active sJIA without MAS at the time of sampling. This difference was particularly evident for CXCL9, whose median levels were approximately 15-fold higher in patients with MAS compared to patients with active sJIA but without MAS.

[0210] In patients with sec-HLH, IFNγ levels and levels of three IFNγ-related chemokines were significantly increased. The levels of IFNγ and IFNγ-related chemokines were nearly indistinguishable from those in patients with MAS, and the differences were not statistically significant. Coincidentally, the levels of IFNγ and the three IFNγ-induced chemokines in patients with active MAS and those with active sec-HLH were comparable in treatment-naive and previously treated patients.

[0211] The levels of IFNγ, as well as CXCL9, CXCL10, and CXCL11, are related to the presence of MAS in individual patients. Figures 8A–8D show the levels of IFNγ, CXCL9, CXCL10, and CXCL11 in individual patients for whom paired samples were available during active MAS and active sJIA without MAS. Consistent with the results obtained in cross-sectional analyses, the levels of IFNγ and the three IFNγ-inducible chemokines were significantly higher in samples obtained during MAS by paired sample analysis. In addition, in several patients, samples were available both before and after the MAS episode, demonstrating that the levels of IFNγ and IFNγ-inducible chemokines returned to normal with the resolution of MAS clinical symptoms. For example, one patient in this study experienced three episodes of MAS, and serum samples were obtained during these episodes as well as during a disease phase without MAS at the time of sampling. Further confirming the relationship between increased production of IFNγ and three IFNγ-induced chemokines and active MAS, in this patient, elevated levels of IFNγ and three IFNγ-related chemokines were only seen during MAS episodes (Figures 9A-9B).

[0212] Levels of IFNγ and IFNγ-related chemokines correlate with laboratory abnormalities in MAS. The correlation of levels of IFNγ and three IFNγ-induced chemokines at the time of sampling with laboratory parameters of MAS was then examined. In patients with active sJIA without MAS, levels of IFNγ and three IFNγ-induced chemokines were not associated with laboratory parameters of MAS, with one exception: levels of CXCL9, CXCL10, and CXCL11 were weakly correlated with ALT levels, and r 2The correlation coefficients ranged from 0.17 to 0.25 (Table 2). It is noteworthy that ALT levels were within the normal range in all patients with active sJIA but without MAS, although the significance of this association is unclear. In patients with MAS at the time of sampling, no significant correlations were found between laboratory features of MAS and IL-1 and IL-6. In contrast, in patients with MAS at the time of sampling, levels of IFNγ and IFNγ-inducible chemokines were associated with ferritin levels, neutrophil and platelet counts, and increased LDH and ALT, all of which are typically abnormal in patients with MAS (Table 2). With the sole exception of the correlation between IFNγ and LDH, which did not reach statistical significance, correlations with laboratory abnormalities were particularly evident for IFNγ and CXCL9 (Table 2 and Figures 10A–10J). Again, as noted above, these correlations were absent in patients with active s-JIA without MAS at the time of sampling. One patient in this group had significantly elevated levels of IFNγ (336.2 pg / ml), CXCL9 (549,400 pg / ml), and CXCL10 (35,066 pg / ml). This patient had particularly severe MAS and was admitted to the intensive care unit with severe central nervous system involvement. This observation provides further support for the hypothesis that there is a strong association between IFNγ and CXCL9 levels and disease severity. Collectively, these results indicate that increased production of IFNγ and IFNγ-related chemokines is a hallmark of active MAS that strongly correlates with the severity of laboratory abnormalities in MAS.

[0213] Table 2. Serum levels of IL-1β, IL-6, IFNγ, and the three IFNγ-related chemokines CXCL9, CXCL10, and CXCL11 in patients with active secondary HLH, patients with active MAS at the time of sampling, patients with active sJIA without MAS at the time of sampling, and patients with clinically inactive sJIA. TIFF2025118898000031.tif140152Values are expressed as median (interquartile range) *Active vs. clinically inactive sJIA: p<0.01

[0214] Table 3. Correlations between laboratory parameters of disease activity and levels of IFNγ, CXCL9, CXCL10, CXCL11, and IL-6 in patients with MAS at the time of sampling and in patients with active sJIA without MAS. TIFF2025118898000032.tif86156NEU=neutrophil count;PLT=platelet count;ALT=alanine aminotransferase;LDH=lactate dehydrogenase; 1 = median (IQR); r*=Spearman's r. Correlation of laboratory parameters of disease activity with IFN-γ, CXCL9, CXCL10, CXCL11, and IL-6 in patients with MAS and those with active sJIA.

[0215] Correlation between IFNγ and the levels of IFNγ-induced chemokines in patients with MAS: To further characterize the relationship between IFNγ and the three IFNγ-induced chemokines in patients with MAS, we assessed the correlation between IFNγ levels and the levels of each chemokine. In particular, CXCL9 appears to be primarily and specifically induced by IFNγ, whereas CXCL10 and CXCL11 are also induced by type I interferons. Consistent with this, in patients with active MAS, circulating levels of IFNγ were significantly correlated with CXCL9 (r = 0.693; r 2 = 0.48; p = 0.001), but had a weaker correlation with CXCL10 levels (r = 0.535; r 2 =0.29; p=0.015) (Figures 11A-11F). The correlation with CXCL11 levels was weaker and did not reach statistical significance (r=0.447; r 2 =0.20; p=0.08) (not shown).

[0216] IFNγ-induced chemokines correlate with disease activity in a mouse model of MAS. To further investigate the relevance of IFNγ-induced chemokine production to MAS, we examined the expression of these chemokines in target tissues (liver and spleen) in a mouse model of MAS. In this model, clinical and laboratory features of MAS are induced by mimicking acute infection with the TLR4 agonist lipopolysaccharide (LPS) in a background of high levels of IL-6 in IL-6 transgenic mice (Strippoli et al., Arthritis Rheum 2012). This approach recapitulates what occurs in patients with sJIA: infection can trigger MAS / HLH in the presence of active disease, which is indeed characterized by high levels of IL-6. Following induction with LPS, high mRNA levels of CXCL9 and CXCL10 were present in the liver and spleen of IL-6 transgenic mice. In particular, serum ferritin levels significantly correlated with the expression levels of CXCL9 in the spleen and liver, and CXCL10 in the liver, indicating a relationship between IFNγ-related upstream events in target tissues (i.e., CXCL9 and CXCL10 production in the liver and spleen) and typical downstream laboratory abnormalities, such as high ferritin levels. Collectively, data from patients with MAS and from MAS mouse models point to a clear relationship between increased production of IFNγ and increased expression of CXCL9, and to a lesser extent, CXCL10, as well as laboratory abnormalities in MAS.

[0217] Studies in both patients and animal models of p-HLH have demonstrated a central role for IFNγ in the pathogenesis. However, the role of IFNγ in sec-HLH, including MAS, in the setting of sJIA, has remained unclear. This study conclusively demonstrates that high levels of IFNγ and IFNγ-inducible chemokines were present in patients with MAS occurring in sJIA. In addition, levels of IFNγ, CXCL9, and CXCL10 strongly correlated with laboratory parameters of MAS severity. This study found that serum levels of IFNγ and three IFNγ-related chemokines were comparable between patients with active sJIA and those with clinically inactive disease. This result argues against a pathogenic role for IFNγ in sJIA and is, indeed, consistent with numerous observations by other authors.Three gene expression studies failed to find a significant IFNγ-inducing signature in peripheral blood mononuclear cells (PBMCs) from patients with active sJIA without MAS at the time of sampling (Fall, N., et al., Gene expression profiling of peripheral blood from patients with untreated new-onset systemic juvenile idiopathic arthritis reveals molecular heterogeneity that may predict macrophage activation syndrome. Arthritis Rheum, 2007. 56(11): pp. 3793-804; Ogilvie, EM, et al., Specific gene expression profiles in systemic juvenile idiopathic arthritis. Arthritis Rheum, 2007. 56(6): pp. 1954-65; Pascual, V., et al., Role of interleukin-1 (IL-1) in the pathogenesis of systemic onset juvenile idiopathic arthritis and clinical response to IL-1 blockade. J Exp Med, 2005. 201(9): pp. 1479-86). After ex vivo stimulation of PBMCs, the number of IFNγ-producing cells in patients with active sJIA was similar to that in controls (Lasiglie, D., et al., Role of IL-1 beta in the development of human T(H)17 cells: lessons from NLPR3 mutated patients. PLoS One, 2011. 6(5): p. e20014).Consistently, patients with both active and inactive SJIA do not show increased serum or synovial fluid levels of IFNγ (de Jager, W., et al., Blood and synovial fluid cytokine signatures in patients with juvenile idiopathic arthritis: a cross-sectional study. Ann Rheum Dis, 2007. 66(5): p. 589-98). Supporting the lack of a role for IFNγ in sJIA arthritis, CXCL9 and CXCL10 are nearly undetectable in synovial tissue from sJIA patients, whereas high levels of these chemokines can be found in synovial tissue from patients with oligoarticular or polyarticular JIA (Sikora, KA, et al., The limited role of interferon-gamma in systemic juvenile idiopathic arthritis cannot be explained by cellular hyporesponsiveness. Arthritis Rheum, 2012. 64(11): pp. 3799-808). Recent data in mice show that immune stimulation of IFNγ knockout mice with Freund's complete adjuvant results in a systemic inflammatory syndrome that includes features of sJIA, further supporting a limited role for IFNγ in sJIA (Avau, A., et al., Systemic juvenile idiopathic arthritis-like syndrome in mice following stimulation of the immune system with Freund's complete adjuvant: regulation by interferon-gamma. Arthritis Rheumatol, 2014. 66(5): p. 1340-51).

[0218] In sharp contrast, the present study demonstrated significantly higher levels of IFNγ and IFNγ-related chemokines in patients with active MAS at the time of sampling compared with those with active sJIA without MAS at the time of sampling. This was also confirmed in individual patients using serial samples obtained during both active MAS and active sJIA without MAS. Coincidentally, the present study did not find significantly increased levels of IL-6 or IL-1β in patients sampled during MAS, nor did they correlate with laboratory parameters of MAS, suggesting that although these cytokines are crucially involved in the pathogenesis of sJIA (De Benedetti, F., et al., Randomized trial of tocilizumab in systemic juvenile idiopathic arthritis. N Engl J Med, 2012. 367(25): pp. 2385-95; Ruperto, N., et al., Two randomized trials of canakinumab in systemic juvenile idiopathic arthritis. N Engl J Med, 2012. 367(25): pp. 2396-406), they may not be important in maintaining MAS. This finding of elevated levels of IFNγ and IFNγ-related chemokines is consistent with several previous observations.Shimizu et al. reported that levels of neopterin, a catabolic product of guanosine triphosphate synthesized by human macrophages upon stimulation with IFNγ, were higher in patients with MAS during sJIA compared with patients with active sJIA but 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). Recently, Put et al. reported elevated levels of IFNγ and CXCL10 in five patients with both primary and secondary HLH, three of whom had MAS during the course of sJIA (Put, K., et al., Cytokines in systemic juvenile idiopathic arthritis and heemophagocytic lymphohistiocytosis: tipping the balance between interleukin-18 and interferon-gamma. Rheumatology (Oxford), 2015). Consistent with these results, five patients with active sJIA without MAS at the time of sampling had significantly lower levels of IFNγ and CXCL10 (Put et al., Rheumatology 2015).

[0219] Interestingly, this study found that not only were IFNγ and IFNγ-related chemokine levels significantly elevated, but their levels, especially CXCL9 levels, closely correlated with the laboratory features of MAS, suggesting a link with disease severity. Further supporting the link with disease severity, this study found significantly elevated levels of IFNγ, CXCL9, and CXCL10 in one patient with severe disease accompanied by multiorgan failure and central nervous system involvement, along with generalized seizures requiring prolonged intensive care unit admission.

[0220] In patients with MAS, among the three IFNγ-inducible chemokines, CXCL9 was found to have the strongest correlation with IFNγ levels. This observation is consistent with the established idea that CXCL9 production appears to be specifically and exclusively induced by IFNγ, in contrast to the production of CXCL10 and CXCL11, which can also be induced by type I interferons (Groom, JR and AD Luster, CXCR3 ligands: redundant, collaborative, and antagonistic functions. Immunol Cell Biol, 2011. 89(2): pp. 207-15). This suggests that CXCL9 levels may serve as a sensitive and specific biomarker for MAS activity. Indeed, using a mouse model of MAS mimicking the triggering of MAS by infectious stimuli in the background of elevated IL-6 levels (Strippoli et al., Arthritis Rheum 2012), this study also found that CXCL9 expression levels in the liver and spleen significantly correlated with circulating levels of ferritin. For CXCL10 expression levels, this correlation was present only at the liver level, not at the spleen level. This is also supported by findings in patients with MAS, where CXCL9 levels were strictly correlated with all laboratory parameters of MAS. Collectively, these observations in humans and mice indicate that CXCL9 is strongly correlated with MAS features and IFNγ production, further supporting the hypothesis that excessive IFNγ production plays a major pathogenic role in MAS. These observations are also consistent with immunohistochemistry data generated by Put et al. using serial lymph node biopsies from the same SJIA patient obtained during active sJIA without MAS, as well as during MAS.They reported that CXCL10 and indoleamine 2,3-dioxygenase, both IFNγ-inducible proteins, were detected at high levels by immunohistochemistry in tissue obtained during MAS but not in tissue obtained during active sJIA without MAS (Put et al., Rheumatology 2015).

[0221] These results in MAS and sec-HLH, together with available literature observations in patients with p-HLH, support the hypothesis that increased IFNγ and IFNγ-related chemokines, especially CXCL9, are a unique feature of HLH, independent of the underlying cause. In this regard, it is interesting to note that high levels of CXCL9 were detected in patients with recurrent MAS caused by NLRC4 gain-of-function mutations (Canna, SW, et al., An activating NLRC4 inflammasome mutation causes autoinflammation with recurrent macrophage activation syndrome. Nat Genet, 2014. 46(10): pp. 1140-1146), suggesting that IFNγ overproduction may be present even in the setting of HLH caused solely by inflammasome dysregulation.

[0222] Data in animal models of p-HLH in both perforin- and Rab27a-knockout mice clearly demonstrate the pathogenic role of IFNγ. Similarly, recent data in a TLR9-induced model of HLH (a model of HLH secondary to infection) also demonstrated a key role for increased IFNγ production (Behrens, EM, et al., Repeated TLR9 stimulation results in macrophage activation syndrome-like disease in mice. J Clin Invest, 2011. 121(6): pp. 2264-77 and (Bautois et al., in progress)). Further studies have recently demonstrated that treatment with anti-IFNγ antibodies in the above-mentioned mouse model of MAS increases survival and reverses the clinical and laboratory features of MAS (Prencipe et al., in progress). Collectively, these animal observations and the results of this study provide a rationale for IFNγ neutralization as a therapeutic approach in MAS.

[0223] Example 7 Safety, tolerability, pharmacokinetics, and efficacy evaluation of multiple intravenous doses of anti-interferon gamma and anti-IFNγ monoclonal antibodies in pediatric patients with primary hemophagocytic lymphohistiocytosis (HLH) The studies presented in this example were designed to determine the safety and tolerability profile of multiple intravenous (IV) administration of an anti-IFNγ antibody, herein designated NI-0501; to determine the NI-0501 efficacy and benefit / risk profile in patients with HLH; to delineate the pharmacokinetic (PK) profile of NI-0501 in patients with HLH; to define an appropriate NI-0501 therapeutic dose regimen for HLH; and to evaluate the immunogenicity of NI-0501.

[0224] Preclinical Studies: Previous studies have demonstrated that NI-0501 exhibits similar binding affinity and blocking activity for IFNγ from non-human primate species, including rhesus and cynomolgus monkeys, but not from dogs, cats, pigs, rabbits, rats, or mice. Toxicology and safety studies in cynomolgus monkeys demonstrated that there was no off-target toxicity attributable to administration of NI-0501, that weekly dosing of NI-0501 was well tolerated, and did not require antibiotic prophylaxis, and no abnormal histopathological or behavioral findings were observed in these preliminary studies.

[0225] Due to its ability to bind to free and IFNγR1-bound IFNγ, studies were conducted to examine the potential of NI-0501 to mediate ADCC and CDC activity in the presence of target. A lack of ADCC activity was demonstrated, and no induction of CDC activity was observed.

[0226] Phase 1 Clinical Study: A Phase 1, randomized, double-blind, placebo-controlled, single ascending dose study in 20 healthy adult volunteers to investigate the safety, tolerability, and pharmacokinetic profile of a single intravenous (IV) dose of NI-0501. During this study, 6 subjects received placebo, while 3, 3, 4, and 4 subjects (14 subjects total) received NI-0501 doses of 0.01, 0.1, 1, and 3 mg / kg, respectively.

[0227] PK analysis of NI-0501 revealed the expected profile for an IgG1 with a long half-life (approximately 22 days), slow clearance (≦0.007 L / h) and low volume of distribution (average <6 L).

[0228] A total of 41 adverse events (AEs) were observed in 14 of 20 subjects (70%) after the start of the drug infusion, 10 of which were reported by the 4 subjects receiving placebo. 36 (87.8%) AEs were mild and 5 (12.2%) were moderate. No severe or life-threatening AEs were reported. 23 AEs (56.1%) in 10 of the 14 subjects who experienced an AE were reported as drug-related (with at least a reasonable possibility). Most AEs were isolated, and no trend associated with increasing NI-0501 dosage was observed. All NI-0501 infusions were event-free.

[0229] In summary, NI-0501 infusions were well tolerated, and the effects observed during 8 weeks of monitoring following drug infusion did not indicate any serious or unexpected off-target safety or immunogenicity concerns.

[0230] Phase 2 / 3 Clinical Study Materials and Methods: These studies are performed in patients with primary HLH. The study is divided into three parts: screening, treatment, and follow-up. An overview is shown in Figure 12.

[0231] In these studies, suitable patients include patients who are naive to HLH treatment (also referred to herein as "first-line patients") or patients who may have already received conventional HLH therapy (also referred to herein as "second-line patients") and who, for example, according to their treating physician, have not responded satisfactorily or have shown signs of intolerance thereto. Patients who receive NI-0501 after failing or showing intolerance to conventional HLH therapy are the core cohort of the study to demonstrate the efficacy of NI-0501 as a second-line treatment for primary HLH. Treatment-naive patients are enrolled for the collection of efficacy and safety data in the first-line setting.

[0232] The following patients will be excluded from this study: patients with a diagnosis of secondary HLH due to documented rheumatic or neoplastic disease; patients who have been previously treated with T-cell depleting agents (e.g., antithymocyte globulin (ATG), anti-CD52 therapy) during the two weeks prior to screening, or with any other biologic drug within five half-lives of its prescribed duration (except for rituximab in cases of documented B-cell EBV infection); patients with active mycobacteria, Histoplasma capsulatum (Histoplasma patients with Salmonella Capsulatum, Shigella, Salmonella, Campylobacter, and Leishmania infections; patients with evidence of a history of tuberculosis or latent tuberculosis; patients with positive serology for HIV antibody, hepatitis B surface antigen, or hepatitis C antibody; patients with existing malignancies; patients with another comorbidity or malformation significantly affecting cardiovascular, pulmonary, hepatic, or renal function; patients with a history of hypersensitivity or allergy to any component of the study regimen; patients who have received a live or live-attenuated (including BCG) vaccine within 12 weeks prior to screening; and / or pregnant or lactating female patients.

[0233] The studies presented in this example use the anti-interferon gamma antibody NI-0501, a fully human IgG1 monoclonal antibody (mAb) directed against human IFNγ. NI-0501 is provided as a sterile concentrate for injection (per mL) as shown in Table 4 below.

[0234] Table 4: NI-0501 formulation TIFF2025118898000033.tif57150

[0235] In these studies, NI-0501 will be administered by IV infusion over a 1-hour period at an initial dose of 1 mg / kg. This dose is expected to inhibit at least 99% of the IFNγ effect for 3 days in patients with baseline IFNγ concentrations of 3400 pg / mL or less. Infusions will be administered every 3 days until study day 15 (SD15) (infusion number 6), and then twice weekly. An NI-0501 dose escalation to 3 mg / kg is permitted at any time during the study according to predefined criteria (as described in Table 5 below) guided by clinical and laboratory response in each patient. After a minimum of two infusions at 3 mg / kg, if reevaluation reveals that the same clinical and laboratory criteria still apply that qualify the patient to receive NI-0501 at 3 mg / kg, the NI-0501 dose may be increased to 6 mg / kg for up to four infusions, with regular monitoring of clinical and laboratory HLH parameters. Based on the evolution of these parameters, the dose of NI-0501 may i) be reduced again to 3 mg / kg, or ii) remain at 6 mg / kg (or be increased above 6 mg / kg) for additional IV infusions if PK and PD evidence indicates excessively high IFNγ production and consequently rapid NI-0501 elimination. Dose escalation may occur at any time during the study if the clinical and laboratory criteria described in this example are met.

[0236] Table 5. Clinical and laboratory criteria guiding dose escalation TIFF2025118898000034.tif56152TIFF2025118898000035.tif241152 a If these criteria are met after SD6, the NI-0501 dose should be increased from 1 to 3 mg / kg. b If the NI-0501 dose has already been increased to SD3, at least two infusions at a dose of 3 mg / kg must be performed before the baseline reassessment. c It depends on whether the dose escalation to 3 mg / kg was performed on SD3 or SD6. d For up to 4 injections. Abbreviations: bsl. = baseline; ANC = absolute neutrophil count; US = ultrasound

[0237] In these studies, NI-0501 was administered for 8 weeks, with the treatment period divided into two separate periods: Treatment Periods 1 and 2, shown in FIG.

[0238] After 8 weeks of NI-0501 administration, a conditioning regimen in preparation for hematopoietic stem cell transplantation (HSCT) may be initiated. If the patient's condition and donor availability permit transplantation, the expected duration of treatment may be shortened to no less than 4 weeks. If a suitable donor has not been identified by week 8, or if the transplant schedule needs to be delayed for reasons unrelated to NI-0501 administration, NI-0501 treatment may be continued in the context of a long-term follow-up study once a favorable benefit / risk profile has been established for the patient.

[0239] In these studies, NI-0501 was administered on a background of dexamethasone, which could be tapered depending on the patient's condition. In treatment-naive patients, NI-0501 was administered at a dose of 10 mg / m 2 In patients receiving NI-0501 as second-line HLH treatment, dexamethasone should be administered at a dose of at least 5 mg / m 2 Patients must be receiving dexamethasone at a dose of 0.01 mg / kg or a higher dose if they were receiving a higher dose before screening. Patients are required to have received dexamethasone from SD-1.

[0240] Dexamethasone may be tapered depending on the patient's condition at the discretion of the attending physician. The tapering scheme may be selected by the attending physician, provided that the dexamethasone dose is not reduced by more than half at each step and the frequency of changes is not more than weekly.

[0241] If the disease worsens after tapering of dexamethasone, the dose of dexamethasone may be increased and maintained according to the attending physician until a satisfactory response is achieved.

[0242] As recommended in HLH treatment guidelines, patients will receive prophylaxis for Pneumocystis jiroveci, fungal, and Herpes Zoster virus infections from the day before NI-0501 treatment initiation until the end of the study. Patients will receive prophylaxis from the day before NI-0501 treatment initiation (i.e., SD-1) until the end of the study. For example, for Pneumocystis jiroveci prophylaxis, patients will receive, for example, 750 mg / m orally provided twice daily in equally divided doses, three consecutive days per week. 2 / day sulfamethoxazole and 150 mg / m 2 For fungal infection prophylaxis, patients may receive, for example, fluconazole 12 mg / kg daily, at a maximum daily dose of 400 mg. For HZ virus prophylaxis, patients may receive, for example, acyclovir 200 mg four times daily for children over 2 years of age and 100 mg four times daily for children under 2 years of age. These treatments are always provided orally whenever possible, otherwise intravenously.

[0243] Patients may also receive any of a variety of concomitant therapies, such as cyclosporine A, intrathecal methotrexate, and glucocorticosteroids. Cyclosporine A (CsA) may be continued if already administered to patients prior to screening. CsA may be discontinued at any time. Once NI-0501 administration has begun, CsA will not be newly introduced during the course of the study.

[0244] If a patient is receiving intrathecal methotrexate and glucocorticoids at the time of initiation of NI-0501 treatment, this treatment will be continued as needed. If CNS symptoms appear before the initiation of NI-0501 treatment, therapy with intrathecal methotrexate and glucocorticoids must be initiated before the first dose of NI-0501.

[0245] IV immunoglobulin (IVIG) is only acceptable as replacement therapy in cases of documented immunoglobulin deficiency. For example, in cases of documented immunoglobulin deficiency that warrants replacement, IVIG may be provided at a dose of 0.5 g / kg every 4 weeks or more frequently to maintain adequate IgG levels. Infusions within 4 weeks prior to screening, as well as during NI-0501 treatment, are both acceptable.

[0246] Analgesic therapy, blood product transfusions, electrolyte and glucose infusions, antibiotic, antifungal and antiviral therapy, and systemic supportive therapy are permitted. Once the maximum NI-0501 dose level is achieved, additional HLH therapy may be permitted in the event of unsustained or limited HLH improvement. As used herein, unsustained HLH improvement refers to patients who are unable to maintain at least a 50% improvement from baseline in three HLH parameters (see Table 6 below). At least two consecutive measurements must document a decline in HLH improvement. As used herein, limited HLH improvement refers to a less than 50% change from baseline in at least three HLH clinical and laboratory criteria. Etoposide should be administered as additional HLH therapy unless clear evidence of lack of response or intolerance to the drug is derived from previous medical history.

[0247] The following therapies should not be used concurrently with NI-0501 administration: etoposide, T-cell depleting agents, or any other biologic drugs are generally not tolerated, with the exception of: G-CSF in cases of prolonged neutropenia; rituximab in cases of documented B-cell EBV infection; and additional HLH treatment in cases of unsustained or limited HLH improvement (as defined herein) at the maximum NI-0501 dose level. Etoposide should be administered unless clear evidence of lack of response or intolerance to this drug is derived from previous medical history. Vaccination with live or attenuated (including BCG) vaccines must be avoided throughout the study, including the 4-week follow-up period. If NI-0501 concentrations remain at therapeutic levels after the end of the study, the vaccination-free period should be extended until measurable concentrations of NI-0501 are no longer detectable.

[0248] The evolution of clinical signs (fever, splenomegaly, CNS symptoms) and laboratory parameters (CBC, fibrinogen, ferritin, sCD25 levels) that characterize the disease will be used to assess the achievement of response and time to response. The primary efficacy endpoint includes the overall response rate, i.e., the achievement of either a complete or partial response or improvement in HLH at the end of treatment (EoT) as defined in Table 6 below. Secondary efficacy endpoints include time to response at any time during the study; durability of response, i.e., maintenance of the response achieved at any time during the study at EoT and beyond (including data collected in any long-term follow-up studies); the number of patients who can reduce glucocorticoids by 50% or more of the baseline dose; the number of patients who can proceed to HSCT, if deemed appropriate; survival at week 8 (or EoT) and end of study; serum concentration of NI-0501 to determine the NI-0501 pharmacokinetic (PK) profile; determination of pharmacodynamic (PD) effects, including levels of circulating total IFNγ and its neutralizing markers, i.e., CXCL9 and CXCL10; and determination of other biomarkers, e.g., sCD25, IL-10.

[0249] Table 6: Response definitions TIFF2025118898000036.tif201152TIFF2025118898000037.tif119152* The following test parameters are specifically considered for determining reactivation: - Platelets - neutrophils - Fibrinogen - Ferritin - soluble CD25 (sCD25; i.e., soluble IL-2 receptor). Assessment of NK function, red blood cell / hemoglobin and triglyceride levels cannot be considered for determining reactivation.

[0250] Safety parameters to be collected and evaluated include the incidence, severity, causality and outcome of adverse events (AEs) (severe and non-severe), with particular focus on infections; the evolution of laboratory parameters such as complete blood count (CBC) focusing on red blood cells (hemoglobin), neutrophils and platelets, liver tests, renal function tests, and coagulation; the number of patients discontinued for safety reasons; and other parameters such as the level of circulating antibodies to NI-0501 (if any) to determine immunogenicity (ADA).

[0251] The primary endpoint (overall response rate) will be assessed using an exact binomial test at the one-sided 0.025 level. Time to response, duration of response, and survival time will be presented using Kaplan-Meier curves, with medians calculated where available. 95% confidence intervals will be calculated for the median for each of these endpoints. Additional endpoints based on binary outcomes, including the number of patients achieving a 50% or greater reduction in glucocorticoids and the number of patients able to proceed to HSCT, will be converted to proportions and the associated 95% confidence intervals calculated. Statistical significance via p-values will be obtained only for the primary endpoint. All other endpoints will be considered supportive of the primary endpoint; as a result, no formal hierarchy of endpoints will be stated.

[0252] Administration of NI-0501 in patients results in rapid normalization of fever within several hours after the first infusion of NI-0501. Figures 13A and 13B depict the effect of NI-0501 infusion on body temperature in two patients with a temperature >37.5°C at the start of NI-0501 treatment. Figure 14 is a series of graphs and tables depicting the effect of NI-0501 administration on neutrophil counts in patients. Figure 15 is a series of graphs and tables depicting the effect of NI-0501 administration on platelet counts in patients. Figure 16 is a series of graphs and tables depicting the effect of NI-0501 administration on serum ferritin levels in patients. Figure 17 is a series of graphs and tables depicting the effect of NI-0501 administration on glucocorticoid tapering in patients. Figure 18 is a graph depicting that administration of NI-0510 maintained IFNγ neutralization until the time of HSCT. The HLH response to NI-0501 treatment also persisted until transplant. Patients were also assessed for CNS involvement after administration of NI-0501. A summary of baseline CNS involvement and status through end of treatment (EOT) is shown below in Table 11.

[0253] Table 11. Response to NI-0501 Treatment - CNS Involvement TIFF2025118898000038.tif89145Note: Patients received IT therapy except for patient number (Pt.#) 4, for whom regular medication application LP was not performed. * Treatment is ongoing $ Treatment began two weeks ago ^ No control was performed at EOT

[0254] Of the 10 patients who underwent hematopoietic stem cell transplantation (HSCT), all patients engrafted; in one patient, a CD34 stem cell boost was required due to mixed chimerism on day +145 after HSCT. In one patient, secondary engraftment failure followed by HLH reactivation occurred. This patient died on day +68 after HSCT due to acute respiratory failure and bacterial infection. Another patient died on day +47 after HSCT (septic shock in the setting of severe GvHD). Mild GvHD was reported in the other three patients and has resolved / is resolving.

[0255] Neutralizing serum concentrations of NI-0501 at the time of HSCT, reflected by levels of CXCL9 (a chemokine induced by IFNγ) below the limit of quantification, were measured in 8 of 10 patients undergoing transplantation. Thus, these data indicate that NI-0501 can avoid the short- and long-term toxicities reported for etoposide-based regimens. This translates into a reduced risk of allo-HSCT-related complications.

[0256] These data demonstrate that NI-0501 treatment can improve and / or eliminate clinical and laboratory abnormalities associated with HLH, including CNS signs and symptoms. Response to NI-0501 is independent of the presence and type of causative mutation and / or the presence and type of infectious trigger. NI-0501 has been well tolerated. No safety concerns have emerged to date (e.g., no myelotoxicity, no widespread immunosuppression). No infections caused by pathogens known to be promoted by IFNγ neutralization have been observed. Neutralization of IFNγ with NI-0501 may provide an innovative and targeted approach to the management of HLH.

[0257] Example 8Safety, tolerability, pharmacokinetics, and efficacy of short-term intravenous administration of anti-interferon gamma (anti-IFNγ) monoclonal antibody NI-0501 in patients with systemic juvenile idiopathic arthritis (sJIA) presenting with macrophage activation syndrome / secondary HLH (MAS / sHLH). The study provided in this example is designed to demonstrate the efficacy and safety of NI-0501 for the treatment of MAS / sHLH in patients with sJIA and is divided into two parts: (i) a pilot study to evaluate the PK profile and dosing strategy of NI-0501 and to preliminary evaluate the NI-0501 benefit / risk in this patient population; and (ii) a pivotal study to demonstrate the efficacy and safety of NI-0501 (study continued with confirmation of the positive benefit / risk profile and dosing regimen of NI-0501). An overview of this study design is shown in Figure 19.

[0258] The primary objectives of the pilot study are: (i) to define an appropriate NI-0501 treatment dose regimen for sJIA patients with MAS / sHLH; (ii) to evaluate the benefit / risk profile of NI-0501 in sJIA patients with MAS / sHLH; and (iii) to delineate the pharmacokinetic (PK) profile of NI-0501 in sJIA patients with MAS / sHLH. The primary objectives of the pivotal study are: (i) to determine the efficacy of NI-0501 in sJIA patients with MAS / sHLH; (ii) to evaluate the safety and tolerability profile of short-term intravenous (iv) administration of NI-0501 in sJIA patients with MAS / sHLH; (iii) to confirm the positive benefit / risk profile of NI-0501 in sJIA patients with MAS / sHLH; (iv) to conduct an exploratory evaluation of the chemokines CXCL9 and CXCL10 as diagnostic biomarkers for MAS / sHLH and as predictors of response to NI-0501 treatment; and (v) to evaluate the immunogenicity of NI-0501 in sJIA patients with MAS / sHLH.

[0259] The study population included sJIA patients with MAS / sHLH who showed an inadequate response to high-dose glucocorticoid treatment. Criteria for inclusion in the study included: (i) gender: males and females; (ii) age: <16 years at the time of sJIA diagnosis; (iii) a diagnosis of active MAS / sHLH confirmed by a treating rheumatologist in the presence of at least two of the following laboratory and clinical criteria: (a) laboratory criteria: platelet count ≤ 262 x 10 9 / L, WBC count ≤ 4.0 x10 9 / L, AST level > 59 U / L, and / or fibrinogen level ≤ 2.5 g / L; (b) clinical criteria: hepatomegaly, bleeding symptoms, and / or CNS dysfunction; (iv) patients with an inadequate response to at least 3 days of high-dose IV glucocorticoid treatment with local standard of care (including, but not limited to, 30 mg / kg mPDN pulses on 3 consecutive days); (v) high-dose IV glucocorticoids should be no less than 2 mg / kg / day mPDN equivalents in two separate daily doses, up to 60 mg / day. In the case of rapid deterioration of the patient's condition and / or laboratory parameters, patients may be included in the study even if they have been on high-dose IV glucocorticoids for less than 3 days; (vi) patient consent (or consent of their legally authorized representative); and (vii) acceptable contraceptive measures if the patient has passed puberty.

[0260] Exclusion criteria included the following: (i) a diagnosis of suspected or confirmed primary HLH or HLH due to neoplastic disease; (ii) patients who had been treated with anakinra, tocilizumab, canakinumab, TNF inhibitors, rituximab, or any other biologic drug within five half-lives of their respective drugs; (iii) active mycobacterial (typical and atypical), Histoplasma capsulatum, Shigella, Salmonella, Campylobacter, and Leishmania infection; (iv) evidence of latent tuberculosis; (v) positive serology for HIV antibodies; and (vi) (vii) patients with another comorbidity or malformation that significantly affects cardiovascular, pulmonary, CNS, hepatic, or renal function that, in the opinion of the investigator, may significantly affect the likelihood of responding to treatment and / or NI-0501 safety assessment; (viii) a history of hypersensitivity or allergy to any component of the study regimen; (ix) BCG vaccination within 12 weeks prior to screening; (x) other live or live-attenuated vaccinations within 6 weeks prior to screening; and / or (xi) pregnant or lactating female patients.

[0261] Dosing Regimen, Administration Frequency, and Treatment Duration: In these studies, NI-0501 will be used in the formulation shown in Example 7. In Part 1, NI-0501 will be administered at an initial dose of 6 mg / kg via infusion over a 1-hour period at SD0. NI-0501 treatment will continue at a dose of 3 mg / kg every 3 days for 4 weeks (i.e., up to SD27). NI-0501 treatment may be shortened upon achievement of a complete clinical response (i.e., MAS remission). After 4 weeks, NI-0501 treatment may be continued as maintenance for up to 4 additional weeks (i.e., up to SD56) as needed until MAS remission is achieved, with the possibility of reducing the dose to 1 mg / kg and extending the interval between infusions to weekly administration. If the PK profile indicates unexpected TMDD (and thus signaling abnormally high IFNγ production), the dose of NI-0501 may be increased to 10 mg / kg, guided by clinical and PK evidence. This dose escalation is approved only upon careful evaluation of the benefit / risk profile in that individual patient.

[0262] If the proposed dosing regimen is confirmed as appropriate and the positive benefit / risk profile of NI-0501 is demonstrated in Part 2, the study will continue. Minor changes to the dosing regimen may be made if warranted based on evidence obtained in Part 1.

[0263] Background Therapy and Concomitant Medications: NI-0501 will be administered at a background dose of at least 2 mg / kg of methylprednisolone (mPDN) equivalent up to 60 mg / day (in patients weighing 30 kg or more), which may be tapered during treatment depending on the patient's condition. Patients will receive prophylactic treatment for herpes zoster infection, preferably beginning the day before (and in any case before initiating NI-0501 treatment) until serum NI-0501 levels are no longer detectable. Cyclosporine A (CsA) may be continued if initiated at least 3 days before the start of NI-0501 treatment. CsA dose adjustments are permitted to maintain therapeutic levels. CsA may be discontinued at any time during the study at the investigator's discretion. Once NI-0501 administration has begun, CsA cannot be newly introduced. If patients are receiving intrathecal methotrexate and glucocorticoids at the time of initiation of NI-0501 treatment, this treatment may be continued as needed. Vaccination with live or attenuated vaccines (including BCG) must be avoided throughout the study and, in any case, until serum NI-0501 levels are no longer detectable. Analgesic therapy, transfusion of blood products, electrolyte and glucose infusion, antibiotic, antifungal, and antiviral therapy, and systemic supportive care are permitted.

[0264] Sample Size: In Part 1, at least 5 evaluable patients will be enrolled. In Part 2, at least 10 evaluable patients will be enrolled during the continuation of the study, achieving a total of 15 evaluable patients. Given the rare nature of the disease and the lack of approved treatments, a sample size of 15 was not formally justified. In any event, based on the assumption that at least 50% of patients respond inadequately to systemic glucocorticoids alone, i.e., 50% of patients receiving glucocorticoids achieve MAS remission by week 8 after initiating treatment, the study has 70% power to detect an improvement from 50% to 77% using a 5% one-sided significance level.

[0265] Study Duration and End-of-Study Definitions: The study duration will be 8 weeks for each patient (+ up to 1 week screening period). End-of-study will be defined as the last visit of the last patient. All patients who receive at least one dose of NI-0501 will be invited to enter the NI-0501-05 study for long-term follow-up.

[0266] Study endpoints :Part 1 (pilot) of the study will evaluate the following to validate dosing regimens in this patient population: (i) the benefit / risk profile of NI-0501; (ii) the PK profile of NI-0501; (iii) levels of chemokines known to be induced by IFNγ (e.g., CXCL9, CXCL10, CXCL11); (iv) the development of distinct features of MAS of cytopenias, liver dysfunction, and coagulopathy at 2, 4, 6, and 8 weeks after initiating NI-0501; and (v) the dose and duration of NI-0501 treatment. In Part 2 (core) of the study, the efficacy study endpoints are as follows: (a) primary efficacy endpoint: number of patients achieving MAS remission by week 8 after initiation of NI-0501 treatment; and (b) secondary efficacy endpoints: time to MAS remission; time to first response according to investigator assessment; number of patients who can be tapered at any time during the study to the same (or lower) dose of glucocorticoids as received before the onset of MAS; time to achieve glucocorticoid tapering; survival at the end of the study; and number of patients who discontinued from the study due to lack of efficacy. In Part 2 (core) of the study, safety study endpoints are: (a) incidence, severity, causality, and outcome of AEs (severe and non-severe), with particular focus on infections; evolution of laboratory parameters, particularly CBC (focusing on hemoglobin, neutrophils, and platelets), LFTs, and coagulation parameters; number of patients discontinued from the study for safety reasons; and levels of circulating antibodies to NI-0501 (if any) to determine immunogenicity (ADA).

[0267] Pharmacokinetics and pharmacodynamics will be assessed by the PK profile of NI-0501; levels of circulating free IFNγ before administration and total IFNγ (free IFNγ + bound to NI-0501) after NI-0501 initiation; levels of chemokines known to be induced by IFNγ (e.g., CXCL9, CXCL10, CXCL11); correlation between chemokine levels (CXCL9, CXCL10) and levels of free NI-0501, free IFNγ (pre-dose), and total IFNγ; correlation of chemokine and total IFNγ levels with laboratory parameters of MAS severity, e.g., ferritin, platelet count, LFTs (exploratory analysis); and other potential disease biomarkers (e.g., sCD25, IL-10, IL-6, IL-18, TNFα, neopterin).

[0268] Other Aspects While the present invention has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative and not limiting of 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 following claims.

[0269] Sequence information SEQUENCE LISTING <110> SWEDISH ORPHAN BIOVITRUM AG <120> METHODS AND COMPOSITIONS FOR DIAGNOSIS AND TREATMENT OF DISORDERS IN PATIENTS WITH ELEVATED LEVELS OF CXCL9 AND OTHER BIOMARKERS <150> US 62 / 158,153 <151> 2015-05-07 <150> US 62 / 221,393 <151> 2015-09-21 <150> US 62 / 246,949 <151> 2015-10-27 <160> 105 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 1 Ser Tyr Ala Met Ser 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 2 Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 3 Asp Gly Ser Ser Gly Trp Tyr Val Pro His Trp Phe Asp Pro 1 5 10 <210> 4 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 4 Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn Tyr Val Gln 1 5 10 <210> 5 <211> 7 <212> PRT 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32 Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn Tyr Val His 1 5 10 <210> 33 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 33 Gln Ser Ser Asp Thr Thr Tyr His Gly Gly Val Val 1 5 10 <210> 34 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 34 Gly Gly Asn Tyr Gly Asp Tyr Phe Asp Tyr Phe Asp Tyr 1 5 10 <210> 35 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 35 Gln Ser Tyr Glu Gly Phe 1 5 <210> 36 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 36 Thr Gly Arg Asn Gly Asn Ile Ala Ser Asn Tyr Val Gln 1 5 10 <210> 37 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 37 Glu Asp Thr Gln Arg Pro Ser 1 5 <210> 38 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 38 Gln Ser Ser Asp Ser Asn Arg Val Leu 1 5 <210> 39 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 39 Asp Phe Trp Val Ile Thr Ser Gly Asn Asp Tyr 1 5 10 <210> 40 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 40 Gln Ser Phe Asp Ser Thr Asn Leu Val Val 1 5 10 <210> 41 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 41 Ala Gly Ser Ser Gly Ser Ile Ala Ser Asn Tyr Val Gln 1 5 10 <210> 42 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 42 Gln Ser Tyr Ser Tyr Asn Asn Gln Val Val 1 5 10 <210> 43 <211> 1362 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 43 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagatggt 300 agcagtggct ggtacgtacc acactggttc gacccctggg gccagggaac cctggtcacc 360 gtctcctcag cctccaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc 420 acctctgggg gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg 480 acggtgtcgt ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta 540 cagtcctcag gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc 600 acccagacct acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaga 660 gttgagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc 720 ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 780 cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 840 ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900 cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960 aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1020 accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gccccatcc 1080 cgggaggaga tgaccagaa ccaggtcagc ctgacctgcc tggtcaagg cttctatccc 1140 agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agacaacta caaccacg 1200 cctcccgtgc tggactccga cggctccttc ttcctctata gcaagctcac cgtggacaag 1260 agcaggtggc agcagggaa cgtctctca tgctccgtga tgcatgaggc tctgcacac 1320 cactacacgc agagagcct ctccctgtct ccgggtaaat ag 1362 <210> 44 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 44 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Ser Ser Gly Trp Tyr Val Pro His Trp Phe Asp Pro 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Asp Val Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 <210> 45 <211> 654 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 45 aattttatgc tgactcagcc ccactgtg tcggagtctc cggggagac ggtaccacc 60 tcctgcactc gcagcagtgg cagcattgcc agcaactatg tgcagtgta ccacagcgc 120 ccggcagtt cccccaccac tgtcatctat gaggatacc agagaccctc tggggtccct 180 gatcggttct ctggctccat cgacagctcc tccaattctg cctccctcac catctctggg 240 ctgaagactg aggacgaggc tgactactac tgtcagtctt atgatggcag caatcgttgg 300 atgttcggcg gagggaccaa gctgaccgtc ctaggtcagc caaggctgc cccctcggtc 360 actctgttcc cgccctcctc tgaggagctt caagccaaca aggccacact ggtgtgtctc 420 ataagtgact tctacccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 480 aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 540 agctacctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 600 acgcatgaag ggagcaccgt ggaagaca gtggccccta cagaatgttc atag 654 <210> 46 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 46 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Thr Thr Val 35 40 45 Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Gly 85 90 95 Ser Asn Arg Trp Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 115 120 125 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 130 135 140 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 145 150 155 160 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 165 170 175 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 180 185 190 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 195 200 205 Lys Thr Val Ala Pro Thr Glu Cys Ser 210 215 <210> 47 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 47 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Ser Ser Gly Trp Tyr Val Pro His Trp Phe Asp Pro 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 48 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 48 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Thr Thr Val 35 40 45 Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Gly 85 90 95 Ser Asn Arg Trp Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 49 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 49 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagatggt 300 agcagtggct ggtacgtacc acactggttc gacccctggg gccggggcac cctggtcacc 360 gtctcgagt 369 <210> 50 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 50 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Ser Ser Gly Trp Tyr Val Pro His Trp Phe Asp Pro 100 105 110 Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 51 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 51 aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60 tcctgcactc gcagcagtgg cagcattgtc agcaactatg tgcagtggta ccaacagcgc 120 ccgggcagtg cccccaccac tgtcatctat gaggataacc ggagaccctc tggggtccct 180 gatcggttct ctggctccat cgacagctcc tccaatactg cctccctcac catctctggg 240 ctggaggctg aggacgaggc tgactactac tgtcagtctt atgatggcag caatcgttgg 300 atgttcggcg gagggaccaa gctgaccgtc ctaggt 336 <210> 52 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 52 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Val Ser Asn 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Thr Val 35 40 45 Ile Tyr Glu Asp Asn Arg Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Thr Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Glu Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Gly 85 90 95 Ser Asn Arg Trp Met Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 53 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 53 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagatcat 300 agcagtggct ggtacgtaat ctccggtatg gacgtctggg gccgagggac aatggtcacc 360 gtctcgagt 369 <210> 54 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 54 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp His Ser Ser Gly Trp Tyr Val Ile Ser Gly Met Asp Val 100 105 110 Trp Gly Arg Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 55 <211> 333 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 55 aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60 tcctgcaccc gcagcagtgg cagcattgcc agcaactatg tgcagtggta ccagcagcgc 120 ccgggcagtt cccccaccac tgtgatctct gaggataacc aaagaccctc tggggtccct 180 gatcggttct ctggctccgt cgacagctcc tccaactctg cctccctcac catttctgga 240 ctgaggactg aggacgaggc tgactattac tgtcagtcta atgattccga caatgtggtt 300 ttcggcggag ggaccaagct gaccgtccta ggt 333 <210> 56 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 56 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Thr Thr Val 35 40 45 Ile Ser Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Val Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Arg Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Asn Asp Ser 85 90 95 Asp Asn Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 57 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 57 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 <h2 style=";text-align:left;direction:ltr">tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gcagactccg tgaagggccg gttcaccatc tccagagaca atcccaagaa cacgctgtat 240<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gaaggaccta 300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> acagtgggtg gtccctggta ctactttgac tactggggcc aaggaaccct ggtcaccgtc 360<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tcgagt 366<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 58<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 122<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> PRT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Artificial Sequence<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <220><h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <223> chemically synthesized<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 58<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1 5 10 15<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 20 25 30<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 35 40 45<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val<h2 style=";text-align:left;direction:ltr"> 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Served Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Leu Thr Val Gly Gly Pro Trp Tyr Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 59 <211> 333 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 59 aattttatgc tgactcagcc ccactgtg tcggagtctc cggggagac ggtaccacc 60 tcctgcaccc gcagcagtgg cagcattgtc agcaactatg tgcagtgta ccagcagcgc 120 ccgggcagtg cccccaccac tgtgatctt gacgatgacc aaagaccctc tggggtccct 180 ggtcggttct ctggctccct cgacagctcc tccactcg cctccctcac catctctggg 240 ctgcagactg aggacgaggc tgactactac tgtcagtctt atgatagcag caatgtgta 300 ttcggcgggg ggaccaaggt caccgtccta ggt 333 <210> 60 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 60 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Val Ser Asn 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Thr Val 35 40 45 Ile Phe Asp Asp Asp Gln Arg Pro Ser Gly Val Pro Gly Arg Phe Ser 50 55 60 Gly Ser Leu Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Gln Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser 85 90 95 Ser Asn Val Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly 100 105 110 <210> 61 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 61 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gaaagatgga 300 tggaacgcgc tgggatggct tgaatcctgg ggccggggca ccctggtcac cgtctcgagt 360 <210> 62 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 62 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Trp Asn Ala Leu Gly Trp Leu Glu Ser Trp Gly Arg 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 63 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 63 aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaggac gataaccatc 60 tcctgcaccc gcagtggtgg cagcattggc agctactatg tgcagtggta ccagcagcgc 120 ccgggcactg cccccaccac tgtgatctat gacgataaaa aaagaccctc tggggtccct 180 gatcggttct ctggctccat cgacagctcc tccaactctg cctccctcac catctctgga 240 ctgaagactg aggacgaggc tgactactat tgtcagtctt atgatagcaa caatcttgtg 300 gttttcggcg gagggaccaa ggtcaccgtc ctaggt 336 <210> 64 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 64 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Arg 1 5 10 15 Thr Ile Thr Ile Ser Cys Thr Arg Ser Gly Gly Ser Ile Gly Ser Tyr 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Thr Ala Pro Thr Thr Val 35 40 45 Ile Tyr Asp Asp Lys Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser 85 90 95 Asn Asn Leu Val Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly 100 105 110 <210> 65 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 65 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagatggt 300 agcagtggct ggtacgtacc acactggttc gacccctggg gcagggggac aatggtcacc 360 gtctcgagt 369 <210> 66 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 66 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Ser Ser Gly Trp Tyr Val Pro His Trp Phe Asp Pro 100 105 110 Trp Gly Arg Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 67 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 67 aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60 tcctgcaccc gcagcagtgg caccattgcc agcaactatg tgcagtggta ccagcagcgc 120 ccgggcagtt cccccaccac tgtgatctat gaggataacc aaagaccctc tggggtccct 180 gatcggttct ctggctccat cgacagctcc tccaactctg cctccctcac catctctgga 240 ctgaagactg aggacgaggc tgactactac tgtcagtctt atgataacag caatcattgg 300 gtgttcggcg gagggaccaa ggtcaccgtc ctaggt 336 <210> 68 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 68 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Thr Ile Ala Ser Asn 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Thr Thr Val 35 40 45 Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Asn 85 90 95 Ser Asn His Trp Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly 100 105 110 <210> 69 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 69 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc caggggggtc cctgaaactc 60 tcctgtgcag cctctggatt cacctttagc agcaatgcca tgagttgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcaact cttactggta gtggtggtac cgcatactac 180 gcagactccg tggagggccg gttcagcatc tccagagaca attccaagaa cacactgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaagggcacg 300 gaactcgtgg gaggaggact tgacaactgg ggccaaggca ccctggtcac cgtctcgagt 360 <210> 70 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 70 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Asn 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Leu Thr Gly Ser Gly Gly Thr Ala Tyr Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Ser Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Thr Glu Leu Val Gly Gly Gly Leu Asp Asn Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 71 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 71 aattttatgc tgactcagcc ccactctctg tcggagtctc cggggaagac ggtgacgatc 60 tcctgcaccg gcagcggagg cagcattgcc accaactatg tgcagtggta tcagcagcgc 120 ccgggcagtg cccccaccac tgtgatccat gaggataacc aaagaccctc tggggtccct 180 gatcggttct ctggctccat cgacggctcc tccaactctg cctccctcac catctctgga 240 ctgcagcctg aggacgaggc tgattactac tgtcagtctt atgatagtga caatcatcat 300 gtggtattcg gcggagggac caagctgacc gtcctaggt 339 <210> 72 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 72 Asn Phe Met Leu Thr Gln Pro His Ser Leu Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Gly Ser Gly Gly Ser Ile Ala Thr Asn 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Thr Val 35 40 45 Ile His Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Gly Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Gln Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser 85 90 95 Asp Asn His His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 Gly <210> 73 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 73 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gaaagatgga 300 tggaacgcgc tgggatggct tgaatcctgg ggcaagggga caatggtcac cgtctcgagt 360 <210> 74 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 74 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Trp Asn Ala Leu Gly Trp Leu Glu Ser Trp Gly Lys 100 105 110 Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 75 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 75 aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60 tcctgcaccg gcagcagtgg cagcattgcc agcaactatg tgcagtggta ccagcagcgc 120 ccgggcagtg cccccaccac tgtgatctat gaggataacc aaagaccctc tggggtccct 180 gatcggttct ctggctccat cgacagctcc tccaactctg cctccctcac catctctgga 240 ctgaagactg aggacgaggc tgactactac tgtcagtctt atgatagcag caatcaagag 300 gtggtattcg gcggagggac caagctgacc gtcctaggt 339 <210> 76 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 76 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Gly Ser Ser Gly Ser Ile Ala Ser Asn 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Thr Val 35 40 45 Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser 85 90 95 Ser Asn Gln Glu Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 Gly <210> 77 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 77 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagatggt 300 agcagtggct ggtacgtacc acactggttc gacccctggg gccagggaac cctggtcacc 360 gtctcgagt 369 <210> 78 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 78 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Ser Ser Gly Trp Tyr Val Pro His Trp Phe Asp Pro 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 79 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 79 aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggttaccatc 60 tcctgcaccc gcagcagtgg cagcattgtc agcaactatg tacagtggta ccagcagcgc 120 ccgggcagtt cccccaccac tgtgatctat gaggataacc aaagaccctc tggggtccct 180 gatcggttct ctggctccat cgacagctcc tccaactctg cctccctcac catctctgga 240 ctgaagactg aggacgaggc tgactactac tgtcagtctt atgatagcaa caatttttgg 300 gtgttcggcg gagggaccaa gctgaccgtc ctaggt 336 <210> 80 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 80 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Val Ser Asn 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Thr Thr Val 35 40 45 Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser 85 90 95 Asn Asn Phe Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 81 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 81 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgt gaaaaggtcc 300 tttgatagtg gtgggtcctt tgagtactgg ggccagggga caatggtcac cgtctcgagt 360 <210> 82 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 82 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Lys Arg Ser Phe Asp Ser Gly Gly Ser Phe Glu Tyr Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 83 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 83 aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtcaccatc 60 tcctgcaccc gcagcagtgg ctacattgcc agctcctatg tgcagtggta ccagcagcgc 120 ccgggcagtt cccccaccac tgtaatcttt gaggatgacc ggagaccctc tggggtccct 180 gatcggttct ctggctccat cgacggctcc tccaactctg cctccctcac catctctgga 240 ctgaggactg aggacgaggc tgactactac tgtcagtctt atgatgacac cactccctgg 300 gtgttcggcg gagggaccaa gctgaccgtc ctaggt 336 <210> 84 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 84 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Tyr Ile Ala Ser Ser 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Thr Thr Val 35 40 45 Ile Phe Glu Asp Asp Arg Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Gly Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Arg Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Asp 85 90 95 Thr Thr Pro Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 85 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 85 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagagtcggc 300 agctggtacc tggaagattt tgatatctgg ggccggggga caatggtcac cgtctcgagt 360 <210> 86 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 86 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Gly Ser Trp Tyr Leu Glu Asp Phe Asp Ile Trp Gly Arg 100 105 110 Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 87 <211> 342 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 87 aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggttaccatc 60 tcctgcaccc gcagcagtgg cagcattgcc agcaactatg ttcactggta tcagcagcgc 120 ccgggcagtt cacccaccac tgtgatctat gaggataacc gaagaccctc tggggtccct 180 gctcggttct ctggctccat cgacagctcc tccaactctg cctccctcac catctctgga 240 ctggagactg acgacgaggc tgactactac tgtcagtctt ctgataccac ctatcatgga 300 ggtgtggtat tcggcggagg gaccaagctg accgtcctag gt 342 <210> 88 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 88 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn 20 25 30 Tyr Val His Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Thr Thr Val 35 40 45 Ile Tyr Glu Asp Asn Arg Arg Pro Ser Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Glu Thr Asp Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ser Asp Thr 85 90 95 Thr Tyr His Gly Gly Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val 100 105 110 Leu Gly <210> 89 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 89 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gaaaggcggt 300 aactacggtg attacttcga ctactttgac tactggggca gagggacaat ggtcaccgtc 360 tcgagt 366 <210> 90 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 90 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Gly Asn Tyr Gly Asp Tyr Phe Asp Tyr Phe Asp Tyr Trp 100 105 110 Gly Arg Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 91 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 91 aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60 tcctgcaccc gcagcagtgg cagcattgcc agcaattatg tgcagtggta ccagcagcgc 120 ccgggcagtg cccccaccat tgtgatctat gaagataacc aaagaccctc tggggtccct 180 catcggttct ctggctccat cgacagctcc tccaactctg cctccctcac catctctgga 240 ctgaagactg aggacgaggc tgactactac tgtcagtctt atgaggggtt cggcggaggg 300 accaagctga ccgtcctagg t 321 <210> 92 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 92 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val 35 40 45 Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val Pro His Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Glu Gly 85 90 95 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 <210> 93 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 93 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg gttcactatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gaaagatgga 300 tggaacgcgc tgggatggct tgaatcctgg ggccagggga caatggtcac cgtctcgagt 360 <210> 94 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 94 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Arg Asp Asn Serves Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Served Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Trp Asn Ala Leu Gly Trp Leu Glu Ser Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 95 <211> 333 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 95 aattttatgc tgactcagcc ccacgctgtg tcggagtctc cggggaagac ggtgaccatt 60 tcctgcaccg gcagaaatgg siacattgcc agcaactag tgcagtgta ccagcagcgc 120 ccggacagtg cccccaccct tataatcttt gaagataccc aaagaccctc tggggtccct 180 actcggctct caggctccat cgacacctcc tccattctg cctcctcat catctctca 240 ttgaggactg aggacgaggc tgattactac tgtcaatctt ctgattccaa cagggtgctg 300 ttcggcggag ggaccaaggt caccgtccta ggt 333 <210> 96 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 96 Asn Phe Met Leu Thr Gln Pro His Ala Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Gly Arg Asn Gly Asn Ile Ala Ser Asn 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Asp Ser Ala Pro Thr Leu Ile 35 40 45 Ile Phe Glu Asp Thr Gln Arg Pro Ser Gly Val Pro Thr Arg Leu Ser 50 55 60 Gly Ser Ile Asp Thr Ser Ser Asn Ser Ala Ser Leu Ile Ile Ser Ser 65 70 75 80 Leu Arg Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ser Asp Ser 85 90 95 Asn Arg Val Leu Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly 100 105 110 <210> 97 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 97 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gaaagatttt 300 tgggttatta cgagtgggaa tgactactgg gggcggggga ccacggtcac cgtctcgagt 360 <210> 98 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 98 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Phe Trp Val Ile Thr Ser Gly Asn Asp Tyr Trp Gly Arg 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 99 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 99 aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtgaccatc 60 tcctgcaccc gcagcagtgg cagcattgct agcaattatg tgcagtggta ccagcagcgc 120 ccgggcagtt cccccaccac tgtgatcttt gaagataacc gaagaccctc tggggtccct 180 gatcggtttt ctggctccat cgacacctcc tccaactctg cctccctcac catctctgga 240 ctgaagactg aggacgaggc tgactactac tgtcagtctt ttgatagcac caatcttgtg 300 gtgttcggcg gagggaccaa gctgaccgtc ctaggt 336 <210> 100 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 100 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Thr Thr Val 35 40 45 Ile Phe Glu Asp Asn Arg Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Thr Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Phe Asp Ser 85 90 95 Thr Asn Leu Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 101 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 101 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gaaagatgga 300 tggaacgcgc tgggatggct tgaatcctgg gggaagggga ccacggtcac cgtctcgagt 360 <210> 102 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 102 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Trp Asn Ala Leu Gly Trp Leu Glu Ser Trp Gly Lys 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 103 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> chemically synthesized <400> 103 aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60 tcctgcgccg gcagcagtgg cagcattgcc agcaactatg tgcagtggta ccagcagcgc 120 ccgggcagtg cccccaccgc tgtgatctat gaggataacc aaagaccctc tggggtccct 180 gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctgga 240 ctgaagactg aggacgaggc tgactactac tgtcaatctt actcttacaa caatcaggtc 300 gtgttcggcg gagggaccaa ggtcaccgtc ctaggt 336 <210> 104 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 104 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Ala Gly Ser Ser Gly Ser Ile Ala Ser Asn 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ala Val 35 40 45 Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Ser Tyr 85 90 95 Asn Asn Gln Val Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly 100 105 110 <210> 105 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> chemically synthesized <400> 105 Thr Arg Ser Ser Gly Tyr Ile Ala Ser Ser Tyr Val Gln 1 5 10

Claims

1. 1. A method of treating hemophagocytic lymphohistiocytosis (HLH) in a subject in need thereof, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof, The antibody or antigen-binding fragment thereof binds to interferon gamma (IFNγ), and a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO: 1); a variable heavy chain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence a variable heavy chain complementarity determining region 3 (VH CDR3) comprising the amino acid sequence of: a variable light chain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of: a variable light chain complementarity-determining region 2 (VL CDR2) region comprising the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and The method according to any one of claims 1 to 4, wherein the variable light chain complementarity determining region 3 (VL CDR3) region comprises the amino acid sequence of:

2. The method of claim 1, wherein the antibody comprises a heavy chain variable amino acid sequence of the amino acid sequence of SEQ ID NO: 47 and a light chain variable amino acid sequence of the amino acid sequence of SEQ ID NO:

48.

3. 10. The method of claim 1, wherein the antibody is formulated as a sterile concentrate for infusion.

4. 4. The method of claim 3, wherein the formulation comprises 5 mg antibody, 1.55 mg L-histidine, 3.14 mg L-histidine monohydrochloride monohydrate, 7.31 mg sodium chloride (NaCl), and 0.05 mg polysorbate 80, wherein the pH is between 5.8 and 6.

2.

5. 5. The method of claim 4, wherein the pH is 6.

0.

6. 10. The method of claim 1, wherein the antibody is administered to a subject in need thereof by IV infusion over a period of 1 hour at an initial dose of 1 mg / kg.

7. 7. The method of claim 6, wherein the antibody is administered to a subject in need thereof by an initial IV infusion over a period of 1 hour at an initial dose of 1 mg / kg followed by at least one additional IV infusion.

8. 8. The method of claim 7, wherein at least one additional IV infusion is at a dose higher than the initial dose of 1 mg / kg.

9. 7. The method of claim 6, wherein at least one additional IV infusion dosage is 3 mg / kg.

10. 10. The method of claim 6 or claim 9, wherein at least one additional IV infusion is administered at least three days after the initial IV infusion.

11. 11. The method of claim 10, wherein at least one additional IV infusion is administered at a time selected from the group consisting of: 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial infusion, and 15 days after the initial infusion.

12. 11. The method of claim 10, wherein at least one additional IV infusion is administered 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial infusion, and 15 days after the initial infusion.

13. 7. The method of claim 6, wherein the antibody is administered to a subject in need thereof by an initial IV infusion over a period of 1 hour at an initial dose of 1 mg / kg, followed by at least one series of additional IV infusions, wherein said series of additional IV infusions comprises at least one series of twice-weekly IV infusions.

14. 14. The method of claim 13, wherein at least one series of twice-weekly IV infusions is administered at a dose higher than an initial dose of 1 mg / kg.

15. 14. The method of claim 13, wherein at least one series of twice-weekly IV infusions is administered at a dose of 3 mg / kg.

16. 16. The method of claim 13 or claim 15, wherein at least one additional IV infusion is administered at least three weeks after the initial IV infusion.

17. 17. The method of claim 16, wherein at least one additional IV infusion is administered at a time selected from the group consisting of 3 weeks after the initial IV infusion, 4 weeks after the initial IV infusion, 5 weeks after the initial IV infusion, 6 weeks after the initial infusion, 7 weeks after the initial infusion, and 8 weeks after the initial infusion.

18. 17. The method of claim 16, wherein at least one additional IV infusion is administered 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.

19. 7. The method of claim 6, wherein the antibody is administered to a subject in need thereof by an initial IV infusion followed by at least two additional IV infusions.

20. 20. The method of claim 19, wherein the at least two additional IV infusions are at doses higher than the initial dose of 1 mg / kg.

21. 21. The method of claim 20, wherein the first additional IV infusion and the second additional IV infusion are administered at the same dosage.

22. 22. The method of claim 20 or 21, wherein the first additional IV infusion and the second additional IV infusion are administered at the same dosage, which is higher than the initial dose.

23. 23. The method of claim 22, wherein at least one of the first and second additional IV infusions is administered at a dosage of 3 mg / kg.

24. 24. The method of claim 19 or claim 23, wherein the first additional IV infusion is administered at least three days after the initial IV infusion.

25. 25. The method of claim 24, wherein the first additional IV infusion is administered at a time selected from the group consisting of: 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial infusion, and 15 days after the initial infusion.

26. 25. The method of claim 24, wherein the first additional IV infusion is administered 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial infusion, and 15 days after the initial infusion.

27. 25. The method of claim 24, wherein the second additional IV infusion is administered at a time selected from the group consisting of 3 weeks after the initial IV infusion, 4 weeks after the initial IV infusion, 5 weeks after the initial IV infusion, 6 weeks after the initial infusion, 7 weeks after the initial infusion, and 8 weeks after the initial infusion.

28. 25. The method of claim 24, wherein the second additional IV infusion is administered 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.

29. 25. The method of claim 24, wherein the first additional IV infusion is administered 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial infusion, and 15 days after the initial infusion, and the second additional IV infusion is administered 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.

30. 21. The method of claim 20, wherein the first additional IV infusion and the second additional IV infusion are administered at different dosages.

31. 31. The method of claim 30, wherein the first additional IV infusion and the second additional IV infusion are administered at different dosages, wherein each dosage is higher than the initial dose.

32. 31. The method of claim 30, wherein the first additional IV infusion is administered at a dosage of 3 mg / kg.

33. 31. The method of claim 30, wherein the second additional IV infusion is administered at a dosage of 6 mg / kg.

34. 31. The method of claim 30, wherein the first additional IV infusion is administered at a dosage of 3 mg / kg and the second additional IV infusion is administered at a dosage of 6 mg / kg.

35. 35. The method of claim 30 or claim 34, wherein the first additional IV infusion is administered at least three days after the initial IV infusion.

36. 36. The method of claim 35, wherein the first additional IV infusion is administered at a time selected from the group consisting of: 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial infusion, and 15 days after the initial infusion.

37. 36. The method of claim 35, wherein the first additional IV infusion is administered 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial infusion, and 15 days after the initial infusion.

38. 36. The method of claim 35, wherein the second additional IV infusion is administered at a time selected from the group consisting of 3 weeks after the initial IV infusion, 4 weeks after the initial IV infusion, 5 weeks after the initial IV infusion, 6 weeks after the initial infusion, 7 weeks after the initial infusion, and 8 weeks after the initial infusion.

39. 36. The method of claim 35, wherein the second additional IV infusion is administered 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.

40. 36. The method of claim 35, wherein a first additional IV infusion is administered 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial infusion, and 15 days after the initial infusion, and a second additional IV infusion is administered 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.

41. 31. The method of claim 30, wherein the first additional IV infusion comprises at least a first series of twice-weekly IV infusions and the second additional IV infusion comprises at least a second series of twice-weekly IV infusions.

42. 42. The method of claim 41, wherein the first series of twice-weekly IV infusions and the second series of twice-weekly IV infusions are administered at a dose higher than an initial dose of 1 mg / kg.

43. 43. The method of claim 42, wherein a first series of twice-weekly IV infusions is administered at a dose of 3 mg / kg and a second series of twice-weekly IV infusions is administered at a dose of 6 mg / kg.

44. 44. The method of claim 41 or 43, wherein the first series of additional IV infusions is administered at least 3 days after the initial IV infusion.

45. 45. The method of claim 44, wherein the first series of additional IV infusions is administered at a time selected from the group consisting of: 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial infusion, and 15 days after the initial infusion.

46. 45. The method of claim 44, wherein 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 infusion, and 15 days after the initial infusion.

47. 45. The method of claim 44, wherein the second series of additional IV infusions is administered at a time selected from the group consisting of 3 weeks after the initial IV infusion, 4 weeks after the initial IV infusion, 5 weeks after the initial IV infusion, 6 weeks after the initial infusion, 7 weeks after the initial infusion, and 8 weeks after the initial infusion.

48. 45. The method of claim 44, wherein the second series of additional IV infusions is administered at 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.

49. 45. The method of claim 44, wherein a 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 infusion, and 15 days after the initial infusion, and a second series of additional IV infusions is administered 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.

50. 10. The method of claim 1, wherein the subject is receiving background dexamethasone.

51. Subjects have not been previously treated for HLH and have received at least 10 mg / m dexamethasone 2 51. The method of claim 50, wherein the compound is administered at a dose of

52. Subjects receiving antibodies as second-line HLH treatment and dexamethasone at least 5 mg / m 2 51. The method of claim 50, wherein the compound is administered at a dose of

53. 10. The method of claim 1, further comprising administering at least a second agent to the subject.

54. 54. The method of claim 53, wherein the second agent is a therapeutic agent, an anti-inflammatory agent, and / or an immunosuppressant.

55. detecting the level of CXCL9, alone or in combination with one or more other biomarkers, in a biological sample from the subject; comparing the detected level of CXCL9 with a control expression level; and If the detected level is elevated, administering to the subject an anti-interferon gamma (IFNγ) antagonist in an amount sufficient to alleviate the symptoms of the disorder. and methods for alleviating the symptoms of the disorder, including:

56. 56. The method of claim 55, wherein the one or more other biomarkers are selected from the group consisting of total IFNγ levels, CXCL10, CXCL11, and combinations thereof.

57. 56. The method of claim 55, wherein the biological sample is blood or is derived from blood.

58. 56. The method of claim 55, wherein the biological sample is serum.

59. 56. The method of claim 55, wherein the anti-IFNγ antagonist is an anti-IFNγ antibody or an immunologically active fragment thereof.

60. The anti-IFNγ antibody or immunologically active fragment thereof comprises a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO: 1); a variable heavy chain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence of: a variable heavy chain complementarity determining region 3 (VH CDR3) comprising the amino acid sequence of: a variable light chain complementarity determining region 1 (VL CDR1) region comprising the amino acid sequence of: A variable heavy chain complementarity determining region 2 (VL CDR2) comprising the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and Variable heavy chain complementarity determining region 3 (VL CDR3) comprising the amino acid sequence 60. The method of claim 59, comprising:

61. 60. The method of claim 59, wherein the anti-IFNγ antibody or immunologically active fragment thereof comprises a heavy chain variable amino acid sequence of SEQ ID NO: 47 and a light chain variable amino acid sequence of SEQ ID NO:

48.

62. 60. The method of claim 59, wherein the anti-IFNγ antibody or immunologically active fragment thereof comprises the heavy chain amino acid sequence of SEQ ID NO: 44 and the light chain amino acid sequence of SEQ ID NO:

46.

63. 56. The method of claim 55, wherein the subject is a human.

64. 56. The method of claim 55, wherein the disorder is an autoimmune disorder or an inflammatory disorder.

65. 56. The method of claim 55, wherein the disorder is primary or secondary hemophagocytic lymphohistiocytosis (HLH).

66. 56. The method of claim 55, wherein the disorder is macrophage activation syndrome (MAS).

67. 56. The method of claim 55, wherein the disorder is MAS in the setting of systemic juvenile idiopathic arthritis (sJIA).

68. (a) a therapeutically effective amount of an isolated antibody or antigen-binding fragment thereof that binds interferon gamma (IFNγ); (b) L-histidine; (c) L-histidine monohydrochloride monohydrate; (d) sodium chloride (NaCl); (e) Polysorbate 80; and having a pH of 5.8 to 6.

2.

69. The isolated antibody comprises a variable heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO: 1); a variable heavy chain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence a variable heavy chain complementarity determining region 3 (VH CDR3) comprising the amino acid sequence of: a variable light chain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of: a variable light chain complementarity-determining region 2 (VL CDR2) region comprising the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and 69. The formulation of claim 68, comprising a variable light chain complementarity determining region 3 (VL CDR3) region comprising the amino acid sequence of:

70. 69. The formulation of claim 68, wherein the antibody comprises a heavy chain variable amino acid sequence of the amino acid sequence of SEQ ID NO: 47 and a light chain variable amino acid sequence of the amino acid sequence of SEQ ID NO:

48.

71. 69. The formulation of claim 68, comprising 5 mg antibody, 1.55 mg L-histidine, 3.14 mg L-histidine monohydrochloride monohydrate, 7.31 mg sodium chloride (NaCl), and 0.05 mg.

72. 72. The formulation of claim 68 or claim 71, wherein the pH of the formulation is 6.

0.

73. 69. The formulation of claim 68, which is a sterile concentrated formulation for injection.

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