Methods and compositions for diagnosing and treating disorders in patients with elevated levels of CXCL9 and other biomarkers

Abstract

The present invention relates to methods and compositions for diagnosing and treating disorders in patients with elevated levels of CXCL9 and other biomarkers. Methods and compositions for treating hemophagocytic lymphohistiocytosis (HLH) are provided. The present disclosure also relates generally to methods and compositions for diagnosing and treating disorders associated with elevated levels of CXCL9, total IFNy, and other biomarkers. The present disclosure also relates to methods of treating, delaying progression of, or otherwise ameliorating symptoms of disorders in patients with elevated levels of CXCL9, total IFNy, and other biomarkers using agents that intervene or otherwise antagonize interferon gamma (IFNy) signaling, including neutralizing anti-IFNy antibodies.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201680040022.8, filed on May 9, 2016.

[0002] Related applications This application claims the benefits 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 which are incorporated herein by reference in their entirety. Technical Field

[0003] This disclosure generally relates to methods and compositions for treating hemophagocytic lymphohistiocytosis (HLH). Methods and compositions are also provided for diagnosing and treating conditions associated with elevated CXCL9 levels, elevated total IFNγ levels, and other biomarkers. This disclosure also relates to methods for treating, delaying the progression of, or otherwise improving the symptoms of conditions in patients with elevated CXCL9 levels, elevated total IFNγ levels, and other biomarkers using agents that intervene in or otherwise antagonize interferon-γ (IFNγ) signaling (including neutralizing anti-IFNγ antibodies). Background Technology

[0004] Human interferon-gamma (IFNγ, IFN-gamma) is a lymphokine produced by activated T lymphocytes and natural killer cells. It exhibits antiproliferative and immunomodulatory activity, binding to IFNγ-R (a heterodimer receptor on most primary cells of the immune system) and triggering a series of inflammatory events. The immunomodulatory activity of IFNγ is known to have beneficial effects in many clinical situations. However, there are numerous clinical contexts where IFNγ activity is known to have detrimental effects. For example, autoimmune diseases are associated with high levels of IFNγ in the blood and diseased tissues of autoimmune patients. IFNγ activity is also associated with disease states such as cachexia and septic shock.

[0005] IFNγ is involved in a variety of diseases; anti-IFNγ agents are being developed as therapeutics. Therefore, there is a need for compositions and methods for identifying biomarkers of IFNγ production in IFNγ-related diseases. Summary of the Invention

[0006] The compositions and methods described herein utilize a fully human IgG1 anti-interferon-γ (IFNγ) monoclonal antibody (mAb), referred to herein as NI-0501, which binds to and neutralizes IFNγ. NI-0501 binds to both the soluble and receptor (IFNγR1) forms of IFNγ. The compositions and methods described herein may be used for the treatment of hemophagocytic lymphohistiocytosis (HLH).

[0007] The anti-IFNγ antibody referred to in this article as NI-051 contains a variable heavy chain complementarity-determining region 1 (VH CDR1) containing the amino acid sequence of SYAMS (SEQ ID NO: 1); a variable heavy chain complementarity-determining region 2 (VH CDR2) containing the amino acid sequence of AISGSGGSTYYADSVKG (SEQ ID NO: 2); a variable heavy chain complementarity-determining region 3 (VH CDR3) containing the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO: 3); a variable light chain complementarity-determining region 1 (VL CDR1) containing the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO: 4); a variable light chain complementarity-determining region 2 (VL CDR2) containing the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and a variable light chain complementarity-determining region 3 (VL CDR3) containing the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6). NI-0501 contains the heavy chain variable amino acid sequence of the amino acid sequence of SEQ ID NO: 47 and the light chain variable amino acid sequence of the amino acid sequence of SEQ ID NO: 48.

[0008] In the compositions and methods provided herein, NI-0501 is formulated as a sterile concentrate (in mL) for infusion. In some embodiments, NI-0501 is formulated as follows: 5 mg NI-051, 1.55 mg L-histidine, 3.14 mg L-histidine hydrochloride monohydrate, 7.31 mg sodium chloride (NaCl), and 0.05 mg polysorbate 80, wherein the pH is between 5.8 and 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 hydrochloride monohydrate, 7.31 mg sodium chloride (NaCl), and 0.05 mg polysorbate 80, wherein the pH is 6.0.

[0009] In the compositions and methods provided herein, NI-0501 is administered to subjects in need for the treatment, prevention, and / or delay of the onset or progression of HLH-related symptoms, or to the relief of HLH-related symptoms. In some embodiments, NI-0501 is administered to subjects in need via intravenous infusion over 1 hour at an initial dose of 1 mg / kg. In certain patient populations, such as those with low body weight and / or very young age, the intravenous infusion may continue for more than 1 hour, such as at least 90 minutes, at least 2 hours, or at least 3 hours or longer.

[0010] In some embodiments, NI-0501 is administered to the recipient at an initial dose of 1 mg / kg over 1 hour following the initial IV infusion via 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 is at a dose of 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 following: 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial IV infusion, and 15 days after the initial IV infusion. In some embodiments, the at least one additional IV infusion is administered at 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial IV infusion, and 15 days after the initial IV infusion.

[0011] In some embodiments, NI-0501 is administered to the recipient at an initial dose of 1 mg / kg over 1 hour following the initial IV infusion via at least a series of additional IV infusions, wherein the series of additional IV infusions comprises at least a series of twice-weekly IV infusions. In some embodiments, at least a series of twice-weekly IV infusions are administered at a dose higher than the initial dose of 1 mg / kg. In some embodiments, at least a series of twice-weekly IV infusions are administered at a dose of 3 mg / kg. In some embodiments, at least one additional IV infusion is administered at least three weeks after the initial IV infusion. In some embodiments, at least one additional IV infusion is administered at a time selected from the following: 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 implementations, at least one additional IV infusion is given 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, and 8 weeks after the initial IV infusion.

[0012] In some embodiments, NI-0501 is administered to the recipient subject with at least two additional IV infusions following the initial IV infusion. In some embodiments, the at least two additional IV infusions are at a dose higher than the initial dose of 1 mg / kg. In some embodiments, the first and second additional IV infusions are administered at the same dose. In some embodiments, the first and second additional IV infusions are administered at the same dose higher than the initial dose. In some embodiments, at least one of the first and second additional IV infusions is administered at a dose of 3 mg / kg. In some embodiments, the first additional IV infusion is administered at least 3 days after the initial IV infusion. In some embodiments, the first additional IV infusion is administered at a time selected from the following: 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial IV infusion, and 15 days after the initial IV infusion. In some implementations, the first additional IV infusion is given 3 days, 6 days, 9 days, 12 days, and 15 days after the initial IV infusion. In some implementations, the second additional IV infusion is given at a time selected from the following: 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, and 8 weeks after the initial infusion. In some implementations, the second additional IV infusion is given 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, and 8 weeks after the initial IV infusion. In some implementations, the first additional IV infusion is given 3 days, 6 days, 9 days, 12 days, and 15 days after the initial IV infusion, and the second additional IV infusion is given 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, and 8 weeks after the initial infusion.

[0013] In some embodiments, the first and second additional IV infusions are administered at different doses. In some embodiments, the first and second additional IV infusions are administered at different doses, wherein the second additional IV infusion dose is higher than the first additional IV infusion dose. In some embodiments, the first and second additional IV infusions are administered at different doses, wherein the second additional IV infusion dose is higher than the first additional IV infusion dose, and wherein both the first and second additional IV infusion doses are higher than the initial dose. In some embodiments, at least one of the first and second additional IV infusions is administered at a dose of 3 mg / kg. In some embodiments, the first additional IV infusion is administered at a dose of 3 mg / kg, and the second additional IV infusion is administered at a dose of 6 mg / kg. In some embodiments, the first additional IV infusion is administered at least 3 days after the initial IV infusion. In some embodiments, the first additional IV infusion is administered at a time selected from the following: 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial IV infusion, and 15 days after the initial IV infusion. In some implementations, the first additional IV infusion is given 3 days, 6 days, 9 days, 12 days, and 15 days after the initial IV infusion. In some implementations, the second additional IV infusion is given at a time selected from the following: 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, and 8 weeks after the initial infusion. In some implementations, the second additional IV infusion is given 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, and 8 weeks after the initial IV infusion. In some implementations, the first additional IV infusion is given 3 days, 6 days, 9 days, 12 days, and 15 days after the initial IV infusion, and the second additional IV infusion is given 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, and 8 weeks after the initial infusion.

[0014] In some embodiments, the first additional IV infusion comprises at least a first series of twice-weekly IV infusions, and the second additional IV infusion comprises at least a second series of twice-weekly IV infusions. In some embodiments, the first series of twice-weekly IV infusions and the second series of twice-weekly IV infusions are administered at a dose higher than the initial dose of 1 mg / kg. In some embodiments, the first series of twice-weekly IV infusions is administered at a dose of 3 mg / kg, and the second series of twice-weekly IV infusions is administered at a dose of 6 mg / kg. In some embodiments, the first series of additional IV infusions is administered at least 3 days after the initial IV infusion. In some embodiments, the first series of additional IV infusions is administered at a time selected from the following: 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial IV infusion, and 15 days after the initial IV infusion. In some implementations, the first series of additional IV infusions is administered 3 days, 6 days, 9 days, 12 days, and 15 days after the initial IV infusion. In some implementations, the second series of additional IV infusions is administered at times selected from the following: 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, and 8 weeks after the initial infusion. In some implementations, the second series of additional IV infusions is administered 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, and 8 weeks after the initial IV infusion. In some implementations, the first series of additional IV infusions are given 3 days, 6 days, 9 days, 12 days, and 15 days after the initial IV infusion, and the second series of additional IV infusions are given 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, and 8 weeks after the initial infusion.

[0015] In some embodiments, infusions are administered every 3 days until 15 days after the initial dose. In some embodiments, infusions are administered every 3 days until 15 days after the initial dose, followed by twice-weekly infusions starting at least 15 days after the initial dose. In some embodiments, the infusion dose is increased to 3 mg / kg at any point after the initial dose. In some embodiments, after at least two 3 mg / kg infusions, the dose of NI-0501 is increased to 6 mg / kg up to four infusions.

[0016] In the compositions and methods provided herein, NI-0501 is administered to subjects in need for the treatment, prevention, and / or delay of the onset or progression of HLH-related symptoms, or to alleviate HLH-related symptoms. In some embodiments, NI-0501 is administered to subjects in need via IV infusion over 1 hour at an initial dose of 1 mg / kg. In some embodiments, infusions are administered every 3 days after the initial dose until 15 days after the initial dose. In some embodiments, infusions are administered every 3 days after the initial dose until 15 days after the initial dose, followed by twice-weekly infusions starting at least 15 days after the initial dose. In some embodiments, the infusion dose is increased to 3 mg / kg at any point after the initial dose. In some embodiments, after at least two 3 mg / kg infusions, the dose of NI-0501 is increased to 6 mg / kg up to 4 infusions.

[0017] In the compositions and methods provided herein, NI-0501 is administered to subjects in need for the treatment, prevention, and / or delay of the onset or progression of HLH-related symptoms, or to alleviate HLH-related symptoms. In some embodiments, NI-0501 is administered to subjects in need via intravenous infusion at a dose greater than 6 mg / kg. In some embodiments, a second dose of NI-0501 greater than 6 mg / kg is administered to subjects in need via intravenous infusion after an initial dose. In some embodiments, the second dose is at least 10 mg / kg. In some embodiments, the second dose is 10 mg / kg. In some embodiments, the second dose is 10 mg / kg, repeated daily. In some embodiments, the second dose is 10 mg / kg, repeated daily for up to 1 week. In some embodiments, the second dose is 10 mg / kg, repeated daily for up to 2 weeks. In some embodiments, the second dose is 10 mg / kg, repeated daily for more than 2 weeks.

[0018] In some embodiments, NI-0501 is administered to subjects in need for the treatment, prevention, and / or delay of the onset or progression of secondary HLH-related symptoms, or to alleviate secondary HLH-related symptoms. In some embodiments, NI-0501 is administered to subjects in need in the context of sJIA for the treatment, prevention, and / or delay of the onset or progression of secondary HLH-related symptoms, or to alleviate secondary HLH-related symptoms. In some embodiments, NI-0501 is administered to subjects in need as an initial dose of 6 mg / kg. In some embodiments, NI-0501 treatment is continued with subsequent doses of NI-0501. In some embodiments, NI-0501 treatment is continued with a subsequent dose of 3 mg / kg of NI-0501 every 3 days for at least 4 weeks (i.e., until SD27).

[0019] In some implementations, NI-0501 treatment is reduced, discontinued, or otherwise shortened upon achieving the desired clinical outcome. In some implementations, NI-0501 treatment is shortened upon evidence of a complete clinical response (i.e., MAS remission).

[0020] In some implementations, after 4 weeks, NI-0501 treatment may be continued as needed for up to an additional 4 weeks (i.e., until SD56) as maintenance until MAS remission is achieved. In some implementations, continuing NI-0501 treatment as needed for up to an additional 4 weeks (i.e., until SD56) after 4 weeks as maintenance until MAS remission has the possibility of reducing the dose to 1 mg / kg and extending the interval between infusions to weekly dosing.

[0021] In the compositions and methods provided herein, NI-0501 is administered to a subject in need for the treatment, prevention, and / or delay of the onset or progression of HLH-related symptoms, or to alleviate HLH-related symptoms, wherein the subject has a history of dexamethasone administration. In some embodiments, the subject is a treatment-naïve patient (i.e., has not previously been treated for HLH), and dexamethasone is administered at a dose of at least 10 mg / m². 2 The dosage was administered. In some implementations, the subject was receiving NI-0501 as second-line HLH treatment, and dexamethasone was administered at a dose ranging from 10 mg / m². 2 -5 mg / m 2 The dosage was administered. In some implementations, the subject was receiving NI-0501 as second-line HLH treatment, and dexamethasone was administered at a dose of at least 5 mg / m². 2 The dose was administered. In some implementations, the subject was receiving NI-0501 as second-line HLH treatment, and dexamethasone was administered at a dose less than 5 mg / m². 2 The prescribed dosage was administered.

[0022] In some embodiments, NI-0501 is administered before and / or during and / or after treatment in combination with one or more other agents (e.g., as a non-limiting example, therapeutic agents, anti-inflammatory drugs, and / or immunosuppressants). In some embodiments, the second agent is an agent known for the treatment of HLH. In some embodiments, the other agents include at least etoposide. In some embodiments, NI-0501 and the other agents are formulated into a single therapeutic composition and administered simultaneously. Alternatively, NI-0501 and the other agents are separated from each other, for example, each formulated into a separate therapeutic composition and administered simultaneously, or administered at different times during the treatment regimen. For example, NI-0501 may be administered before, after, or alternately with the other agents. As described herein, NI-0501 and the other agents may be administered in single or multiple doses.

[0023] In some embodiments, NI-0501 and other agents are administered simultaneously. For example, NI-0501 and other agents may be formulated in a single composition or administered as two or more separate compositions. In some embodiments, NI-0501 and other agents are administered sequentially, or at different times during a treatment regimen.

[0024] In some embodiments, the other agents are immunosuppressants. In some embodiments, the immunosuppressant is cyclosporine A (CsA). In some embodiments, the subject had been receiving CsA prior to administration of NI-0501. In some embodiments, the other agents include at least etoposide. In some embodiments, the subject had been receiving etoposide prior to administration of NI-0501.

[0025] In some implementations, the other agents are intrathecal methotrexate and / or glucocorticoids. In some implementations, the subject had been receiving intrathecal methotrexate and / or glucocorticoids prior to administration of NI-0501.

[0026] In some implementations, the other agent is IV immunoglobulin (IVIG). In some implementations, IVIG is administered as an alternative treatment in subjects with confirmed immunoglobulin deficiency. In some implementations where the subject has confirmed immunoglobulin deficiency, IVIG is administered at a dose of 0.5 g / kg every 4 weeks or more to maintain appropriate IgG levels.

[0027] In some implementations, one or more other agents are analgesic treatment, blood product infusions, electrolyte and glucose infusions, antibiotics, antifungal and antiviral treatments, and / or routine supportive care.

[0028] This disclosure also provides compositions and methods for identifying or otherwise refining patient populations with a disease in which the patient's CXCL9 level is elevated alone or in conjunction with one or more other interferon-γ (IFNγ)-related biomarkers. Specifically, this disclosure provides compositions and methods for detecting CXCL9 levels as a biomarker of IFNγ production in patients with or suspected of having hemophagocytic lymphohistiocytosis (HLH). More specifically, this disclosure provides compositions and methods for detecting CXCL9 levels as a biomarker of IFNγ production in patients with or suspected of having secondary hemophagocytic lymphohistiocytosis (HLH). In some embodiments, the compositions and methods are used to detect CXCL9 levels as a biomarker of IFNγ production in patients with or suspected of having macrophage activation syndrome (MAS). In some embodiments, the compositions and methods are used to detect CXCL9 levels as a biomarker of IFNγ production in patients with or suspected of having MAS in the context of autoimmune diseases or inflammatory conditions. In some embodiments, the composition and method are used to detect CXCL9 levels as a biomarker of IFNγ production in patients with MAS in the context of having or suspected of having systemic autoimmune disease or inflammatory conditions. In some embodiments, the composition and method are used to detect CXCL9 levels as a biomarker of IFNγ production in patients with MAS in the context of having or suspected of having systemic juvenile idiopathic arthritis (sJIA). In some embodiments, the composition and method are used to detect CXCL9 levels as a biomarker of IFNγ production in patients with MAS in the context of having or suspected of having systemic lupus erythematosus (SLE).

[0029] Patients identified as having elevated CXCL9 levels were identified as suitable candidates for treatment with agents 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 (e.g., antibodies or other peptide-based therapeutics, peptide-based therapeutics, small molecule inhibitors, nucleic acid-based therapeutics, and their derivatives).

[0030] In patients with or suspected of having the condition, fluid and other biological samples contained elevated levels of CXCL9, either alone or in conjunction with other IFNγ-related biomarkers, such as CXCL10 and / or CXCL11.

[0031] CXCL9 and these other biomarkers are indicators of IFNγ production in the body. Therefore, the use of anti-IFNγ antagonists that interfere with, inhibit, reduce, or otherwise antagonize IFNγ signaling, such as neutralizing anti-IFNγ antibodies or other peptide-based therapeutics, peptide-based therapeutics, small molecule inhibitors, nucleic acid-based therapeutics, and their derivatives, blocks or otherwise inhibits IFNγ activity. Therefore, the compositions and methods described herein can be used to treat patients exhibiting elevated levels of CXCL9 and / or other biomarkers by administering anti-IFNγ antagonists, such as neutralizing anti-IFNγ antibodies or other peptide-based therapeutics, peptide-based therapeutics, small molecule inhibitors, nucleic acid-based therapeutics, and their derivatives, to treat symptoms of a condition dependent on, delayed in the progression of, or otherwise improved in the condition, IFNγ expression and / or activity abnormalities (e.g., elevated levels), abnormal pro-inflammatory cytokine production and / or combinations thereof, driven by, associated with, or otherwise affected by, IFNγ. Patients who may be suitable candidates for treatment with anti-IFNγ antagonists (such as neutralizing anti-IFNγ antibodies, as described herein) are identified by detecting the level of CXCL9 alone or in conjunction with one or more IFNγ-related ligands or other biomarkers. In some embodiments, patients without elevated levels of CXCL9 alone or in conjunction with other IFNγ-related biomarkers may still be treated with anti-IFNγ antagonists, including neutralizing anti-IFNγ antibodies described herein or other peptide-based therapeutics, peptide-based therapeutics, small molecule inhibitors, nucleic acid-based therapeutics, and their derivatives.

[0032] Patients with elevated CXCL9 levels, alone or in combination with one or more other IFNγ-related biomarkers, were identified as suitable candidates for therapy with one or more anti-IFNγ antagonists, such as the neutralizing anti-IFNγ antibody described herein. As used herein, “elevated expression level” means an expression level greater than the baseline expression level of CXCL9, alone or in combination with one or more other biomarkers, in samples from patients who do not have or are not suspected of having primary or secondary HLH or HLH-related conditions, or in another control sample. In some embodiments, the elevated expression levels of CXCL9 and / or other biomarkers are significant elevations.

[0033] The detected level of CXCL9, alone or in conjunction with one or more other IFNγ-related biomarkers, can be used to refine or otherwise stratify a patient population. In some embodiments, the detected level is used to determine the dosage of an anti-IFNγ antagonist to be administered to a given patient. In some embodiments, the detected level is used to classify or otherwise stratify a patient population. For example, based on the CXCL9 detection level, a patient may be classified as having “severe” or high-grade MAS, or conversely, as having non-severe or low-grade MAS.

[0034] The sample is, for example, blood or a blood component, such as serum or plasma. In some embodiments, the sample is another bodily fluid, such as, by non-limiting example, 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 an HLH patient.

[0035] In addition to detecting levels of IFNγ and / or other IFNγ-related biomarkers, suitable patients for treatment with anti-IFNγ antagonists can be identified by evaluating any of a variety of other biological and clinical parameters that can improve the sensitivity and specificity of biomarkers used to identify or otherwise refine patient populations. Alternatively, these other biological and clinical parameters can be used alone as a means of identifying suitable candidates for treatment with anti-IFNγ antagonists or other suitable therapies. By way of non-limiting examples, these biological and clinical parameters include any of the following: ferritin levels, neutrophil count, platelet count, alanine aminotransferase levels, and / or lactate dehydrogenase levels.

[0036] The compositions and methods of the present invention are useful for conditions including any condition in which IFNγ expression and / or activity is abnormal (e.g., elevated), particularly HLH, including secondary HLH, MAS, and / or sJIA.

[0037] By way of non-limiting example, the methods and compositions provided herein are suitable for diagnosing and / or treating conditions such as primary and / or secondary HLH conditions. By way of non-limiting example, suitable autoimmune and / or inflammatory conditions include primary and / or secondary HLH conditions associated with abnormal IFNγ activity and / or expression.

[0038] Once a patient is identified as having elevated CXCL9 levels, alone or in conjunction with one or more IFNγ-related biomarkers, they are treated with an anti-IFNγ antagonist. For example, an anti-IFNγ antagonist is a neutralizing anti-IFNγ antibody or a fragment of it with immune activity (e.g., antigen binding). Suitable neutralizing anti-IFNγ antibodies include any of the anti-IFNγ antibodies described herein.

[0039] In some embodiments, the anti-IFNγ antibody or its immunoactive fragment contains a variable heavy chain complementarity-determining region 1 (VH CDR1) comprising an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or higher identity with 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 having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or higher identity with the amino acid sequence of AISGSGGSTYYADSVKG (SEQ ID NO: 2); and a variable heavy chain complementarity-determining region 3 (VH CDR3) comprising an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or higher identity with the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO: 3); and a variable heavy chain complementarity-determining region 3 (VH CDR3) comprising an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or higher identity with the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO: 1). 4) A variable light chain complementarity-determining region 1 (VL CDR1) containing an amino acid sequence with at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or higher identity; a variable light chain complementarity-determining region 2 (VL CDR2) containing an amino acid sequence with at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or higher identity with the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and a variable light chain complementarity-determining region 3 (VLCDR3) containing an amino acid sequence with at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or higher identity with the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6).

[0040] In some embodiments, the anti-IFNγ antibody or its immunoactive fragment contains a VH CDR1 region containing the amino acid sequence of SYAMS (SEQ ID NO: 1); a VHCDR2 region containing the amino acid sequence of AISGSGGSTYYADSVKG (SEQ ID NO: 2) and a VH CDR3 region containing the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO: 3); a variable light chain complementarity-determining region 1 (VL CDR1) region containing the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO: 4); a VL CDR2 region containing the amino acid sequence of EDNQRPS (SEQ ID NO: 5) and a VL CDR3 region containing the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6).

[0041] In some embodiments, the anti-IFNγ antibody or its immunologically active fragment contains a heavy chain comprising a combination of VH CDR1, VHCDR2 and VH CDR3 sequences, 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.

[0042] In some embodiments, the anti-IFNγ antibody or its immunologically active fragment contains a light chain comprising a combination of VL CDR1, VLCDR2 and VL CDR3 sequences, wherein the combination is a combination of the three light chain CDR sequences (VL CDR1, VL CDR2, VL CDR3) shown in a single row in Table 1B.

[0043] In some embodiments, the anti-IFNγ antibody or its immunologically active fragment contains a heavy chain comprising a combination of VH CDR1, VHCDR2, and VH CDR3 sequences, 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 VL CDR1, VL CDR2, and VL CDR3 sequences, wherein the combination is a combination of the three light chain CDR sequences (VL CDR1, VL CDR2, VLCDR3) shown in a single row in Table 1B.

[0044] In some embodiments, the anti-IFNγ antibody or its immunoactive fragment comprises an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or higher identity with the heavy chain variable amino acid sequence of SEQ ID NO: 47.

[0045] In some embodiments, the anti-IFNγ antibody or its immunoactive fragment comprises an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or higher identity with the amino acid sequence of SEQ ID NO: 48.

[0046] In some embodiments, the anti-IFNγ antibody or its immunoactive fragment comprises an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or higher identity to the heavy chain variable amino acid sequence of the amino acid sequence of SEQ ID NO: 47 and an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or higher identity to the light chain variable amino acid sequence of the amino acid sequence of SEQ ID NO: 48.

[0047] In some embodiments, the anti-IFNγ antibody or its immunoactive fragment comprises a heavy chain variable amino acid sequence of the amino acid sequence of SEQ ID NO: 47.

[0048] In some embodiments, the anti-IFNγ antibody or its immunoactive fragment comprises 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 its immunomodulatory fragment 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.

[0050] In some embodiments, the anti-IFNγ antibody or its immunoactive fragment contains an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or higher identity to the heavy chain amino acid sequence of the amino acid sequence of SEQ ID NO: 44.

[0051] In some embodiments, the anti-IFNγ antibody or its immunoactive fragment comprises an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or higher identity 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 its immunoactive fragment comprises an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or higher identity to the heavy chain amino acid sequence of SEQ ID NO: 44 and an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or higher identity to the light chain amino acid sequence of SEQ ID NO: 46.

[0053] In some embodiments, the anti-IFNγ antibody or its immunoactive fragment comprises the heavy chain amino acid sequence of the amino acid sequence of SEQ ID NO: 44.

[0054] In some embodiments, the anti-IFNγ antibody or its immunoactive fragment comprises the light chain amino acid sequence of the amino acid sequence of SEQ ID NO: 46.

[0055] In some embodiments, the anti-IFNγ antibody or its immunologically active fragment comprises the heavy chain amino acid sequence of the amino acid sequence of SEQ ID NO: 44 and the light chain amino acid sequence of the amino acid sequence of SEQ ID NO: 46.

[0056] In some embodiments, the anti-IFNγ antibody or its immunoactive fragment comprises an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or higher identity with a heavy chain variable amino acid sequence selected from SEQ ID NO: 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98 and 102.

[0057] In some embodiments, the anti-IFNγ antibody or its immunoactive fragment comprises an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or higher identity with a light chain variable amino acid sequence selected from SEQ ID NO: 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100 and 104.

[0058] In some embodiments, the anti-IFNγ antibody or its immunomodulatory fragment comprises an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or higher identity with a heavy chain variable amino acid sequence selected from SEQ ID NO: 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, and 102, and an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or higher identity with a light chain variable amino acid sequence selected from SEQ ID NO: 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, and 104.

[0059] In some embodiments, the anti-IFNγ antibody or its immunoactive fragment contains a heavy chain variable amino acid sequence selected from SEQ ID NO: 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98 and 102.

[0060] In some embodiments, the anti-IFNγ antibody or its immunoactive fragment contains a light chain variable amino acid sequence selected from SEQ ID NO: 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, and 104.

[0061] In some embodiments, the anti-IFNγ antibody or its immunoactive fragment contains a heavy chain variable amino acid sequence selected from SEQ ID NO: 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 SEQ ID NO: 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100 and 104.

[0062] In some embodiments, an anti-IFNγ antibody or its immunologically active fragment is administered in a therapeutically effective amount. The therapeutically effective amount of the antibody of the present invention generally refers to the amount required to achieve a therapeutic purpose. This therapeutic purpose may be a binding interaction between the antibody and its target antigen, which in some cases interferes with the function of the target. By non-limiting example, a common range of therapeutically effective doses of the antibody or antibody fragment of the present invention is about 0.1 mg / kg body weight to about 50 mg / kg body weight. A common dose frequency range is, for example, twice daily to once weekly.

[0063] In some embodiments, the anti-IFNγ antibody or its immunologically active fragment is administered at an initial dose (i.e., a loading dose) in the range of about 0.5 mg / kg to about 2 mg / kg, for example, in the range of 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 its immunologically active fragment is administered at an initial dose of about 1.0 mg / kg.

[0064] In some embodiments, the anti-IFNγ antibody or its immunologically active fragment is administered as an initial loading dose followed by one or more maintenance doses. In some embodiments, the one or more maintenance doses are doses substantially similar to the initial loading dose. In some embodiments, the one or more maintenance doses are doses less than the initial loading dose. In some embodiments, the one or more maintenance doses are doses greater than the initial loading dose.

[0065] In some embodiments, one or more maintenance doses comprise at least two or more doses, wherein each maintenance dose is the same dose. In some embodiments, two or more maintenance doses are substantially similar to the initial loading dose. In some embodiments, two or more maintenance doses are greater than the initial loading dose. In some embodiments, two or more maintenance doses are less than the initial loading dose.

[0066] In some embodiments, one or more maintenance doses comprise at least two or more doses, wherein each maintenance dose is a different dose. In some embodiments, two or more maintenance doses are administered in an escalating dose pattern. In some embodiments, two or more maintenance doses are administered in a decelerating dose pattern.

[0067] In some embodiments, one or more maintenance doses comprise at least two or more doses, wherein each maintenance dose is administered at periodic intervals. In some embodiments, the two or more doses are administered at increasing intervals. In some embodiments, the two or more doses are administered at decreasing intervals.

[0068] In some embodiments, the anti-IFNγ antibody or its immunologically active fragment is administered at an initial loading dose ranging from about 0.5 mg / kg to about 2 mg / kg, for example, from about 0.5 mg / kg to about 1.5 mg / kg and / or from about 0.5 mg / kg to about 1.0 mg / kg; followed by at least one, for example, two or more, three or more, four or more, or five or more maintenance doses. In some embodiments, the anti-IFNγ antibody or its immunologically active fragment is administered at an initial loading dose of about 1.0 mg / kg; followed by at least one, for example, two or more, three or more, four or more, or five or more maintenance doses.

[0069] The pharmaceutical compositions of the present invention may include the anti-IFNγ antibody and the carrier of the present invention. These pharmaceutical compositions may be included in kits (e.g., diagnostic kits).

[0070] The present invention also provides kits for implementing any of the methods provided herein. For example, in some embodiments, the kit includes a detection reagent specific for CXCL9, alone or in conjunction with one or more IFNγ-related biomarkers, and tools for detecting the detection reagent. Attached Figure Description

[0071] Figure 1 This is a graph illustrating the correlation between pre-drug serum CXCL9 levels and total IFNγ levels 24 hours after NI-0501 antibody infusion in patients with primary HLH in an ongoing phase 2 trial.

[0072] Figure 2 This is a graph illustrating the correlation between serum CXCL9 levels 24 hours after NI-0501 antibody infusion and total IFNγ levels before administration in patients with primary HLH in an ongoing phase 2 trial.

[0073] Figure 3A and 3B This is a series of graphs depicting the correlation between serum CXCL9 and IFNγ levels in patients with systemic juvenile idiopathic arthritis (sJIA) secondary to macrophage activation syndrome (MAS) and in patients with active sJIA.

[0074] Figures 4A-1, 4A-2, 4B-1, 4B-2, 4C-1, 4C-2, 4D-1, and 4D-2 are a series of figures depicting the correlation between IFNγ and serum CXCL9 levels and clinical parameters in patients with active sJIA and sJIA-secondary MAS.

[0075] Figure 5This is a diagram illustrating the complete neutralization of IFNγ as indicated by the undetectable level of IFNγ-induced chemokines.

[0076] Figure 6 This is a graphic depicting the improvement in HLH disease activity during NI-0501 treatment (2 weeks and end of treatment): platelet count >100 x 10⁻⁶. 9 / L, neutrophil count >1 x 10 9 The percentage of patients with a blood plasma density of 1 / L, fibrinogen >1.5 g / L, and ferritin reduction of at least 25%.

[0077] Figure 7A and 7B This is a series of graphs describing the correlation between CXCL9 levels before drug administration and total IFNγ levels 24 hours after NI-0501 infusion. Figure 7B The illustration depicts examples of the distribution of each IFNγ and CXCL9 during NI-0501 treatment.

[0078] Figure 8A , 8B Figures 8C and 8D are a series of graphs describing serum levels of IFNγ and CXCL9, CXCL10, and CXCL11 in individual patients during active MAS and during active sJIA without MAS (Act sJIA) at the time of sampling. Significance was determined using the Wilcoxon rank test for paired samples.

[0079] Figure 9A and 9B This describes a patient's white blood cell (WBC) and platelet (PLT) counts and ferritin levels during his sJIA and 3 MAS episodes. Figure 9A Changes in IFNγ, CXCL9, CXCL10, and CXCL11 ( Figure 9B A series of graphs showing changes in serum levels of ).

[0080] Figures 10A, 10B, 10C, 10D, 10E, 10F, 10G, 10H, 10I, and 10J are a series of graphs depicting the correlations between IFNγ and CXCL9 levels and ferritin levels, neutrophil and platelet counts, and LDH and ALT levels in patients with active MAS at sampling time (red circles) and patients with active sJIA but no MAS at sampling time (black triangles). Spearman correlation coefficients (Rs) and significance levels (p) for each correlation are shown in Table 3.

[0081] Figure 11A , 11BFigures 11C, 11D, 11E, and 11F are a series of graphs describing the relationship between IFNγ and CXCL9 and CXCL10 in MAS. Figure A: Correlation between IFNγ levels and CXCL9 and CXCL10 levels in patients with MAS at sampling time. Spearman correlation coefficients (Rs) and significance levels (p) for each correlation are shown in Table 3.

[0082] Figure 12 This is an illustration of the screening, treatment, and follow-up sections of the study presented in Example 7.

[0083] Figure 13A and 13B This is a diagram illustrating the effect of NI-0501 on body temperature in two patients with a body temperature >37.5°C at the start of NI-0501 treatment.

[0084] Figure 14 This is a series of charts describing the effect of NI-0501 administration on neutrophil counts in patients.

[0085] Figure 15 This is a series of charts describing the effect of NI-0501 administration on platelet count in patients.

[0086] Figure 16 This is a series of charts describing the effect of NI-0501 administration on serum ferritin levels in patients.

[0087] Figure 17 This is a series of charts describing the effect of NI-0501 administration on the gradual reduction of glucocorticoid levels in patients.

[0088] Figure 18 This is a diagram illustrating the effect of NI-0510 in maintaining IFNγ neutralization until HSCT. The response of HLH to NI-0501 treatment also persists until transplantation.

[0089] Figure 19 This is an illustration of the screening, treatment, and follow-up portion of the study provided in Example 8. Detailed Implementation

[0090] The compositions and methods described herein utilize a fully human IgG1 anti-interferon-γ (IFNγ) monoclonal antibody (mAb) (referred to herein as NI-0501) that binds to and neutralizes IFNγ. NI-0501 binds to both the soluble and receptor (IFNγR1) forms of IFNγ. Because NI-0501 is human IgG1, it retains the properties of this immunoglobulin isotype, including the ability to occupy Fcγ receptors and bind complement. IFNγ is one of the most potent and pleiotropic cytokines of the immune system. It is crucial for both innate and adaptive immunity against viral and intracellular bacterial infections. Upon binding to its receptor, IFNγ plays a role in generating various physiological and cellular responses. Numerous studies over the past 20 years have linked IFNγ to the pathogenesis and maintenance of inflammatory diseases (see, for example, Billiau A. “Interferon-gamma: biology and role in pathogenesis.” Adv. Immunol. 1996; 62:61-130; Schoenborn JR, Wilson CB. “Regulation of interferon-gamma during innate and adaptive immune responses.” Adv. Immunol. 2007;96:41-101; and Zhang SY, Boisson-Dupuis S, Chapgier A et al., “Inborn errors of interferon (IFN)-mediated immunity in humans: insights into the respective roles of IFN-alpha / beta, IFN-gamma, and IFN-lambda in host defense.” Immunol. Rev. 2008;226:29-40). Once antigen-binding specific immunity occurs, IFNγ primarily functions through natural killer (NK) and natural killer T cells. (NKT) cells (as part of the innate immune response) and effector T cells produced through CD4 Th1 and CD8 cytotoxic T lymphocytes (CTLs).

[0091] The compositions and methods provided herein can be used to treat hemophagocytic lymphohistiocytosis (HLH). HLH is a syndrome characterized by severely impaired or absent cytotoxic function of NK and CD8+ T cells in response to significant activation of the immune system.

[0092] HLH includes primary (hereditary / familial) HLH and secondary HLH, both clinically described as immune system dysregulation leading to severe hypercytokineemia with detrimental outcomes to 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: Table 9. HLH Classification Primary HLH is a heterologous autosomal recessive disorder. Primary HLH is most common in infancy and early childhood, with an estimated incidence of 1 in 50,000 live births in Europe (Henter JI, Elinder G, Soder O, Ost A. Incidence in Sweden and clinical features of familial hemophagocytic lymphohistiocytosis. Acta Paediatr. Scand. 1991;80:428-435). This disease is fatal, and without treatment, the median survival is less than 2 months after the onset of symptoms (Janka GE. Familialhemophagocytic lymphohistiocytosis. Eur. J. Pediatr. 1983;140:221-230; and Aricò 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).

[0093] Impaired cytotoxic function in HLH leads to hypercytokineemia and hemophagocytosis. These, in turn, cause all the typical symptoms of HLH (Dhote R, Simon J, Papo T et al. Reactive hemophagocytic syndrome in adult systemic disease: report of twenty-six cases and literature review. Arthritis Rheum. 2003;49:633-639; Risdall RJ, McKenna RW, Nesbit ME et al. Virus-associated hemophagocytic syndrome: a benignhistiocytic 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, phagocytic activity, hypercytokineemia and / or lymphohistiocytic infiltration, bone marrow hypoplasia, and meningeal infiltration.

[0094] The cytokines that are elevated in HLH patients include: IFNγ, interleukin-6 (IL-6), IL-10, tumor necrosis factor (TNF) α, IL-8, macrophage colony-stimulating factor (MCSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0095] HLH can also occur during infections, rheumatic diseases, or neoplastic diseases; in this case, it is called secondary HLH. Secondary HLH presents with the same signs and symptoms as the primary form and may be just as severe. Current treatments for secondary HLH aim to address the underlying cause. This is certainly the case for HLH caused by infections (such as leishmaniasis). Notably, the presence of certain infections, particularly viral infections (such as those caused by CMV or EBV), is very often the trigger for the primary form of HLH. This observation is further supported by the following evidence in animal models of primary HLH: infection with lymphocytic choroid plexus meningitis virus (LCMV) is essential for the development of the disease (Jordan MB, Hildeman D, Kappler J, Marrack P. An animal model of hemophagocytic lymphohistiocytosis (HLH): CD8+ T cells and interferon gamma are essential for the disorder. Blood 2004;104:735-743; Pachlopnik SJ, Ho CH, Chretien F et al. Neutralization of IFNgamma defeats haemophagocytosis in LCMV-infected perforin-and Rab27a-deficient mice. EMBO Mol. Med. 2009;1:112-124; Kögl T, Müller 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, Menasché G et al. Distinct severity of HLHin both human and murine mutants with complete loss of cytotoxic effectorPRF1, RAB27A, and STX11 Blood. 2013;121:595-603).

[0096] When HLH occurs during oncology, especially hematologic malignancies, the severity of the patient's condition often necessitates immediate treatment of the HLH before addressing the underlying disease.

[0097] The presence of signs and symptoms of HLH in patients with rheumatic diseases (such as systemic juvenile idiopathic arthritis (sJIA) and systemic lupus erythematosus (SLE)) is often referred to by rheumatologists as macrophage activation syndrome (MAS) and can precede the rheumatic disease itself. Most patients with MAS have impaired NK cells, and perforin function tests and a significant number of patients show polymorphisms or heterozygous mutations in PRF1 and UNC13D. Although it is an extremely serious and life-threatening condition, it usually resolves when appropriate treatment is initiated (in most cases consisting of corticosteroids and cyclosporine). However, in approximately 15% of patients who develop MAS, the disease may be difficult to control, and etoposide may be considered (Minoia F, Davi S, Horne AC et al. Clinical Features, Treatment, and Outcome of Macrophage Activation Syndrome Complicating Systemic Juvenile Idiopathic Arthritis: A Multinational, Multicenter Study of 362 Patients. Arthritis & Rheumatism 2014;66: 3160-3169).

[0098] Although primary HLH is primarily recognized as a childhood disease, HLH is a condition that can occur in adults, and there is a growing awareness that it occurs more frequently than previously thought. In most adult patients, the disease occurs during periods of malignancy (primarily non-Hodgkin's lymphoma), infection, autoinflammatory or autoimmune diseases, and iatrogenic immunodeficiency.

[0099] There are currently no approved drugs for the treatment of HLH. However, experts in this field have established guidelines for the management of HLH patients (Henter JI, Horne AC, Arico' M, Egeler RM, Filipovich AH, Imashuku SLadisch 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 bonemarrow transplantation. Blood 2002; 100:2367-2373; and Jordan MB, Allen CE, Weitzman S, Filipovich AH, McClain KL. How I treat hemophagocytic lymphohistiocytosis. Blood 2011; 118:4041-4052).

[0100] The management of patients with primary HLH currently includes the following steps (Henter et al. Blood Cancer 2007): (i) an 8-week induction therapy with a combination of corticosteroids and immunosuppressive drugs (e.g., etoposide, CsA, alenzumab, antithymocyte globulin); (ii) maintenance therapy until transplantation; and (iii) transplantation for all patients with identified genetic defects and ultimately no disease-related mutations in cases of extremely severe HLH.

[0101] The primary goal of induction therapy is to suppress the life-threatening inflammatory process characterized by HLH, enabling transplantation in patients who require it (Horne A, Janka G, Maarten ER et al. Haematopoietic stem cell transplantation in haemophagocytic lymphohistiocytosis. Br. J. Haematol. 2005;129:622-630). Transplantation is the only therapeutic treatment for HLH associated with high penetrance genetic mutations (Henter et al. Blood 2002).

[0102] Despite the adoption of such guidelines, the overall mortality rate of primary HLH remains at 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).

[0103] During induction, the use of drugs associated with serious short-term and long-term safety concerns further contributes to the already high mortality rate. The compositions and methods presented herein have been developed as targeted therapies, ensuring efficacy with low toxicity.

[0104] In recent years, increasing evidence has emerged regarding the crucial role of IFNγ in the development of HLH (Henter JI, Elinder G, Soder O et al. Hypercytokinemia in familial hemophagocytic lymphohistiocytosis. Blood 1991;78:2918-2922; Jordan MB, Hildeman D, Kappler J, Marrack P. An animal model of hemophagocytic lymphohistiocytosis (HLH): CD8+ T cells and interferon gamma are essential for the disorder. Blood 2004;104:735-743; Pachlopnik SJ, Ho CH, Chretien F et al. Neutralization of IFNgamma defeats haemophagocytosis in LCMV-infected perforin-and Rab27a-deficient mice. EMBO Mol. Med. 2009;1:112-124; Behrens). EM, Canna SW, Slade K etal. Repeated TLR9 stimulation results in macrophage activation syndrome-likedisease in mice. J. Clin. Invest 2011;121:2264-2277; Cytokine Pattern in 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).

[0105] Gene mutations characterizing the primary form of HLH all affect proteins involved in the same process, ultimately impairing cytotoxic activity. Perforin mutations were first identified in HLH patients.

[0106] Perforin knockout (KO) mice are considered a model of human disease. In fact, once infected with LCMV, these mice exhibit all the diagnostic features of the human disease and many clinically and laboratory-specific characteristics, and they die if left untreated. For these reasons, perforin KO mice have been used to study the pathophysiology of HLH. The HLH-like pathology they exhibit depends on the production of CD8+ T cells and IFNγ in response to antigen-binding stimuli.

[0107] It was confirmed that when high circulating levels of IFNγ were neutralized, administration of anti-IFNγ antibodies not only restored clinical and laboratory abnormalities but also significantly improved survival. Conversely, the elimination of any other cytokines had no effect on survival (Jordan et al., Blood 2004; Pachlopnik et al., EMBO Mol. Med. 2009).

[0108] Two secondary HLH models were investigated under the NI-0501 research project. In one model, repeated administration of CpG (inducing TLR9 stimulation) was used to model chronic severe hyperstimulation as infection-induced HLH in healthy mice (i.e., those with normal genetics and cytotoxic pathways). Although these mice did not necessarily die, they developed typical clinical and laboratory features of HLH. When IFNγ was neutralized with anti-IFNγ antibody, the clinical and laboratory features of the disease were restored. Notably, in this model, administration of anti-IFNγ antibody was also demonstrated to lead to complete neutralization of IFNγ effects in relevant target tissues (e.g., liver and spleen) (draft in preparation).

[0109] To investigate the pathophysiology of secondary HLH occurring in the context of rheumatic disease, an animal model was created using IL-6 transgenic mice expressing high levels of IL-6, similar to those produced in patients with sJIA (the rheumatic disease most commonly associated with secondary forms of HLH). These mice died with multiple features of the human disease when induced with Toll-like receptor (TLR) ligands (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). In these mice, survival was significantly improved and laboratory parameters were restored when IFNγ was neutralized with anti-IFNγ antibodies (Prencipe G et al., draft in progress).

[0110] The importance of IFNγ in HLH is further enhanced by the high levels of circulating IFNγ in patients with primary HLH (Henter et al., Blood 1991; Xu et al., J Pedatr 2011). In a series of 71 patients from HLH diagnosis to treatment and follow-up monitoring, IFNγ levels exceeded the upper limit of normal (17.3 pg / mL) in all patients, particularly exceeding 1000 pg / mL in 53.5%. Rapid increases in IFNγ levels in the early stages have also been reported, and with effective treatment of HLH, levels can decrease from >5000 pg / mL to normal within 48 hours.

[0111] Recent observational studies in patients with secondary forms of HLH have confirmed high levels of IFNγ in both patients with infection-related HLH and those with HLH occurring in the context of sJIA. Levels of CXCL9, CXCL10, and CXCL11 (three known IFNγ-induced chemokines) were also significantly elevated. Notably, levels of IFNγ and the three IFNγ chemokines were found to be significantly correlated with laboratory parameters of disease severity, such as ferritin, platelet count, and transaminases (Bracaglia et al., manuscript submitted).

[0112] Hypercytokineemia and organ infiltration caused by activated lymphocytes and histiocytes are the causes of all HLH symptoms, and depend on excessive CD8+ T cell activity and high IFNγ levels. IFNγ neutralization constitutes a reasonable treatment approach. In fact, there are currently no available agents specifically targeting CD8+ T cells, and targeting the various cytokines downstream of IFNγ is not necessarily feasible.

[0113] Therefore, based on data from animal models of primary and secondary HLH and observations in patients with both primary and secondary HLH, the key role of IFNγ in the pathogenesis of the disease is confirmed. Neutralization of IFNγ provides a solid foundation for the development of targeted therapies for HLH, which must be effective without or with limited toxicity.

[0114] This disclosure also provides compositions and methods for identifying or otherwise refining patient populations with conditions in which CXCL9 levels are elevated alone or in conjunction with one or more other interferon-γ (IFNγ)-related biomarkers. Specifically, this disclosure provides compositions and methods for detecting CXCL9 levels as a biomarker of IFNγ production in hemophagocytic lymphohistiocytosis (HLH), secondary HLH, and / or macrophage activation syndrome (MAS).

[0115] Extensive evidence from animal models suggests a key pathogenic role for IFNγ in primary hemophagocytic lymphohistiocytosis (HLH). High levels of IFNγ are also present in individuals with HLH. Previously, high levels of IFNγ and three IFNγ-related chemokines, CXCL9, CXCL10, and CXCL11, have been reported in patients with active MAS (a secondary form of HLH occurring in cases of systemic juvenile idiopathic arthritis (sJIA)) (see, for example, Bracaglia C., Caiello I, De Graaf K. et al. Pediatric Rheumatology 2014, 12(Suppl1):O3). Indirect evidence in mice suggests that IFNγ is primarily produced in peripheral tissues, and blood concentrations may be relatively low.

[0116] Macrophage activation syndrome (MAS) is a potentially fatal and severe complication of chronic inflammatory rheumatic diseases. It typically occurs in cases of systemic juvenile idiopathic arthritis (sJIA), with 10–20% of patients experiencing the syndrome during the course of the disease. Although rare, it can also occur in systemic lupus erythematosus, Kawasaki disease, and other autoimmune and autoinflammatory diseases. In sJIA, MAS usually occurs during active disease phases, including disease flare-ups. Infectious triggers can be identified in a high proportion of patients. Typical features of MAS include fever, splenomegaly, hemorrhage and signs of liver disease, central nervous system involvement, and kidney involvement that can lead to multiple organ failure. Laboratory abnormalities include decreased white blood cell, platelet, and hemoglobin levels, decreased transaminase levels, significantly 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 considerable portion of deaths from sJIA (Minoia, F., et al., Clinical features, treatment, and outcome of macrophage activation syndrome complicating systemic juvenile idiopathic arthritis: a multinational, multicenter study of 362 patients. Arthritis Rheumatol, 2014.66(11): p. 3160-9; Hashkes, PJ, et al., Mortality outcomes in pediatric rheumatology in the US. Arthritis Rheum, 2010. 62(2): p. 599-608). A better understanding of the pathogenesis of the disease, along with the identification of new therapeutic targets and the development of possible targeted therapies, could lead to significant improvements in the management and outcomes of MAS.

[0117] MAS exhibits most of the clinical features and laboratory abnormalities of hemophagocytic lymphohistiocytosis (HLH), and is currently actually classified as secondary or reactive HLH (sec-HLH) (Jordan, MB, et al., How I treat hemophagocytic lymphohistiocytosis.Blood, 2011. 118(15): p. 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 result in defective cytotoxic activity of CD8+ lymphocytes and NK cells. HLH is defined as secondary or reactive in the absence of an identifiable genetic cause and / or familial inheritance, according to existing classifications. Sec-HLH can occur in the absence of a verifiable trigger, or in the presence of infection, malignancy, or rheumatic disease, the latter often referred to as MAS. The genetic basis of MAS is being elucidated, and multiple studies have shown an association between MAS and, in general, sec-HLH with heterozygosity of low penetrance variants or mutations in the same pathogenic genes as p-HLH (Kaufman, KM et al., Whole-exome sequencing reveals overlap between macrophage activation syndrome in systemic juvenile idiopathic arthritis and familial hemophagocytic lymphohistiocytosis. Arthritis Rheumatol, 2014. 66(12): p. 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 macrophageactivation syndrome (MAS) occurring in systemic juvenile idiopathic arthritis (sJIA). Pediatric Rheumatology 2014, 12(Suppl 1): P53). These similarities in the genetic background between p-HLH and MAS further support a shared pathogenic mechanism.

[0118] Studies in p-HLH patients and p-HLH mouse models support the hypothesis that defective cytotoxic activity and aberrations in antigen-presenting cell (APC)-CD8+ T cell crosstalk lead to defective silencing of immune responses and aberrant T cell activation. This results in uncontrolled immune activation and the production of pro-inflammatory cytokines by T lymphocytes and macrophages, leading to organ damage. Studies in p-HLH animal models, specifically in perforin and Rab27-deficient mice, have shown the crucial role of interferon-γ (IFNγ) produced by activated CD8+ T cells. In perforin-deficient mice, IFNγ neutralization leads to survival in otherwise lethal syndrome, where biochemical and hematological abnormalities are restored (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): p.735-43; Pachlopnik Schmid, J. et al., Neutralization of IFNgamma defeats haemophagocytosis in LCMV-infected perforin-and Rab27a-deficient mice. EMBOMol Med, 2009. 1(2): p. 112-24). In Rab27-deficient mice, where the disease does not lead to death, neutralization of IFNγ resulted in significant improvement in peripheral organ involvement, including the central nervous system (Pachlopnik 2009). High circulating IFNγ levels were also present in HLH patients diagnosed according to the HLH 2004 diagnostic criteria (My, LT et al., Comprehensive analyzes and characterization of haemophagocytic lymphohistiocytosis in Vietnamese children.Br J Haematol, 2010. 148(2): p.301-10; Takada, H. et al., Increased serum levels of interferon-gamma-inducible protein 10 and monokine induced by gamma interferon in patients with haemophagocytic lymphohistiocytosis. Clin Exp Immunol, 2003. 133(3): p. 448-53; Tang, Y. et al., Early diagnostic and prognostic significance of a specific Th1 / Th2 cytokine pattern in children with haemophagocytic syndrome. Br JHaematol, 2008. 143(1): p. 84-91; Xu, XJ et al., Diagnostic accuracy of a specific cytokine pattern in hemophagocytic lymphohistiocytosis in children. JPediatr, 2012. 160(6): p. 984-90 e1), therefore it is not necessarily based on the presence of a genetic mutation. It should be noted that these studies include a considerable (but variable) proportion of patients without a verifiable genetic cause (ibid.).

[0119] This study was designed to evaluate the correlation between serum levels of IFNγ and three IFNγ-related chemokines in patients with active MAS and their correlation with laboratory parameters of disease activity to identify biomarkers of IFNγ production in vivo. Specifically, circulating levels of IFNγ, CXCL9, CXCL10, CXCL11, and IL-6 were measured in patients with sJIA, approximately 37% (20 / 54) of whom had MAS at the time of sampling. The relationship between circulating levels and disease activity parameters was also evaluated, as was the correlation between IFNγ levels and CXCL9, CXCL10, and CXCL11 levels. In some implementations, the biomarker is total IFNγ level, which can be used as a pharmacodynamic biomarker.

[0120] As demonstrated in this study, compared with active sJIA without MAS at the time of sampling, the levels of IFNγ and three IFNγ-related chemokines, CXCL9, CXCL10, and CXCL11, were significantly elevated in active MAS. Laboratory parameters of disease severity, such as ferritin, neutrophils, platelets, alanine aminotransferase, and lactate dehydrogenase, were found to be significantly correlated with IFNγ and CXCL9 in active MAS; less correlated with CXCL10 and CXCL11; and not correlated with IL-6 levels. In patients with active sJIA but without MAS, no significant correlation was found between laboratory parameters and cytokine levels. In active MAS, IFNγ levels were significantly correlated with CXCL9 levels, less correlated with CXCL10 levels, and not correlated with CXCL11 levels.

[0121] High levels of IFNγ and CXCL9 in patients with active MAS are significantly associated with laboratory parameters of disease severity. IFNγ and CXCL9 are closely related in patients with active MAS. Since it has been shown that CXCL9 is 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 results presented in this paper confirm that CXCL9 is a biomarker for IFNγ production in MAS.

[0122] The study presented in this article also confirms that the levels of IFNγ and chemokine (CXC motif) ligand 9 (CXCL9), CXL10, and CXCL11 (three known chemokines induced by IFNγ) are elevated in patients with MAS complicated by sJIA, but not in patients with active sJIA without MAS. However, in these patients, the levels of IFNγ, CXCL9, CXCL10, and CXCL11 correlated with laboratory parameters of disease severity.

[0123] The neutralizing anti-IFNγ antibody of the present invention includes, for example, the heavy chain complementarity-determining region (CDR) shown in Table 1A below, the light chain CDR shown in Table 1B, and combinations thereof. Amino acids including the complementarity-determining region (CDR), as defined by Chothia et al., 1989; EA Kabat et al., 1991, are highlighted below with underlined and italicized text. (See Chothia, C et al., Nature 342:877-883 (1989); Kabat, EA et al., Sequences of Protein of immune interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)).

[0124] Table 1A. VHCDR sequences from antibody clones that bind to and neutralize IFNγ Table 1B. VL CDR sequences from antibody clones that bind to and neutralize IFNγ Exemplary antibodies of the present invention include, for example, the anti-IFNγ antibody described in PCT Publication No. WO2006 / 109191, the contents of which are incorporated herein by reference in their entirety.

[0125] Exemplary antibodies of the present invention include, for example, an antibody referred to herein as NI-0501, which binds to human IFNγ. The following represents the heavy chain, light chain, variable heavy (VH) chain, and variable light (VL) chain sequences of the NI-0501 antibody, having the CDR sequences indicated by underlined lines in the VH and VL amino acid sequences: NI-0501 heavy chain nucleic acid sequence: NI-0501 heavy chain amino acid sequence: EVQLLESSGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYCAK DGSSGWYVPHWFDP WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 44) NI 0501 light chain nucleic acid sequence: AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCATCTCCTGCACTCGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGTACCAACAGCGCCCGGGCAGTTCCCCCACCACTGTCATCTATGAGGATAACCAGAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAATTCTGCCTCCCTCACCATCTCTGGGCTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGATGGCAGCAATCGTTGGATGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCATAG (SEQ ID NO: 45) NI 0501 light chain amino acid sequence: NFMLTQPHSVSESPGKTVTISC TRSSGSIASNYVQ WYQQRPGSSPTTVIY EDNQRPS GVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC QSYDGSNRWM FGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID NO: 46) NI-0501 heavy chain variable region amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DGSSGWYVPHWFDP WGQGTLVTVSS (SEQ ID NO: 47) NI 0501 light chain variable region amino acid sequence NFMLTQPHSVSESPGKTVTISC TRSSGSIASNYVQ WYQQRPGSSPTTVIY EDNQRPS GVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC QSYDGSNRWM FGGGTKLTVL (SEQ ID NO: 48) Suitable anti-IFNγ antibodies include those described in U.S. Patent 7,700,098, which is incorporated herein by reference in its entirety. Several exemplary antibodies include those referred to herein as 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. Antibody (“G10”).

[0126] The following shows the sequences of these antibodies.

[0127] A6 VH nucleic acid sequence: GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGATGGTAGCAGTGGCTGGTACGTACCACACTGGTTCGACCCCTGGGGCCGGGGCACCCTGGTCACCGTCTCGAGT(SEQ ID NO: 49) A6 VH amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DGSSGWYVPHWFDP WGRGTLVTVSS (SEQ ID NO: 50) A6 VL nucleic acid sequence: AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCATCTCCTGCACTCGCAGCAGTGGCAGCATTGTCAGCAACTATGTGCAGTGGTACCAACAGCGCCCGGGCAGTGCCCCCACCACTGTCATCTATGAGGATAACCGGAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAATACTGCCTCCCTCACCATCTCTGGGCTGGAGGCTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGATGGCAGCAATCGTTGGATGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT (SEQ ID NO: 51) A6 VL amino acid sequence: NFMLTQPHSVSESPGKTVTISC TRSSGSIVSNYVQ WYQQRPGSAPTTVIY EDNRRPS GVPDRFSGSIDSSSNTASLTISGLEAEDEADYYC QSYDGSNRWM FGGGTKLTVLG (SEQ ID NO: 52) B4 VH nucleic acid sequence: GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGATCATAGCAGTGGCTGGTACGTAATCTCCGGTATGGACGTCTGGGGCCGAGGGACAATGGTCACCGTCTCGAGT(SEQ ID NO: 53) B4 VH amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DHSSGWYVISGMDV WGRGTMVTVSS (SEQ ID NO: 54) B4 VL nucleic acid sequence: AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGTACCAGCAGCGCCCGGGCAGTTCCCCCACCACTGTGATCTCTGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCGTCGACAGCTCCTCCAACTCTGCCTCCCTCACCATTTCTGGACTGAGGACTGAGGACGAGGCTGACTATTACTGTCAGTCTAATGATTCCGACAATGTGGTTTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT (SEQ ID NO: 55) B4 VL amino acid sequence: NFMLTQPHSVSESPGKTVTISC TRSSGSIASNYVQ WYQQRPGSSPTTVIS EDNQRPS GVPDRFSGSVDSSSNSASLTISGLRTEDEADYYC QSNDSDNVV FGGGTKLTVLG (SEQ ID NO: 56) B9 VH nucleic acid sequence: GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATCCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAGGACCTAACAGTGGGTGGTCCCTGGTACTACTTTGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGT (SEQID NO: 57) B9 VH amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNPKNTLYLQMNSLRAEDTAVYYCAK DLTVGGPWYYFDY WGQGTLVTVSS (SEQ ID NO: 58) B9 VL nucleic acid sequence: AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGTCAGCAACTATGTGCAGTGGTACCAGCAGCGCCCGGGCAGTGCCCCCACCACTGTGATCTTTGACGATGACCAAAGACCCTCTGGGGTCCCTGGTCGGTTCTCTGGCTCCCTCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGGCTGCAGACTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGATAGCAGCAATGTGGTATTCGGCGGGGGGACCAAGGTCACCGTCCTAGGT (SEQ ID NO: 59) B9 VL amino acid sequence: NFMLTQPHSVSESPGKTVTISC TRSSGSIVSNYVQ WYQQRPGSAPTTVIF DDDQRPS GVPGRFSGSLDSSSNSASLTISGLQTEDEADYYC QSYDSSNVV FGGGTKVTVLG (SEQ ID NO: 60) C9 VH nucleic acid sequence: GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAAGATGGATGGAACGCGCTGGGATGGCTTGAATCCTGGGGCCGGGGCACCCTGGTCACCGTCTCGAGT (SEQ ID NO:61) C9 VH amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DGWNALGWLES WGRGTLVTVSS (SEQ ID NO: 62) C9 VL nucleic acid sequence: AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAGGACGATAACCATCTCCTGCACCCGCAGTGGTGGCAGCATTGGCAGCTACTATGTGCAGTGGTACCAGCAGCGCCCGGGCACTGCCCCCACCACTGTGATCTATGACGATAAAAAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGAGGACGAGGCTGACTACTATTGTCAGTCTTATGATAGCAACAATCTTGTGGTTTTCGGCGGAGGGACCAAGGTCACCGTCCTAGGT (SEQ ID NO: 63) C9 VL amino acid sequence: NFMLTQPHSVSESPGRTITISC TRSGGSIGSYYVQ WYQQRPGTAPTTVIY DDKKRPS GVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC QSYDSNNLVV FGGGTKVTVLG (SEQ ID NO: 64) C10 VH nucleic acid sequence: GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGATGGTAGCAGTGGCTGGTACGTACCACACTGGTTCGACCCCTGGGGCAGGGGGACAATGGTCACCGTCTCGAGT(SEQ ID NO: 65) C10 VH amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DGSSGWYVPHWFDP WGRGTMVTVSS (SEQ ID NO: 66) C10 VL nucleic acid sequence: AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCATCTCCTGCACCCGCAGCAGTGGCACCATTGCCAGCAACTATGTGCAGTGGTACCAGCAGCGCCCGGGCAGTTCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGATAACAGCAATCATTGGGTGTTCGGCGGAGGGACCAAGGTCACCGTCCTAGGT (SEQ ID NO: 67) C10 VL amino acid sequence: NFMLTQPHSVSESPGKTVTISC TRSSGTIASNYVQ WYQQRPGSSPTTVIY EDNQRPS GVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC QSYDNSNHWV FGGGTKVTVLG (SEQ ID NO: 68) D3 VH nucleic acid sequence: GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCAGGGGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCAATGCCATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTCTTACTGGTAGTGGTGGTACCGCATACTACGCAGACTCCGTGGAGGGCCGGTTCAGCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAGGGCACGGAACTCGTGGGAGGAGGACTTGACAACTGGGGCCAAGGCACCCTGGTCACCGTCTCGAGT (SEQ ID NO:69) D3 VH amino acid sequence: EVQLLESGGGLVQPGGSLKLSCAASGFTFS SNAMS WVRQAPGKGLEWVS TLTGSGGTAYYADSVEG RFSISRDNSKNTLYLQMNSLRAEDTAVYYCAK GTELVGGGLDN WGQGTLVTVSS (SEQ ID NO: 70) D3 VL nucleic acid sequence: AATTTTATGCTGACTCAGCCCCACTCTCTGTCGGAGTCTCCGGGGAAGACGGTGACGATCTCCTGCACCGGCAGCGGAGGCAGCATTGCCACCAACTATGTGCAGTGGTATCAGCAGCGCCCGGGCAGTGCCCCCACCACTGTGATCCATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACGGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGCAGCCTGAGGACGAGGCTGATTACTACTGTCAGTCTTATGATAGTGACAATCATCATGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT (SEQ ID NO: 71) D3 VL amino acid sequence: NFMLTQPHSLSESPGKTVTISC TGSGGSIATNYVQ WYQQRPGSAPTTVIH EDNQRPS GVPDRFSGSIDGSSNSASLTISGLQPEDEADYYC QSYDSDNHHVV FGGGTKLTVLG (SEQ ID NO: 72) D6 VH nucleic acid sequence: GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAAGATGGATGGAACGCGCTGGGATGGCTTGAATCCTGGGGCAAGGGGACAATGGTCACCGTCTCGAGT (SEQ ID NO:73) D6 VH amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DGWNALGWLES WGKGTMVTVSS (SEQ ID NO: 74) D6 VL nucleic acid sequence: AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCATCTCCTGCACCGGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGTACCAGCAGCGCCCGGGCAGTGCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGATAGCAGCAATCAAGAGGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT (SEQ ID NO: 75) D6 VL amino acid sequence: NFMLTQPHSVSESPGKTVTISC TGSSGSIASNYVQ WYQQRPGSAPTTVIY EDNQRPS GVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC QSYDSSNQEVV FGGGTKLTVLG (SEQ ID NO: 76) D8 VH nucleic acid sequence: GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGATGGTAGCAGTGGCTGGTACGTACCACACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT(SEQ ID NO: 77) D8 VH amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DGSSGWYVPHWFDP WGQGTLVTVSS (SEQ ID NO: 78) D8 VL nucleic acid sequence: AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTTACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGTCAGCAACTATGTACAGTGGTACCAGCAGCGCCCGGGCAGTTCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGATAGCAACAATTTTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT (SEQ ID NO: 79) D8 VL amino acid sequence: NFMLTQPHSVSESPGKTVTISC TRSSGSIVSNYVQ WYQQRPGSSPTTVIY EDNQRPS GVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC QSYDSNNFWV FGGGTKLTVLG (SEQ ID NO: 80) E1 VH nucleic acid sequence: GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGTGAAAAGGTCCTTTGATAGTGGTGGGTCCTTTGAGTACTGGGGCCAGGGGACAATGGTCACCGTCTCGAGT (SEQ ID NO:81) E1 VH amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCVK RSFDSGGSFEY WGQGTMVTVSS (SEQ ID NO: 82) E1 VL nucleic acid sequence: AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTCACCATCTCCTGCACCCGCAGCAGTGGCTACATTGCCAGCTCCTATGTGCAGTGGTACCAGCAGCGCCCGGGCAGTTCCCCCACCACTGTAATCTTTGAGGATGACCGGAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACGGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAGGACTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGATGACACCACTCCCTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT (SEQ ID NO: 83) E1 VL amino acid sequence: NFMLTQPHSVSESPGKTVTISC TRSSGYIASSYVQ WYQQRPGSSPTTVIF EDDRRPS GVPDRFSGSIDGSSNSASLTISGLRTEDEADYYC QSYDDTTPWV FGGGTKLTVLG (SEQ ID NO: 84) F8 VH nucleic acid sequence: GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGTCGGCAGCTGGTACCTGGAAGATTTTGATATCTGGGGCCGGGGGACAATGGTCACCGTCTCGAGT (SEQ ID NO:85) F8 VH amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR VGSWYLEDFDI WGRGTMVTVSS (SEQ ID NO: 86) F8 VL nucleic acid sequence: AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTTACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAACTATGTTCACTGGTATCAGCAGCGCCCGGGCAGTTCACCCACCACTGTGATCTATGAGGATAACCGAAGACCCTCTGGGGTCCCTGCTCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGGAGACTGACGACGAGGCTGACTACTACTGTCAGTCTTCTGATACCACCTATCATGGAGGTGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT (SEQ ID NO: 87) F8 VL amino acid sequence: NFMLTQPHSVSESPGKTVTISC TRSSGSIASNYVH WYQQRPGSSPTTVIY EDNRRPS GVPARFSGSIDSSSNSASLTISGLETDDEADYYC QSSDTTYHGGVV FGGGTKLTVLG (SEQ ID NO: 88) F9 VH nucleic acid sequence: GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAAGGCGGTAACTACGGTGATTACTTCGACTACTTTGACTACTGGGGCAGAGGGACAATGGTCACCGTCTCGAGT (SEQID NO: 89) F9 VH amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK GGNYGDYFDYFDY WGRGTMVTVSS (SEQ ID NO: 90) F9 VL nucleic acid sequence: AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAATTATGTGCAGTGGTACCAGCAGCGCCCGGGCAGTGCCCCCACCATTGTGATCTATGAAGATAACCAAAGACCCTCTGGGGTCCCTCATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGAGGGGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT (SEQ ID NO: 91) F9 VL amino acid sequence: NFMLTQPHSVSESPGKTVTISC TRSSGSIASNYVQ WYQQRPGSAPTIVIY EDNQRPS GVPHRFSGSIDSSSNSASLTISGLKTEDEADYYC QSYEGF GGGTKLTVLG (SEQ ID NO: 92) G7 VH nucleic acid sequence: GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACTATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAAGATGGATGGAACGCGCTGGGATGGCTTGAATCCTGGGGCCAGGGGACAATGGTCACCGTCTCGAGT (SEQ ID NO:93) G7 VH amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DGWNALGWLES WGQGTMVTVSS (SEQ ID NO: 94) G7 VL nucleic acid sequence: AATTTTATGCTGACTCAGCCCCACGCTGTGTCGGAGTCTCCGGGGAAGACGGTGACCATTTCCTGCACCGGCAGAAATGGCAACATTGCCAGCAACTATGTGCAGTGGTACCAGCAGCGCCCGGACAGTGCCCCCACCCTTATAATCTTTGAAGATACCCAAAGACCCTCTGGGGTCCCTACTCGGCTCTCAGGCTCCATCGACACCTCCTCCAATTCTGCCTCCCTCATCATCTCTTCATTGAGGACTGAGGACGAGGCTGATTACTACTGTCAATCTTCTGATTCCAACAGGGTGCTGTTCGGCGGAGGGACCAAGGTCACCGTCCTAGGT (SEQ ID NO: 95) G7 VL amino acid sequence: NFMLTQPHAVSESPGKTVTISC TGRNGNIASNYVQ WYQQRPDSAPTLIIF EDTQRPS GVPTRLSGSIDTSSNSASLIISSLRTEDEADYYC QSSDSNRVL FGGGTKVTVLG (SEQ ID NO: 96) G9 VH nucleic acid sequence: GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAAGATTTTTGGGTTATTACGAGTGGGAATGACTACTGGGGGCGGGGGACCACGGTCACCGTCTCGAGT (SEQ ID NO:97) G9 VH amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DFWVITSGNDY WGRGTTVTVSS (SEQ ID NO: 98) G9 VL nucleic acid sequence: AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTGACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCTAGCAATTATGTGCAGTGGTACCAGCAGCGCCCGGGCAGTTCCCCCACCACTGTGATCTTTGAAGATAACCGAAGACCCTCTGGGGTCCCTGATCGGTTTTCTGGCTCCATCGACACCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGTCTTTTGATAGCACCAATCTTGTGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT (SEQ ID NO: 99) G9 VL amino acid sequence: NFMLTQPHSVSESPGKTVTISC TRSSGSIASNYVQ WYQQRPGSSPTTVIF EDNRRPS GVPDRFSGSIDTSSNSASLTISGLKTEDEADYYC QSFDSTNLVV FGGGTKLTVLG (SEQ ID NO: 100) G10 VH nucleic acid sequence: GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAAGATGGATGGAACGCGCTGGGATGGCTTGAATCCTGGGGGAAGGGGACCACGGTCACCGTCTCGAGT (SEQ ID NO:101) G10 VH amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DGWNALGWLES WGKGTTVTVSS (SEQ ID NO: 102) G10 VL nucleic acid sequence: AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCATCTCCTGCGCCGGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGTACCAGCAGCGCCCGGGCAGTGCCCCCACCGCTGTGATCTATGAGGATAACCAAAGACCC TCTGGGGTCCCTGATCGATTCTCTGGCTCCATCGACAGTCCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGAGGACGAGGCTGACTACTACTGTCAATCTTACTCTTACAACAATCAGGTCGTGTTCGGCGGAGGGACCAAGGTCACCGTCCTAGGT (SEQ ID NO: 103) G10 VL amino acid sequence: NFMLTQPHSVSESPGKTVTISC AGSSGSIASNYVQ WYQQRPGSAPTAVIY EDNQRPS GVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC QSYSYNNQVV FGGGTKVTVLG (SEQ ID NO: 104) In some embodiments, the IFNγ antibody is engineered in the form of an IgG isotype. In some embodiments, the IFNγ antibody is engineered in the form of an IgG1 isotype.

[0128] 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 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.

[0129] definition: Unless otherwise defined, scientific and technical terms used in connection with this invention shall have the same meaning as commonly understood by one of ordinary skill in the art. Furthermore, unless otherwise required herein, singular terms shall include plural terms, and plural terms shall include singular terms. Generally, terms used with respect to cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry, hybridization, or other techniques described herein are well-known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., perforation, lipid transfection). Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions or as commonly practiced in the art or as described herein. The above techniques and procedures are generally performed according to conventional methods well-known in the art and according to the various general or more specific references cited and discussed in this specification. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Hrbor, NY (1989)). Terms used with respect to laboratory procedures and techniques in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry described herein are well-known and commonly used in the art. Standard technologies are used in chemical synthesis, chemical analysis, drug preparation, formulation, delivery, and patient treatment.

[0130] Administration of anti-IFNγ antibody It should be recognized that the therapeutic entities of the present invention are administered together with suitable carriers, excipients, and other active agents incorporated into the formulation to provide improved delivery, transport, tolerability, etc. A variety of suitable formulations are available in all pharmacists' known formulations: Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 edited by Blaug and Seymour. These formulations include, for example, powders, pastes, ointments, gels, wax formulations, oil formulations, lipids, lipid-containing (cationic or anionic) vesicles (e.g., Lipofectin™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbon waxes (polyethylene glycol of varying molecular weights), semi-solid gels, and semi-solid mixtures containing carbon waxes. Any of the above mixtures may be suitable for the treatments and therapies of the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation and that 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 deliverysome 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 their references to additional information regarding formulations, excipients, and carriers familiar to pharmacists.

[0131] The efficacy of a treatment is determined by combining it with any known methods used to diagnose or treat a specific immune-related condition. Relief of one or more symptoms of an immune-related condition indicates that the antibody provides clinical benefit.

[0132] The antibodies of this invention, including polyclonal, monoclonal, humanized, and fully human antibodies, can be used as therapeutic agents. These therapeutic agents are generally used to treat or prevent diseases or pathologies associated with the abnormal expression or activation of a specified target in a subject. The antibody formulation, preferably one exhibiting high specificity and high affinity for its target antigen, is administered to the subject; the antibody formulation generally exerts its effect due to its binding to the target. Administration of the antibody may eliminate, inhibit, or interfere with the target's signal transduction function. Administration of the antibody may eliminate, inhibit, or interfere with the binding of the target to endogenous ligands naturally bound to the target.

[0133] The therapeutically effective amount of the antibody of the present invention generally relates to the amount required to achieve a therapeutic purpose. As mentioned above, this can be a binding interaction between the antibody and its target antigen, which in some cases interferes with the function of the target. The required amount to be administered will also depend on the binding affinity of the antibody to its specific antigen, and will also depend on the rate at which the administered antibody is depleted from the free volume of the other subject to which it was administered. By way of non-limiting example, a common range of therapeutically effective doses of the antibody or antibody fragment used in the present invention is about 0.1 mg / kg body weight to about 50 mg / kg body weight. A common range of dosing frequencies is, for example, from twice daily to once weekly.

[0134] The antibodies or fragments thereof of the present invention can be administered in the form of pharmaceutical compositions for the treatment of various diseases and conditions. Principles and considerations involved in the preparation of such compositions, as well as guidance in component selection, are provided in, for example, *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.

[0135] The formulation may also contain one or more active compounds, such as anti-IFNγ antagonists required for the specific indication to be treated, preferably those that have complementary activities and do not adversely affect each other. Alternatively or additionally, the composition may contain agents that enhance its function, such as cytotoxic agents, cytokines, chemotherapeutic agents, or growth inhibitors. Such molecules are suitably present in a combination in amounts effective for the intended purpose.

[0136] In one embodiment, the active compound, such as an anti-IFNγ antagonist, is administered in combination therapy, i.e., in combination with one or more other agents that can be used to treat a pathological condition or disease. The term "combination" herein means agents administered substantially simultaneously (simultaneously or sequentially). If administered sequentially, it is preferable that the first of the two compounds is still detectable at an effective concentration at the treatment site when the second compound is initiated.

[0137] For example, combination therapies may include one or more neutralizing anti-IFNγ antibodies of the present invention, which are 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 agents or cell growth inhibitors as described in more detail below. Such combination therapies can advantageously utilize lower doses of the administered therapeutic agents, thereby avoiding the potential toxicities or complications associated with various monotherapies.

[0138] When using antibody fragments, minimally inhibitory fragments that specifically bind to the binding domain of the target protein and / or minimally inhibitory fragments that interfere with or otherwise antagonize IFNγ signaling are preferred. For example, peptide molecules that retain the ability to bind to the target protein sequence can be designed based on the variable region sequence of the antibody. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology (see, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). The formulation may also contain one or more active compounds that are essential for the specific indication to be treated, preferably those that do not adversely affect each other and have complementary activities. Alternatively or additionally, the composition may contain agents that enhance its function, such as cytotoxic agents, cytokines, chemotherapeutic agents, or growth inhibitors. Such molecules are suitably combined in an amount effective for the intended purpose.

[0139] The levels of CXCL9 and other biomarkers are detected using any of a variety of standard detection techniques. Assay reagents can be used to detect the presence of a given target (or a fragment thereof) in a sample. In some embodiments, the assay reagent contains a detectable label. In some embodiments, the assay reagent is an antibody (or a fragment thereof) or a probe. In some embodiments, the reagent or probe is labeled. The term "labeled" with respect to a probe or antibody is intended to include both direct labeling of a probe or antibody by conjugating (i.e., physically linking) a detectable substance to the probe or antibody, and indirect labeling by reacting a probe or antibody with another directly labeled reagent. Examples of indirect labeling include using a fluorescently labeled second antibody to detect a first antibody and labeling the ends of a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin.

[0140] 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 the subject. Therefore, blood and fractions or components of blood, including serum, plasma, or lymph, are included within the usage of the term "biological sample." Body fluids can be fluids isolated from any location within the subject's body (preferably a peripheral location), including but not limited to, for example, blood, plasma, serum, synovial fluid, urine, sputum, cerebrospinal fluid, pleural fluid; fluids of the respiratory, intestinal, and genitourinary tracts; saliva, fluids within organ systems, ascites, tumor cyst fluid, amniotic fluid, and combinations thereof. A biological sample also includes experimentally isolated portions of all the aforementioned fluids. A biological sample also includes solutions or mixtures comprising homogenized solid materials such as feces, tissue, and biopsy samples. The detection methods of the present invention can be used to detect analytes mRNA, protein, or genomic DNA in biological samples in vitro and in vivo. For example, in vitro techniques for detecting analyte mRNA include RNA hybridization and in situ hybridization. In vitro techniques for detecting analyte proteins include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. In vitro techniques for detecting analyte genomic DNA include DNA hybridization. Methods for performing immunoassays are described, for example, in "ELISA: Theory and Practice: Methods in Molecular Biology", Vol. 42, JR Crowther (Ed.) Human Press, Totowa, NJ, 1995; "Immunoassay", E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and "Practice and Theory of Enzyme Immunoassays", P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for detecting analyte proteins involve introducing labeled anti-analyte protein antibodies into the subject. For example, the antibody may be labeled with a radiolabel, the presence and location of which in the subject can be detected using standard imaging techniques.

[0141] The compositions and methods described herein can be used to treat any of a variety of conditions associated with interferon-γ (IFNγ) expression and / or activity (including aberrant IFNγ expression and / or activity). The compositions and methods of this disclosure can be used to treat hemophagocytic lymphohistiocytosis (HLH). HLH is a rare and serious life-threatening disease characterized by extremely inflammatory clinical signs and symptoms (fever, splenomegaly, cytopenia, coagulopathy), leading to an abnormal immune-mediated pathology that, through tissue damage, can ultimately cause multiple organ failure and death (Henter JI, Elinder G, Söder O, Hansson M et al: Hypercytokinemia in familialhemophagocytic lymphohistiocytosis. Blood 1991, 78:2918-2922). HLH includes primary (hereditary / familial) HLH and secondary HLH.

[0142] Primary HLH is a heterologous autosomal recessive disorder that mostly occurs in infancy and early childhood, with an estimated incidence of 1 in 50,000 live births in Europe (Janka GE: Familial hemophagocytic lymphohistiocytosis. Eur. J. Pediatr. 1983, 140:221-230). Without treatment, the disease is fatal, with a median survival of less than 2 months after symptom onset (Filipovich AH: Hemophagocytic lymphohistiocytosis (HLH) and related disorders. Hematology Am Soc HematolEduc Program 2009:127-131).

[0143] Genetic defects in primary HHL all affect cytotoxic pathways involving NK cells and / or cytotoxic lymphocytes required to clear activated macrophages, proteins encoding perforin synthesis, cytolytic granule maturation, granule exocytosis, and genes involved in granule release or exocytosis (Filipovich, A., K. McClain and A. Grom. 2010. Histiocytic disorders: recent insights into pathophysiology and practical guidelines. Biol. Blood Marrow Transplant. 16(1 Suppl):S82-S89). In approximately 20–40% of patients with primary HLH, the impaired cytotoxic function characteristic of HLH syndrome is due to mutations in the gene encoding perforin (PRF1), a cytolytic protein of cytotoxic granules and a key regulator of T-cell and natural killer cell-mediated cytolysis. In approximately 10% of patients, the disease is caused by mutations in the UNC13D gene, which encodes proteins involved in the release of perforin into target cells. In addition, some immunodeficiency syndromes, such as Gricecelli syndrome type 2 (GS-2) and Chediak-Higashi syndrome (CHS), often coexist with HLH (Janka GE, Lehmberg K: Hemophagocytic lymphohistiocytosis: pathogenesis and treatment. Hematology Am Soc Hematol Educ Program 2013, 2013:605-611).

[0144] Secondary forms of HLH can occur during infections, autoimmune / rheumatic diseases, or in conjunction with malignancies. Secondary forms present with the same signs and symptoms as primary HLH and can be just as severe.

[0145] The compositions and methods disclosed herein can be used to treat secondary HLH. The compositions and methods disclosed herein can be used to treat macrophage activation syndrome (MAS).

[0146] MAS is a potentially life-threatening complication of a rheumatic disease caused by the overactivation and proliferation of T lymphocytes and macrophages. The uncontrolled proliferation of these immune cells leads to marked hypercytokineemia and a hyperinflammatory state associated with fever, cytopenia, hepatosplenomegaly, liver dysfunction, coagulation abnormalities, and hyperferritinemia, and 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).

[0147] Because of its strong clinical and pathological similarity to HLH, MAS is classified as a secondary or acquired form of HLH. In fact, recent studies have confirmed that most MAS patients have impaired NK and perforin function tests, and many 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).

[0148] MAS most commonly occurs in patients with sJIA and is less common in patients with systemic lupus erythematosus (SLE). Although rare, it has also been reported in patients with vasculitis (especially Kawasaki disease). Approximately 7–17% of patients with SJIA develop significant MAS (Sawhney S, Woo P, Murray KJ: Macrophage activation syndrome: a potentially fatal complication of rheumatic disorders. Arch. Dis. Child. 2001, 85:421–426; Moradinejad MH, Ziaee V: The incidence of macrophage activation syndrome in children with rheumatic disorders. Minerva Pediatr. 2011, 63:459–466). Some evidence suggests that subclinical MAS can be seen in up to one-third of patients with active systemic diseases (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).

[0149] Because macrophage activation syndrome (MAS) can be fatal, timely diagnosis and prompt intervention are essential for proper disease management. The reported mortality rate for MAS is 20-30%, and it remains a 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).

[0150] Different sets of diagnostic criteria have been proposed for MAS in patients with sJIA. The HLH-2004 diagnostic criteria, developed primarily for the primary (genetic) form of HLH, are sometimes recommended (Henter J, Horne A, Aricó M, Egeler RM et al: HLH-2004: Diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer 2007, 48:124-131). However, these criteria have several limitations and may not be suitable for patients with sJIA. For example, criteria (such as cytopenia and hypofibrinogen below the thresholds required by HLH-2004) only become apparent in the later stages of MAS, because these patients often have elevated white blood cell and platelet counts and elevated serum fibrinogen levels as part of the sJIA inflammatory response (SchulertGS, Grom AA: Pathogenesis of macrophage activation syndrome and potential for cytokine-directed therapies. Annu. Rev. Med. 2015, 66:145-159). Phagocytic activity may not be present in most patients with MAS (Minoia F, Davì 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, phagocytic activity, NK cell activity, and sCD25 are not routinely evaluated in the case of MAS.

[0151] The alternative approach is based on the application of the Preliminary Diagnostic Criteria (PDG) for MAS complicated with sJIA, which was established through an analysis comparing the MAS patient population with the patient population with sudden sJIA1.

[0152] Recently, a large-scale study compared the HLH-2004 diagnostic criteria and preliminary diagnostic criteria for sJIA-related MAS with the ability to differentiate sJIA / MAS from sJIA (in the absence of MAS) and systemic infection (Davì S, Minoia F, Pistorio A, Horne A et al: Performance of current guidelines for diagnosis of macrophage activation syndrome complicating systemic juvenileidiopathic arthritis. Arthritis & rheumatology (Hoboken, NJ) 2014, 66:2871-2880). Despite some limitations due to its retrospective nature, this study appears to indicate that the preliminary MAS criteria strike an optimal balance between sensitivity and specificity, and achieve maximum consistency with the diagnosis made by the treating physician. The sensitivity of the HLH-2004 complete set of criteria is <30%. However, it has also been reported that the proportion of patients meeting each individual criterion of PDG is highly variable, and some clinical features (such as CNS dysfunction and bleeding) may appear in the late stages of MAS, making its sensitivity low in early MAS (Lehmberg K, Pink I, Eulenburg C, Beutel K et al: Differentiating macrophage activation syndrome in systemic juvenile idiopathic arthritis from other forms of hemophagocytic lymphohistiocytosis. The Journal of pediatrics 2013, 162:1245-1251).

[0153] Recently, a diagnostic score (HS score) was developed and validated in a retrospective cohort of 312 patients, 162 of whom were diagnosed with reactive hemophagocytic syndrome (Fardet L, Galicier L, Lambotte O, Marzac C, Aumont C, Chahwan D, Coppo P, Hejblum G: Development and validation of the HS score, 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, thermoregulation, organ megagenesis], five biological [i.e., triglycerides, ferritin, serum glutamate-oxaloacetate transaminase, fibrinogen levels, and cytopenia], and one cytological [i.e., phagocytic activity characteristics of bone marrow aspirate]) remained in the HS score, with the probability of having hemophagocytic syndrome ranging from <1% with an HS score ≤90 to >99% with an HS score ≥250.

[0154] Until a final consensus is reached on proven diagnostic criteria for MAS, clinical diagnosis by skilled physicians remains crucial in the challenge of differentiating MAS from conditions with overlapping features, such as sudden onset of SJIA or sepsis-like syndrome.

[0155] There are currently no approved drugs for the treatment of MAS. High-dose glucocorticoids are typically the first-line treatment for MAS. Cyclosporine A (CsA) has been proposed as an alternative treatment for patients who do not respond to glucocorticoids (Stéphan JL, Koné-Paut I, Galambrun C, Mouy R, Bader-Meunier B, Prieur AM: Reactivehaemophagocytic syndrome in children with inflammatory disorders. Aretrospective study of 24 patients. Rheumatology (Oxford, England) 2001, 40:1285-1292).

[0156] As part of the development of the HLH-94 treatment regimen for pHLH, etoposide is also being considered in patients who do not respond to high-dose glucocorticoids. However, the potential toxicity of the drug remains a major concern. Other existing first-line treatments for HLH 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; for example, alenzusmab / ATG treatments are extremely immunosuppressive, and survival with these treatments is considered very poor.

[0157] The efficacy of biologics that inhibit the IL-1, IL-6R, or TNFα pathways in the treatment of MAS remains unclear. Although biologics that inhibit these pathways have been reported to be effective in isolation, some patients have developed macrophage syndrome (MAS) in the context 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, The findings of 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, 367:2385-2395, and patient reports of non-responsiveness to these treatments, suggest that inhibition of IL-1, IL-6R, or TNFα does not provide protection against the development of MAS, nor does it provide effective treatment for a fully developed syndrome.

[0158] A large, retrospective, multicenter study investigated the clinical, laboratory, and histopathological features, current treatments, and outcomes of macrophage-induced idiopathic arthritis (MAS / sJIA) in 362 patients (Minoia F, Davì 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). In approximately half of the patients, MAS occurred against a background of active sJIA or during disease flare-ups, occurring during 30% of sJIA outbreaks. Infectious causative agents were identified in one-third of the patients. Of the 24 patients, the type of infection was reported, with EBV being the most common causative agent (25%). In 11 patients (3.8%), MAS was considered to be related to treatment side effects: 8 of these involved biologics targeting the IL-6 (N=4), IL-1 (N=3), or TNFα (N=1) pathways. Almost all patients received glucocorticoids. Cyclosporine, biologics, and etoposide were administered to 61%, 15%, and 12% of patients, respectively.

[0159] The identification of effective treatment options for MAS thus represents a significant unmet medical need. More than 50% of patients with sJIA and MAS do not respond to systemic glucocorticoids alone, or may require high-dose, long-term treatment with significant morbidity. When patients do not respond to glucocorticoids, adequately evidence-based data on the efficacy of other treatments (such as CsA or etoposide) are unavailable. The progression of MAS can become immediately irreversible, leading to fatal outcomes. Current data indicate an 8% mortality rate for sJIA-related MAS, with approximately one-third of patients requiring ICU admission. Recent findings regarding the crucial role of IFNγ in the pathogenesis of this disease suggest that IFNγ blockade may represent a novel therapeutic target.

[0160] The compositions (including the NI-0501 composition) and methods disclosed herein are advantageous for current treatments of primary and secondary HLH.

[0161] MAS and HLH are characterized by a persistent immune cell activation and a cytokine storm associated with the overproduction of pro-inflammatory cytokines, including IFNγ, TNFα, IL-1, and IL-6 (Henter JI, Elinder G, Söder O, Hansson M et al: Hypercytokinemia in familial hemophagocytic lymphohistiocytosis. Blood 1991, 78:2918-2922; Imashuku S, Hibi S, Fujiwara F, Todo S: Hyper-interleukin(IL)-6-naemia in haemophagocytic lymphohistiocytosis. Br. J. Haematol. 1996,93:803-807; Xu X, Tang Y, Song H, Yang S et al: Diagnostic accuracy of a specific cytokine pattern in hemophagocytic lymphohistiocytosis in children. J. Pediatr. 2012, 160:984-90.e1; Put K, Avau A, Brisse E, Mitera T et al: Cytokines in systemic juvenile idiopathic arthritis and haemophagocyticlymphohistiocytosis: tipping the balance between interleukin-18 and interferon-γ. Rheumatology (Oxford) 2015).Over the past few years, evidence has accumulated to support the role of IFNγ in 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, Chrétien F, Lefebvre JM et al: Neutralization of IFNgamma defeats hemophagocytosis in LCMV-infected perforin-and Rab27a-deficient mice. EMBOMol 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, The key roles of both TLR9 stimulation results in macrophage activation syndrome-like disease in mice. J. Clin. Invest. 2011, 121:2264-2277.

[0162] For primary HLH, perforin knockout mice are considered an associated model because these mice, once infected with LCMV, exhibit all the diagnostic features of the human disease and many clinically and laboratory-specific characteristics. The HLH-like disease they develop depends on the production of CD8+ T cells and IFNγ in response to antigen stimulation (Imashuku S, Hibi S, Fujiwara F, Todo S: Hyper-interleukin (IL)-6-naemia in haemophagocytic lymphohistiocytosis. Br. J. Haematol. 1996, 93:803-807). It has been demonstrated that when high circulating IFNγ levels are neutralized by administration of anti-IFNγ antibodies, not only do clinical and laboratory abnormalities resolve, but survival is also significantly improved. Conversely, the elimination of many other cytokines had no effect on survival (Imashuku S, Hibi S, Fujiwara F, Todo S: Hyper-interleukin (IL)-6-naemia in haemophagocytic lymphohistiocytosis. Br. J. Haematol. 1996, 93:803-807; Xu X, Tang Y, Song H, Yang S et al: Diagnostic accuracy of a specific cytokine pattern inhemophagocytic lymphohistiocytosis in children. J. Pediatr. 2012, 160:984-90.e1). Further emphasizing the importance of IFNγ in HLH is the presence of high levels of circulating IFNγ in these patients (Henter JI, Elinder G, Söder 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 to treatment and follow-up, IFNγ levels exceeded the upper limit of normal (17.3 pg / mL) in all patients, with 53.5% exceeding 1000 pg / mL. Rapid increases in IFNγ levels were also reported in the early stages, and a decrease from >5000 pg / mL to normal within 48 hours with effective treatment of HLH.

[0163] Two animal models of secondary HLH were investigated under the NI-0501 research project to elucidate the potential pathogenic role of IFNγ. First, in a mouse model of infection-driven HLH simulating infection, repeated administration of CpG induced hypercytokinemia via TLR9 activation, leading to the clinical (e.g., weight loss, splenomegaly) and laboratory (e.g., cytopenia, hyperferritinemia) features of HLH33. When IFNγ was neutralized by administration of an anti-IFNγ antibody, the clinical and laboratory features of the disease were restored. Neutralization of IFNγ was shown to be complete in relevant target tissues (e.g., liver and spleen). Notably, administration of the anti-IFNγ antibody revealed IFNγ levels 500–2,000 times higher than those measured in blood, potentially better reflecting IFNγ production in tissues. Upon TLR9 stimulation, incremental regulation of two IFNγ-inducible chemokines (CXCL9 and CXCL10) was observed in both blood and liver, and a significant correlation was observed between serum IFNγ levels and serum CXCL9 and CXCL10 concentrations. Neutralization of IFNγ induces a significant decrease in serum CXCL9 and CXCL10 mRNA levels as well as in the liver (Buatois V, Chatel L, Cons L, Lory S et al: IFNγ drives disease in the TLR9-mediated secondary HLH in the liver: rationale for a new therapeutic target in secondary HLH, in preparation).

[0164] Second, an IL-6 transgenic mouse model expressing high levels of IL-6 was investigated, as it mimics the condition of patients with sJIA (the rheumatic disease most commonly associated with the secondary form of HLH). Increased lethality, increased production of inflammatory cytokines, and overactivation of inflammatory signaling pathways were observed when induced with a Toll-like receptor (TLR) ligand. However, these mice exhibited decreased platelet and neutrophil counts; elevated sCD25, ferritin, and LDH levels, similar to many features commonly found in patients with MAS (Strippoli R, Carvello F, Scianaro R, De Pasquale L et al: Amplification of the response to Toll-like receptor ligands by prolonged exposure to interleukin-6 in mice: implication for the pathogenesis of macrophage activation syndrome. Arthritis Rheum. 2012, 64:1680-1688). In these mice, survival was significantly improved and laboratory parameters were restored when IFNγ was neutralized by administration of anti-IFNγ antibody (Prencipe G et al., draft in progress).

[0165] Similar evidence has recently been gathered in observational studies of patients with secondary forms of HLH secondary to infection or of unknown cause (excluding HLH by normal cytotoxic activity, lack of mutations in known genes that cause pHLH, and lack of family history) or patients with MAS occurring in the context of sJIA.

[0166] Serum samples were analyzed in 14 patients with secondary HLH (7 of whom had confirmed underlying infections) during both the active, fully developed disease phase and the remission phase. Compared to remission, levels of IFNγ, CXCL9, and CXCL10 were significantly higher during the active phase (IFNγ: 34.7 vs. <3.5 pg / ml; CXCL9: 33598 vs. 745 pg / ml; CXCL10: 4420 vs. 132 pg / ml; median). IFNγ levels were significantly correlated with CXCL9 levels (p = 0.0018) and less significantly correlated with CXCL10 levels (p = 0.014). The levels of IFNγ and chemokines (especially CXCL9) were significantly correlated with parameters of disease severity (e.g., neutrophil and platelet counts, ferritin, and ALT), which further supports the pathogenic role of IFNγ in secondary HLH and the potential use of chemokines as disease-related biomarkers (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).

[0167] Similar findings were presented in patients with MAS occurring in patients with sJIA. Serum concentrations of IFNγ, IFNγ-inducible chemokines (CXCL9, CXCL10, CXCL11), and IL-6 were measured in 54 patients with sJIA, 20 of whom had MAS. IL-6 levels were comparable in patients with fully developed MAS and those with active sJIA but no MAS at the time of sampling. Conversely, circulating IFNγ and chemokine levels were significantly higher in MAS, particularly CXCL9, with the median level being approximately 15-fold higher than in patients with active sJIA but no MAS (13392 vs. 837 pg / mL; p = 0.005). Notably, only in MAS patients did a significant correlation be shown 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). IFNγ levels were also correlated with laboratory parameters of disease severity, with the exception of LDH, for which no statistical significance was reached (Bracaglia et al., draft pending).

[0168] In summary, these data provide a solid foundation for the neutralization of IFNγ as a targeted therapy for secondary HLH and MAS, and for its study in a clinical context.

[0169] The compositions (including the NI-0501 composition) and methods of this disclosure are advantageous compared to existing treatments for sJIA. For example, the compositions (including the NI-0501 composition) and methods of this disclosure can be used to treat MAS / sHLH in patients with sJIA, with the primary objective of achieving MAS remission.

[0170] The underlying principles for identifying the patient population that would benefit from NI-0501 treatment and for evaluating the efficacy of NI-0501 in MAS / sHLH are based on multiple factors. First, preclinical data from relevant animal models of MAS / sJIA showed that IFNγ neutralization significantly improved survival and restored changes in laboratory parameters. Second, observational data in MAS / sHLH patients showed the presence of high levels of IFNγ, and more importantly, extremely high levels of IFNγ-induced chemokines CXCL9, CXCL10, and CXCL11. Third, in MAS / sHLH patients, the concentrations of IFNγ and CXCL9 were significantly correlated with disease parameters such as ferritin, platelet count, and transaminases. Next, the favorable tolerability and lack of associated safety concerns observed in previously studied pHLH patients for all infusions administered confirmed the observational studies in healthy volunteers, where infections caused by pathogens known to be facilitated by IFNγ neutralization were not reported, and none of the infections occurring in some pHLH patients were considered related to NI-0501 treatment, but rather to their immune status, disease duration, and prior or concurrent treatment. Fifth, preliminary data from previous clinical studies showed a favorable effect on disease parameters, with a considerable effect in the first few days of treatment: typical clinical signs and symptoms of HLH began to improve rapidly after each administration of NI-0501 (fever within hours, splenomegaly / hepatomegaly within days); among the 18 evaluable patients at the cutoff, NI-0501 treatment led to 10 patients switching to HSCT. Next, evidence from PK modeling and simulation methods showed a predictable pharmacokinetic profile of NI-0501, achieving and maintaining IFNγ neutralization. Finally, when NI-0501 is insufficient to control the disease, conventional therapies (such as CsA) can be started immediately without an interval.

[0171] In summary, based on preclinical and clinical evidence, there is a solid underlying principle for the neutralization of IFNγ in MAS / sHLH secondary to rheumatic diseases, and preliminary data from pHLH patients suggest a favorable benefit-risk profile for NI-0501, which significantly improves HLH characteristics to normalization.

[0172] Therefore, NI-0501 represents an innovative and effective treatment approach in the management of this serious and life-threatening complication of rheumatic disease, potentially limiting the side effects of long-term high-dose glucocorticoid therapy.

[0173] Pharmaceutical Composition The antibodies or soluble chimeric peptides (also referred to herein as “active compounds”) and their derivatives, fragments, analogs, and homologs of the present invention may be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the antibody or soluble chimeric peptide and a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” as used herein is intended to include any and all solvents, dispersion media, coating materials, antibacterial and antifungal agents, isotonic agents, and absorption delay agents compatible with drug administration. Suitable carriers are described in standard reference texts in the art: the latest edition of Remington's Pharmaceutical Sciences, incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, glucose solution, and 5% human serum albumin. Liposomes and non-aqueous carriers, such as fixed oils, may also be used. The use of these media and reagents for pharmaceutically active substances is well known in the art. Their use in the composition should be considered unless any conventional media or reagent is incompatible with the active compound. Additional active compounds may also be incorporated into the composition.

[0174] The pharmaceutical compositions of the present invention are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following components: a sterile diluent, such as water for injection, saline solution, fixative oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methylparaben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid (EDTA); a buffer, such as acetate, citrate, or phosphate; and a reagent for adjusting the pH, such as sodium chloride or glucose. The pH may be adjusted with an acid or base (e.g., hydrochloric acid or sodium hydroxide). Parenteral formulations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0175] Suitable pharmaceutical compositions for injection include sterile aqueous solutions (wherein being water-soluble) or dispersions and sterile powders prepared ex-situ for use of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be present in a fluid state sufficient for easy injection. It must be stable under manufacturing and storage conditions and must be preserved against contamination by microorganisms (e.g., bacteria and fungi). The carrier can be a solvent or dispersion medium comprising, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using coating materials such as lecithin, in the case of dispersions, by maintaining the desired particle size, and by using surfactants. Antimicrobial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars, polyols like mannitol and sorbitol, and sodium chloride, will preferably be included 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.

[0176] Sterile injectable solutions can be prepared by incorporating the active compound in the desired amount with one or a combination of the ingredients listed above (if necessary) into a suitable solvent, followed by sterile filtration. Typically, dispersions are prepared by incorporating the active compound into a sterile solvent containing a base dispersion medium and any other desired ingredients from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, the preparation method is vacuum drying and freeze-drying to obtain a powder of the active ingredient plus any other desired ingredients from its previously sterile filtered solution.

[0177] Oral compositions generally include an inert diluent or an edible carrier. They may be encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic administration purposes, the active compound may be blended with excipients and used in tablet, lozenge, or capsule form. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and rinsed and spat out or swallowed. Pharmaceutically compatible binders and / or excipients may be included as part of the composition. The tablets, pills, capsules, lozenges, etc., may contain any of the following components or compounds with similar properties: binders, such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients, such as starch or lactose; disintegrants, such as alginic acid, Primogel, or corn starch; lubricants, such as magnesium stearate or sterotes; flow aids, such as colloidal silica; sweeteners, such as sucrose or saccharin; or flavorings, such as peppermint, methyl salicylate, or orange flavorings.

[0178] For administration by inhalation, the compound is delivered from a pressurized container or dispenser in the form of an aerosol spray containing a suitable propellant (e.g., a gas such as carbon dioxide) or a nebulizer.

[0179] Systemic administration can also be achieved via mucosal or transdermal routes. For mucosal or transdermal administration, a penetrant suitable for penetrating the barrier is used in the formulation. Such penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives used for mucosal administration. Mucosal administration can be achieved using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, creams, gels, or creams generally known in the art.

[0180] The compound can also be prepared in the form of suppositories (e.g., together with a conventional suppository base, such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0181] In one embodiment, the active compound is prepared together with a carrier that protects the compound from rapid elimination from the body, such as in controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. This material is also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (including liposomes targeting infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, such as those described in U.S. Patent No. 4,522,811.

[0182] Formulating oral or parenteral compositions in dosage units is particularly advantageous for ease of administration and to ensure dosage uniformity. As used herein, dosage unit form refers to a physically discrete unit suitable for use as a single dose in a subject to be treated; each unit contains a predetermined amount of an active compound, calculated to be combined with a desired drug carrier to produce the desired therapeutic effect. The specifications of the dosage unit form of the present invention are determined by, and directly depend on, the unique properties of the active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations in the field of formulating such an active compound for individual treatment.

[0183] The pharmaceutical composition may be included in a container, package, or dispenser along with instructions for use.

[0184] The present invention also relates to the following embodiments: 1. A method of treating hemophagocytic lymphohistiocytosis (HLH) in a subject of need, the method comprising administering a therapeutically effective amount of an antibody or an antigen-binding fragment thereof, which binds to interferon-γ (IFNγ) and contains a variable heavy chain complementarity-determining region 1 (VH CDR1) comprising the amino acid sequence SYAMS (SEQ ID NO: 1); a variable heavy chain complementarity-determining region 2 (VH CDR2) comprising the amino acid sequence AISGSGGSTYYADSVKG (SEQ ID NO: 2) and a variable heavy chain complementarity-determining region 3 (VH CDR3) comprising the amino acid sequence DGSSGWYVPHWFDP (SEQ ID NO: 3); a variable light chain complementarity-determining region 1 (VL CDR1) comprising the amino acid sequence TRSSGSIASNYVQ (SEQ ID NO: 4); a variable light chain complementarity-determining region 2 (VL CDR2) comprising the amino acid sequence EDNQRPS (SEQ ID NO: 5) and a variable light chain complementarity-determining region 2 (VL CDR2) comprising the amino acid sequence QSYDGSNRWM (SEQ ID NO: 5). 6) Variable light chain complementarity determination region 3 (VL CDR3).

[0185] 2. The method of embodiment 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.

[0186] 3. The method of implementation scheme 1, wherein the antibody is formulated into a sterile concentrated preparation for infusion.

[0187] 4. The method of embodiment 3, wherein the formulation comprises 5 mg of antibody, 1.55 mg of L-histidine, 3.14 mg of L-histidine hydrochloride monohydrate, 7.31 mg of sodium chloride (NaCl) and 0.05 mg of polysorbate 80, wherein the pH is between 5.8 and 6.2.

[0188] 5. The method of implementation scheme 4, wherein the pH is 6.0.

[0189] 6. The method of implementation scheme 1, wherein the antibody is administered to the subject in need via IV infusion at an initial dose of 1 mg / kg over 1 hour.

[0190] 7. The method of implementation scheme 6, wherein the antibody is administered to the subject in need at an initial dose of 1 mg / kg within 1 hour following the initial IV infusion, via at least one additional IV infusion.

[0191] 8. The method of embodiment 7, wherein the at least one additional IV infusion is at a dose higher than the initial dose of 1 mg / kg.

[0192] 9. The method of embodiment 6, wherein the at least one additional IV infusion dose is 3 mg / kg.

[0193] 10. The method of implementation scheme 6 or implementation scheme 9, wherein the at least one additional IV infusion is given at least 3 days after the initial IV infusion.

[0194] 11. The method of embodiment 10, wherein the at least one additional IV infusion is given at a time selected from the following: 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial IV infusion, and 15 days after the initial IV infusion.

[0195] 12. The method of implementation scheme 10, wherein the at least one additional IV infusion is given 3 days, 6 days, 9 days, 12 days and 15 days after the initial IV infusion.

[0196] 13. The method of embodiment 6, wherein the antibody is administered to the subject in need at an initial dose of 1 mg / kg over 1 hour following an initial IV infusion via at least a series of additional IV infusions, wherein the series of additional IV infusions comprises at least a series of twice-weekly IV infusions.

[0197] 14. The method of embodiment 13, wherein the at least one series of twice-weekly IV infusions are administered at a dose higher than the initial dose of 1 mg / kg.

[0198] 15. The method of embodiment 13, wherein the at least one series of twice-weekly IV infusions are administered at a dose of 3 mg / kg.

[0199] 16. The method of implementation scheme 13 or implementation scheme 15, wherein the at least one additional IV infusion is given at least three weeks after the initial IV infusion.

[0200] 17. The method of embodiment 16, wherein the at least one additional IV infusion is given at a time selected from the following: 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.

[0201] 18. The method of implementation scheme 16, wherein the at least one additional IV infusion is given 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks and 8 weeks after the initial IV infusion.

[0202] 19. The method of implementation scheme 6, wherein the antibody is given to the subject in need by at least two additional IV infusions following the initial IV infusion.

[0203] 20. The method of embodiment 19, wherein the at least two additional IV infusions are at a dose higher than the initial dose of 1 mg / kg.

[0204] 21. The method of embodiment 20, wherein the first additional IV infusion and the second additional IV infusion are administered at the same dose.

[0205] 22. The method of embodiment 20 or 21, wherein the first additional IV infusion and the second additional IV infusion are administered at the same dose higher than the initial dose.

[0206] 23. The method of embodiment 22, wherein at least one of the first and second additional IV infusions is administered at a dose of 3 mg / kg.

[0207] 24. The method of implementation scheme 19 or implementation scheme 23, wherein the first additional IV infusion is given at least 3 days after the initial IV infusion.

[0208] 25. The method of implementation scheme 24, wherein the first additional IV infusion is given at a time selected from the following: 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial IV infusion, and 15 days after the initial IV infusion.

[0209] 26. The method of implementation scheme 24, wherein the first additional IV infusion is given 3 days, 6 days, 9 days, 12 days and 15 days after the initial IV infusion.

[0210] 27. The method of implementation scheme 24, wherein the second additional IV infusion is given at a time selected from the following: 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.

[0211] 28. The method of implementation scheme 24, wherein the second additional IV infusion is given 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks and 8 weeks after the initial IV infusion.

[0212] 29. The method of implementation scheme 24, wherein the first additional IV infusion is given 3 days, 6 days, 9 days, 12 days and 15 days after the initial IV infusion, and the second additional IV infusion is given 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks and 8 weeks after the initial IV infusion.

[0213] 30. The method of embodiment 20, wherein the first additional IV infusion and the second additional IV infusion are administered at different doses.

[0214] 31. The method of embodiment 30, wherein the first additional IV infusion and the second additional IV infusion are administered at different doses, wherein each dose is higher than the initial dose.

[0215] 32. The method of embodiment 30, wherein the first additional IV infusion is administered at a dose of 3 mg / kg.

[0216] 33. The method of embodiment 30, wherein the second additional IV infusion is administered at a dose of 6 mg / kg.

[0217] 34. The method of embodiment 30, wherein the first additional IV infusion is administered at a dose of 3 mg / kg and the second additional IV infusion is administered at a dose of 6 mg / kg.

[0218] 35. The method of implementation scheme 30 or implementation scheme 34, wherein the first additional IV infusion is given at least 3 days after the initial IV infusion.

[0219] 36. The method of embodiment 35, wherein the first additional IV infusion is given at a time selected from the following: 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial IV infusion, and 15 days after the initial IV infusion.

[0220] 37. The method of implementation scheme 35, wherein the first additional IV infusion is given 3 days, 6 days, 9 days, 12 days and 15 days after the initial IV infusion.

[0221] 38. The method of embodiment 35, wherein the second additional IV infusion is given at a time selected from the following: 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.

[0222] 39. The method of implementation scheme 35, wherein the second additional IV infusion is given 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks and 8 weeks after the initial IV infusion.

[0223] 40. The method of implementation scheme 35, wherein the first additional IV infusion is given 3 days, 6 days, 9 days, 12 days and 15 days after the initial IV infusion, and the second additional IV infusion is given 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks and 8 weeks after the initial IV infusion.

[0224] 41. The method of embodiment 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.

[0225] 42. The method of embodiment 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 the initial dose of 1 mg / kg.

[0226] 43. The method of embodiment 42, wherein 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.

[0227] 44. The method of implementation scheme 41 or 43, wherein the first series of additional IV infusions is given at least 3 days after the initial IV infusion.

[0228] 45. The method of implementation scheme 44, wherein the first series of additional IV infusions are given at times selected from the following: 3 days after the initial IV infusion, 6 days after the initial IV infusion, 9 days after the initial IV infusion, 12 days after the initial IV infusion, and 15 days after the initial IV infusion.

[0229] 46. ​​The method of implementation scheme 44, wherein the first series of additional IV infusions are given 3 days, 6 days, 9 days, 12 days and 15 days after the initial IV infusion.

[0230] 47. The method of implementation scheme 44, wherein the second series of additional IV infusions is given at times selected from the following: 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.

[0231] 48. The method of implementation scheme 44, wherein the second series of additional IV infusions are given 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks and 8 weeks after the initial IV infusion.

[0232] 49. The method of implementation scheme 44, wherein the first series of additional IV infusions are given 3 days, 6 days, 9 days, 12 days and 15 days after the initial IV infusion, and the second series of additional IV infusions are given 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks and 8 weeks after the initial infusion.

[0233] 50. The method of implementation scheme 1, wherein the subject is administered dexamethasone in the context of administration.

[0234] 51. The method of implementation scheme 50, wherein the subject has not previously been treated for HLH, and wherein the dexamethasone is administered at a dose of at least 10 mg / m². 2 The prescribed dosage was administered.

[0235] 52. The method of implementation scheme 50, wherein the subject is receiving the antibody as second-line HLH treatment, and dexamethasone is administered at a dose of at least 5 mg / m². 2 The prescribed dosage was administered.

[0236] 53. The method of implementation scheme 1, wherein the method further comprises administering at least a second dose to the subject.

[0237] 54. The method of embodiment 53, wherein the second agent is a therapeutic agent, an anti-inflammatory drug, and / or an immunosuppressant.

[0238] 55. A method for alleviating symptoms of a disease, the method comprising detecting the level of CXCL9 alone or together with one or more other biomarkers from a biological sample from a subject, comparing the detected level of CXCL9 expression with the level of control expression, and administering an interferon-gamma (IFNγ) antagonist to the subject in an amount sufficient to alleviate the symptoms of the disease when the detected level is elevated.

[0239] 56. The method of embodiment 55, wherein the one or more other biomarkers are selected from total IFNγ levels, CXCL10, CXCL11, and combinations thereof.

[0240] 57. The method of embodiment 55, wherein the biological sample is blood or derived from blood.

[0241] 58. The method of embodiment 55, wherein the biological sample is serum.

[0242] 59. The method of embodiment 55, wherein the anti-IFNγ antagonist is an anti-IFNγ antibody or an immunologically active fragment thereof.

[0243] 60. The method of embodiment 59, wherein the anti-IFNγ antibody or its immunologically active fragment contains 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 AISGSGGSTYYADSVKG (SEQ ID NO: 2); a variable heavy chain complementarity-determining region 3 (VH CDR3) comprising the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO: 3); a variable light chain complementarity-determining region 1 (VL CDR1) comprising the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO: 4); a variable heavy chain complementarity-determining region 2 (VL CDR2) comprising the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and a variable heavy chain complementarity-determining region 3 (VL CDR3) comprising the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6).

[0244] 61. The method of embodiment 59, wherein the anti-IFNγ antibody or its immunoactive fragment comprises the heavy chain variable amino acid sequence of SEQ ID NO:47 and the light chain variable amino acid sequence of SEQ ID NO:48.

[0245] 62. The method of embodiment 59, wherein the anti-IFNγ antibody or its immunologically active fragment comprises the heavy chain amino acid sequence of SEQ ID NO:44 and the light chain amino acid sequence of SEQ ID NO:46.

[0246] 63. The method of implementation scheme 55, wherein the subject is a human being.

[0247] 64. The method of implementation plan 55, wherein the condition is an autoimmune or inflammatory condition.

[0248] 65. The method of implementation scheme 55, wherein the condition is primary or secondary hemophagocytic lymphohistiocytosis (HLH).

[0249] 66. The method of implementation scheme 55, wherein the condition is macrophage activation syndrome (MAS).

[0250] 67. The method of implementation scheme 55, wherein the condition is MAS in the case of systemic juvenile idiopathic arthritis (sJIA).

[0251] 68. A formulation comprising: (a) A therapeutically effective amount of an isolated antibody or its antigen-binding fragment that binds to interferon-γ (IFNγ); (b) L-histidine; (c) L-histidine hydrochloride monohydrate; (d) Sodium chloride (NaCl); (e) Polysorbate 80; The pH of the formulation is between 5.8 and 6.2.

[0252] 69. The formulation of embodiment 68, wherein the isolated antibody comprises a variable heavy chain complementarity-determining region 1 (VH CDR1) containing the amino acid sequence of SYAMS (SEQ ID NO: 1); a variable heavy chain complementarity-determining region 2 (VH CDR2) containing the amino acid sequence of AISGSGGSTYYADSVKG (SEQ ID NO: 2); a variable heavy chain complementarity-determining region 3 (VH CDR3) containing the amino acid sequence of DGSSGWYVPHWFDP (SEQ ID NO: 3); a variable light chain complementarity-determining region 1 (VL CDR1) containing the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO: 4); a variable light chain complementarity-determining region 2 (VL CDR2) containing the amino acid sequence of EDNQRPS (SEQ ID NO: 5); and a variable light chain complementarity-determining region 3 (VL CDR3) containing the amino acid sequence of QSYDGSNRWM (SEQ ID NO: 6).

[0253] 70. The formulation of embodiment 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.

[0254] 71. The formulation of embodiment 68, wherein the formulation comprises 5 mg of antibody, 1.55 mg of L-histidine, 3.14 mg of L-histidine hydrochloride monohydrate, 7.31 mg of sodium chloride (NaCl) and 0.05 mg of [unclear text - possibly related to a specific formulation].

[0255] 72. The formulation of embodiment 68 or embodiment 71, wherein the pH of the formulation is 6.0.

[0256] 73. The formulation of embodiment 68, wherein the formulation is a sterile concentrated formulation for infusion.

[0257] The invention will be further described in the following embodiments, which do not limit the scope of the invention as described in the claims. Example

[0258] Example 1 CXCL9 levels, a biomarker for IFNγ production, in macrophage activation syndrome (MAS) The study presented in this article was designed to evaluate the correlation between serum levels of IFNγ and three IFNγ-related chemokines in patients with active MAS and laboratory parameters related to their own disease activity, thereby seeking potential biomarkers for IFNγ production in vivo.

[0259] In sJIA patients (n=54), 20 of whom had MAS at sampling time, circulating levels of IFNγ, CXCL9, CXCL10, CXCL11, and IL-6 were measured using the Luminex multiplex assay. The relationships between these circulating levels and disease activity parameters, as well as the correlations between IFNγ levels and CXCL9, CXCL10, and CXCL11 levels, were evaluated.

[0260] Compared with active sJIA without MAS at the time of sampling, the levels of IFNγ and three IFNγ-related chemokines (CXCL9, CXCL10, and CXCL11) were significantly elevated (all p < 0.005). In active MAS, laboratory parameters of disease severity (ferritin, neutrophils, platelets, alanine aminotransferase, and lactate dehydrogenase) were found to be significantly correlated with IFNγ and CXCL9, and less correlated with CXCL10 and CXCL11; no correlation was found with IL-6 levels. In patients with active sJIA but without MAS, no significant correlation was found between laboratory parameters and cytokine levels (see Table 7 below). In active MAS, IFNγ levels were significantly correlated with CXCL9 levels (r = 0.69; r < 0.005). 2 =0.47; p=0.001), which was associated with a low degree of correlation with CXCL10 levels (r=0.53; r=0.47; p=0.001). 2 =0.28; p=0.015), which was not correlated with the CXCL11 level (r=-0.04; p=0.886).

[0261] Table 7. Correlation between laboratory parameters of disease activity and IFNγ, CXCL9, CXCL10, CXCL11 and IL-6 in patients with MAS and active sJIA.

[0262] N = neutrophil count; PLT = platelet count; ALT = alanine aminotransferase. 1 =Median (IQR); r*=Spearman r High levels of IFNγ and CXCL9 in patients with active MAS were significantly correlated with laboratory parameters of disease severity. IFNγ and CXCL9 were closely associated in patients with active MAS. Since CXCL9 has been shown to be induced only by IFNγ and not by other interferons (see, for example, Groom JR and Luster AD Immunol Cell Biol 2011, Feb; 89(2):207-15), these findings confirm CXCL9 as a biomarker for IFNγ production in MAS.

[0263] Example 2 Correlation between CXCL9 and IFNγ levels in patients with primary hemophagocytic lymphohistiocytosis (HLH) The studies presented in this article are from an ongoing phase 2 trial in patients with primary HLH who received the NI-0501 antibody and patients who received the NI-0501 antibody for compassionate use.

[0264] like Figure 1 As shown, serum levels of CXCL9 and IFNγ were measured using Luminex and Meso Scale Discovery (MSD) technologies, respectively, in samples obtained from 6 patients with primary HLH and 3 patients with special use. Correlation analysis was performed between CXCL9 and total IFNγ concentrations. Statistical analysis was conducted, and Spearman's test was used to obtain p-values.

[0265] like Figure 2 As shown, serum levels of CXCL9 and IFNγ were measured before drug administration using Luminex and MSD techniques in samples obtained from 6 patients with primary HLH and 3 patients receiving special treatment. Correlation analyses were performed between CXCL9 and total IFNγ concentrations. Statistical analyses were conducted, and p-values ​​were obtained using the Spearman test.

[0266] Example 3 Correlation between CXCL9 and IFNγ levels in patients with secondary hemophagocytic lymphohistiocytosis (HLH) The studies presented in this article are from observational studies of secondary HLH patients who received NI-0501 antibody and from patients who received the specially prescribed NI-0501 antibody.

[0267] Specifically, these are patients with systemic juvenile idiopathic arthritis (sJIA) that has developed macrophage activation syndrome (MAS, a form of secondary HLH). For these patients, there was also a correlation between CXCL9 or IFNγ and disease parameters such as ferritin, platelet count (PLT), neutrophil count (Neu), and alanine aminotransferase (ALT).

[0268] like Figure 3A and 3B As shown, serum levels of CXCL9 and IFNγ in samples from 19 patients with secondary MAS following sJIA and 24 patients with active sJIA at the time of sampling were measured using Luminex technology via a multiplex assay. Correlation analysis was performed between CXCL9 and IFNγ concentrations. Statistical analysis was conducted, and Spearman's test was used to obtain p-values.

[0269] 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 using Luminex technology via a multiplex assay in patients with secondary metastatic arterial disease (sJIA) and those with active sJIA at the time of sampling. Correlation analysis was performed between IFNγ or CXCL9 levels and ferritin, platelet count, neutrophil count, or ALT (alanine aminotransferase). Statistical analysis was conducted, and Spearman's test was used to obtain p-values.

[0270] Example 4. Correlation between CXCL9 and IFNγ levels in patients with severe hemophagocytic lymphohistiocytosis (HLH) This study presents a case from a patient who received the specially prescribed NI-0501 antibody. The patient presented with symptoms of NLRC4-related disease and severe hemophagocytic lymphohistiocytosis (HLH). (Recently reported...) NLRC4 Gene mutations cause recurrent macrophage activation syndrome and increased IL-18 production, the latter of which is known to induce IFNγ.

[0271] Patients in this study exhibited the following characteristics: onset at 20 days of age, accompanied by fever, rash, significant hepatosplenomegaly, pancytopenia, hypofibrinogen, hypertriglyceridemia, and significant increases in ferritin and sCD25. This was followed by multiple organ failure requiring ICU admission. HLH diagnosis was based on 6 of the 8 HLH-2004 criteria. Genes causing primary HLH (PRF1, UNC13D, STXBP2, STX11, RAB27A, XIAP) and functional tests (perforin expression, degranulation, and cytotoxicity) were negative. High-dose intravenous corticosteroids and intravenous cyclosporine A provided gradual improvement in overall condition and laboratory abnormalities. The infection was caused by Candida albicans (…). Candida Albicans ) and Klebsiella pneumoniae ( Klebsiella Pneumoniae Sepsis can lead to HLH reactivation, rapid deterioration of overall condition, and new admission to the ICU. Treatment with etoposide and / or ATG is not considered in immunocompromised subjects due to the presence of active infection.

[0272] Measurable serum levels of IFNγ and high serum levels of IFNγ-induced chemokines CXCL9 and CXCL10, as well as significantly elevated serum levels of IL-18, were confirmed (Table 8).

[0273] Table 8. Levels of IFNγ, IFNγ-related chemokines, and IL-18 at the start of NI-0501 treatment and during NI-0501 treatment. NI-0501 is a specific treatment for dexamethasone (13.6 mg / m²). 2 The treatment began with oral cyclosporine-A and intravenous cyclosporine-A. NI-0501 was administered every 3 days, followed by every 7 days, according to pharmacokinetics. No infusion response was observed. NI0501 was well tolerated. Clinical features and laboratory abnormalities of HLH progressively improved. The active, progressive infection was rapidly cleared. After 5 months of treatment, the patient remained in excellent condition. The patient continued to receive oral cyclosporine-A (6 mg / kg) and prednisone (0.3 mg / kg is equivalent to 0.9 mg / m² dexamethasone). All HLH parameters normalized.

[0274] The subjects still experienced unexplained inflammatory episodes. NLRC4 Analysis revealed a newly discovered missense mutation (T337N). Elevated serum IL-18 levels were recorded, confirming the mutation. NLRC4 The association with mutations. High levels of IFNγ associated with NI-0501 indicate high IFNγ production. As shown by the undetectable levels of IFNγ-inducible chemokines, IFNγ was completely neutralized ( Figure 5 (See Table 8). Circulating levels of IL-18 continued to rise.

[0275] Therefore, this study indicates that in patients with severe refractory HLH (due to...) NLRC4 In patients with HLH (caused by mutation), blocking IFNγ with NI-0501 was well tolerated, with no safety concerns, resulting in control of all HLH characteristics, rapid reduction of glucocorticoids, and was associated with the resolution of progressive active infection.

[0276] Example 5 Targeted therapy for hemophagocytic lymphohistiocytosis (HLH) using NI-0501 This article presents a phase 2 trial study from children with primary HLH. Primary HLH (pHLH) is a rare immunomodulatory disorder that is fatal if left untreated. It is driven by pathological immune activation, leading to fever, splenomegaly, cytopenia, 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 HLH patients, high IFNγ production is considered a key driver of the disease. Immunochemotherapy, primarily based on etoposide regimens, is currently the only pharmacological approach to control HLH and offers patients the potential for curative allogeneic hematopoietic stem cell transplantation (allogeneic HSCT). Despite recent attempts to further intensify treatment regimens, mortality and morbidity remain high, partly due to drug-related toxicities.

[0277] As described above, NI-0501 is a high-affinity fully human anti-IFNγ mAb that binds to and neutralizes human IFNγ, providing a new targeting approach for controlling HLH.

[0278] method: An open-label phase 2 study was conducted in the United States and Europe to evaluate the safety and efficacy of NI-0501 in children with confirmed or suspected pHLH. NI-0501 was administered as an initial dose of 1 mg / kg every 3 days, followed by dexamethasone at a starting background of 5–10 mg / m². 2 Dosage may be increased in individual patients based on PK data and / or clinical response guidance. Treatment duration ranged from 4 to 8 weeks. The ability to switch to allogeneic HSCT, relevant HLH disease parameters, and 8-week survival were evaluated.

[0279] Study population Thirteen patients were recruited: 8 fetal / mature (F / M), with a median age of 1.0 year (range 2.5 months–13 years). Twelve patients received NI-0501 as second-line therapy after receiving standard therapy and subsequent reactivation, unsatisfactory response, or intolerance to other treatments. One patient received NI-0501 as first-line therapy. Nine patients carried known HLH genetic defects (3 FHL2, 2 FHL3, 2 GS-2, 1 XLP1, 1 XLP2). Most patients were at the severe end of the HLH range, with impaired overall condition and significant toxicity from previous HLH treatments. Elevated ferritin was observed in 12 / 13 patients, elevated sCD25 in 8, cytopenia in 10 patients, splenomegaly in 8, hypofibrinogen and hypertriglyceridemia in 9. Hepatic impairment and CNS involvement were present in 7 and 3 patients, respectively.

[0280] result: Overall, NI-0501 treatment significantly improved parameters of HLH disease activity ( Figure 6 Of the 13 patients, 9 achieved a satisfactory response. Six patients underwent HSCT. Two patients with good HLH control were scheduled for HSCT upon identification of a suitable donor. In one patient (who achieved disease control with first-line NI-0501), HSCT could not be planned due to the absence of the pathogenic HLH gene mutation. Eleven of the 13 patients were alive at 8 weeks. CNS signs and symptoms resolved in two evaluable patients. A reduction of dexamethasone dose by more than 50% was possible in more than 50% of patients during the first 4 weeks of NI-0501 treatment.

[0281] Biomarker evaluation, particularly CXCL9, a chemokine known to be strongly induced by IFNγ, not only confirms complete IFNγ neutralization but also shows promise as a novel parameter for the diagnosis of HLH associated with IFNγ production. Figure 7A and 7B ).

[0282] NI-0501 was well tolerated, and no safety concerns were identified. No infections known to be induced by IFNγ neutralization have been reported, and no infections occurred in patients who had not received prior chemotherapy. Seven patients reported at least one SAE, all of which were assessed by DMC as unrelated to NI-0501 administration. No unexpected events attributable to NI-0501's "off-target" effects (e.g., myelotoxicity, hemodynamic effects) were observed.

[0283] in conclusion: IFNγ, through targeted neutralization with NI-0501, offers a revolutionary and potentially less toxic approach to HLH management. Results from this study demonstrate that NI-0501 is a safe and effective treatment option in patients with primary HLH who have not responded satisfactorily to or are intolerant of conventional therapies. Furthermore, treatment with NI-0501 was not associated with any of the typical short- or long-term toxicities associated with etoposide-based regimens. Evaluations of NI-0501 as first-line therapy in patients with pHLH are ongoing, with similar significant clinical benefits anticipated.

[0284] Example 6 Elevated circulating levels of interferon-γ and interferon-induced chemokines were observed in patients with macrophage activation syndrome complicated by systemic JIA. Interferon-γ (IFNγ) is a key mediator in a mouse model of primary hemophagocytic histiocytosis (HLH). Given the similarities between primary and secondary HLH (sec-HLH), including macrophage activation syndrome (MAS), IFNγ levels and their biological activities were analyzed in patients with systemic juvenile idiopathic arthritis (sJIA) and MAS.

[0285] In the study presented in this article, serum levels of IL-1β, IL-6, IFNγ, and IFN-induced and / or IFN-associated chemokines CXCL9, CXCL10, and CXCL11 were evaluated using the Luminex multiplex assay in sec-HLH patients (n=11) and 20 of them in sJIA patients with MAS at sampling time (n=54). The expression of IFNγ-induced chemokines (CXCL9 and CXCL10 mRNA levels in the liver and spleen) and their correlation with ferritin levels were evaluated in an IL-6 transgenic mouse model (where MAS characteristics were induced by LPS stimulation of TLR4).

[0286] As described in more detail below, circulating levels of IFNγ and IFN-induced chemokines were significantly elevated during MAS (also referred to in this paper as active MAS and sec-HLH). Levels of IFNγ and IFN-induced chemokines were significantly higher in MAS patients compared to those with active sJIA but without MAS. In the group with active sJIA but without MAS, IFNγ and IFN-induced chemokines were comparable to those in patients with clinically inactive sJIA. During MAS, laboratory abnormalities characterizing the syndrome (including ferritin and alanine transferase levels and neutrophil and platelet counts) were significantly correlated with IFNγ and CXCL9 levels. In a mouse model of MAS, serum ferritin levels in the liver and spleen were significantly correlated with mRNA CXCL9 levels.

[0287] Therefore, the research presented below suggests that high levels of IFNγ and IFN-induced chemokines, and their correlation with the severity of MAS laboratory abnormalities, indicate that IFNγ plays a crucial role in MAS. Elevated circulating levels of interferon-γ and interferon-induced chemokines characterize patients with macrophage activation syndrome complicated by systemic JIA.

[0288] Materials and Methods: Patients and Samples. Peripheral blood samples were collected from patients with or without MAS in sJIA at the following three pediatric rheumatology centers: Ospedale Pediatrico Bambino Gesù in Rome, Istituto Giannina Gaslini in Genoa, and Cincinnati Children's Hospital Medical Centre. Fifty-four patients with sJIA meeting the ILAR classification criteria for systemic arthritis (age at onset 7.9 years, interquartile range 4.6–13.6 years; 48% female) were studied (Petty, RE et al., International Leagues of Associations for Rheumatology classification of juvenile idiopathic arthritis: second revision, Edmonton, 2001.J Rheumatol, 2004, 31(2): p. 390-2). Samples were collected from 20 patients with SJIA during episodes of active, fully developed MAS diagnosed by the treating physicians at each of the three centers. Post-hoc analysis showed that 17 of these 20 episodes (85%) met the newly proposed classification criteria for MAS (Minoia F, Davì S, Bovis F et al. Development of new classification criteria for macrophageactivation syndrome complicating systemic juvenile idiopathic arthritis. Pediatric Rheumatology 2014, 12(Suppl 1):O1.). Samples were also available from 28 patients with active SJIA but no evidence of MAS. During clinically inactive disease as defined by Wallace criteria, 35 samples were available from 35 patients with SJIA (with or without MAS in their medical history) (Wallace, CA et al., Preliminary criteria for clinical remission for select categories of juvenile idiopathic arthritis. J Rheumatol, 2004.31(11): p. 2290-4).

[0289] Because increased IFNγ has been shown in patients with sec-HLH (excluding rheumatic diseases), samples were also collected from 11 sec-HLH patients observed at Ospedale Pediatrico Bambino Gesù (age at onset 8.6 years, interquartile range 4.1–12.9 years; 36% female) and used as positive controls. All sec-HLH patients met the 2004-HLH diagnostic criteria (Henter, JI et al., HLH-2004: Diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer, 2007, 48(2): p. 124-31): Six patients met five criteria, and five patients met four criteria. It should be noted that sCD25 levels (U / ml) were not obtained because the test was not routinely performed at the institution that recruited these patients. Diagnosis of primary HLH was excluded based on the lack of family history, the lack of known pathogenic mutations causing HLH, and the presence of normal functional studies (including NK activity, perforin expression, and CD107 degranulation). All 11 sec-HLH patients contributed one sample obtained during the active period of the disease.

[0290] Researchers at various centers collected information on diagnosis and the clinical and laboratory characteristics of all patients at the time of sampling from a centralized World Wide Web database. Of the 20 MAS patients sampled during active disease, 6 were not receiving any treatment at the time of sampling, while the remaining 14 had received one of the specific treatments for MAS, including glucocorticoid pulse therapy, cyclosporine A, anaerobic acid, or cyclophosphamide. Of the 11 sec-HLH patients with active disease, 6 were not receiving specific treatment at the time of sampling, while the remaining 5 had received at least one of the aforementioned treatments. The study was approved by the Ethics Committee of Ospedale Pediatrico Bambino Gesù. Written consent was collected from all participants.

[0291] Quantitative determination of cytokines. Levels of IL-6, IL-1β, IFNγ, CXCL9, CXCL10, and CXCL11 were analyzed using Luminex® multiplex bead technology. Reagents were purchased from Millipore and all reagents were provided using the Milliplex® MAP kit. Reagents were prepared according to the manufacturer's instructions. Two copies of 25 μl / well of standards, blanks, and quality control samples were added to each well of a Milliplex MAP 96-well plate, followed by 25 μl of serum matrix. 25 μl of assay buffer was added to each well, followed by 25 μl of sample. Samples were added in duplicate or triplicate, depending on the available sample volume. The plates were measured using a Luminex 200® system (Luminex Corp.). Raw data were obtained using x 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 from the Milliplex Analyst software was then further analyzed in dedicated macros used for Luminex analysis (NI-Sc-ESM-MAC-012-v01 and Sc-ESM-MAC-013-v01).

[0292] Animal experiments. The generation and phenotype of IL-6 transgenic mice, as well as the characteristics of 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: 对巨噬细胞活化综合征发病机制的提示。ArthritisRheum, 2012, 64(5): p. 1680-8). Mice were kept under dedicated pathogen-free conditions and handled in accordance with national policy. The study protocol was approved by the local ethics committee. All experiments were performed on mice at 10 and 14 weeks of age. Mice were given a single intraperitoneal dose of lipopolysaccharide (LPS) at 5 µg / g body weight (E. coli serotype O55: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). The results were obtained using TaqMan UniversalPCR Master Mix (Applied Biosystems) with mice. Cxcl9 and Cxcl10 Gene expression assays (Applied Biosystems) were used for real-time PCR assays. Mice were used. Hprt (Applied Biosystems) normalizes gene expression data. Data is expressed in arbitrary units (AU), using 2... -∆ct Methods: Serum ferritin concentration was determined using a commercially available ELISA kit (ALPCODiagnostics) according to the manufacturer's instructions.

[0293] Statistical analysis was performed using GraphPad Prism 5 software. Continuous variables (quantitative demographic, clinical, and laboratory data) were expressed as medians and interquartile ranges (IQRs) and compared using the Mann-Whitney U test. Wilcoxon's notated rank test was used to compare paired groups, assuming the distribution of differences before and after the hypothesis did not follow a Gaussian distribution. Spearman's rank correlation was used to assess the relationship with laboratory parameters. A p-value < 0.05 was considered statistically significant.

[0294] Results: Levels of IFNγ and IFNγ-induced chemokines are elevated in patients with MAS. This is as expected when comparing patients with active sJIA without MAS at the time of sampling to those sampled during clinically inactive disease (deBenedetti, F. et al.). Correlation of serum interleukin-6 levels with joint involvement and thrombocytosis in systemic juvenile rheumatoid arthritis.A study published in *Arthritis Rheum* (1991, 34(9): p. 1158-63) found that IL-6 levels were significantly higher in patients with active sJIA compared to those in patients with clinically inactive disease (p<0.01). As reported in several previous studies on active SJIA, serum IL-1β levels were below the limit of detection in most patients, independent of disease activity. Notably, there was no difference in the levels of IFN and the three IFNγ-induced chemokines γ between patients with clinically active sJIA and those with clinically inactive disease.

[0295] When patients with MAS at sampling were compared to those with active sJIA without MAS at sampling, IL-1β and IL-6 levels were comparable, indicating that the levels of these two cytokines, known to play a key role in active sJIA, are not increased in fully developed MAS. It should be noted that circulating IL-1β levels were below the limit of quantification (i.e., 3.5 pg / ml) in most patients with sJIA, whether or not they had MAS. In contrast, circulating IFNγ levels were significantly higher in patients with active MAS compared to those with active sJIA without MAS at sampling. The levels of the three IFNγ-related chemokines, CXCL9, CXCL10, and CXCL11, were also significantly higher in patients with active MAS compared to those with active sJIA without MAS at sampling. This difference was particularly pronounced for CXCL9, with its median level being approximately 15 times higher in MAS patients compared to those with active sJIA without MAS.

[0296] In patients with sec-HLH, the levels of IFNγ and the three IFNγ-related chemokines were significantly increased. The levels of IFNγ and IFNγ-related chemokines were mostly indistinguishable from those in MAS patients, and the differences were not statistically significant. Incidentally, in patients with active MAS and active sec-HLH, the levels of IFNγ and the three IFNγ-induced chemokines were comparable in untreated and treated patients.

[0297] The levels of IFNγ and CXCL9, CXCL10 and CXCL11 were associated with the presence of MAS in each patient. Figures 8A-8DThe levels of IFNγ and CXCL9, CXCL10, and CXCL11 were shown in each patient, with paired samples available from these patients during active MAS and during active sJIA without MAS. Consistent with results obtained in representative analyses, the levels of IFNγ and the three IFNγ-induced chemokines were significantly higher in samples obtained during MAS, as shown by paired sample analysis. Additionally, in several patients, samples were available both before and after MAS attacks, confirming that IFNγ and IFNγ-induced chemokine levels returned to normal as MAS clinical symptoms subsided. For example, one patient in this study experienced three MAS attacks, with serum samples obtained during these attacks and at the time of sampling during the disease-free period without MAS. This further confirmed the relationship between increased production of IFNγ and the three IFNγ-induced chemokines and active MAS; in this patient, elevated levels of IFNγ and the three IFNγ-related chemokines were only present during MAS attacks. Figures 9A-9B ).

[0298] The levels of IFNγ and IFNγ-related chemokines were associated with laboratory abnormalities in MAS. The correlation between the levels of IFNγ and three IFNγ-induced chemokines and laboratory parameters of MAS at sampling time was examined. In patients with active sJIA but without MAS, the levels of IFNγ and the three IFNγ-induced chemokines were not associated with laboratory parameters of MAS, with one exception: the levels of CXCL9, CXCL10, and CXCL11 were weakly correlated with ALT levels, with a r 2The range was 0.17–0.25 (Table 2). The significance of this association is unclear; however, it should be noted that ALT levels were within the normal range in all patients with active s-JIA without MAS. In patients with MAS at sampling time, no significant correlation was found between the laboratory characteristics of MAS and IL-1 and IL-6. In contrast, in patients with MAS at sampling time, the levels of IFNγ and IFNγ-induced chemokines were associated with ferritin levels, neutrophil and platelet counts, and elevated LDH and ALT, all of which are typically abnormal in MAS patients (Table 2). The correlation with laboratory abnormalities was particularly pronounced for IFNγ and CXCL9, with the exception of the correlation between IFNγ and LDH, which did not reach statistical significance (Table 2 and Figures 10A-10J). Furthermore, as mentioned above, these correlations were not present in patients with active s-JIA without MAS at sampling time. 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 with severe central nervous system involvement and was admitted to the intensive care unit. This observation provides further support for the hypothesis that there is a strong correlation between IFNγ and CXCL9 levels and disease severity. In summary, these results demonstrate that increased production of IFNγ and IFNγ-related chemokines is a characteristic feature of active MAS strongly correlated with the severity of laboratory abnormalities in MAS.

[0299] Table 2. Serum levels of IL-1β, IL-6, IFNγ, and three IFNγ-related chemokines CXCL9, CXCL10, and CXCL11 in patients with active secondary HLH, active MAS at sampling time, active sJIA without MAS at sampling time, and clinically inactive sJIA.

[0300] Values ​​are displayed as median (interquartile range). *Active sJIA compared to clinically inactive sJIA: p<0.01 Table 3. Correlation between laboratory parameters of disease activity and levels of IFNγ, CXCL9, CXCL10, CXCL11 and IL-6 in patients with MAS and patients with active sJIA but no MAS at the time of sampling.

[0301] NEU = Neutrophil count; PLT = Platelet count; ALT = Alanine aminotransferase; LDH = Lactate dehydrogenase; 1 = Median (IQR); r*= Spearman r.

[0302] Correlation between laboratory parameters of disease activity and IFN-γ, CXCL9, CXCL10, CXCL11, and IL-6 in patients with MAS and active sJIA. The correlation between IFNγ and IFNγ-induced chemokine levels in MAS patients. To further characterize the relationship between IFNγ and the three IFNγ-induced chemokines in MAS patients, the correlation between IFNγ levels and the levels of each individual chemokine was evaluated. Notably, CXCL9 showed that it was primarily induced by IFNγ, while CXCL10 and CXCL11 were also induced by type I interferon. Consistent with this, in patients with active MAS, circulating IFNγ levels were significantly correlated with CXCL9 (r=0.693; r 2 =0.48; p=0.001), but showed a weak correlation with CXCL10 levels (r=0.535; r 2 =0.29; p=0.015) Figures 11A-11F The correlation with CXCL11 levels was also weak and did not reach statistical significance (r=0.447; r 2 =0.20; p=0.08) (not shown).

[0303] IFNγ-induced chemokines are associated with disease activity in a MAS mouse model. To further investigate the association between IFNγ-induced chemokine production and MAS, the expression of these chemokines in target tissues (liver and spleen) was studied in a MAS mouse model. In this model, clinical and laboratory characteristics of MAS were induced by simulating acute infection with the TLR4 agonist lipopolysaccharide (LPS) against a background of high IL-6 levels in IL-6 transgenic mice (Strippoli et al. Arthritis Rheum 2012). This method reproduces what occurs in sJIA patients: infection can induce MAS / HLH in the presence of active disease, characterized by high levels of IL-6. Following LPS induction, high levels of CXCL9 and CXCL10 mRNA were present in the liver and spleen of IL-6 transgenic mice. Notably, serum ferritin levels were significantly correlated with CXCL9 expression in the spleen and liver, and CXCL10 expression in the liver, indicating a relationship between upstream IFNγ-related events in target tissues (i.e., CXCL9 and CXCL10 production in the liver and spleen) and typical downstream laboratory abnormalities (e.g., high ferritin levels). In summary, data from MAS patients and MAS mouse models clearly demonstrate that increased IFNγ production is associated with increased CXCL9 expression, CXCL10 (with a lower degree of correlation), and laboratory abnormalities in MAS.

[0304] Studies in both p-HLH patients and animal models have confirmed the important role of IFNγ in disease pathogenesis. However, the role of IFNγ in sec-HLH, including MAS in the context of sJIA, remains unclear. This study convincingly demonstrates the presence of high levels of IFNγ and IFNγ-induced chemokines in patients with MAS occurring in sJIA. Furthermore, levels of IFNγ, CXCL9, and CXCL10 were strongly correlated with laboratory parameters of MAS severity. The study found that serum levels of IFNγ and the three IFNγ-related chemokines were comparable between patients with active sJIA and those with clinically inactive disease. These results do not indicate a pathogenic role of IFNγ in sJIA and are indeed consistent with several observations from other authors. Three gene expression studies did not identify significant IFNγ-inducible markers in peripheral blood mononuclear cells (PBMCs) from patients with active sJIA but without MAS at sampling time (Fall, N. et al., Gene expression profiling of peripheral blood from patients with untreated new-onset systemic juvenile idiopathic arthritis reveals molecular heterogeneity that may predict macrophage activation syndrome. Arthritis Rheum, 2007. 56(11): p.3793-804; Ogilvie, EM et al., Specific gene expression profiles in systemic juvenile idiopathic arthritis.Arthritis Rheum, 2007. 56(6): p. 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): p. 1479-86). Following ex vivo stimulation of PBMCs, the number of cells producing IFNγ in patients with active sJIA was similar to that in controls (Lasiglie, D. et al., Role of IL-1 beta in the development of human T(H)17 cells: lesson from NLPR3 mutated patients. PLoS One, 2011. 6(5): p. e20014). Consistently, patients with both active and inactive SJIA did not show elevated 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). CXCL9 and CXCL10 were almost undetectable in the synovial tissue of patients with sJIA, while high levels of these chemokines were present in the synovial tissue of patients with oligoarticular or polyarticular JIA, supporting the view that IFNγ has no role in joint inflammation of sJIA (Sikora, KA et al., The limited role of interferon-gamma in systemic juvenile idiopathic arthritis cannot be explained by cellular hyporesponsiveness. Arthritis Rheum, 2012. 64(11): p. 3799-808. Recent data in mice show that IFNγ knockout mice immunized with Freund's complete adjuvant exhibit a systemic inflammatory syndrome including features of sJIA, further supporting the limited role of IFNγ in sJIA (Avau, A. et al., Systemic juvenile idiopathic arthritis-like syndrome in mice following stimulation of the immune system with Freund's complete adjuvant: regulation by interferon- gamma. Arthritis Rheumatol, 2014. 66(5): p. 1340-51).

[0305] In stark contrast, this study showed significantly higher levels of IFNγ and IFNγ-related chemokines in patients with active sJIA but without MAS at the time of sampling compared to patients with active sJIA but without MAS at the time of sampling. This was also confirmed in each patient using serial samples obtained during both periods of active MAS and active sJIA without MAS. Incidentally, in patients sampled during MAS, this study did not find a significant increase in IL-6 or IL-1β levels, nor did it find any association with MAS laboratory parameters, suggesting that these cytokines, despite their crucial involvement 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): p. 2385-95; Ruperto, N. etal., Two randomized trials of canakinumab in systemic juvenile idiopathic arthritis. N Engl J Med, 2012. 367(25): p. 2396-406), but may not be crucial in maintaining MAS. This finding of elevated levels of IFNγ and IFNγ-related chemokines is consistent with previous findings. Shimizu et al. reported that neopterin (a metabolite of guanosine triphosphate synthesized by human macrophages upon stimulation with IFNγ) levels were higher in MAS patients during sJIA compared to patients with active sJIA without MAS (Shimizu, M. et al., Distinct cytokine profiles of systemic-onset juvenile idiopathic arthritis-associated macrophage activation syndrome with particular emphasis on the role of interleukin-18 in its pathogenesis. Rheumatology (Oxford), 2010. 49(9): p. 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 progression of sJIA (Put, K. et al., Cytokines in systemic juvenile idiopathic arthritis and haemophagocytic lymphohistiocytosis: tipping the balance between interleukin-18 and interferon-gamma. (Rheumatology (Oxford), 2015). Consistent with these results, the levels of IFNγ and CXCL10 were significantly lower in the five patients with active sJIA but no MAS at the time of sampling (Put et al., Rheumatology 2015).

[0306] Notably, this study found that not only were the levels of IFNγ and IFNγ-related chemokines significantly elevated, but their levels, particularly CXCL9, were also strongly correlated with the laboratory characteristics of MAS, indicating an association with disease severity. Further supporting this association with disease severity, the study found significantly higher levels of IFNγ, CXCL9, and CXCL10 in one patient with severe illness, multiple organ failure, central nervous system involvement, and systemic seizures, requiring long-term intensive care.

[0307] In MAS patients, CXCL9 was found to be most strongly correlated with IFNγ levels among the three IFNγ-induced chemokines. This observation is consistent with the established view that, unlike the production of CXCL10 and CXCL11, which can also be induced by type I interferon, CXCL9 production shows a unique and specific induction by IFNγ (Groom, JR and ADLuster, CXCR3 ligands: redundant, collaborative and antagonistic functions. Immunol Cell Biol, 2011. 89(2): p. 207-15). This suggests that CXCL9 levels can be used as a sensitive and specific biomarker for MAS activity. In fact, using a mouse model of MAS induced by infectious stimuli in the context of high IL-6 levels (Strippoli et al., Arthritis Rheum 2012), this study also found that CXCL9 expression levels in the liver and spleen were significantly correlated with circulating ferritin levels. For CXCL10 expression levels, this correlation was only present in the liver, but not in the spleen. This is also supported by findings in MAS patients, where CXCL9 levels were closely correlated with all laboratory parameters of MAS. In conclusion, these observations in humans and mice show that CXCL9 is strongly correlated with MAS characteristics and IFNγ production, further supporting the hypothesis that excessive IFNγ production plays a major pathogenic role in MAS. These observations are also consistent with immunohistochemical data generated by Put et al. using a series of lymph node biopsy samples from the same SJIA patient during active sJIA without MAS and during MAS. They reported high levels of CXCL10 and indoleamine 2,3-dioxygenase, both IFNγ-induced proteins, in tissues obtained during MAS but not during active sJIA without MAS (Put et al., Rheumatology 2015).

[0308] These results in MAS and sec-HLH, along with the observations available in p-HLH patients in the references, support the hypothesis that increased IFNγ and IFNγ-related chemokines, particularly CXCL9, are a distinctive feature of HLH independent of underlying etiology. In this regard, it is noteworthy that high levels of CXCL9 were detected in patients with recurrent MAS induced by NLRC4 gain-of-function mutations (Canna, SW et al., An activating NLRC4 inflammasome mutation causes autoinflammation with recurrent macrophage activation syndrome. Nat Genet, 2014. 46(10): p. 1140-6), indicating that IFNγ overproduction may occur even in the context of abnormally induced HLH regulated only by inflammasome.

[0309] In p-HLH animal models, data from both perforin and Rab27a knockout mice clearly demonstrate the pathogenic role of IFNγ. Similarly, recent data from TLR9-induced HLH and infection-secondary HLH models also show a major role in increased IFNγ production (Behrens, EM et al., Repeated TLR9 stimulation results in macrophage activation syndrome-like disease in mice. J Clin Invest, 2011. 121(6): p. 2264-77 and (Bautois et al., ongoing). Other studies in the aforementioned MAS mouse model have recently confirmed that treatment with anti-IFNγ antibodies improves survival and restores the clinical and laboratory characteristics of MAS (Prencipe et al., ongoing). In summary, these findings and observations in animals provide a basis for IFNγ neutralization as a treatment for MAS.

[0310] Example 7 Safety, tolerability, pharmacokinetics, and efficacy evaluation of multiple intravenous administrations of anti-interferon-γ-Gamma anti-IFNγ monoclonal antibody in pediatric patients with primary hemophagocytic lymphohistiocytosis (HLH). The study presented in this article was designed to determine the safety and tolerability profile of multiple intravenous (IV) administrations of an anti-IFNγ antibody referred to herein as NI-0501; to determine the efficacy and benefit / risk profile of NI-0501 in patients with HLH; to describe the pharmacokinetic (PK) profile of NI-0501 in patients with HLH; to determine the appropriate NI-0501 treatment dosage regimen for HLH; and to evaluate the immunogenicity of NI-0501.

[0311] Preclinical studyPrevious studies have shown that NI-0501 exhibits similar binding affinity and blocking activity for IFNγ derived from non-human primate species (including rhesus macaques and cynomolgus monkeys) but not from dogs, cats, pigs, rabbits, rats, or mice. Toxicological and safety studies in cynomolgus monkeys have shown no off-target toxicity attributable to NI-0501, weekly administration of NI-0501 is well tolerated, no antibiotic prophylaxis is required, and no abnormal histopathological or behavioral findings have been observed in these previous studies.

[0312] Due to NI-0501's ability to bind free and IFNγR1-bound FNγ, this study aimed to investigate its potential to mediate ADCC and CDC activities in the presence of a target. No ADCC activity was confirmed, and no CDC activity induction was observed.

[0313] Phase I clinical study This study investigated the safety, tolerability, and pharmacokinetic profile of a single intravenous (IV) administration of NI-0501 in 20 healthy adult volunteers, using a phase 1 randomized, double-blind, placebo-controlled, single-escalation dose. During the study, 6 participants received placebo, while 3, 3, 4, and 4 participants (a total of 14 participants) received NI-0501 doses of 0.01, 0.1, 1, and 3 mg / kg, respectively.

[0314] PK analysis of NI-0501 revealed the expected profile of IgG1, which has a long half-life (approximately 22 days), slow clearance (≤0.007 L / h), and low volume of distribution (average <6 L).

[0315] Of the 20 participants, 14 (70%) experienced a total of 41 adverse events (AEs) after the start of drug infusion, 10 of which were reported by the 4 participants receiving placebo. 36 AEs (87.8%) were of mild severity, and 5 (12.2%) were of moderate severity. No serious or life-threatening AEs were reported. Of the 14 participants who experienced AEs, 23 were drug-related (56.1%) (at least reasonably likely). Most AEs were observed to be isolated and did not show a trend associated with NI-0501 dose escalation. All NI-0501 infusions were event-free.

[0316] In summary, NI-0501 infusion was well tolerated, and the effects observed during 8 weeks of post-infusion monitoring did not indicate any serious or unexpected off-target safety or immunogenicity concerns.

[0317] Materials and Methods for Phase 2 / 3 Clinical Study These studies were conducted in patients with primary HLH. The studies were divided into three parts: screening, treatment, and follow-up. See overview below. Figure 12 .

[0318] In these studies, suitable patients included those newly treated with HLH (also referred to as “first-line patients”) or those who had received conventional HLH therapy (also referred to as “second-line patients”) but who, for example, did not achieve a satisfactory response by their treating physician or showed signs of intolerance. Patients who received NI-0501 after unsuccessful or intolerable conventional HLH therapy represented the key cohort of this study to demonstrate the efficacy of NI-0501 as a second-line treatment for primary HLH. Patients who had received initial treatment were recruited to collect efficacy and safety data in the first-line context.

[0319] This study excludes the following patients: those diagnosed with confirmed rheumatic disease or neoplasia complicated by secondary HLH; those who have been pretreated with any T-cell depletion agent (e.g., anti-thymocyte globulin (ATG), anti-CD52 therapy) or any other biological agent (except rituximab in documented B-cell EBV infection) within 2 weeks prior to screening; and those with active mycobacteria, Histoplasma capsulatum (HMP), or other similar pathogens. Histoplasma Capsulatum ), Shigella spp. Shigella Salmonella ( Salmonella ), Campylobacter spp. Campylobacter ) and Leishmania ( Leishmania Patients with the following conditions are not eligible for screening: 1) infection; 2) evidence of tuberculosis or latent tuberculosis; 3) positive serum for HIV antibodies, hepatitis B surface antigen binding, or hepatitis C antibodies; 4) malignant tumors; 5) comorbidities or malformations that seriously affect cardiovascular, pulmonary, hepatic, or renal function; 6) a history of hypersensitivity or allergic reactions to any component of the study protocol; 7) patients who received live or attenuated live virus (including BCG) vaccines within the 12 weeks prior to screening; and 8) pregnant or lactating women.

[0320] The study presented in this article used anti-interferon-γ antibody NI-0501, a fully human IgG1 monoclonal antibody (mAb) targeting human IFNγ. NI-0501 was provided as a sterile concentrate (per mL) for infusion, as shown in Table 4 below.

[0321] Table 4. NI-0501 Formulation Element Volume (per mL) NI-0501 5 mg L-histidine 1.55 mg L-histidine hydrochloride monohydrate 3.14 mg Sodium chloride (NaCl) 7.31 mg Polysorbate 80 0.05 mg pH 6.0 ± 0.2 In these studies, NI-0501 was administered via IV infusion over 1 hour at an initial dose of 1 mg / kg. This dose was expected to inhibit at least 99% of IFNγ activity for 3 days in patients with a baseline IFNγ concentration of ≤3400 pg / mL. Infusions were administered every 3 days until day 15 of the study (SD15) (infusion #6), and thereafter twice weekly. At any time during the study period, the NI-0501 dose could be increased to 3 mg / kg based on predetermined criteria guided by clinical and laboratory responses in individual patients (as described in Table 5 below). Following at least two 3 mg / kg infusions, upon re-evaluation, if the same clinical and laboratory criteria for patient eligibility to receive 3 mg / kg of NI-0501 still apply, the NI-0501 dose may be increased to 6 mg / kg up to 4 infusions, subject to periodic monitoring of clinical and laboratory HLH parameters. Based on the progress of these parameters, the dose of NI-0501 may: i) be reduced back to 3 mg / kg, or ii) if PK and PD evidence indicates extremely high IFNγ production and therefore the fastest NI-0501 clearance, be maintained at 6 mg / kg for additional IV infusions (or increased by more than 6 mg / kg). Dose increases may occur at any time during the study period if the clinical and laboratory criteria listed herein are met.

[0322] Table 5. Clinical and laboratory criteria for guiding dose increases a If these standards still apply after SD6, the NI-0501 dose is increased from 1 mg / kg to 3 mg / kg.

[0323] b If the NI-0501 dose has been increased in SD3, at least two infusions at a dose of 3 mg / kg must be performed before standard reassessment.

[0324] c The dose may be increased to 3 mg / kg depending on whether it occurs in SD3 or SD6.

[0325] d For a maximum of 4 bets.

[0326] Abbreviations: bsl. = baseline; ANC = absolute neutrophil count; US = ultrasound In these studies, NI-0501 was administered for up to 8 weeks, and the treatment period could be divided into two separate phases: treatment phase 1 and phase 2, as shown below. Figure 12 As shown.

[0327] Eight weeks after NI-0501 administration, preparation for hematopoietic stem cell transplantation (HSCT) can begin. If the patient's condition and donor availability permit transplantation, the expected duration of treatment may be shortened, but not to less than four weeks. If a suitable donor has been identified by week 8, or if the transplant schedule is delayed for reasons unrelated to NI-0501 administration, NI-0501 treatment may continue in long-term follow-up studies, provided a favorable benefit / risk ratio is established for the patient.

[0328] In these studies, NI-0501 was administered in the context of dexamethasone, which was gradually reduced based on patient condition. In newly treated patients, NI-0501 could be administered at 10 mg / m². 2 Dexamethasone should be administered in the context of HLH treatment. In patients receiving NI-0501 as second-line therapy, dexamethasone must be administered at a dose of at least 5 mg / m². 2 The dose should be increased, or if higher, the same dose as before screening. Patients need to start receiving dexamethasone from SD-1.

[0329] Dexamethasone may be gradually reduced based on the patient's condition, as determined by the treating physician. The tapering schedule can be chosen by the treating physician, provided that the dexamethasone dose is not halved at any step and the frequency of changes does not exceed once a week.

[0330] If the disease worsens after the dexamethasone dosage has been gradually reduced, the dosage of dexamethasone can be increased as directed by the treating physician and maintained until a satisfactory response is achieved.

[0331] As recommended in the HLH treatment guidelines, from the day before starting NI-0501 treatment until the end of the study, patients received treatment against Pneumocystis jirovecii (Pneumocystis jirovecii). Pneumocystis jiroveci Fungi and shingles Herpes Zoster Prophylactic treatment for viral infection. Patients received prophylactic treatment starting the day before the start of NI-0501 treatment (i.e., SD-1) until the end of the study. For example, for Pneumocystis jirovecii prophylaxis, patients may receive, for example, 750 mg / m² orally twice daily for three consecutive weeks in equal portions. 2 / sulfamethoxazole with 150 mg / m 2 / day of trimethoprim. For fungal infection prophylaxis, patients may receive, for example, fluconazole 12 mg / kg daily, up to 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 will be administered orally whenever possible, otherwise intravenously.

[0332] Patients may also receive any of the various concomitant therapies, such as cyclosporine A, intrathecal methotrexate, and glucocorticoids. Cyclosporine A (CsA) may continue if it was administered to the patient prior to screening. CsA may be withdrawn at any time. Once NI-0501 is started, CsA cannot be reintroduced during the study.

[0333] If the patient is receiving intrathecal methotrexate and glucocorticoids at the start of NI-0501 treatment, the treatment can be continued as needed. If CNS symptoms appear before the start of NI-0501 treatment, intrathecal methotrexate and glucocorticoid therapy must be started before the first NI-0501 administration.

[0334] In cases of confirmed immunoglobulin deficiency, IV immunoglobulin (IVIG) is permitted only as a replacement therapy. For example, in cases of confirmed immunoglobulin deficiency (deemed to warrant replacement therapy), IVIG may be administered at a dose of 0.5 g / kg every 4 weeks or more to maintain adequate IgG levels. Any infusions within the preceding 4 weeks prior to screening and any infusions during NI-0501 treatment are acceptable.

[0335] Analgesic therapy, blood product infusions, electrolyte and glucose infusions, antibiotics, antifungal and antiviral therapy, and general supportive care are permitted. Once the maximum NI-0501 dose level is reached, additional HLH treatments are permitted if HLH improvement is not maintained or is limited. As used herein, unmaintained HLH improvement is defined as a patient who cannot maintain at least 50% improvement from baseline for three HLH parameters (see Table 6 below). Loss of HLH improvement must be recorded at least twice consecutively. As used herein, limited HLH improvement is defined as a change of less than 50% from baseline across at least three clinical and laboratory criteria for HLH. Etoposide should be given as additional HLH treatment unless there is clear evidence of lack of response or intolerance to the drug from the patient's medical history.

[0336] The following therapies may not be concomitant with NI-0501 administration: Etoposide, T-cell depletion agents, or any other biological agents are generally not permitted, except for: G-CSF, in cases of prolonged neutropenia; rituximab, in cases of confirmed B-cell EBV infection; and other HLH treatments in cases where HLH improvement has not been maintained or is limited at the maximum NI-0501 dose level (as defined herein). Etoposide should be administered unless there is clear evidence of lack of response or intolerance to the drug from a medical history. Live or attenuated (including BCG) vaccines should be avoided throughout the study period, including the 4-week follow-up period. If NI-0501 concentrations remain at therapeutic levels at the end of the study, the period without vaccination should be extended until measurable concentrations of NI-0501 are no longer detectable.

[0337] The progression of clinical signs (fever, splenomegaly, CNS symptoms) and laboratory parameters (CBC, fibrinogen, ferritin, sCD25 levels) characterizing the disease was used to evaluate the achievement of a response and the time to a response. The primary efficacy endpoint included the overall response rate, i.e., the achievement of 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 period; duration of response, i.e., maintenance of the response achieved at any time during the study period up to EoT and beyond (including data collected in any long-term follow-up studies); number of patients who were able to reduce glucocorticoids to 50% or more of the baseline dose; number of patients who were able to undergo HSCT when deemed necessary; survival at week 8 (or EoT) and at the end of the study; serum concentrations of NI-0501 to determine the pharmacokinetic (PK) profile of NI-0501; determination of pharmacodynamic (PD) effects, including circulating total IFNγ and its neutralizing markers (i.e., CXCL9 and CXCL10); and determination of other biomarkers such as sCD25 and IL-10.

[0338] Table 6. Definition of a reaction Safety parameters to be collected and evaluated include the incidence, severity, precipitating factors, and outcomes (serious and non-serious) of adverse events (AEs), with particular attention to infections; laboratory parameters such as progression of complete blood count (CBC), with a focus on red blood cells (hemoglobin), neutrophils and platelets, liver function tests, kidney function tests, and coagulation; the number of patients who withdrew for safety reasons; and other parameters such as the level of circulating antibodies against NI-0501 (if any) to determine immunogenicity (ADA).

[0339] The primary endpoint (overall response rate) was evaluated using an exact binomial test at a one-sided 0.025 level. Time to response, response durability, and survival were provided using Kaplan-Meier curves, and, where feasible, as calculated medians. 95% confidence intervals were calculated for the medians of each of these endpoints. Other endpoints based on binary outcomes (including the number of patients with a 50% or higher reduction in glucocorticoids and the number of patients eligible for HSCT) were converted into proportions and their corresponding 95% confidence intervals were calculated. Statistical significance for the primary endpoint was obtained only in terms of p-value. All other endpoints were considered supportive of the primary endpoint; therefore, a formal endpoint hierarchy was not published.

[0340] Administration of NI-0501 in patients resulted in rapid normalization of fever within hours of the first NI-0501 infusion. Figure 13A and 13B This indicates the effect of NI-0501 infusion on body temperature in two patients with a body temperature >37.5℃ at the start of NI-0501 treatment. Figure 14 This is a series of charts describing the effect of NI-0501 administration on neutrophil counts in patients. Figure 15 This is a series of charts describing the effect of NI-0501 administration on platelet count in patients. Figure 16 This is a series of charts describing the effect of NI-0501 administration on serum ferritin levels in patients. Figure 17 This is a series of charts describing the effect of NI-0501 administration on the gradual reduction of glucocorticoid levels in patients. Figure 18 This is a diagram illustrating the maintenance of IFNγ neutralization with NI-0510 until HSCT. The response of HLH to NI-0501 treatment continued until transplantation. Patients were also evaluated for any CNS involvement following NI-0501 administration. A summary of baseline CNS involvement and status at the end of treatment (EOT) is shown in Table 11 below.

[0341] Table 11. Response to NI-0501 treatment - CNS involvement Note: Patients receiving IT therapy are excluded, except for patient #4, who did not receive routine IP administration. * Treatment continues $ Treatment will begin 2 weeks later. ^ Control group without EOT Of the 10 patients who underwent hematopoietic stem cell transplantation (HSCT), all were successfully engrafted; in one patient, CD34 stem cell enhancement was required at D+145 post-HSCT due to mixed chimerism. One patient experienced second transplant failure followed by HLH reactivation. This patient died at D+68 post-HSCT due to acute respiratory failure and bacterial infection. Another patient died at D+47 post-HSCT (septic shock in severe GvHD). Mild GvHD was reported in three other patients and was resolving / resolving.

[0342] Neutralizing serum concentrations of NI-0501 at HSCT were measured in 8 of the 10 transplant recipients, as reflected by CXCL9 levels (a chemokine strongly induced by IFNγ) below the limit of quantitation. Therefore, these data suggest that NI-0501 can eliminate the short- or long-term toxicities reported with etoposide-based regimens. This reduces the risk of translating into allogeneic HSCT-related complications.

[0343] These data indicate that NI-0501 treatment improves and / or eliminates clinical and laboratory abnormalities associated with HLH, including CNS signs and symptoms. Response to NI-0501 is independent of the presence and type of pathogenic mutations and / or the presence and type of infectious agents. NI-0501 is well tolerated. No safety concerns have been reported to date (e.g., no myelotoxicity, no widespread immunosuppression). No infections caused by pathogens known to be promoted by IFNγ neutralization have been observed. IFNγ neutralization with NI-0501 provides a novel targeted approach for the management of HLH.

[0344] Example 8 Safety, tolerability, pharmacokinetics, and efficacy of short-term intravenous administration of NI-0501 (an anti-interferon-γ (anti-IFNγ) monoclonal antibody) in patients with systemic juvenile idiopathic arthritis (sJIA) who have developed macrophage activation syndrome / secondary HLH (MAS / sHLH). This study was designed to demonstrate the efficacy and safety of NI-0501 in the treatment of MAS / sHLH in patients with sJIA. The study was divided into two parts: (i) a trial study to evaluate the pharmacokinetics (PK) profile and dosing strategy of NI-0501 and to preliminarily assess the benefits / risks of NI-0501 in this patient population; and (ii) a pivotal study to demonstrate the efficacy and safety of NI-0501 (to be continued once the dosing regimen and positive benefit / risk profile of NI-0501 are confirmed). An overview of the study design can be found in [link to study design]. Figure 19 .

[0345] The primary objectives of the trial were: (i) to determine the appropriate NI-0501 treatment dosage 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 disclose the pharmacokinetic (PK) profile of NI-0501 in sJIA patients with MAS / sHLH. The primary objectives of the key studies were: (i) to determine the efficacy of NI-0501 in sJIA patients with MAS / sHLH; (ii) to evaluate the safety and tolerability profile of short-term intravenous (iv) administration of NI-0501 in sJIA patients with MAS / sHLH; (iii) to confirm the positive benefit / risk profile of NI-0501 in sJIA patients with MAS / sHLH; (iv) to conduct exploratory evaluation of 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.

[0346] The study population included patients with sJIA and concomitant MAS / sHLH who showed inappropriate response to high-dose glucocorticoid therapy. Inclusion criteria included the following: (i) sex: male and female; (ii) age: <16 years at diagnosis of sJIA; (iii) a diagnosis of active MAS / sHLH confirmed by a treating rheumatologist with at least two of the following laboratory and clinical criteria: (a) laboratory criteria: platelet count ≤ 262 x 10⁻⁶. 9 / L, WBC count ≤ 4.0 x10 9 (a) AST levels >59 U / L and / or fibrinogen levels ≤ 2.5 g / L; (b) Clinical criteria: hepatomegaly, bleeding, and / or CNS dysfunction; (iv) Patients with an inappropriate response to high-dose IV glucocorticoid therapy for at least 3 days (including but not limited to 3 consecutive days of 30 mg / kg mPDN pulses), according to local standards of care; (v) High-dose IV glucocorticoids should not be lower than 2 mg / kg / day mPDN, equivalent to two separate daily doses up to 60 mg / day. In cases of rapid deterioration in patient condition and / or laboratory parameters, inclusion may occur within 3 days of the start of high-dose IV glucocorticoids; (vi) Patient consent (or consent of a legally authorized representative); and (vii) Patients who have been using contraception since puberty.

[0347] Exclusion criteria include: (i) a diagnosis of suspected or confirmed primary HLH or HLH occurring with neoplasia; (ii) patients treated with anaspirin, tocilizumab, canakinumab, TNF inhibitors, rituximab, or any other biological agent with a duration of 5 times their prescribed half-life; (iii) active mycobacterial (typical and atypical), Histoplasma capsulatum, Shigella spp., Salmonella spp., Campylobacter spp., and Leishmania spp. infections; (iv) evidence of latent tuberculosis; and (v) HIV antibody response. Positive serum; (vi) presence of malignancy; (vii) another comorbidity or malformation that seriously affects cardiovascular, pulmonary, CNS, liver, or kidney function, and in the investigator's opinion may seriously affect the likelihood of response to treatment and / or evaluation of the safety of NI-0501; (viii) history of hypersensitivity or allergic reaction to any component of the study protocol; (ix) received the BCG vaccine within 12 weeks prior to screening; (x) received another live or live attenuated vaccine within 6 weeks prior to screening; and / or (xi) pregnant or lactating women.

[0348] Dosing regimen, dosing frequency and duration of treatment In these studies, NI-0501 was used in the formulation shown in Example 7. In Part 1, NI-0501 was administered via infusion at an initial dose of 6 mg / kg over a 1-hour time interval at SD0. Treatment with NI-0501 was continued at a dose of 3 mg / kg every 3 days for 4 weeks (i.e., until SD27). NI-0501 treatment may be shortened upon achieving a complete clinical response (i.e., MAS remission). After 4 weeks, NI-0501 treatment may be continued as needed for up to another 4 weeks (i.e., until SD56) as maintenance until MAS remission is achieved, with the possibility of reducing the dose to 1 mg / kg and extending the interval between infusions to weekly dosing. If the PK profile shows unexpected TMDD (and therefore signals abnormally high IFNγ production), the dose of NI-0501 may be increased to 10 mg / kg as guided by clinical and PK evidence. This dose increase is only approved after careful evaluation of the benefit / risk profile in the individual patient.

[0349] In Part 2, the study may proceed only after the proposed dosing regimen is confirmed to be appropriate and a positive NI-0501 benefit / risk is demonstrated. Minor modifications to the dosing regimen may be made as needed, based on the evidence obtained in Part 1.

[0350] Background therapy and concomitant therapy:NI-0501 is administered against a background of at least 2 mg / kg methylprednisolone (mPDN) equivalent up to 60 mg / day (in patients weighing 30 kg or more) (which may be gradually reduced during treatment depending on the patient's condition). Patients receive prophylactic treatment for herpes zoster infection, preferably starting the day before (and in any case prior to initiating NI-0501 treatment) until serum NI-0501 levels are no longer detectable. Cyclosporine A (CsA) may be continued if started at least 3 days before initiating NI-0501 treatment. CsA dose adjustments are permitted to maintain therapeutic levels. CsA may be withdrawn at any time during the study period at the investigator's discretion. CsA cannot be reintroduced once NI-0501 administration has begun. If the patient is receiving intrathecal methotrexate and glucocorticoids at the start of NI-0501 treatment, treatment may be continued as needed. Throughout the study and under no circumstances should live or attenuated (including BCG) vaccines be administered until serum NI-0501 levels are no longer detectable. Analgesic therapy, blood product infusions, electrolyte and glucose infusions, antibiotics, antifungal and antiviral therapy, and general supportive care are permitted.

[0351] Sample size In Part 1, at least 5 evaluable patients may be recruited. In Part 2, at least 10 evaluable patients may be recruited to reach a total of 15 evaluable patients as the study continues. Given the rare orphan nature of this disease and the lack of any approved treatment, a formal validation sample size of 15 patients is not possible. However, based on the assumption that at least 50% of patients respond inadequately to systemic glucocorticoids alone—that is, 50% of patients using glucocorticoids achieve MAS remission by week 8 after initiation of treatment—and applying a 5% unilateral significance level, the study could have 70% power to detect improvements ranging from 50% to 77%.

[0352] Study duration and end-of-study definition: The study duration may be 8 weeks for each patient (plus a maximum of 1 week for screening). The end of the study is defined as the last follow-up of the last patient. All patients required to receive at least one dose of NI-0501 are eligible to participate in the NI-0501-05 study for long-term follow-up.

[0353] Study endpointsIn Part 1 (experimental), the following were evaluated to confirm the dosing regimen for this patient population: (i) the benefit / risk profile of NI-0501; (ii) the pharmacokinetic profile of NI-0501; (iii) the levels of known IFNγ-induced chemokines (e.g., CXCL9, CXCL10, CXCL11); (iv) the progression of distinctly different characteristics of MAS in cytopenia, hepatic dysfunction, and coagulopathy at 2, 4, 6, and 8 weeks after NI-0501 initiation; and (v) the dose and duration of NI-0501 treatment. In Part 2 (Critical) of the study, the efficacy endpoints were as follows: (a) primary efficacy endpoint: number of patients achieving MAS remission at week 8 after initiation of NI-0501 treatment; and (b) secondary efficacy endpoints: time to MAS remission as assessed by the investigator; time to initial response; number of patients at any time during the study period who could be progressively reduced to the same (or lower) dose given before the onset of MAS; time to progressive reduction of glucocorticoids; survival at the end of the study; and number of patients who withdrew from the study due to lack of efficacy. In Part 2 (Critical) of the study, the safety endpoints were as follows: (a) incidence, severity, precipitating factors, and outcomes (serious and non-serious), with particular attention to infections; progression of laboratory parameters, particularly CBC (focusing on hemoglobin, neutrophils, and platelets), LFTs, and coagulation parameters; number of patients who withdrew from the study for safety reasons; and the level of circulating antibodies against NI-0501 (if any) used to determine immunogenicity (ADA).

[0354] Pharmacokinetics and pharmacodynamics were evaluated using the PK profile of NI-0501; the levels of circulating free IFNγ before administration and total IFNγ (free IFNγ+ bound to NI-0501) after initiation of NI-0501; the levels of known IFNγ-induced chemokines (e.g., CXCL9, CXCL10, CXCL11); the correlation between chemokine levels (CXCL9, CXCL10) and levels of free NI-0501, free IFNγ (before administration), and total IFNγ; the correlation between chemokine and total IFNγ levels and laboratory parameters of MAS severity (e.g., ferritin, platelet count, LFTs (experimental analysis)); and the levels of other potential disease biomarkers (e.g., sCD25, IL-10, IL-6, IL-18, TNFα, neopterin).

[0355] Other implementation plans Although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. A method of treating hemophagocytic lymphohistiocytosis (HLH) in a subject of need, the method comprising administering a therapeutically effective amount of an antibody or an antigen-binding fragment thereof, which binds to interferon-γ (IFNγ) and contains a variable heavy chain complementarity-determining region 1 (VH CDR1) comprising the amino acid sequence SYAMS (SEQ ID NO: 1); a variable heavy chain complementarity-determining region 2 (VH CDR2) comprising the amino acid sequence AISGSGGSTYYADSVKG (SEQ ID NO: 2) and a variable heavy chain complementarity-determining region 3 (VH CDR3) comprising the amino acid sequence DGSSGWYVPHWFDP (SEQ ID NO: 3); a variable light chain complementarity-determining region 1 (VL CDR1) comprising the amino acid sequence TRSSGSIASNYVQ (SEQ ID NO: 4); a variable light chain complementarity-determining region 2 (VL CDR2) comprising the amino acid sequence EDNQRPS (SEQ ID NO: 5) and a variable light chain complementarity-determining region 2 (VL CDR2) comprising the amino acid sequence QSYDGSNRWM (SEQ ID NO: 5). 6) Variable light chain complementarity determination region 3 (VL CDR3).

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. The method of claim 1, wherein the antibody is formulated into a sterile concentrated preparation for infusion.

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

2.

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

0.

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

7. The method of claim 6, wherein the antibody is administered to the subject in need at an initial dose of 1 mg / kg within 1 hour following the initial IV infusion via at least one additional IV infusion.

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

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

10. The method of claim 6 or claim 9, wherein the at least one additional IV infusion is given at least 3 days after the initial IV infusion.

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